Bioethanol Author

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BIOETHANOL
Biochemistry and Biotechnological Advances
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BIOETHANOL
Biochemistry and Biotechnological Advances
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Edited by
Ayerim Y. Hernández Almanza, PhD
Nagamani Balagurusamy, PhD
Héctor Ruiz Leza, PhD
Cristóbal N. Aguilar, PhD
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First edition published 2023
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Library and Archives Canada Cataloguing in Publication

Title: Bioethanol : biochemistry and biotechnological advances / edited by Ayerim Y. Hernández Almanza, PhD, Nagamani
Balagurusamy, PhD, Héctor Ruiz Leza, PhD, Cristóbal N. Aguilar, PhD.
Other titles: Bioethanol (Palm Beach, Fla.)
Names: Hernández Almanza, Ayerim Y., editor. | Balagurusamy, Nagamani, editor. | Leza, Héctor Ruiz, editor. | Aguilar,
Cristóbal N., editor.
Description: First edition. | Includes bibliographical references and index.
Identifiers: Canadiana (print) 20220137609 | Canadiana (ebook) 20220137617 | ISBN 9781774638491 (hardcover) |
ISBN 9781774634875 (softcover) | ISBN 9781003277132 (ebook)
Subjects: LCSH: Biomass conversion. | LCSH: Ethanol as fuel. | LCSH: Ethanol.
Classification: LCC TP248.B55 B56 2022 | DDC 662/.88—dc23
Library of Congress Cataloging-in-Publication Data
CIP data on file with US Library of C

ongress
ISBN: 978-1-77463-849-1 (hbk)

ISBN: 978-1-77463-487-5 (pbk)
ISBN: 978-1-00327-713-2 (ebk)
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About the Editors
Ayerim Y. Hernández Almanza, PhD

Full Professor, School of Biological Sciences,
Autonomous University of Coahuila, Torreón, Coahuila, México
Ayerim Y. Hernández Almanza, PhD, is Professor (full-time) at the School
of Biological Science, the Autonomous University of Coahuila, since 2019.
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Prof. Hernández Almanza’s experience is in extraction and purification of
bioactive compounds, fermentation process, microbial pigments production,
and biological characterization. She is recognized as a member (candidate)
of the National System of Research (SNI) by the National Council of Science
and Technology of the Government of México. In 2019 she obtained the
“Mujer Universitaria” award in Scientific Ambit given by the Autonomous
University of Coahuila. Prof. Hernández-Almanza has published over 10
original research papers in indexed journals and 12 book chapters and has
participated and contributed in more than five scientific meetings. She has
realized two research stays: at Università degli Studi di Perugia, Perugia,
Italia (2013), and at Gachon University, Seongnam, Gyeonggi-do, South
Korea (2016).
Nagamani Balagurusamy, PhD

Full Professor, Bioremediation Lab, Faculty of Biological Sciences,
Autonomous University of Coahuila, Torreón, Coahuila, México
Nagamani Balagurusamy, PhD, is Professor (full-time) at the Autonomous
University of Coahuila, Maxico, since September 2001. He is head of the
bioremediation lab of the Faculty of Biological Sciences, Torreon campus,
and coordinator of the graduate program on biochemical engineering, which is
recognized as of the National Post-Graduate Programs of Quality (PNPC) of
the Council of Science and Technology of the Federal Government of Mexico.
He is also a visiting Professor of the Graduate Program on Biotechnology of the
DepartmentofBiology,WestVirginiaStateUniversity,WV,USA.Hehaspublished
more than 70 scientific articles in international and national indexed journals,
6 books, and more than 40 chapters in books published by Springer, Elsevier,
CRC, and others. He has participated in more than 75 international and national
conferences. He is a member of the National Researchers Council of Mexico
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vi About the Editors
(SNI-CONACYT, Level I distinction) and also a regular member of Mexican

Academy of Sciences
Héctor Ruiz Leza, PhD

Full Professor, Biorefinery Group, School of Chemistry,
Autonomous University of Coahuila, Saltillo, Coahuila, México
Héctor Ruiz Leza, PhD, is currently a full Professor in the Biorefinery
Group of the Faculty of Chemistry Sciences at the Autonomous University
of Coahuila. He is the founder of Biorefinery Group, principal coordinator
of the biorefinery pilot plant in the Food Research Department, and leader of
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the biomass pretreatment stage in the Cluster of Bioalcoholes in the Mexican
Centre for Innovation in Bioenergy (Cemie-Bio) from the Secretary of
Energy in Mexico. Dr. Ruiz obtained his PhD in Chemical and Biological
Engineering from the Center of Biological Engineering at the University of
Minho, Portugal, in 2011 and was a postdoctoral researcher at the University
of Minho (Portugal) and University of Vigo (Spain).
Dr. Ruiz is currently working on the development of biorefinery strategies
for the production of high added-value compounds and biofuels from ligno-
cellulosic, micro, and macroalgal biomass in the context of the bioeconomy.
He is technically responsible for several research projects (CONACYT) and
a technical consultant for biomass conversion companies in Mexico and
Europe. Dr. Ruiz has conducted several research stays and technical visits at
the University of North Texas (USA), Federal University of Sergipe (Brazil),
Brazilian Bioethanol Science and Technology Laboratory-CTBE (Brazil),
Chemical and Biological Engineering Department at the University of British
Columbia (Canada), CIEMAT-Renewable Energy Division, Biofuels Unit
(Spain), University of Jaén (Spain), Sadar Swaran Singh National Institute
of Bio-Energy (India), Tokyo Institute of Technology (Japan), University
of Concepción (Chile), Umeå University (Sweden), National Laboratory
of Energy and Geology-LNEG (Portugal), CSIR-National Institute for
Interdisciplinary Science and Technology (India), University of Florida-Stan
Mayfield Biorefinery Plant (USA), University of Kannur (India), Federal
University of Rio Grande do Norte (Brazil).
He has authored or co-authored 75 publications, including papers in
indexed journals and chapters, with over 2300 citations and an h-index of
28 (Google Scholar Citations). Currently, Dr. Ruiz is Editor-in-Chief of
BioEnergy Research Journal (Springer Publishing) and Associate Editor of
Biotechnology for Biofuels (BioMed Central-Part of Springer Nature), and
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About the Editors vii
he participates in the Editorial Advisory Board of Industrial Crops and Prod-

ucts (Elsevier Editorial) and Biofuel Research Journal. He was the editor of
the book Hydrothermal Processing in Biorefineries published by Springer
Publishing in 2017. Dr. Ruiz was awarded with the prize “Dr. Carlos Casas
Campillo” of the Mexican Society of Biotechnology and Bioengineering in

2016. This award aims to give recognition and encourage young researchers
for their contribution to the development of biotechnology and bioengi-
neering in Mexico. Dr. Ruiz has been a member of the Mexican Academy of
Science in the engineering section since 2020. He received a Young Scientist
Award at the Autonomous University of Coahuila for the year 2019, and he is
a member of the National Researchers Council in Mexico (SNI-CONACYT,
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Level II distinction).
Cristóbal N. Aguilar, PhD

Full Professor, Associate Editor of Heliyon (Microbiology) and
Frontiers in Sustainable Food Systems (Food Processing), Bioprocesses,
and Bioproducts Research Group, Food Research Department,
School of Chemistry, Autonomous University of Coahuila, Saltillo, Mexico
Cristóbal N. Aguilar, PhD, is a Director of Research and Postgraduate
Programs at the Autonomous University of Coahuila, Mexico. His scientific
impact has an index of more than 41 h. He has been awarded several awards,
the most important of which are the Outstanding Scientific Award by the
International Bioprocessing Association 2018; the State Prize for Science,
Technology, and Innovation Coahuila 2019; the National Research Award
2010 of the Mexican Academy of Sciences; the Carlos Casas Campillo 2008
Award from the Mexican Society of Biotechnology and Bioengineering;
AgroBio National Prize-2005; and the Mexican Food Science and Technology
Award.
Dr. Aguilar is a member of the Mexican Academy of Sciences, the Inter-
national Bioprocessing Association, the Mexican Academy of Sciences, the
Mexican Society of Biotechnology and Bioengineering, and the Mexican
Association of Food Science and Biotechnology. He has developed more
than 50 research projects, including 20 international exchange projects.
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Contents
Contributors..............................................................................................................xi
Abbreviations......................................................................................................... xvii
Acknowledgment..................................................................................................... xxi
Preface..................................................................................................................xxiii
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1. Physiology of Ethanol Production by Yeasts.................................................1
Miriam Soledad Valenzuela Gloria, Diana Laura Alva-Sánchez,
M. P. Luévanos Escareño, Cristóbal N. Aguilar, Nagamani Balagurusamy,
and Ayerim Hernández-Almanza
2. Physiology of Ethanol Production by Zymomonas mobilis.........................21

Laura Andrea Pérez-García, Cindy Nataly Del Rio-Arellano,
David Francisco Lafuente Rincón, and Norma M. De La Fuente-Salcido
3. Physiology of Ethanol Production by Clostridium thermocellum..............43

D. B. Arya, Salom Gnana Thanga Vincent, and Nagamani Balagurusamy
4. Genetic Regulation of Principal Microorganisms

(Yeast, Zymomonas mobilis, and Clostridium thermocellum)
Producing Bioethanol/Biofuel.......................................................................53
Dania Sandoval-Nuñez, Teresa Romero-Gutiérrez, Melchor Arellano-Plaza,
Anne Gschaedler, and Lorena Amaya-Delgado
5. Metabolic Engineering of Yeast, Zymomonas mobilis, and Clostridium

thermocellum to Increase Yield of Bioethanol.............................................97
S. Sánchez-Muñoz, M. J. Castro-Alonso, F. G. Barbosa, E. Mier-Alba,
T. R. Balbino, D. Rubio-Ribeaux, I. O. Hernández-De Lira, J. C. Santos,
C. N. Aguilar, and S. S. Da Silva
6. Increasing Ethanol Tolerance in Industrially Important Ethanol
Fermenting Organisms................................................................................141
Kalyanasundaram Geetha Thanuja and Subburamu Karthikeyan
7. Challenges in Developing Sustainable Fermentable Substrate

for Bioethanol Production...........................................................................161
Charu Saraf and Kakoli Dutt
8. Emerging Strategies for Ethanol Purification...........................................195

Alan D. Perez, Javier Quintero, and Juan A. León
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x Contents
9. Design and Engineering Parameters of Bioreactors for

Production of Bioethanol.............................................................................233
David Francisco Lafuente-Rincón, Inty Omar Hernández-De Lira,
Héctor Hernández-Escoto, María Alejandra Sánchez-Muñoz,
Héctor Hugo Molina Correa, Cristian Emanuel Gámez-Alvarado,

Perla Araceli Meléndez-Hernández, and Javier Ulises Hernández-Beltrán
10. Integrated Production of Ethanol from Starch and Sucrose...................... 271
C. A. Prado, S. Sánchez-Muñoz, R. T. Terán-Hilares, L. T. Carvalho,
L. G. De Arruda, M. L. Silva da Cunha, P. Abdeshahian, S. S. Da Silva,
N. Balagurusamy, and J. C. Santos
11. Conversion of Sweet Sorghum Juice to Bioethanol..................................315
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Iosvany López-Sandin, Francisco Zavala-García, Guadalupe Gutiérrez-Soto,
and Héctor Ruiz Leza
12. Biotechnology Development of Bioethanol from Sweet

Sorghum Bagasse.........................................................................................339
Daniel Tinôco
13. Bioethanol Production Using Agave americana L. Leaves

Wastes From Coahuila................................................................................371
César D. Pinales-Márquez, Shiva, Rohit Saxena, Rosa M. Rodríguez-Jasso, and
Héctor Ruiz Leza
14. Sustainable Ethanol Production from Lignocellulosic Biomass:

A Water Footprint Analysis Over Pretreatment Technologies.................401
Oznur Yildirim, Dogukan Tunay, Bestami Ozkaya, and Ahmet Demir
15. Biodegradation and Biocatalysis Aspects of Direct Bioethanol

Production by Fungi in a Single Step Named
Consolidated Bioprocessing........................................................................419
Luis Fernando Amador Castro and Danay Carrillo Nieves
16. Bioelectrosynthesis of Ethanol via Bioelectrochemical Systems:

Future Perspectives......................................................................................445
Silvia Yudith Martínez Amador, Leopoldo Javier Ríos González,
Miguel Angel Medina Morales, Mónica Margarita Rodríguez Garza,
Ileana Mayela María Moreno Dávila, Thelma Karina Morales Martínez, and
José Antonio Rodríguez de la Garza
17. Challenges and Perspectives on Application of Biofuels in the

Transport Sector..........................................................................................463
F. G. Barbosa, S. Sánchez-Muñoz, E. Mier-Alba, M. J. Castro-Alonso,
R. T. Hilares, P. R. F. Marcelino, C. A. Prado, M. M. Campos, A. S. Cardoso,
J. C. Santos, and S. S. Da Silva
Index......................................................................................................................501
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Contributors
P. Abdeshahian
Biopolymers, Bioprocesses, Process Simulation Laboratory, Department of Biotechnology,
Engineering School of Lorena, University of São Paulo (EEL-USP), Lorena–12.602.810, SP, Brazil
Cristóbal N. Aguilar
Bioprocesses and Bioproducts Research Group, BBG-DIA, Food Research Department,
School of Chemistry, Autonomous University of Coahuila (UAdeC), Saltillo–25280, Coahuila, Mexico
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Diana Laura Alva-Sánchez
School of Biological Science, Autonomous University of Coahuila, Torreón–27000, Coahuila, Mexico
Silvia Yudith Martínez Amador

Botany Department, Agronomic Division, Antonio Narro Autonomous Agrarian University, Mexico
Lorena Amaya-Delgado
Industrial Biotechnology Unit, Center for Research and Assistance in Technology and Design of the State
of Jalisco A.C., Camino Arenero–1227, El Bajio del Arenal, C.P.–45019, Zapopan, Jalisco, Mexico
Melchor Arellano-Plaza
L. G. De Arruda
D. B. Arya
Department of Environmental Sciences, University of Kerala, Thiruvananthapuram, Kerala, India
Nagamani Balagurusamy
Bioremediation Laboratory, Biological Science Faculty, School of Biological Science,
Autonomous University of Coahuila (UAdeC), Torreón–27000, Coahuila, Mexico
T. R. Balbino
Bioprocesses and Sustainable Products Laboratory, Department of Biotechnology,
F. G. Barbosa
M. M. Campos
A. S. Cardoso
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xii Contributors
L. T. Carvalho
Bioprocesses and Sustainable Products Laboratory, Department of Biotechnology, Engineering School
of Lorena, University of São Paulo (EEL-USP), Lorena–12.602.810, SP, Brazil
Luis Fernando Amador Castro

Tecnologico de Monterrey, Escuela de Ingenieria y Ciencias, Av. General Ramon Corona No. 2514,
Zapopan–45201, Jal., Mexico
M. J. Castro-Alonso
Héctor Hugo Molina Correa

Bioremediation Laboratory, Biological Sciences Faculty, Autonomous University of Coahuila,
Torreón–27000, Coahuila, México
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M. L. Silva da Cunha
Engineering School of Lorena, University of São Paulo (EEL-USP), 12.602.810, Lorena, SP, Brazil
Ileana Mayela María Moreno Dávila
Department of Biotechnology, School of Chemistry, Autonomous University of Coahuila, Mexico
Ahmet Demir
Department of Environmental Engineering, Yildiz Technical University, Turkey, Davutpasa Campus,
Istanbul–34220, Turkey
Kakoli Dutt
Department of Bioscience and Biotechnology, Banasthali Vidyapith, Rajasthan–304022, India,
Tel.: 9887398384, E-mail: kakoli_dutt@rediffmail.com
M. P. Luévanos Escareño
Norma M. De La Fuente-Salcido
Faculty of Biological Sciences, Autonomous University of Coahuila, Torreón, Coahuila, México,
E-mail: normapbr322@gmail.com
Cristian Emanuel Gámez-Alvarado

José Antonio Rodríguez de la Garza

Department of Biotechnology, School of Chemistry, Autonomous University of Coahuila, Mexico,
Phone: +52 (844) 415-5752–ext 120, E-mail: antonio.rodriguez@uadec.edu.mx
Mónica Margarita Rodríguez Garza

Miriam Soledad Valenzuela Gloria

Leopoldo Javier Ríos González

Anne Gschaedler
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Contributors xiii
Guadalupe Gutiérrez-Soto
University of Nuevo León, Faculty of Agronomy, Francisco Villa S/N Col. Ex Hacienda
El Canadá–66415, General Escobedo, N. L., México
Ayerim Hernández-Almanza
School of Biological Science, Autonomous University of Coahuila, Torreón–27000, Coahuila, Mexico,

E-mail: ayerim_hernandez@uadec.edu.mx
Javier Ulises Hernández-Beltrán
Torreón–27000, Coahuila, México, Tel.: +52-871-7571-785, E-mail: ulises.hernandez@uadec.edu.mx
Héctor Hernández-Escoto
Chemical Engineering Department, University of Guanajuato, Noria Alta s/n, Guanajuato, 36050, Mexico
R. T. Hilares
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Material Laboratory, Catholic University of Santa Maria (UCSM), Yanahuara, AR–04013, Peru
Subburamu Karthikeyan
Department of Renewable Energy Engineering, Tamil Nadu Agricultural University, Coimbatore,
Tamil Nadu, India, E-mail: skarthy@tnau.ac.in
David Francisco Lafuente-Rincón
Bioprocesses and Bioprospecting Laboratory, Biological Sciences Faculty,
Autonomous University of Coahuila, Torreón–27000, Coahuila, México
Juan A. León
Laboratory of Process Intensification and Hybrid System (LIPSH), Department of Chemical Engineering,
National University of Colombia, Manizales, Caldas, Colombia, E-mail: jaleonma@unal.edu.co
Inty Omar Hernández-De Lira

Bioremediation Laboratory, Faculty of Mechanical and Electrical Engineering, Biological Science
Faculty, Autonomous University of Coahuila (UAdeC), Torreón–27000, Coahuila, México
Iosvany López-Sandin
El Canadá–66415, General Escobedo, N. L., México
P. R. F. Marcelino
Thelma Karina Morales Martínez

Bioprocess and Microbial Biochemistry Group, School of Chemistry,
Autonomous University of Coahuila, Mexico
Perla Araceli Meléndez-Hernández

Chemical Engineering Department, University of Guanajuato, Noria Alta s/n, Guanajuato, 36050, Mexico
E. Mier-Alba
Miguel Angel Medina Morales

Danay Carrillo Nieves

Tecnologico de Monterrey, Escuela de Ingenieria y Ciencias, Av. General Ramon Corona No. 2514,
Zapopan–45201, Jal., Mexico
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xiv Contributors
Bestami Ozkaya
Alan D. Perez
Sustainable Process Technology (SPT), University of Twente, Enschede, Overijssel, The Netherland,
E-mail: a.d.perezavila@utwente.nl
Laura Andrea Pérez-García
Faculty of Biological Sciences, Autonomous University of Coahuila, Torreón, Coahuila, México
César D. Pinales-Márquez
Biorefinery Group, Food Research Department, Faculty of Chemistry Sciences,
Autonomous University of Coahuila, Saltillo–25280, Coahuila, Mexico
C. A. Prado
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Javier Quintero
Department of Chemical Engineering, National University of Colombia, Manizales, Caldas, Colombia,
E-mail: jaquinteroj@unal.edu.co
David Francisco Lafuente Rincón

Cindy Nataly Del Rio-Arellano

Rosa M. Rodríguez-Jasso
Biorefinery Group, Food Research Department, Faculty of Chemistry Sciences, Autonomous University
of Coahuila, Saltillo–25280, Coahuila, Mexico, Phone: (+52)-1-844-416-12-38,
E-mail: rrodriguezjasso@uadec.edu.mx
Teresa Romero-Gutiérrez
Computer Sciences Department, Exact Sciences and Engineering University Centre, Universidad de
Guadalajara, Blvd. Gral. Marcelino Garcia Barragan–1421, Olimpica. Guadalajara, Mexico
D. Rubio-Ribeaux
Héctor Ruiz Leza

Biorefinery Group, Food Research Department, Faculty of Chemistry Sciences, Autonomous University
of Coahuila, Saltillo–25280, Coahuila, Mexico, E-mail: hector_ruiz_leza@uadec.edu.mx
María Alejandra Sánchez-Muñoz

S. Sánchez-Muñoz
Engineering School of Lorena, University of São Paulo (EEL-USP), Lorena–12.602.810, SP, Brazil,
E-mails: salvador.sanchez@usp.br; sanchezmunoz.ssm@gmail.com
Dania Sandoval-Nuñez
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Contributors xv
J. C. Santos
Biopolymers, Bioreactors, and Process Simulation Laboratory, Department of Biotechnology,
Engineering School of Lorena, University of São Paulo (EEL-USP), 12.602.810. Lorena, SP, Brazil,
E-mail: jsant200@usp.br
Charu Saraf
Department of Bioscience and Biotechnology, Banasthali Vidyapith, Rajasthan–304022, India,
Tel.: 9910331924, E-mail: charusaraf17@gmail.com
Rohit Saxena
Shiva
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S. S. Da Silva
Engineering School of Lorena, University of São Paulo (EEL-USP), 12.602.810. Lorena, SP, Brazil,
E-mail: silviosilverio@gmail.com
R. T. Terán-Hilares
Material Laboratory, Catolic University of Santa Maria (UCSM), Yanahuara–04013, AR, Perú
Kalyanasundaram Geetha Thanuja

Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore,
Tamil Nadu, India
Daniel Tinôco
Biochemical Engineering Department, School of Chemistry, Federal University of Rio de Janeiro,
Rio de Janeiro–21941909, RJ, Brazil, E-mail: dneto@peq.coppe.ufrj.br
Dogukan Tunay
Salom Gnana Thanga Vincent

Department of Environmental Sciences, University of Kerala, Thiruvananthapuram, Kerala, India
Oznur Yildirim
Francisco Zavala-García
El Canadá–66415, General Escobedo, N. L., México, E-mail: francisco.zavala.garcia@gmail.com
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Abbreviations
AAA aromatic amino acids

ABTS 2,20-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)
AD anaerobic digestion
ADH alcohol dehydrogenase
ADH2 alcohol dehydrogenase II
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AFEX ammonia fiber expansion
ALDH acetaldehyde by aldehyde dehydrogenase
ARP ammonia recycling percolation
ATPS aqueous two-phase systems
BFP biofuture platform
C4H cinnamate 4-hydroxylase
CBP consolidated bioprocessing
CCCF closed-circulating fermentation system
CEMIE-Bio Mexican Center for Innovation in Bioenergy
CEs carbohydrate esterases
CMFS continuous membrane fermenter separator
CNOOC China National Offshore Oil Corporation
CO2 carbon dioxide
COMT caffeic acid 3-O-methyltransferase
CRISPR clustered regularly interspaced short palindromic repeats
CS chitosan
CSIR Industrial Scientific Research Council
CSTR continuous stirred tank reactors
CWI cell wall integrity
DDGs distillers dried grains
DDG dry distillation grain
DESs deep eutectic solvents
DHAP dihydroxyacetone phosphate
DMC direct microbial conversion
DP degree of polymerization
DSSF delayed simultaneous saccharification and fermentation
DyP dye decolorizing peroxidase
EA ethanol adaption
ED Entner-Doudoroff
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xviii Abbreviations
ED extractive distillation
EISA energy independence and security act
EMP Embden-Meyerhof-parnas
EPA energy policy act
EPS exopolysaccharides
ESR environmental stress response
EtOH alcohol
EU European Union
FISH fluorescent in situ hybridization
FST fiber separation technology
G3P glycerol-3-phosphate
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GAP glyceraldehyde-3-phosphate
GBEP global bioenergy partnership
GHG greenhouse gas
GRAS generally recognized as safe
gTME global transcription machinery engineering
GW global warming
H2O2 hydrogen peroxide
HAA 3-hydroxyanthranilic acid
HAD homogenous azeotropic distillation
HC hydrodynamic cavitation
HCl hydrochloric acid
HCR hydrodynamic cavitation reactors
HCT hydroxycinnamoyl transferase
HER hydrogen evolution reaction
HMF 5-hydroxymethylfurfural
HOG high osmolarity glycerol
HPLC high-performance liquid chromatography
HR homologous recombination
HSP heat shock proteins
HTAD heterogeneous azeotropic distillation
HTLR hydrothermal liquefaction reactors
HyPol hyperbranched polymer
iHG integrated high gravity
IL ionic liquids
IMHeRE intelligent microbial heat-regulating engine
ISC iron-sulfur clusters
ISPR in situ product recovery
KDPG 2-keto-3-deoxy-6-phosphogluconic acid
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Abbreviations xix
LBG liquefied biogas

LCA life cycle assessment
LCB lignocellulosic biomass
LDH lactate dehydrogenase
LHW liquid hot water

LiP lignin peroxidase
LLX liquid-liquid extraction
MAVS membrane assisted vapor stripping
MDC microbial desalination cell
Mdh malate dehydrogenase
MEC microbial electrolysis cell
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MEDC microbial electrodialysis cell
MES microbial bioeletroshyntesis
MFC microbial fuel cell
MnP manganese peroxidase
MREC microbial reverse electrodialysis electrolysis cell
MWIR microwave irradiation reactor
MWR microwave reactors
NGS next-generation sequencing
NRC national research council
OD oven-dried
OPEC Organization of Arab Petroleum Exporting Countries
PA phosphatidic acid
PDC pyruvate decarboxylase
PDMS polydimethylsiloxane
PFD prefoldin
Pgd phosphogluconate dehydrogenase
PKA protein kinase A
PM particle matters
PPP pentose phosphate pathway
PSA pressure swing adsorption
PSSF pre-saccharification and fermentation strategy
PTA phosphotransacetylase
PTMSP poly(1-trimethylsily-1-propyne)
PV pervaporation
RDB renewable diesel blendstock
REFINE RNA seq examiner for phenotype-informed network engineering
RFS renewable fuel program
RGI rhamnogalacturonan I
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xx Abbreviations
RKI1 ribose-5-phosphate isomerase

RNAi RNA-interference
ROS reactive oxygen species
SAA soaking aqueous ammonia
SAGA Spt-Ada-Gcn-5 acetyltransferase

SARSH sulfuric acid-treated rice straw hydrolysate
SBP sugar beet pulp
SBR sequencing batch reactor
SE steam explosion
SHCF separate hydrolysis and co-fermentation
SHF separate hydrolysis and fermentation
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SMT selective milling technology
sRNA small RNA
SSCF simultaneous saccharification and co-fermentation
SSF simultaneous saccharification and fermentation
STBR stirred tank bioreactors
STR stirred tank reactor
Tal transaldolase
TALEN’s transcription activator-like effector nuclease
TAME TALEN assisted multiplex editing
TEM transmission electron microscopy
TEMPOL 4-Hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl
TF transcription factors
TiO2 titanium dioxide
TKL1 transketolase
TPP trehalose-6-phosphate phosphatase
TPS trehalose-6-phosphate synthase
VP versatile peroxidase
WW water washing
XDH xylitol dehydrogenase
XGA xylogalacturonan
XI xylose isomerase
XOs xylooligosaccharides
XR xylose reductase
XYL1 xylose reductase heterologous
ZFN zinc finger nuclease
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Acknowledgment
The editors thank the researchers who participated as reviewers of the

submitted chapters. Their dedicated time in the revision and the experience
that each one has in the different topics contributed to the improvement and
quality of this book.
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• Raúl Rodríguez Herrera
• Arturo Sócrates Palacios
ESPOL Polytechnic University, Ecuador
• Leopoldo Javier Ríos González
• Alejandra Alvarado
University of Tübingen, Germany
• Danay Carrillo Nieves
Tecnológico de Monterrey, Mexico
• Aldo Ricardo Almeida Robles
University of Copenhagen, Denmark
• Miguel Ángel Medina Morales
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Preface
Worldwide, various industries generate waste during the production processes

they implement. These wastes range from vegetable material (such as seeds,
skin, bagasse, among others) to synthetic material (such as plastics). In most
cases, it is a serious environmental problem; however, some plant residues,
mainly generated by food industries, have been analyzed due to their high
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biological potential. A clear example is the use of corn and sugarcane
bagasse, rich in sugars, to obtain bioethanol. Bioethanol is one of the most
interesting biofuels since it has a positive characteristic on the environment.
During the last decades, there has been a great interest in the production
and use of biodiesel or bioethanol as promising alternatives to replace fossil
fuels. For this reason, the development of biorefineries and processes, as well
as the design of bioreactors, have been fundamental issues to understand this
area. Likewise, the study of metabolic and physiological processes that are
carried out by some bioethanol-producing microorganisms is also necessary
to know. Yeast, like Zymomonas mobilis and Clostridium thermocellum,
have been demonstrated to have a prominent role in bioethanol production.
However, it is necessary to delve into factors such as increasing the tolerance
of these strains to produce bioethanol, improve genetic regulation and
implement genetic engineering methods that favor production yields.
On the other hand, the development of tools and strategies that allow
obtaining bioethanol of major quality, viable alternatives for purification of
the product are factors that still have to be improved. Bioethanol production
has proven to be a promising option to reduce the damage caused by the
conventional processes used to obtain fuels; therefore, there are still some
challenges to overcome.
This book includes the advances and perspectives within the bioethanol
industry, also describes some biochemical and physiological parameters
carried out by the main bioethanol producing microorganisms as well as the
potential applications that this bioproduct can have and the advantage that it
would generate.
—Editors
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CHAPTER 1
Physiology of Ethanol Production by

Yeasts
MIRIAM SOLEDAD VALENZUELA GLORIA,1
DIANA LAURA ALVA-SÁNCHEZ,1 M. P. LUÉVANOS ESCAREÑO,1
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CRISTÓBAL N. AGUILAR,2 NAGAMANI BALAGURUSAMY,1 and
AYERIM HERNÁNDEZ-ALMANZA1
1
School of Biological Science, Autonomous University of Coahuila,
Torreón–27000, Coahuila, Mexico,
E-mail: ayerim_hernandez@uadec.edu.mx (A. Hernández-Almanza)
2
Bioprocesses and Bioproducts Research Group, BBG-DIA, Food Research
Department, School of Chemistry, Autonomous University of Coahuila,
Saltillo–25280, Coahuila, Mexico
ABSTRACT
Yeasts have been gaining popularity due to their ability to produce a series
of compounds of interest such as pigments, phenolic compounds, fatty acids,
enzymes, and even under the right conditions, and they are ethanol producers.
The latter is characterized by producing ethanol and carbon dioxide (CO2)
under anaerobic conditions using fermentable sugars as substrates. To
consider that yeast is suitable for the production of ethanol, it is desirable
that it meets some characteristics of adaptability to the use of different
sources of carbon and nitrogen, to acidity or low availability of glycerol, and
even tolerance to high levels of ethanol concentration. In addition to these
criteria, it is also important to consider that the fermenting strain must be
able to use hexose and pentose and tolerate the inhibitory by-products of the
pretreatment. In such a way that by manipulating both carbon and nitrogen
sources, as well as environmental factors, it would be possible to increase
or decrease ethanol production, which requires knowledge of the species to
be used. This is widely used in the industry, in which over time, the use of
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2 Bioethanol: Biochemistry and Biotechnological Advances
ethanol has been diversifying more and more, so what began to be used in
the food industry for the production of alcoholic beverages, today it is used
for the production of sustainable alternative fuels, which have gained impact
in recent years thanks to their profitable production thanks to the low cost of
substrates and their efficient fermentation.
1.1 INTRODUCTION
Yeast can produce compounds of interest such as pigments, phenolic

compounds, fatty acids, enzymes, among others. Also, yeasts have been used
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in various industrial fermentation processes due to their ability to convert high
concentrations of sugars into ethanol and CO2; for example, Saccharomyces
cerevisiae has been exploited for centuries for the production of alcoholic
beverages. Currently, ethanol obtained from this via is an alternative to be
used as a substitute for gasoline [1–3].
The ethanol that is produced through fermentation represents a positive
alternative as fuel to petroleum, as a source of energy for batteries through
electrochemical effects, as energy in the generation of energy by thermal
combustion, etc. [4]. Ethanol is significantly less toxic to humans than is
gasoline in such a way that it reduces air pollution thanks to its low volatility,
photochemical, and waste activity [5]. Ethanol obtained from waste materials
from biomass or renewable sources is called bioethanol, and it can be used as
fuel, chemical raw material, and solvent in various industries.
The production of ethanol as liquid fuel is obtained by fermentation
from biomass, sugar cane, cereal grains, and sugar beets, and it is gaining
a lot of popularity around the world [4, 6]. If you have greater control of
the fermentation conditions, this can contribute to the reduction of stress
towards yeast cells and decrease contamination by bacteria and wild yeasts.
Therefore, having large information gaps around the ethanol production
processes, leading to large investigations in order to achieve the generation
of higher quality products and more optimized processes [7].
1.2 PRINCIPAL ETHANOL-PRODUCING YEASTS
Ethanol-producing yeasts are characterized for producing ethanol and

CO2 in anaerobic conditions using fermentable sugars as substrate [8].
The principal attributes for considering a good ethanol-producing yeast
for their use in an industry are diverse as high tolerance levels of ethanol
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Physiology of Ethanol Production by Yeasts 3
concentration, acidity, high temperature, low glycerol formation, capacity

for use different sources of carbon and nitrogen. Also, it is important
to observe the tolerance and inhibitors of the yeast in production about
biomass hydrolyzed [9–12].
Saccharomyces cerevisiae is the more globally used yeast for industrial

production of ethanol [13, 14]; Brazil, the United States, European Union
(EU), and China being the main producers of ethanol (Renewable Fuels
Association). Furthermore, S. cerevisiae is an ideal yeast for the industry due
to easy manipulation with molecular methods such as genetic engineering
since its genome has been extensively studied [15, 16]. The studies have
been characterized for the improvement of the strains using by-products
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as substrate rich in sugars for the production of ethanol (Table 1.1). For
example, yeasts such as Schizosaccharomyces pombe, Candida krusei,
Kluyveromyces marxianus, Dekkera bruxellensis, Pichia striptis, Pichia
kudriavzevii, Wickerhamomyces anomalous, among others; they have been
isolated and identified as producing ethanol with good behavior and tolerant
to high concentrations of alcohol (EtOH) [17–19].
The main factors that contribute to the low growth of yeasts in the process
and that lead to high ethanol yields characterized in 90–92% of the theoretical
conversion of sugar to ethanol are high cell densities, cell recycling, and high
ethanol concentration [20–22].
1.3 BIOCHEMISTRY OF ETHANOL PRODUCTION
The generation of ethanol from lignocellulosic biomass (LCB) is possible

through three stages. The first of them consists in allowing the biomass to have
a better management through a treatment for the next stage, which consists of
subjecting the treated biomass to an enzymatic hydrolysis process in order to
improve the disposition of simple sugars, such as glucose and xylose. Finally,
thanks to the availability of sugars, it is possible to carry out fermentation
through the use of different microorganisms [9, 11, 24]. A generally simpli-
fied representation of the process for ethanol production from lignocellulosic
materials by chemical hydrolysis is shown in Figure 1.1.
The treatment given to the biomass prior to enzymatic hydrolysis is
nothing more than through different methods it is possible to modify its
physicochemical properties in order to facilitate the enzymatic work.
However, this can also bring consequences such as crystallization [24].
Parallel that, there is an increase in both the size of the internal surface and
its pore volume; this being also an adjuvant for the enzymatic work [25]. In
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4
TABLE 1.1 Ethanol Production by Yeast Under Different Conditions
Yeast Substrate (%) pH Temperature (°C) Ethanol (%, v/v) Condition Reference
Saccharomyces cerevisiae Glucose (5) 5.5 30 19.8 Electrochemical [18]
(CDBT2) cell (4V1)
Saccharomyces cerevisiae Molasses medium – 37 10.3 UV-C2 radiation [22]
(UVNR56) (28)
Saccharomyces cerevisiae Molasses media Adjusted – 12.2 VHG3 technology [23]
UAF-1 (27) 4.0–4.5
Wickerhamomyces anomalous Glucose (5) 5.5 30 23.7 Electrochemical [18]
(CDBT7) cell (4V)
Kluyveromyces marxianus Xylose (5) – 45 5.2 Modified strain [19]
(YZB014) (recombinant)
Pichia stipitis (PXF58) Xylose (11.4) – 30 4.3 UV-mutagenesis4 [23]
1
V = VOLTS;
2
VGH = Very high gravity;
3
UV-C = Ultraviolet-C;
4
UV-mutagenesis = Ultraviolet mutagenesis.
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response to all of the above, there is a significant improvement in the yield

rate of monomeric sugars [11].
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FIGURE 1.1 Rough diagram of the production of ethanol from lignocellulosic materials.
The complex and irregular reaction by which insoluble cellulose manages

to defragment into solid-liquid interfaces thanks to the simultaneous action
of cellobioses, exoglucanases, and endoglucanases, is better known as the
enzymatic hydrolysis process. This primer reaction is accompanied by
further liquid-phase hydrolysis of soluble intermediates, such as celluloli-
gosaccharides and cellobiose, mainly, which through catalytic reactions are
ungrouped in order to produce glucose through the action of β-glucosidase
[27]. In the short term, enzymatic hydrolysis is in charge to convert LCB to
fermentable sugars through a series of biochemical processes.
Fermentation in this context comprises the action of submitting the
LCB through a catabolic process of incomplete oxidation, which does not
require oxygen, and which final product is an organic compound. Across this
process, the pentoses and hexoses obtained from hydrolysis get fractioned
thanks to the presence of fermenting microorganisms, such as some bacteria,
algae, yeasts, and even some fungi, whether natural or recombinant [28].
In order to explain this complex process in a more comprehensible way
we can say that central metabolism begins with the basic conversion of
sugars to pyruvate, producing energy in the form of ATP and reduced NADH
cofactors, where pyruvate divergence after glycolysis acts as an essential
regulatory point in metabolism [11]. As a result of this process, pyruvate
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manages to have the choice of either following the fermentation route or

breathing. In the case of eukaryotes, this depends on the presence of oxygen.
That is, under aerobic conditions, pyruvate will be converted to acetyl-CoA
by the actions of a pyruvate dehydrogenase and will be directed toward the
citric acid cycle. In counterpart, under anaerobic conditions, pyruvate is

diverted to fermentation [13]. Where, the conversion of pyruvate to ethanol
is a two-step process [1, 25, 29]. First, through the action of pyruvate
decarboxylase (PDC) on pyruvate, acetaldehyde, and CO2 are obtained, the
latter as waste. In this section, three enzymes come into action, which are
encoded in the yeast genome in question, which is their importance in that
they act as a key point of metabolic branching between fermentation and
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respiration. In this process, a direct competition is generated in the action of
PDC, between its use for the production of ethanol and the maintenance of
the balance of pyruvate availability in the metabolic pathway. Subsequently,
acetaldehyde is converted to ethanol by an alcohol dehydrogenase (ADH)
[13, 25]. Due to its nature as an oxidoreductase it manages to promote the
reversible interconversion of alcohols and the aldehydes/ketones themselves
[25]. ADH has available a number of substrates present in the different
metabolic pathways, which results in the need for a strict order in order to
preserve the homeostasis of the intermediates and products [1]. Therefore,
this is why eukaryotes and even humans have numerous ADH enzymes. Of
which it is possible to mention the enzymes Adh1, Adh2 and Adh3 [11].
The Adh1 enzyme is crucial during fermentation, since it replenishes the
NAD + assembly in order to produce ethanol; In addition, glucose manages
to suppress the enzyme Adh2, thus allowing the oxidation of ethanol, this
happens only under certain conditions of need. And last but not least, Adh3 is
expressed in the mitochondria allowing to carry out its main function which
is to maintain the redox balance of the process [1, 25, 29].
1.4 PHYSIOLOGICAL GROWTH AND SUBSTRATE UTILIZATION
Remarkably, under aerobic conditions and with the correct supplies of

glucose, ammonium salts, and inorganic ions, most of the yeast species
studied could survive successfully, being organisms with relatively simple
nutritional needs. Where for its adequate growth it would be necessary to
involve macronutrients and micronutrients, administered in millimolar and
micromolar concentrations, respectively. We commonly call macronutrients
sources of carbon, nitrogen, among others; and micronutrients include trace
elements, such as calcium [30].
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In consequence, nutritional requirements and specific characteristics of

yeasts vary considerably according to the substrates used for their growth
and/or fermentation, plus the environmental conditions in which it is found,
in addition to the variety of the strain in question [30–32]. Some simple
sugars as glucose or fructose can be easily harnessed by yeasts since these are
assimilated right after being hydrolyzed either inside or outside the cell [33].
For the growth process of yeasts not all sugars are used simultaneously,
the process is started with the fermentation of sucrose with the help of the
enzyme invertase, they cause a hydrolysis allowing the generation of glucose
and fructose for the use of yeast, during the initiation phase within fermenta-
tion, glucose is used in a greater proportion than fructose [34], due to the
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glycolytic property of yeast. When the values of these saccharides decrease
under among concentration threshold maltose consumption increases if its
available, which is variable due to its entry into the cell thanks to the action
of maltose-permease enzyme; when it is present, it proceeds to the gradual
use of maltotriose [34, 35].
To mention some of the yeasts capable of fermenting maltotriose, maltose,
sucrose, glucose, and even fructose, they are Saccharomyces cerevisiae and
Saccharomyces uvarum. The importance of fermentable sugars derives from
their usefulness for the production of ethanol, CO2, and some representative
amino acids [36, 37].
The presence of carbon sources in a critical level turns cells metabolism
from respiratory growth into fermentative growth. Among of fermentable
sugars, glucose is the one who suppresses the enzyme that intervenes in the
metabolism of other sugars and in respiratory growth. Then when glucose
finds at critic concentration is converted into ethanol and CO2, this in spite of
a surplus of air; the optimal range for this to occur would be from 35 to 280
mg/L, which is equivalent to 5% of concentration [35, 38, 39].
Over the course of fermentation, a small amount of oxygen is present
in the broth, which goes hand in hand with the tolerance to ethanol and the
viability of the cell, a fact that allows the synthesis of sterols and is related to
fatty acids unsaturated around the membrane [30, 35].
In a broader perspective on the carbon sources, there may be decomposed
into four different sections; starting with the simple disaccharides also
known as hexoses, continuing with the starches, mainly of vegetable origin,
to this triad are also joined the lignocellulosic materials, and finally the agro-
industrial waste [25, 33, 36]. It is important to mention that each one has its
own pros and cons, for example, in terms of simple sugar-based materials,
they can be easily and immediately used as the sole substrate by microbial
strains capable of producing ethanol [25]. On the other hand, due to the excess
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of agricultural production, the use of starch for the production of ethanol has
emerged, however, it is recognized that the levels of performance of this are
too low to achieve the absolute replacement of gasoline [36]. After this is
mandatory to mention the use of lignocellulosic materials as very rentable
source for its low cost and easy obtention, without leaving aside the energy
required to obtain the necessary sugars from LCB, it is important to mention
that the main disadvantage is the availability of said substrates [25, 36].
Furthermore, the complexity of the substrate is what will dictate whether
a hydrolysis is used, either enzymatic or acidic, or if it is worked in a crude
way, the latter being applicable to agro-industrial waste. Therefore, for this
to suck, it is necessary to provide a rich source of nitrogen, since not all
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yeasts are capable of using mineral nitrogen [33, 34]. Even within these there
is a classification; first, we have the sources of mineral nitrogen where both
ammonium salts and urea are located, as they have an assimilation capacity
almost on par; second, there are the sources of organic nitrogen, where the
presence of glutamic and aspartic acids are noted, as well as their amines
(asparagine) in both D and L forms [35]. The aim of nitrogen present in
nitrogenous compounds is that it dissociates in the form of ammonium
ions and hydrogens that provide alkaline pH values to the growth medium,
achieving this by adding such compounds.
It is important to mention that some yeasts and even filamentous fungi
use nitrate as a source of nitrogen, whose assimilation happens thanks
to the reduction to ammonia due to the action of the enzyme ammonium
reductase [40].
1.5 YEAST PHYSIOLOGY AND ENVIRONMENT PARAMETERS
The environmental parameters, better known as extrinsic factors, are those

that, despite not being part of the study subject, are still capable of causing
changes in their properties. As for yeasts, the most notable could be said to
be the temperature, pH, and surface tension of oxygen, in some cases the
activity of water is considered. These parameters make the yeasts modify
both their structure and some defense mechanisms, all in order to survive
under the new conditions. In other words, these adaptations could be done
by two ways, either by changes in their genetic makeup or by changes in
their phenotype [35, 41]. In addition, the extreme exposure to these factors
can have serious consequences for the ethanol production process, where the
least of them would be a low yield in the recovery of ethanol and the greatest
could be to kill the yeast [41].
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Thus, at the time when the yeasts are subjected to certain light doses of
tension, some of their cells express a characteristic called cross-protection,
with which they become resistant to large and generally lethal doses of
other tensions. This characteristic is developed due to the activation of a
specific and general stress response program, known as the environmental

stress response (ESR), which is in charge of regulating the genes necessary
to survive in adverse conditions along with providing defense mechanisms
against changing conditions. Furthermore, this mechanism plays a potential
role in the induction of mutagenesis in the presence of stresses that can cause
genetic instability in microorganisms, in other words, genetic mutation [42,
43]. Within the concept of ESR, it is important to mention cell viability
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and vitality, which are defined as the ability to reproduce and the metabolic
activity of a culture, respectively. So, for these two conditions to occur during
propagation, the participation of storage carbohydrates, such as glycogen and
trehalose, is necessary. Glycogen serves as the main carbohydrate that stores
energy in yeast allows the synthesis of sterols, trehalose, and fatty acids,
throughout the delay stage. Due to this, it is desirable that the growth media
contain high concentrations of said polysaccharide so that the synthesis of
sterols and fatty acids is carried out, which represent a high expenditure of
energy. Glycogen accumulation is the consequence of the limited availability
of nitrogen or carbon. As for trehalose, it is considered to be a protective
oxidative disaccharide in situations of cellular stress such as high levels of
osmolarity, decreased nutrients, hunger, high, and low temperatures, and high
concentration of ethanol, which in high levels intracellular concentration
allows cell viability through the initial stages of fermentation and, therefore,
increases the rates of carbohydrate utilization [42, 44].
1.5.1 TEMPERATURE AS AN EXTRINSIC FACTOR
Temperature is the main factor that affects the production of ethanol, due
to the fermentation process is monitored based on the development of heat,
that is, the increase in heat energy in response to the metabolic activities
of microorganisms. One of the ways of inducing heat stress would be the
absence of cooling, which would lead to reduced growth of microorganisms
and fermentation defects. Therefore, the production of ethanol in tropical
countries has been managed at high temperatures in order to achieve a
reduction in costs in the cooling process. In addition, high temperature
management during fermentation provides a number of advantages, such
as efficient saccharification and fermentation, a constant change from
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fermentation to distillation and even minimal risk of contamination. To

achieve this type of fermentation it is essential to use a competent yeast
strain that can tolerate stress conditions, so it is essential to know the strain
to work in order to make a correct adaptation [1, 42, 45].
Warm, sugary, acid, and aerobic is as it would be described as the ideal

medium for growing most yeast species and some fungi. According to the
literature, the optimal growth temperature of yeasts is between 5°C and 37°C
in general, since this varies according to the physiology of each strain [11, 35,
41, 44, 47], these values cannot be literally applied in practice because there
are other factors that exert changes in the times of generation or growth. As
a result of the fact that this range is quite wide, we could shorten it by saying
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that in general most species grow very well around 25°C [30].
As mentioned in paragraphs preceding high temperature stress (or heat
shock) in yeast cells, heat damage can alter hydrogen bonds and even
hydrophobic interactions, leading to the unfolding of proteins and nucleic
acids. Therefore, anaerobic fermentation, being an exothermic reaction,
ends up increasing the temperature in some species, which are known
as thermotolerant, thus reaching fermentation temperatures above 40°C.
The term “thermotolerant” is used for those yeasts that have the transient
ability in their cells to survive subsequent lethal exposures at elevated
temperatures, that is, after a sudden thermal shock. Heat shock responses
in yeast occur when cells move rapidly at elevated temperatures, and if
this is near-fatal, it will produce “heat shock proteins (HSPs)” with a high
level of conservation, in response to the induced synthesis of a specific
set of proteins. HsP perform numerous physiological functions, including
thermo-protection [11, 30, 41, 44].
1.5.2 INFLUENCE OF pH
Fermentative yeasts are acidophilic mesophiles; therefore, they manage to

ferment under a pH of acid range (4 to 6). This characteristic provides us
with a great advantage by acting as a growth inhibitor of some agene micro-
organism in the medium, that is, it acts as a microbiological control agent
[28, 30]. This is why yeast culture media acidified with organic acids (for
example, acetic, lactic acid) are better at inhibiting growth compared to those
acidified with mineral acids (for example, hydrochloric, phosphoric acids),
since organic acids are responsible for lowering the intracellular pH just after
its translocation through the fungal plasma membranes [28, 47]. Exposure
to organic acids causes cells to deplete their energy (ATP) when they strive
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to maintain pH homeostasis through the activities of the proton-pumping

ATPase complex in the plasma membrane. This forms the basis for the action
of weak acidic preservatives to inhibit the growth of fungi and/or yeast from
food breakdown [47]. S. cerevisiae is the most commonly used yeast in the
industrial production of ethanol, since it tolerates a wide pH range, making

the process less susceptible to infection [11].
1.5.3 OXYGEN SURFACE TENSION
A factor that influences fermentation and improves biochemical processes is
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the aeration of the must, since the presence of oxygen in the initial fermen-
tation phase is essential for the rapid reproduction of yeast and the complete
restoration of sugar. By maintaining the correct amount of glycogen and
trehalose, the correct aeration improves the vitality of the biomass and the
immunity of the yeast cells to different tensions; carrying out in this way a
selective exchange of metabolites and a correct extraction of nutrients from
the environment. When a low amount of oxygen is present in the medium,
a delay in fermentation or a change in the sensory properties of the product
is caused [48].
The aeration of a culture has the advantage of providing the necessary
oxygen for respiration and the elimination of CO2 produced by the metabolism
of carbonate compounds. Therefore, the supply of oxygen through aeration
must be sufficient to avoid the generation of alcohol (EtOH) throughout
the fermentation process and it is recommended that the added air be rich
in oxygen. Then, during growth and metabolism, the transfer of nutrients,
and solid, solid, and gaseous metabolites between the environment and the
cell is essential and continuous, this is where the solubility of the gas enters
the liquid phase, so that it allows the exchange between phases. liquid and
gaseous [35, 49, 52].
Yeasts require appropriate amounts of oxygen to carry out their oxidative
metabolism and thus proceed with the optimal production of ethanol; by
using this gas, they are able to synthesize unsaturated fatty acids and sterols,
which are necessary for continuous anaerobic growth and cell division.
Even S. cerevisiae, which is anaerobic in growth, requires small amounts of
molecular oxygen for the synthesis of fatty acids and sterols. Furthermore,
pentose fermentation yeasts require very small amounts of oxygen, which
must be constantly monitored. So, since the fermentation rate is proportional
to the number of metabolically active yeasts present, this means that the
attenuation of the medium goes hand in hand with the availability of oxygen
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[28, 48]. In consequence, insufficient aeration can lead to insufficient yeast

revitalization, increasing deficiency, and low fermentation rates. In another
hand, excessive aeration can lead to a large amount of biomass. Therefore,
when yeasts are not aerated, they only grow during fermentation, and sugar
consumption is also low [48].

Surprisingly, sterols and fatty acids become a limiting factor because
industrial fermentation takes place without aeration. Whereas if the inhibitors
present in the lignocellulosic hydrolysates are used in high concentrations of
cell mass, this will affect the volumetric productivity of those fermentations
inoculated with lower cell concentrations [44]. This is because optimization
of aeration not only leads to an increase in the speed ratio of the fermentation
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process, but also improves the final characteristics of the product. Yeast with
low oxygen requirements generally produces a slightly higher number of
esters, especially the desired isoamyl acetate [48].
1.6 YEAST PHYSIOLOGY AND ETHANOL PRODUCTION PROCESSES
The process of producing ethanol through yeasts is dictated by the raw

material to be used. Hence, this process is mainly divided into three phases:
• Obtaining fermentable sugars;
• Fermentation of sugars (production of ethanol); and
• Separation and purification of ethanol.
Raw materials are pre-treated in order to reduce their size and therefore
facilitate subsequent processes. The hemicellulose and cellulose will then
hydrolyze to fermentable sugars. The yeasts then begin to work to ferment
these sugars into ethanol. Finally, the recovery of ethanol is carried out by
means of various separation technologies, and thus it can be used as fuel [28].
Over time fermentation under high levels of stress has been improving
in terms of efficiency and tolerance, so some strains of yeast have begun
to gain strength within the industry. According to the literature, it is said
that there is a close relationship between fermentation efficiency and
resistance to stress, that is, the ability of a yeast strain to develop adaptability
to inappropriate environments and growth conditions. Thus, industrial
fermentation occasionally does not complete or progress at a slower rate.
This is mainly due to slow and stagnant fermentations. High concentrations
of ethanol, carbohydrate stress and/or heat are some of the factors that can
cause abnormal and non-optimal fermentation [42].
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A typical batch growth curve is composed of lag, exponential, and

stationary phases, this is a response to the inoculation of fungal cells in
media with the necessary nutrients and conditions [42]. First, the period of
zero population growth is called the lag phase, where the inoculated cells
begin a stage of adjustment to their new chemical and physical environment

(synthesizing ribosomes and enzymes) [44]. Second, the exponential phase is
a period of doubling of logarithmic cells (or mycelial biomass in the case of
filamentous growth) and a constant maximum specific growth rate (µmax, in
reciprocal dimensions of time, per hour), the value of which accurate depends
on the prevalence of growing conditions [45]. During the exponential phase
of balanced growth, cells carry out a rudimentary metabolism, that is, they
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begin to run those metabolic pathways essential for cell growth [42]. When the
final objective of fermentation is both to achieve the highest levels of biomass
production and the optimal extraction of primary metabolites, what is sought
is to extend the growth phase, regularly by means of batch or continuous fed
cultures. Consequently, they begin with the stationary phase, in which fungal
biomass manages to remain constant and the growth rate returns to zero [41].
After prolonged periods in the stationary phase, individual cells can die and
self-autolyze [48]. The stationary phase is characterized by being the stage in
which the microorganism reaches long periods of survival without the need
for the addition of nutrients. Not only the absence of nutrients can induce the
stationary phase, there are other facilitating causes such as the presence of
toxic metabolites (for example, ethanol in the case of yeast), low pH, high
CO2, variable O2 and high temperature [30]. During the stationary phase of
unbalanced growth, fungi can undergo secondary metabolism, specifically
initiating metabolic pathways that are not essential for cell growth but are
involved in the organism’s survival. Therefore, the industrial production
of secondary fungal metabolic compounds such as penicillin and ergot
alkaloids essentially compromise the stability of the cell population during
the stationary phase [30, 41].
There are three processes that are commonly used in the production
of bioethanol which are separate hydrolysis and fermentation (SHF),
simultaneous saccharification and fermentation (SSF), and simultaneous
saccharification and co-fermentation (SSCF). In SHF, the hydrolysis of
lignocellulosic materials is separated from ethanol fermentation, in which
the separation of enzymatic hydrolysis and fermentation makes it easier for
the enzyme to operate at high temperature for better performance, whereas
fermentation organisms can be operated at a moderate temperature to
optimize the use of sugar. In contrast, the other two methods (SSF and SSCF)
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have a short general process since the enzymatic hydrolysis and fermentation
process occurs simultaneously to keep the glucose concentration low; that is,
for SSF, the fermentation of glucose is separated from the pentose while the
SSCF ferments the glucose and the pentose in the same reactor. In turn, these
processes offer a series of benefits such as lower cost, higher ethanol yield
and less processing time [49–52].
Bioethanol fermentation can be carried out in batches, batch feeds, repeat
batches or in continuous mode. In batch process, the substrate is provided at
the beginning of the process without adding or removing the medium; this
system is known as simpler bioreactor with multi-vessel control process, flex-
ible, and easy; where, the fermentation process is carried out in a closed-circuit
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system with a high concentration of sugars and inhibitors at the beginning
and ends with a high concentration of product. Therefore, the batch system
offers a number of benefits, which include complete sterilization, requires
no job skills, is easy to handle raw materials, can be easily controlled, and
flexible to various product specifications. However, productivity is low and
requires high and intensive labor costs. The presence of a high concentration
of sugar in the fermentation medium can lead to inhibition of the substrate
and inhibit cell growth and the production of ethanol [1, 11, 41].
Cellular Recycling Batch Fermentation is a strategic method for effec-
tive ethanol production, as it reduces the time and cost of inoculum prepara-
tion; some of its advantages are easy cell harvesting, stable operation, and
long-term productivity. Besides, sugar materials and immobilized yeast
cells are used as facilitators in the cell separation necessary to carry out cell
recycling [37, 48].
1.7 INDUSTRIAL IMPORTANCE OF ETHANOL
Ethanol is a chemical compound that has been produced for thousands

of years for human consumption, the fermentation process of ethanol is
obtained from raw materials. In recent years, the fermentation process for
the production of ethanol has received great attention for its chemical and
edible purposes. The best-known use of ethanol is as an alcoholic beverage
[55], the increasing demand for various industrial applications that include
its use to preserve biological samples, as a solvent in the manufacture of
perfumes, varnishes, adhesives, pesticides, in the preparation of pharma-
ceutical products such as medicines and drugs, essences, deodorizers, and
as a disinfectant, among many others, that has requited increasing produc-
tion of ethanol [54]. In a relatively short period of time, technologies have
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been developed for the production of a wide range of bio-based products.

Consequently, making the production of liquid fuels the focus of attention
due to the great market potential for the replacement of oil in the fuel supply.
Ethanol has the longest history and is the first commercially produced
biomass-derived liquid fuel [51].

In Brazil the application of ethanol as fuel has obtained a great boom,
since it is possible to manufacture at a low cost through the fermentation
of sugar cane [56], and in the US, corn is the dominant biomass feedstock
[55]. For its application fails to be more profitable various mixtures of
ethanol are used with oil [57]. It can be implemented in neat form or in
a mixture with gasoline. It can be used in percentages up to 10% with
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gasoline in common spark-ignition engines without further modification or
can be operated at higher percentages in mixtures with gasoline in slightly
modified engines which are called flexi-fuel vehicles. It is possible to use
pure gasoline or ethanol blends up to 85% (even 100% ethanol in Brazil)
in flex-fuel engines [58].
The manufacture of these biofuels has increased since 2000 [60]. This
being the case, ethanol is more significant when it is considered according
to the volume produced. Today, the United States and Brazil are the world’s
largest ethanol producers, able to generate ethanol production exceeding 94
billion liters per year, which represents around 85% of world production
[61]. In particular, Brazil is the leading manufacturer of ethanol for auto-
mobiles, with an annual production rate of 4 billion gallons of ethanol from
sugar [62].
In 2016, biofuels supplied around 4.5% of total fuel for road transpor-
tation worldwide and its planned share of world transport fuel by 2050 is
estimated at 25%. [53]. As the lowest-cost manufacturer globally, the United
States remains to retain its place as the most reliable and affordable source of
ethanol internationally, reaching up to 57.8 billion liters of global manufacture
in 2016. Brazil, which produced roughly 27.6 billion L, is accountable for
about 27% of world production, while the EU follows with 5%. Other proper
leaders to mention are China and Canada about ethanol manufacture [57].
The geographic distribution of the production and consumption of ethanol
is related to many factors, such as manufacturing destinations, government
policies, natural resources availability, and environmental regulations.
Different world regions can be perceived as distinct markets with diverse
demands and supply. Estimates denote that the USA is clearly the largest
manufacturer and consumer of ethanol and it is followed by Brazil [63]. With
regards to assessing these main producers, a difference from the perspective
of the increase in production by 2020 is noted [59].
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1.8 FINAL REMARKS
Finally, it is important to reaffirm the ability of yeast cells to grow, metabo-

lize complex industrial raw materials and withstand the hostile environments
of large-scale fermenters. Throughout this chapter, important considerations

of yeast stress and nutritional physiology that influence yeast fermenta-
tive activities have been addressed. In addition to the yeast optimization
and improvement strategies used in alcohol production, it is a priority to
know the yeast strain to use in order to carry out the correct approach when
carrying out the fermentation process. Even today, yeasts continue to be one
of the least understood organisms, and in the same way they are among the
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most important as input in ethanol production processes. Even so, under
the correct conditions of both feeding and environment, it is currently
possible to obtain volumes of more than 20% of ethanol production, when
these conditions are not met, leading to stress in the lead, stuck, slow, and
inefficient fermentations are obtained. Therefore, it is essential to optimize
alcoholic fermentations to understand aspects of yeast cell physiology,
particularly when lignocellulosic substrates are used for the production of
second-generation bioethanol.
KEYWORDS
• alcohol dehydrogenase
• bioethanol production
• biorefinery
• growth conditions
• lignocellulosic biomass
• yeast physiology
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CHAPTER 2

Zymomonas mobilis
LAURA ANDREA PÉREZ-GARCÍA, CINDY NATALY DEL RIO-ARELLANO,
DAVID FRANCISCO LAFUENTE RINCÓN, and
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NORMA M. DE LA FUENTE-SALCIDO
Faculty of Biological Sciences, Autonomous University of Coahuila,
Torreón, Coahuila, México, E-mail: normapbr322@gmail.com
(N. M. D. L. Fuente-Salcido)
ABSTRACT
The bacteria Zymomonas mobilis is a natural ethanologenic with important

desirable physiological features suitable for industrial production of bioethanol.
This anaerobic bacterium is considered a powerful biocatalyst system for
biofuel production and here will be compared with typical microorganisms
involved for ethanol production. The main comparison focuses on the typical
model ethanologenic, Saccharomyces cerevisiae, which uses the Embden-
Meyerhof-Parnas (EMP) pathway for glucose fermentation because Z. mobilis
uses the Entner-Doudoroff (ED) pathway, and also, the energy production in
both will be described. Also, the utilization of carbohydrates, fermentative
process conditions, yield, and perspectives will review, as well as some
genetic modifications to optimize the production of bioethanol by harnessing
the Zymomonas metabolism. Finally, diverse industrial perspectives for Z.
mobilis application will be mentioned briefly.
2.1 INTRODUCTION
Since ancient civilization, the use of alcoholic beverages has taken part of the
universal culture, traditions, and the economic strength of nations. However,
that is why they thought that initially fermentations were considered a
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spontaneous process, normally depending on yeasts and bacteria that were

already present in the raw material, the equipment, or introduced by insects.
Nowadays, the production of ethanol is one of the significant biotechnological
processes in the world in many areas like production of beverages, economic
importance, and research. For this process, there are different sources of
sugars such as cereals, fruits, and other carbon sources that can be used by
various microorganisms to obtain products through the fermentation process
such as wine, beer, or even biofuel that are economically essential products
(Figure 2.1).
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FIGURE 2.1 The fermentation process for the elaboration of alcoholic beverages and biofuel
with different carbon sources.
Bioethanol production uses different substrates. The products derived

from corn or based on starch, are produced primarily by the United States
to be used in the transport sector. Another substrate for the production of
ethanol derived from sugar cane or based on sucrose is produced mainly in
Brazil. These biofuels are of the first generation because they are derived
from the fermentation of hydrolyzed edible starch materials. Nowadays,
there are other generations of bioethanol production that can be used without
involving the socio-economic troubles, and especially second-generation
bioethanol derived from the recovery-conversion of various waste streams
are the most promising substrate applied around the world. These kinds
of processes also contribute to enhancing the economy of many countries
through renewable energy production [1–2].
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Physiology of Ethanol Production by Zymomonas mobilis 23
The simple or complex substrates have the required quality to be used as

an excellent source of carbon for microbial growth and to be converted into
ethanol. Both substrates result in the generation of various carbohydrates
as hexoses, pentoses, or glycerol that will be fermented by microorgan-
isms to be converted into ethanol. The most recognized and important

ethanol producers are the yeast S. cerevisiae and the bacterium Z. mobilis.
The utilization of both microorganisms for the production of alcoholic
beverages or biofuel for the industry is exposed to severe environmental
changes and fermentation processes. There exist different factors that are
influenced the process like temperature shock, osmotic stress caused by the
high concentration of carbon sources and ethanol tolerance [4]. In relation
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to the yield, the factors mentioned previously can change depending on
the microorganism used, as well as the type of fermentation that is carried
out, and the parameters that are used to obtain the highest production of
ethanol per gram of carbon source. In this chapter aims to integrate the
current knowledge about the differences in the yeast S. cerevisiae and the
bacterium Z. mobilis regarding to ethanolic fermentation. Also, the central
role, the potential use of biological features of Z. mobilis and the common
biotechnological mechanisms and tools for the production of bioethanol
will be discussed.
2.2 FERMENTATIVE BIOPROCESS TO PRODUCE ETHANOL
Ancient civilizations brewed beer around the year 6000 A.C. through the
fermentation process. For 2017, the average ethanol production is more
than 1 million barrels (159 million liters) per day, with an annual rate of
16 million gallons (60 million liters). There are many microorganisms that
produce ethanol but in each one has certain limitations due to economic
problems of the production of bioethanol, such as the use of different
substrates and yield. Within the microorganisms, yeasts are leaders in the
production of bioethanol, however, bacteria such as Z. mobilis, Escherichia
coli, or Clostridium sp., are being developed to increase the production
of bioethanol and eliminate the limitations such as the kind of substrate
or tolerance to different factors that can change with the help of genetic
engineering [5].
In addition, ethanol derived from yeast fermentation represents more
than 80% of the world’s renewable fuels and more than 85% of the alcohol
produced is provided by the United States and Brazil. The S. cerevisiae
produces more than 100 billion liters of ethanol per year [6, 7].
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One of the best prospects to the production of ethanol is Z. mobilis

because have several important and distinctive physiological features that
give it advantages for bioethanol production. Among these characteristics is
growth at high concentrations of six-carbon sugar in the medium, tolerance
to high concentrations of ethanol, biological activity at a wide pH range for

the production of bioethanol. In the fermentation process the cells of some
strains of Zymomona sp. can grow up on glucose concentrations above to 400
g of glucose L–1 [5, 8]. On the other hand, the yeast cells undergo osmotic
stress when inoculated into the grape must (generally above 200 g of sugar
L–1). This induces the yeast to synthesize glycerol, in this way it reduces the
permeability of the glycerol and consequently, buffer the osmotic gradient
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between the inside and the outside of the cell [9].
Both Z. mobilis and S. cerevisiae have a different pathway to use a carbon
source during the bioethanol production. Alcoholic fermentation commonly
includes diverse biochemical degradation pathways as glycolysis, alcoholic
fermentation, glycerol-pyruvic fermentation. Also, respiration processes
for utilization of hexoses, xylose catabolic pathways for utilization of
pentoses and glycerol assimilation, and glycolysis for glycerol-converting
microorganisms, and regulation between fermentation and respiration. In
the particular case of Z. mobilis uses ED pathway to anaerobically ferment
glucose for ethanol production. But S. cerevisiae uses EMP pathway for
glycolysis [2]. Fermentation process consists for Z. mobilis 1 mole of ATP is
yielded per mole of glucose through the ED pathway required less enzymatic
protein because together with Pdc and two alcohol dehydrogenases (Adh)
“form the backbone” of this glycolysis metabolic pathway. On the other
hand, S. cerevisiae one molecule of glucose (C6H12O6) is converted into two
molecules of pyruvic acid (C3H4O3) during the process of glycolysis. Pyruvic
acid is further decarboxylated to generate two molecules of acetaldehyde
(CH3CHO), which is reduced to ethanol (C2H5OH). The process described
produce two molecules of ATP as gain, two molecules of ethanol and two
molecules of CO2 [10]. In the conversion of glucose to ethanol and CO2
during fermentation, a total of 12 yeast enzymes are involved, 10 to degrade
glucose to pyruvate with the generation of ATP. After that, pyruvic acid is
converted to acetaldehyde through PDC and the final stage ADH converts
acetaldehyde to ethanol in this way NAD+ is regenerated to allow glycolysis
to continue (Figure 2.2) [7]. This has a big importance because Zymomonas
use less energy than Saccharomyces to make the fermentation process and
these can be traduced to “higher production of ethanol, using less energy to
make the process” depending on the strains.
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FIGURE 2.2 Glycolysis and fermentation in yeast.
2.3 BIOETHANOL PRODUCTION (SUBSTRATES, MICROORGANISMS,

PROCESS, YIELD)
2.3.1 SUBSTRATES
The utilization of biomass such as cellulosic agricultural residues are valu-

able, economical, profitable, and sustainable renewable natural sources that
are used for the production of bioenergy such as biodiesel, biohydrogen,
and bioethanol [11]. The carbon source influences the quality of the ethanol
produced from the fermentation, since the ethanol yield will depend on the
type of sugar obtained [12]. The use of different substrates such as ligno-
cellulosic residues are considered a viable alternative for the production of
biodiesel [13, 14].
2.3.2 MICROORGANISMS
A wide variety of microorganisms’ ferment hexose and pentose sugars such

as; Bacillus, Klebsiella, Escherichia coli, Thermoanerobacter, Aeromonas,
S. cerevisiae, Candida shehatae, Pichia stipitis, C. brassicae, Pachysolen
tannophilus, Fusarium, Mucor indicus, Neurospora, Monilia, Z. mobilis,
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and Rhizopus mobilis are capable to produce ethanol [11, 15]. However, not
all microorganisms can produce the same amount of ethanol, because each
one has their own limitations, like the type of fermentation or the different
tolerance like high sugar concentration or ethanol tolerance.
2.3.3 PROCESS
A fermentation process is sought that is as efficient as possible. Several

methods have been developed to integrate hydrolysis and fermentation to
improve cellulose availability [15]. There are new technologies that have been
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found that make it possible to carry out the viable hydrolysis and fermenta-
tion process, the most common include separate hydrolysis and fermentation
(SHF), simultaneous saccharification and fermentation (SSF), simultaneous
saccharification and fermentation (SSCF), consolidated bioprocessing
(CBP), pre-saccharification followed by simultaneous saccharification and
fermentation (PSSF), separate hydrolysis and co-fermentation (SHCF), and
finally, pre-saccharification followed by simultaneous saccharification and
fermentation (PSSF) [16, 17].
2.3.4 YIELD
The yield depends on the amount of metabolite required, the amount produced
by the native organism and the biological activity of the compound [18].
2.4 GENERAL OVERVIEW OF Zymomonas mobilis
Biotechnology has allowed the increase of innovative fermentation processes

to develop clean, almost inexhaustible, and highly competitive energies such
as renewable energy. One of the most outstanding renewable energies is the
production of ethanol by fermentation, a fuel product for motor vehicles,
which contributes to mitigating the planet’s climate change.
During the last decades, alcoholic fermentation has been extensively
studied to improve and optimize the production of bioethanol, an economically
profitable and eco-friendly biofuel. In this sense, the unit operations, culture
media (substrates, organic waste) and microbial growth conditions involved
in the fermentation process have been improved to increase bioethanol yield.
However, the success of the synthesis of bioproducts (such as bioethanol)
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focuses mainly on the physiological characteristics and fermentative capaci-

ties of the producing microorganism. The microorganisms commonly used
to produce ethanol were yeasts, mainly S. cerevisiae, however, successful
processes have been achieved with extraordinary facultative bacteria such
as Zymomonas mobilis [5]. This bacterium has been attracting significant

attention for usage in large-scale biofuels bioprocessing.
The Zymomonas is a bacterial genus isolated frequently from some
fermented beverages or plants surface, including the succulent Maguey
cactus indigenous from México, African palm wine and also is considered
as spoilage organism in beer or cider ("cider sickness") from Europe [19,
20]. It was first found in 1924 and reported in 1928 by Linder in pulque
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isolates, a popular Mexican fermented pre-Hispanic beverage and reported
as “Thermobacterium mobile.” However, until 1936 Taxon Z. mobilis subsp.
mobilis according to the proposal of Kluyver and van Niel, and formalized in
1976 by the classification proposal and reported by De Ley, J., and J. Swings.
The commonly known taxonomic hierarchy of Zymomonas is described
as follows:
Phylum Proteobacteria
Class Alphaproteobacteria
Order Sphingomonadales
Family Sphingomonadaceae
Genus Zymomonas
Species Zymomonas mobilis
Subspecies Zymomonas mobilis francensis
Zymomonas mobilis mobilis
Zymomonas mobilis pomaceae
The bacteria description included in the Bergey’s Manual of Systematic
Bacteriology listed the characteristics of the bacterium [21].
This facultative aerobic Z. mobilis is, gram-negative bacteria, a non-spore-
forming, rod-shaped grouped in pairs and size of 2–6 × 1.0–1.4 µm.
Commonly non-motile but some exceptions may be possessed one to four
polar flagella. Growth of Z. mobilis on standard medium agar [D-glucose (20
g L–1), yeast extract (5 g L–1)], grows forming glistening colonies, regularly
edged, white to cream-colored, 1–2 mm in diameter after 48 h at 30°C
incubation. Growth optimal conditions includes 30°C and at pH 3.5–7.5 and
vitamins (biotin, pantothenate) as micronutrients [70]. Their nutrition includes
fermentable sugars (glucose, fructose, sucrose), is chemoorganotrophic
and highly efficient ethanol producer through the ED pathway [22]. Shows
constitutive ethanol-tolerance (5%) and also is acid tolerance, but is sensible
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to novobiocin. The distinctive features of Z. mobilis are the genotypically

their DNA content mol %G+C is 47.5–49.5, and phenotypically, the cell
membrane contains pentacyclic triterpenoids of the hopane series and lack of
fatty acid C14:0 2OH (nonhydroxy myristic acid), commonly present in all
others α-Sphingomonas species.

Physiologically Zymomonas is facultatively anaerobic and has a strictly
fermentative metabolism, with multiple applications in biotechnology [23–27].
2.5 AMAZING FEATURES OF Zymomonas mobilis FOR

BIOTECHNOLOGICAL APPLICATIONS
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The physiology of a microorganism is decisive key to ensure the synthesis of
several high added value products by advanced fermentation technologies.
Z. mobilis physiology is particularly fascinating, mostly by their capacity
to synthesize economically and sustainably important products such as
bioethanol among others biochemical as bionic acid, levan, sorbitol, among
others [5, 28].
In this sense, the ED metabolic pathway of Zymomonas mobilis strains is
considered a unique ethanologenic pathway due to its efficiency, character-
ized by a decrease in cellular ATP consumption, which promotes an increase
in the transformation of sugars into biomass, and even with better yields and
productivity of the bioethanol found in S. cerevisiae [23]. The ethanologenic
Zymomonas strains are recognized as the most efficient bacterial ethanol
producer, mainly due to that employ the ED pathway as a strictly step during
a fermentative process, in addition to having a physiology perfectly adapted
to industrial-scale bioethanol production. Sequential steps to glucose trans-
formation by the ED pathway, the glucose phosphate is catabolized into
2-keto-3-deoxy-6-phosphogluconic acid (KDPG) and then is cleaved by
KDPG aldolase to pyruvate and glyceraldehyde-3-phosphate (GAP). The
GAP is oxidized to pyruvate by glycolytic enzymes and ATP produced by
substrate-level phosphorylation. The next reaction is reduction of pyruvic
acid to ethanol and CO2. The overall reaction is:
C6H12O6 → C2H5OH
Glucose → 2 Ethanol + 2 CO2 + 1 ATP
As a catabolic main route exclusive of prokaryotes, the enzymatic divergence
of the ED pathway is it uses 6-phosphogluconate dehydratase and 2-keto-3
deoxyphosphogluconate aldolase to synthesize pyruvate from glucose. There-
fore, the pathway rises a yield of 1 ATP for each glucose molecule catabolized,
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as well as 1 NADH and 1 NADPH. The obvious comparison with the EMP
pathway, indicates that glycolysis rises a yield of 2 ATP and 2 NADH for each
glucose molecule processed [71]. This physiological feature distinguishes Z.
mobilis as a profitable strain for industrial applications.
Metabolic efficiency of Z. mobilis as ethanol producer has broad appli-

cation in industrial-scale ethanol production. Bioethanol is considered an
alternative renewable energy source, indispensable, and urgently necessary
in the very short term [29, 30]. However, it is very important to consider the
advantages and disadvantages of using outstanding ethanologenic bacteria.
The main advantage of using Zymomonas (and yeast) is the clean
production of ethanol during the fermentative process, while the most
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microorganisms generate mixtures of metabolic products in addition to
alcohol. The disadvantage of the use of Zymomonas lies in its specificity
for glucose as the only fermentable sugar and its lacks the enzymes to
break down another carbon source, including the enzymes to degrade
carbohydrate polymers such as starch and cellulose [31], and this would
limit its biotechnological application.
Different strategies, including kinetic modeling and rational metabolic
engineering to understand how central metabolism of Zymomonas work, and
novel genome sequence are widely studied and applied to improve ethanol
synthesis. In addition, the techniques promoted by the application of synthetic
biology, metabolic engineering, and the ongoing world trend of industrial
bioethanol production from the use of agro-industrial waste by the metabolism
of Zymomonas mobilis has facilitated the development of genetically modified
bacteria for co-culturing in cellulose-rich media [14, 32, 72–75].
2.6 FERMENTATIVE PATHWAYS OF Zymomonas mobilis AND

Saccharomyces cerevisiae
Particularly, Z. mobilis uses the metabolic pathway of ED, which is a modified

form of the glycolysis pathway that produces only one molecule of ATP per
metabolized glucose unlike the EMP pathway that produces two glycolytic
ATP molecules used by yeast. The bacterial pathway of ED also generates
reducing equivalents as NADH and NADPH (Figure 2.3) [7].
Alcohols are derived from the catabolism of amino acids through a metabolic
pathway. The amino acids, such as valine, leucine, isoleucine, methionine, and
phenylalanine, are absorbed slowly throughout the fermentation process by the
Ehrlich pathway. After a transamination reaction, the keto acids produced are
converted into alcohols or acids by this pathway [33].
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FIGURE 2.3 Metabolic pathway of Saccharomyces cerevisiae and Zymomonas mobilis.
The metabolism of aromatic amino acids (AAA) in yeast directly influ-

ences a more significant alcohol formation. The repression of nitrogen
metabolites (NCR) can control the amino acid catabolism, which functions
like a complex regulatory system that confers to S. cerevisiae to use sources
with a high concentration of nitrogen to the yeast. Studies showed that the
enzymes involved in the three principal stages of the Ehrlich pathway, which
are transamination, decarboxylation, and reduction, are encoded by the Bat2,
Pdc1 and Adh1 genes. These genes in the process of alcoholic fermenta-
tion, present similar expression profiles. Overexpression of these two genes
generates significant increases in two different alcohols isobutanol and
isoamyl alcohol [33].
2.7 PHYSIOLOGICAL ADVANTAGES IN BIOETHANOL PRODUCTION

OF Zymomonas mobilis
Knowing the differences between the different metabolic routes that exist
between the bacteria Z. mobilis and the yeast S. cerevisiae. Lack of different
enzymes in the Zymomonas metabolic pathway is found as phosphofruc-
tokinase (Pfk) in the EMP pathway, phosphogluconate dehydrogenase
(Pgd) and transaldolase (Tal) in the PPP pathway, as well as 2-oxoglutarate
dehydrogenase complex (sucABCD) and malate dehydrogenase (Mdh)
in the TCA cycle and this enzyme deficiency carry more carbon into the
ethanol production pathway and highly efficient glycolysis resulting in the
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theoretical maximum ethanol production [34]. Zymomonas sp. strains have

a temperature range of 25 to 31°C but there are strains reported that can
grow at temperatures of 40°C. It has a wide range of pH 3.7–7.5 so it has
a high tolerance to acidic pH´s. However, most strains of Zymomonas sp.
grow up at an optimal pH of 5–7. Some strains of Zymomonas also can grow

up at high concentration of glucose medium more than 200 gL–1 but there is
other that can grow up in 400 gL–1 glucose medium [8]. Another important
characteristic is that the ZM4 strain is facultative aerobic, so this bacterium
can perform the metabolism for the production of bioethanol under aerobic
conditions, however it is reported that the production is lower than in the
anaerobic condition [35]. Also, Z. mobilis possess a high tolerance of ethanol
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like 11–16% v/v [29].
2.8 GENETIC FEATURE OF Zymomonas mobilis AND Saccharomyces

cerevisiae
According to previous reports about the complete genome of Z. mobilis

ZM4 corresponds to a circular chromosome with 2,056,416 bp and harbor
five circular plasmids [34]. The chromosome and five plasmids have GC
contents of 46.22%, 43.46%, 45.41%, 43.23%, 41.79%, 37.63% and 41.31%
respectively. The genetic material includes 1,875 protein-encoding genes, 48
tRNA and 6 rRNA genes [19]. The most established product by Z. mobilis
recombinant strains is ethanol, which has been extensively investigated. The
most important detected genes are pdc and adh for ethanol production by
Zymomonas and these genes have been inserted in diverse microorganisms
modified by molecular biology. For ED pathway genes (glk, zwf, pgl, pgk,
and eno) and the PDC-encoding pdc gene were observed to be more abundant
under anaerobic conditions than aerobic conditions depending on the strain
[5]. Otherwise, S. cerevisiae has a genome size of 12 Mb distributed among
16 chromosomes. The entire genome encodes 6,000 genes, of which 5,000
are individually nonessential [36]. The sequence of the S. cerevisiae has
about 12,068 Kb of which defines 5,885 potential protein-encoding genes,
140 genes specifying ribosomal RNA, 40 genes for small nuclear RNA
molecules, and 275 transfer RNA genes. Also, the advantage to have the
complete sequence S. cerevisiae provides information about the higher-order
organization of yeast’s 16 chromosomes and allows some insight into their
evolutionary history [37].
Among the most important genes of Z mobilis are Pdc, this gen synthe-
sizes the protein that transforms pyruvate into acetaldehyde and the gen
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Adh synthesizes the protein that transforms acetaldehyde into ethanol in the
metabolic pathway ED, however, genetic engineering is looking for different
ways to increase the production of ethanol and inhibit acetate production. On
the other hand, there are reports of strains that are already modified with the
inclusion of different gene such as XylA, XylB, talB, and tktA that give them
the ability to use 5 C sugars and thus, be able to use expanding the range of
raw material used as a carbon source [38].
Regarding to S. cerevisiae have only 5,538 genes encoding 100 amino
acids and contains 18 genes for which orthologs are not identified. These
may be species-specific genes in S. cerevisiae, but alternatively, they could
reflect the gaps in the draft genomic sequences available [39, 40]. Yeast cells
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exposed to ethanol synthesize a range of Hsps, including Hsp104, Hsp82,
Hsp70, Hsp26, Hsp30, and Hsp12. The Hsp104 and Hsp12 physiologically
influence the ethanol tolerance in yeast [40]. Cell wall from S. cerevisiae
contains 85% polysaccharides and 15% proteins approximately. If there is
an increase in the level of ethanol, the stability of the membrane will be
affected, which will cause damage to the proteins and, therefore, endocytosis
is inhibited through the membrane [10].
In Figure 2.4, the yeast the Hog1 gene is responsible for encoding the
high osmolarity glycerol pathway (HOG), this pathway mainly encodes two
enzymes GPD1 and GPD2 that catalyze the conversion of dihydroxyacetone
phosphate (DHAP) through glycerol-3-phosphate (G3P) to glycerol. The
Fps1 channel helps to glycerol accumulation and contributes to raising the
osmotic pressure within the cell. Within the stress response are the proteins
trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phospha-
tase (TPP), enzymes responsible for the synthesis of trehalose. In the same
way, heat shock transcription factor (Hsf1) induces the production of HSPs
[40]. Also, in Figure 2.4 in Z. mobilis the Zms4 and Zms6 genes are involved
in direct sRNA and target mRNA interactions. The accumulation of ethanol
within cells under stress increases the expressions of these genes. Increasing
the level of Zms4 accelerates ethanol catabolism through upregulation of
the aldehyde dehydrogenase gene (SsdA/ZmsZMO1754) directly and the
alcohol dehydrogenase 1 gene (AdhA) indirectly to mask ethanol to other
carboxylic acids. On the other hand, Zms6 upregulated under ethanol stress
regulates the expression of the lysine export Zms1437 gene and negatively
regulates the expression of the methylase gene of Zms1934 N-6 DNA to
improve ethanol tolerance and avoid import of methylated DNA created by
ethanol damage, respectively. However, several regulatory functions of this
Zms6 gene are still unknown. Otherwise, Zms16 gene interacts directly as a
target only with the gen Zms6 [41].
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FIGURE 2.4 Metabolic pathway of Saccharomyces cerevisiae and Zymomonas mobilis with
principal genes.
2.9 BIOETHANOL YIELD PRODUCTION OF Zymomonas mobilis

AND Saccharomyces cerevisiae
Currently ethanol production depends on the fermentation of various carbon

sources, which is why continuous improvement in industrial production
is necessary [42]. Usually, ethanol production is using strains that have
specific characteristics, such as tolerant to high temperatures, acidity, and
ethanol [43]. To produce ethanol with Zymomonas strains, various culture
media and fermentation processes, both batch and continuous, have been
used. Different fermentation strategies have been used, as well as various
culture practices for the evaluation of the alcohol fermentation by Z.
mobilis the most common is batch fermentation [5]. The Zymomonas is a
highly biotechnologically relevant ethanogenic bacterium, because it has
a larger cell surface than S. cerevisiae, consumes glucose faster, leading to
a higher production of ethanol, being up to 12% v/v and the tolerance of
ethanol is 16% v/v. While Z. mobilis uses the ED pathway that results in 1
ATP per mole of glucose, S. cerevisiae obtains 2 ATP per mole of glucose.
Therefore, Z. mobilis work with 50% less ATP, which leads to a better
ethanol yield [5, 41, 44].
The TJ14 hybrid used by Benjaphokee in 2012 [43] produce 46.6 gL–1 of
ethanol with 10% glucose medium at 41°C and pH 3, a considerable yield,
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considering tolerance to high temperatures. Most yeast prefers ferment glucose

than fructose when both are in the culture medium [5, 42, 43, 45, 46, 77].
The aforementioned yield is similar to reported by Shihui Yang [78] with
production of 0.50 g g–1 at pH 4.5 and 37C and 100 gL–1 of glucose with 12
hours of fermentation, using Z. mobilis (ATCC 10988) strain, which uses the
ED fermentation pathway, and has a larger surface area than Saccharomyces.
Fatty acids are produced by yeast and bacteria in response to stress. In this
study, Wang et al. in 2016 [45] modified an operon (CP4) that induced their
overproduction and high ethanol yield of 78 gL–1 at 32°C in 60 h. Davis
in 2006 [42] evaluated the ethanol production by Z. mobilis ZM4 and, the
comparison of S. cerevisiae in the same conditions, using the same substrate.
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Ethanol production was 0.49 gg–1 with 100 g of glucose at 30°C with Z.
mobilis ZM4 strain and 0.46 gg–1 with S. cerevisiae, this can be attributed
to the medium supplementing with KH2PO4 and (NH4) 2SO4 among other
substances [5, 42]. Mazaheri and Pirouzi in 2020 [46], evaluate Z. mobilis
for bioethanol production from potato peel waste, obtaining 23.3 gL–1 with
initial sugar concentration of 61.3 gL–1. The combination of enzymes for the
hydrolysis process could have effectively released the fermentable sugars
from the solids of the potato peel, however, the final concentration of ethanol
obtained is not high enough for industrial scales as it causes high energy
consumption in the purification stage [46].
2.10 STRESS CONDITIONS IN FERMENTATION IN Zymomonas

mobilis AND Saccharomyces cerevisiae
The four major stress responses studied during fermentation are tempera-
ture stress, osmotic stress, lack of nutrients and ethanol produced during
fermentation [40].
2.10.1 TOLERANCE TO HIGH CONCENTRATIONS OF GLUCOSE
There are different strains to Zymomonas that can support high concentration
of glucose to grow up in 400 gL–1 medium, but the optimal glucose concentra-
tion is 200 gL–1 to obtain the maximum concentration of ethanol, 96 gL–1. This
bacterium has a higher glucose tolerance than S. cerevisiae and, therefore,
highly concentrated glucose media can potentially be used. The bacterium
metabolizes glucose, sucrose, and fructose. However, there are some strains
that are modified genetically to use other carbon sources like xylose [47].
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The yeasts are classified according to the respiratory-fermentative

metabolism regulation. The Crabtree-positive species ferment under aerobic
conditions, and contrary, the extent of fermentative metabolism for Crabtree-
negative species is very limited under sufficient oxygen conditions [48].
Hypothetically, in high sugar levels, Crabtree-positive yeast can adapt and

exploit sugars faster than Crabtree-negative competitors [49].
2.10.2 ETHANOL TOLERANCE
Osmotic pressure and temperature are environmental factors that influence
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ethanol tolerance in yeast, as well as available nutrients and growth substrates
[50]. High ethanol tolerance is a hallmark of Saccharomyces cerevisiae and a
wide variety of genes are responsible for a moderate ethanol tolerance ranging
from 6 to 12% in laboratory strains and 16 to 20% in industrial strains, respec-
tively [79]. According to Ming [80], wild-type Z. mobilis has an adaptive
mechanism in response to ethanol stress and could tolerate up to 13%. There-
fore, with these results, we can see that Zymomonas has a great advantage
compared to yeast for tolerance to ethanol in the medium, this being one of
the most important factors to consider choosing a microorganism to carry out
the fermentation process in this bioprocess for ethanol production [81].
2.10.3 TEMPERATURE TOLERANCE
Temperature is one of the most important factors that affect the production
of ethanol by different microorganisms. The fermentation without cooling,
produces microbial heat shock stress, the microbial growth is reduced, and
ethanol production yield is affected. There are some Zymomonas strains
thermotolerant, can growth even at 39°C, that is, 5–10°C higher than the
optimum temperature. This Z. mobilis harbor degP gene from heat shock
proteins (HSP) family [51, 52]. The HSF family of proteins (heat shock
transcription factor) are primary modulators of heat shock response (HSR),
as well as the MSN2 and MSN4 genes of significant use in the expression of
the heat shock gene. Heat shock factor in S. cerevisiae (Hsf1) is an essential
protein that assists HSR in adapting to oxidative stress and glucose defi-
ciency [40].
It is noted that despite the fact that some Saccharomyces strains can grow
at high temperatures, ethanol production is lower than Zymomonas strains
that grow at the same range of temperatures [43, 52–56].
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2.11 CONCLUSION
Currently, innovative, and more efficient microbial alternatives for ligno-

cellulosic biorefineries are needed to improve the biofuels (bioethanol)
production worldwide. Physiologically, Z. mobilis is an excellent alternative

by both ethanologenic nature and highly efficient capacity to biodegrade
several lignocellulosic residues in bioethanol. In order to apply Zymomonas
in industrial fermentations, multiple genetic tools, and metabolic engineering
methods, as well as biofilm reactor implementation to enhance and increase
the bioethanol production sustainably have been investigated.
Finally, the results of decades of multidisciplinary scientific research
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strongly suggest that the unique and amazing physiology of Z. mobilis provides
efficient biosynthetic machinery for industrial-scale biotechnological produc-
tion of bioethanol.
KEYWORDS
• bioethanol
• biofuels
• heat shock proteins
• Saccharomyces cerevisiae
• trehalose-6-phosphate phosphatase
• Zymomona mobilis
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CHAPTER 3

Clostridium thermocellum
D. B. ARYA,1 SALOM GNANA THANGA VINCENT,1 and
NAGAMANI BALAGURUSAMY2
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1
Department of Environmental Sciences, University of Kerala,
Thiruvananthapuram, Kerala, India
2
Bioremediation Laboratory, Autonomous University of Coahuila, México
ABSTRACT
Clostridium thermocellum has been extensively studied as a model organism

for microbial cellulose degradation due to its ability to rapidly solubilize
biomass and use cellulose as a carbon and energy source, which is attributed to
the presence of cellulosome, an extracellular multienzyme complex. C. ther-
mocellum is a thermophilic, rod-shaped anaerobe and is capable of producing
ethanol directly from a wide range of substrates. It was first isolated in 1926,
followed by detailed characterization studies in the subsequent years. Recently
this organism has received increased attention as a potential candidate for the
consolidated bioprocessing (CBP) of lignocellulosic biomass (LCB) into
biofuels such as ethanol, where CBP is a single step deconstruction and conver-
sion of lignocellulose into useful products, like acetate, lactate, hydrogen, and
ethanol. Although engineered C. thermocellum can tolerate ethanol concentra-
tions up to 80 g/L, the ethanol production under normal conditions is <30
g/L. This is because of the inefficiency of the ethanol production pathway of
C. thermocellum, wherein, the key enzyme for ethanol production, ferredoxin
oxidoreductase has lower affinity than lactate dehydrogenase (LDH) and
phosphotransacetylase (PTA) resulting in the diversion of carbon flux towards
production of more undesirable by-products like acetic and lactic acids. Never-
theless, several reports describe the successful attempts to increase ethanol
production involving pathway modifications, strain improvement, or genetic
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engineering to achieve a significant increase in ethanol yield up to 75% of the

theoretical maximum.
3.1 INTRODUCTION
Renewable energy sources receive much attraction in the present scenario

due to the reduced supply and high cost of available nonrenewable resources.
The alternative fuel for nonrenewable resources must contain some essential
characteristics: they have to be sufficient to meet the worlds energy demand,
should be environment friendly and cost-effective. Biofuels are one of the
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promising alternatives for nonrenewable resources and hence, significant
technologies should be explored to meet world energy demand for low carbon
fuels [1]. Cellulosic biofuels can make a significant contribution to rural
economy [2]. Ethanol from lignocellulosic material is a very cost-effective
alternative. Due to the complex structure and recalcitrant nature of LCB,
the production of bioethanol from lignocellulosic material needs expensive
chemical or physical pretreatments along with enzyme treatment that has a
negative impact on its industrial large-scale production [3, 4].
Pretreatment is necessary to convert cellulosic material to fermentable
form, such treatment enable the feedstock readily available for the enzyme
action [5]. Acid or hydrolytic enzymes can be used as pretreatment, in which
acid or enzyme degrade complex cellulose to glucose monomers by disrupting
the hydrolytic linkage [6]. The main problem associated with the produc-
tion of microbial ethanol from lignocellulosic material is the conversion of
cellulosic material to fermentable form [7]. It is important to overcome these
impacts for an economically feasible sustainable production by integrating
all processes into CBP and genetic manipulation of ethanol producers [8].
This review consolidates the characteristic features and the physiology of C.
thermocellum that makes it an attractive and potential candidate for ethanol
production.
3.2 WHY Clostridium thermocellum?
For the first-generation bioethanol production, mesophilic organisms such as

Saccharomyces cerevisiae and Zymomonas mobilis were used. The growing
interest of second-generation ethanol due to the obstacles of first-generation
ethanol led to the genetic modification of S. cerevisiae and Z. mobilis and the
use of other microbe which can utilize the feedstock. In 1980, the idea of using
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Physiology of Ethanol Production 45
thermophiles for second generation ethanol production was put forth [8]. The
main advantage of thermophiles over mesophiles is that the thermophiles can
utilize a wide range of substrate and allow direct-ethanol recovery through
in situ vacuum distillation. Moreover, they have the advantages of tolerating
extreme pH and salt concentration and low nutritional requirement [4].

Cellulolytic bacteria are efficient among the microbes for the direct
conversion of LCB into ethanol. The important ethanologenic thermophilic
anaerobe include members of the genera Clostridium, Caldanaerobacter,
Thermoanaerobacter, Bacillus, Geobacillus, Pencibacillus, and Caloramator
[4, 9–11, 42]. Among these, Clostridium is widely studied due to its strain
diversity. They are strict anaerobes and capable of utilizing a wide spectrum
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of substrates and thrive under mesophilic or thermophilic temperature
conditions [12]. The widely explored Clostridium species is C. thermocellum,
which could grow at a temperature range of 50 and 68°C and are able to
degrade complex cellulosic structures with the presence of cellulosome,
a complex hydrolytic enzyme [13] which has demonstrated remarkable
hydrolysis efficiency of cellulose [14]. Moreover, thermophilic cellulolytic
microorganisms like C. thermocellum are regarded as potential candidates
because their growth at high temperatures reduces the risk of contamination
and also increases the substrate solubility [15].
Clostridium thermocellum is an anaerobic, rod-shaped, Gram-positive
thermophilic microbe which is able to produce ethanol directly from cellu-
lose. C. thermocellum is an ideal bacterium for synthesizing various alcohols
including ethanol and isobutanol [16, 17]. Thermophilic cellulolytic micro-
organisms are well suited for this process because thermophilic condition
reduces the risk of contamination and increases the solubility of feedstock
[13]. C. thermocellum is an ideal candidate for CBP which includes a single
step which includes the degradation of lignocellulosic material and their
conversion to useful products [16]. In 1926, Viljoen et al. [18] attempted
to identify the organism capable of degrading cellulose, which lead to the
basic identification of C. thermocellum. Further, McBee [19] character-
ized C. thermocellum and observed that they can grow in between 50 to
60°C in substrates such as cellulose, cellobiose, and hemicellulose. The
various byproducts were CO2, hydrogen gas, acetic acid, succinic acid, and
ethanol. After these studies, Freier et al. [20] revealed that the optimum pH
and temperature for these organisms are 6 to 7 and 55°C respectively. C.
thermocellum can be cultured in both batch and continuous culture, with
growth rates of 0.10/h and 0.16/h, respectively [21]. The attractive feature
of C. thermocellum is the presence of cellulosome, which is an extracel-
lular multienzyme complex containing around 20 enzymes able to reduce
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the resistance of lignocellulosic material from degradation [22]. Also, they

possess multiple carbohydrate esterases (CEs) which help in the reduction of
LCB in a concentrated enzyme reaction [23–25].
3.3 PHYSIOLOGY OF C. thermocellum
The central metabolism of thermophilic anaerobic bacteria like C. thermocellum

is the bioconversion of sugars. Through modified Embden-Meyerhof-parnas
(EMP) glycolytic pathway C. thermocellum converts glucose and cellodexins
to pyruvate by using mixed acid fermentation [26] (Figure 3.1). The entire
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pathway is controlled by a set of cofactors such as NADH, ATP, etc., through
glycolysis and hexose is converted to pyruvate. After that pyruvate is reduced
to acetyl coA by pyruvate ferredoxin oxidoreductase, that leads to the further
reduction of acetyl coA to ethanol by alcohol dehydrogenase (ADH). Final
end products are mixed acids. The formation of these byproducts is the main
obstacle for high ethanol yield. Understanding the electron transfer mechanism
is very important for the metabolic engineering of desired organism [27].
3.4 STRAIN IMPROVEMENT FOR ENHANCED ETHANOL

PRODUCTION
One of the important by-products of cellulose fermentation by C. thermocellum

is ethanol, which attracts attention as a renewable energy resource. The
formation of other by-products like acetate and lactate and their inability to
utilize xylose is a major drawback. In order to avoid this, Shaw et al. [28]
attempted to delete the genes encoding lactate dehydrogenase (LDH) and
phosphotransacetylase (PTA) to increase ethanol production. Wild type C.
thermocellum can only tolerate ethanol up to 5 g/L, above which, the organism
is signiﬁcantly inhibited [29]. Several studies were carried out to overcome this
sensitivity problem. The sensitivity occurs due to the presence of specialized
lipids in the membrane structure. So, the wild type strain must be genetically
modified to tolerate large amounts of ethanol. Ethanol production improved
after the adaptation of strain for improved growth and high yield [30]. Studies
were also focused to increase ethanol production by disrupting the normal
pathways [31]. Recent studies showed that thermophilic anaerobes, especially
C. thermocellum is more effective for the degradation of LCB than industrial
standard fungal cellulases in a wide range of conditions [1]. In 2015, Biswas et
al. [31] and Rydzak et al. [32] attempted to increase the ethanol production by
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disrupting competitive pathways for the electric flux by hydrogenase deletion

to increase the yield to 64% of theoretical maximum. Papanek et al. [33]
attempted to increase ethanol production by deleting genes encoding for lactate,
formate, and other compounds and showed similar output of hydrogenase
deletion. Other attempts were carried out to introduce n-butanol/isobutanol

synthesis pathway to native species [34], which achieved a production rate of
5.5 g/L [35].
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FIGURE 3.1 Simplified scheme of glucose degradation to various end products by strictly
anaerobic bacteria. [Enzyme abbreviations: ALDH: acetaldehyde dehydrogenase; ADH:
alcohol dehydrogenase; AK: acetate kinase; FNOR: ferredoxin oxidoreductase; H2-ase:
hydrogenase; LDH: lactate dehydrogenase; PFOR: pyruvate ferredoxin oxidoreductase; and
PTA: phosphotransacetylase].
Genetically engineered C. thermocellum exhibit high ethanol tolerance

as compared to wild strains of C. thermocellum and other thermophiles [1].
Recently developed engineered strain of C. thermocellum produced ethanol
at 75% of theoretical yield and titer of 25 g/L. Since microbial tolerance to
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ethanol is complex, related to membrane alterations and limited by electron

flux, further studies are required using approaches to genetically modify C.
thermocellum. A recombinant strain of C. thermocellum by introduction of
PDC gene from Zymomonas mobilis enhanced ethanol production ability
which can be further increased by improvements involving bioprocess

optimizations [36]. Genes encoding NfnAB and Rnf are studied widely for
biofuel production due to their presence in several anaerobic microorgan-
isms and their importance in bioethanol production [27]. Introducing four
genes: adhE, nfnA, nfnB, and adhA involved in ethanol production in Ther-
moanaerobacterium saccharolyticum were insufficient to achieve expected
ethanol yields in C. thermocellum [37], which suggests the presence of
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missing components and requires further investigations.
3.5 CONSOLIDATED BIOPROCESSING (CBP)
Consolidated bioprocessing (CBP) technologies combine the enzyme produc-

tion, hydrolysis, and fermentation stages into a single step, which helps to
increase processing efﬁciencies, eliminating the need for added exogenous
hydrolytic enzymes, and reducing the sugar inhibition of cellulases [38]. CBP,
formerly known as direct microbial conversion (DMC), is identified as an
economic process for the production of second-generation biofuel based on a
candidate CBP microorganism or group of microorganisms having the ability
for combined hydrolysis and fermentation. The process is economic due to
simpler feedstock processing, lower energy inputs and higher conversion
efficiencies. Relevant CBP microorganism is required for an economically
feasible production and C. thermocellum is identified as a potential candi-
date for CBP by enhancing pathway thermodynamic function and feasibility
due to the presence of complex cellulosome [39], its fast rate of cellulose
digestion and also the ability to hydrolyze both hemicellulose and cellulose
in a broad range of substrates. Moreover, owing to the higher fermentation
temperature, the fermentation process is not easily polluted and ethanol is
easily separated. CBP is cost-effective because the CBP microorganisms do
not require exogenous saccharifying enzymes and they produce their own
cellulolytic and hemicellulolytic enzymes for the degradation of LCB [14].
The cost-effectiveness and industrial relevance of CBP is determined based
on the target ethanol yield of >90%. For CBP to be economically feasible, the
CBP microorganisms should have an enzyme system capable of fermenting
sugars to ethanol at a rate of more than 1 g/L/h [43]. As no microorganisms
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with such efficiency is discovered, genetic engineering technologies can

be employed for creating efficient CBP microorganisms. The fermentation
process can also be improved through mutagenesis as well as improvement
of technological and culture conditions.
The ideal microorganisms for CBP should have several essential char-
acteristics including simultaneous utilization and conversion of multiple
sugars like cellobiose, glucose, and xylose and also the ability to tolerate
toxic by-products. Most of the studies involving CBP is based on ethanol
fermentation using model substrates like cellulose and xylan and studies
pertaining to real substrate-based ethanol fermentation is limited. Hence,
enhanced ethanol production from native LCB like rice straw biomass is
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the goal of sustainable CBP. Co-culture of different cellulolytic and sugar-
fermenting thermophilic anaerobic bacteria has been widely studied to
achieve improved ethanol production using CBP. Singh et al. [40] demon-
strated the direct fermentation ability of a thermophilic anaerobic cellulolytic
bacteria, C. thermocellum DSM 1313 isolated from Himalayan hot spring,
to convert various cellulosic and hemicellulosic substrates into ethanol
using a CBP based approach without addition of any exogenous enzymes.
This strain showed good ethanol production on pretreated rice straw. Use
of CBP in butanol production from LCB is a good option due to the high
energy density of butanol. A co-culture of C. thermocellum and C. saccha-
roperbutylacetonicum has been reported to produce a significant amount of
butanol using crystalline cellulose as substrate [41], and further research is
also focused on developing new pathways of butanol production by genetic
engineering of C. thermocellum.
3.6 CONCLUSION
The use of biofuels is a suitable alternative to cope up with the increasing

energy demand. Second-generation biofuel production technologies make
use of lignocellulosic feedstock to produce ethanol. Microorganisms
can degrade these feedstocks in several ways. Thermophilic cellulolytic
bacteria are well-known candidates for ethanol yield, among which C.
thermocellum is well known for the degradation of lignocellulosic mate-
rials as well as ethanol production and thus can be used to solve the dual
problem of waste disposal and energy production. The genetic alteration of
the native strains improves ethanol production and opens a way towards a
sustainable world.
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KEYWORDS
• C. thermocellum
• consolidated bioprocessing
• ethanol production
• lactate dehydrogenase
• phosphotransacetylase
• strain improvement
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CHAPTER 4
Genetic Regulation of Principal

Microorganisms (Yeast, Zymomonas
mobilis, and Clostridium thermocellum)
Producing Bioethanol/Biofuel
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DANIA SANDOVAL-NUÑEZ,1 TERESA ROMERO-GUTIÉRREZ,2
MELCHOR ARELLANO-PLAZA,1 ANNE GSCHAEDLER,1 and
LORENA AMAYA-DELGADO1
1
Industrial Biotechnology Unit, Center for Research and Assistance in
Technology and Design of the State of Jalisco A.C., Camino Arenero–1227,
El Bajio del Arenal, C.P.–45019, Zapopan, Jalisco, Mexico
2
Computer Sciences Department, Exact Sciences and Engineering
University Centre, Universidad de Guadalajara, Blvd. Gral. Marcelino
Garcia Barragan–1421, Olimpica. Guadalajara, Mexico
ABSTRACT
Interest in ethanol production has been increasing since the 1980s because it is
considered an alternative source of clean energy. The search for new and cheaper
raw materials drove the development of new processes to obtain ethanol, such
as the fermentation of lignocellulosic hydrolysates. In lignocellulosic ethanol
production, microbial cells are under a stress-inducing environment, and
their physiological behavior is altered significantly, indicating that different
gene regulatory mechanisms are activated from those in non-stress-inducing
conditions. Ethanologenic yeasts and bacteria tune gene expression to regulate
response mechanisms and quickly adapt their global metabolic networks to
grow and produce ethanol under unfavorable circumstances. Understanding
the regulatory mechanism of gene expression during ethanol production has
been a main topic in molecular and cellular biology with the goal of developing
more efficient and resistant ethanol-producing microorganisms.
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In this chapter, we present the state of knowledge in the field of genetic

regulatory mechanisms in the most important microorganisms used for
ethanol production, the yeasts Saccharomyces cerevisiae and Kluyvero-
myces marxianus, and the bacteria Zymomonas mobilis, and Clostridium
thermocellum.
4.1 INTRODUCTION
Ethanol is becoming increasingly important in energy supply and economic

development. Industrial ethanol production is commonly carried out by the
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yeast Saccharomyces cerevisiae, which is capable of fermenting C6 sugars
only [1]. However, the degradation of other materials such as lignocellulosic
biomass (LCB) also produces C5 sugars; this problem has been mitigated by
using recombinant strains of S. cerevisiae and bacteria such as Z. mobilis and
some thermophiles such as Clostridium thermocellum. Interestingly, micro-
organisms such as C. thermocellum can not only efficiently degrade cellulose
and hemicellulose but also ferment hexose sugars to ethanol. Regardless
of the microorganism used for ethanol production, fermentation efficiency
depends on cellular metabolism, which is directly related to gene regulation.
Genetic regulation is essential for microorganisms in several biological
processes, including the generation of biomolecules such as ethanol. During
ethanol production, gene regulation plays a critical role in the cellular
capacity to adapt quickly to the physicochemical conditions of the process,
the ability to utilize carbon sources, and the ability to use alternative
metabolic pathways to overcome nutrient-limiting conditions or respond to
stressors [2]. Genetic regulation can be exerted on different levels in the cell;
the most basic level is transcription. Regulation in eukaryotic cells requires
the coordination of a whole set of genes that are scattered over the genome.
This mechanism is different in bacteria, where genes, which are regulated
in the same way, are often organized into operons, and transcribed from one
regulatory sequence. When ethanol is produced, two types of regulation can
be present: stimulation of the expression of specific genes (positive regula-
tion) or inhibition of their expression (negative regulation) [1, 2]. Several
studies have demonstrated that some restrictions are present in bioethanol
production when wild-type microorganisms are used. These restrictions
involve different biotic (formation of unwanted products) and abiotic effects
(source of carbon, nitrogen, nutrients, temperature, oxygen, and the presence
of stressful molecules) on the microorganisms. These restrictions may lead
to a low conversion rate and low yield in industrial ethanol production. Thus,
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Genetic Regulation of Principal Microorganisms 55
it is critical to know and control genetic regulation in yeast and bacteria to

improve ethanol production.
The regulation of genes involved in the production of ethanol has been
extensively studied. For example, alcohol dehydrogenase II (ADH2) is
encoded by an ADH gene, and its primary function is to catalyze acetaldehyde

conversion to ethanol. Therefore, it may be possible to improve ethanol
production by modifying the function of ADH2. To date, many genetic
engineering methods have been applied to disrupt the expression of the ADH2
gene in S. cerevisiae, Z. mobilis, and C. thermocellum [2–4]. Therefore,
knowing the regulatory mechanisms and generation of intermediary
molecules during ethanol production allows the establishment of strategies
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for the positive or negative regulation of specific genes that increase ethanol
yields. This chapter presents the state of knowledge in the field of genetic
regulatory mechanisms in the most important microorganisms used to
produce ethanol. It is based on experimental evidence of how microorganisms
combine different regulatory mechanisms to coordinate multiple metabolic
pathways during ethanol production.
4.2 GENE REGULATION IN ETHANOLOGENIC YEAST
Gene regulation is a cellular process consisting of activating or deactivating

genes, which can occur at any point in the transcription-translation process.
Gene regulation occurs most frequently at the transcriptional level. The
regulation of gene expression in yeast can take place in different stages
(Figure 4.1). In the nucleus, the chromatin remodeling process regulates the
availability of a gene for transcription. Once transcribed, the primary mRNA
transcript, or pre-mRNA, undergoes RNA processing, which involves
splicing and adding a 5’ cap and 3’ poly (A) tail to produce a mature mRNA
in the nucleus. Mature mRNA is exported from the nucleus to the cytoplasm,
where its lifespan varies. Outside the nucleus, localization factors can direct
mature mRNAs to specific regions of the cytoplasm where they are translated
into polypeptides. The resulting polypeptides can undergo posttranslational
modifications, which can regulate protein folding, glycosylation, intracellular
transport, and protein activation and degradation.
Gene regulation is crucial for all eukaryotic organisms, such as yeast, and
it is mainly required to adapt rapidly to environmental changes and condi-
tions. This regulation may involve adaptation to different carbon sources,
the ability to use alternative metabolic pathways to overcome limiting
nutrient or environmental conditions, and responses to stress factors such as
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temperature, cold, chemical agents, ethanol, and the generation of metabolites

[1]. Gene regulation in yeast is crucial during the generation of products or
molecules of industrial interest, such as ethanol. Because ethanol production
requires a series of biochemical stages, it is imperative to know the regula-
tory mechanisms carried out in yeasts during the fermentation process. This
knowledge allows control of the fermentation processes to achieve higher
yields and productivities. Some of the primary yeast taxa most studied in
ethanol production processes are S. cerevisiae and K. marxianus.
FIGURE 4.1 Representation of levels of genetic regulation in yeasts. Author Copy

4.2.1 SACCHAROMYCES CEREVISIAE AS A MODEL STRAIN IN
ETHANOL PRODUCTION
Saccharomyces cerevisiae is an undisputed model yeast in ethanol produc-

tion at the industrial level. The production of first-generation ethanol (1G
ethanol) is carried out mainly by various strains of S. cerevisiae, which are
used by large ethanol-producing companies such as Lallemand Biofuels
and Distilled Spirits, ABMauri Biotek, and Lesaffre Advanced Fermenta-
tion. First-generation ethanol is produced mainly from sucrose from sugar
cane and starches from cereal grains such as corn [5]. The integration of
consolidated processes is being sought where the use of other types of
carbon sources is required, such as residues from LCB for the production of
second-generation ethanol (2G ethanol).
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One of the main challenges faced in the fermentation process for the
production of 2G ethanol is the presence of various toxic compounds that
affect yeast physiology and metabolism, inhibiting or reducing ethanol yields.
S. cerevisiae has been studied in several fermentation processes where stress
factors such as high temperatures, the absence of nutrients, and the presence
of cell growth inhibitors such as organic acids, furans, phenols, and aldehydes
intervene [6–9]. The presence of inhibitory compounds causes changes in
yeast gene regulation, mainly during the transcription process, where the
overexpression or inactivation of genes is involved in the consumption of the
carbon source and therefore in ethanol production. However, the glycolysis
pathway is not the only pathway affected by the presence of inhibitors, since
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for the carbon source to be consumed and subsequently converted to ethanol,
the inhibitors present in the medium must be metabolized by yeast [10, 11].
Once the inhibitors are metabolized, the yeast is able to consume the sugars
quickly.
Nevertheless, inhibitors activate a series of defense mechanisms and
homeostatic balance in yeasts, achieving an ideal intracellular balance for
ethanol production [9]. The detoxification process in S. cerevisiae employs
a series of oxidation-reduction reactions to remove the high concentration
of reactive oxygen species (ROS) in the mitochondria. On the other hand,
S. cerevisiae uses a series of metabolic pathways to synthesize amino acids,
transporters, and membrane lipids, produce secondary metabolites, and
overexpress enzymes that facilitate the detoxification process and ethanol
generation (Figure 4.2).
4.2.1.1 TRANSCRIPTION FACTOR GENES AND INTERMEDIATE

ENZYMES IN ETHANOL PRODUCTION
Alcohol dehydrogenase (ADH) enzymes are the most studied enzymes in

ethanol-producing yeasts since they catalyze acetaldehyde reduction to
ethanol. In S. cerevisiae, seven ADH isozymes (ADH1 to ADH7) have been
identified that have different physiological roles and functions. The ADH1
enzyme catalyzes acetaldehyde reduction to ethanol during glucose fermen-
tation; it can also catalyze the reverse reaction. ADH1 is constitutively
expressed, while the reduction of intracellular glucose concentration induces
the expression of ADH2. The main function of ADH2 is to oxidize ethanol to
acetaldehyde. On the other hand, ADH3 is a mitochondrial isozyme with an
important role under anaerobic conditions: ADH3 forms part of the ethanol
acetaldehyde shuttle, helping to shuttle mitochondrial NADH to the cytosol
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for NAD+ regeneration. ADH3 is repressed by glucose, and its expression

is derepressed after glucose depletion. ADH4 and ADH5 are associated with
ethanol production, and ADH4 expression is upregulated under low zinc
concentrations or zinc starvation. The ADH5 isozyme might be expressed
under conditions in which none of the other four ADHs (ADH1 to ADH4)
are functional. In fermentation processes in the presence of furan aldehydes
(furfural and HMF), the overproduction of ATP and NADPH has been
observed since the responses to stress by furan aldehydes generate oxida-
tive stress, which provokes a considerable increase in the net production of
NADPH in strains of S. cerevisiae (> 4-fold). In this condition, the additional
NADPH could be used by ADHs (ADH6 and ADH7) to convert furan alde-
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hydes into less toxic compounds, lowering the toxicity of the medium for the
yeast [12].
FIGURE 4.2 Schematic representation of the modulation pathways and genes involved
in ethanol production under stress conditions. [Abbreviations: HXK: hexokinase; GLK:
glucokinase; MDH: malate dehydrogenase; THI: thiamine metabolism regulatory protein; PDR:
transcription factor; YOR: oligomycin resistance ATP-dependent permease; STB: protein STB,
DNA-binding transcription factor; YAP: AP-1-like transcription factor; GZF: DNA-binding
transcription factor; LEU: regulatory protein; PUT: proline utilization trans-activator; WAR: weak
acid resistance protein; SFA: S-(hydroxymethyl)glutathione dehydrogenase; ALD4: potassium-
activated aldehyde dehydrogenase; ALD6: magnesium-activated aldehyde dehydrogenase;
ADH: alcohol dehydrogenase; RHR: glycerol 3-phosphate phosphohydrolase; HOR: glycerol-
1-phosphate phosphohydrolase; CHO: phosphatidylethanolamine N-methyltransferase; ARI:
NADPH-dependent aldehyde reductase; OYE: NADPH dehydrogenase; ALD: aldehyde
dehydrogenase; TFs: transcription factors].
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Multiple sequence alignment analysis of the ADHs of S. cerevisiae shows

the similarities and differences between the seven ADHs (Figure 4.3). ADH1
and ADH2 present the highest amino acid sequence similarity of 93%. ADH3,
ADH5, ADH6 and ADH7 maintain 79, 76, 29, and 27% similarity with
ADH1, respectively. Amino acid sequence analysis of the ADH4 isozyme

revealed a very low similarity (18%) with ADH1.
Advances in synthetic biology have focused on reengineering the ADH gene
to achieve higher substrate specificity and improved catalytic activity. In addition,
genome engineering methods allow us to obtain yeasts that overexpress genes
encoding enzymes associated with the ethanol production process, improve
ethanol tolerance, and assimilate a wide range of carbon sources [13, 14].
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In addition to the study of the expression of various critical genes involved
in ethanol production, transcription factors (TFs) play an essential role in the
production of 2G ethanol, participating in processes of adaptation and yeast
resistance to stressful conditions. TFs such as YAP1, STB5, DAL81, GZF3,
LEU3, PUT3, and WAR1 have been widely studied because they are associ-
ated with various stress response mechanisms. These TFs confer appropriate
biological characteristics on yeast to carry out ethanol production [15]. For
example, YAP1 is associated with the glutathione pathway and is essential in
the intracellular detoxification of ROS. On the other hand, some TFs, such as
LEU3 and DAL81, are involved in carbon metabolism, amino acid biosyn-
thesis, and nitrogen catabolism, allowing yeasts to use the available energy
in the form of ATP and NADPH to carry out detoxification processes [7, 15].
4.2.1.2 GENETIC REGULATION OF SACCHAROMYCES CEREVISIAE

DURING ETHANOL PRODUCTION UNDER STRESS CONDITIONS
During the saccharification process of LCB used as raw material for 2G

ethanol production, several inhibitors are generated, limiting the metabolic
activity of yeasts in the fermentation process. Among the main inhibitory
compounds produced are furfural and 5-hydroxymethylfurfural (HMF).
Even today, the genetic mechanisms involved in tolerance, adaptation, and
resistance to stress caused by these compounds are not fully understood. The
search for strains with higher tolerance to inhibitors promises to guarantee
the desired concentrations and productivities of the processes. However,
finding or generating resistant strains is not easy, not only because all the
mechanisms for tolerance to furan-type inhibitors are unknown but also
because the inhibitors do not act independently but synergistically, even
with ethanol [9, 16]. The TFs involved in different stress responses are
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Apple Academic Press 60
FIGURE 4.3 Multiple sequence alignment analysis of the alcohol dehydrogenases (ADH1 to ADH7) identified in Saccharomyces cerevisiae.
The conserved residues are highlighted in purple. Residues boxed in red correspond to the catalytic zinc-binding motif. Residues boxed in orange
belong to the binding site with NADH/NADPH. The multiple sequence alignment was obtained with the MUSCLE algorithm (https://www.ebi.
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ac.uk/Tools/msa/muscle/), using the ADHs sequences of Saccharomyces cerevisiae (strain ATCC 204508/S288c). Alignments were edited with
Jalview software (https://www.jalview.org/).
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activated or repressed by other TFs, forming a complex regulatory network

(Figure 4.4). This complexity can be exemplified by the function of the YAP1
transcription factor. This transcription activator is related to the oxidative
stress response and redox homeostasis. Its function is the regulation of genes
encoding antioxidant enzymes, including the thioredoxin system (TRX2,

TRR1), the glutaredoxin system (GSH1, GLR1), superoxide dismutase
(SOD1, SOD2), glutathione peroxidase (GPX2), and thiol-specific peroxi-
dases (TSA1, AHP1), which are components of the cellular thiol-redox
pathways. YAP1 acts in conjunction with the transcription factor SKN7 to
induce the expression of antioxidant enzymes. YAP1 is induced by oxida-
tive and carbon stress but is not activated by high temperature, acidic pH, or
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ionic stress. On the other hand, SKN7 forms part of the SLN1-YPD1-SNN7
two-component regulatory system, which is related to the expression control
of genes involved in the response to changes in extracellular osmolarity.
As mentioned above, gene regulation occurs most frequently at the
transcriptional level. The regulation of gene expression in S. cerevisiae
under stress-inducing conditions can affect several biological processes and
trigger a series of regulatory events mediated by numerous TFs (Figure 4.4).
Genes related to the processes of detoxification, adaptation, and tolerance in
S. cerevisiae are regulated by key TFs, such as the YAP gene family, PDR
gene family, RPN4, and HSF1 [7, 9, 15].
The primary genes activated during 2G ethanol production in the pres-
ence of inhibitors such as furfural and HMF are described below.
4.2.1.3 ADAPTATION AND TOLERANCE OF SACCHAROMYCES

CEREVISIAE TO ACETIC ACID, FURFURAL, AND HMF
In recent years, various studies have been carried out to find strategies
to understand the molecular mechanisms involved in the adaptation and
tolerance of S. cerevisiae with respect to the main groups of growth and
fermentation inhibitors, such as aldehydes, phenols, ketones, and weak
organic acids. Furfural, HMF, and acetic acid are the three main compounds
that participate in the inhibition of the 2G ethanol production process [17].
For ethanol production, fermentation is carried out at pH values between
4 and 5, causing the dissociation of acetic acid (pKa = 4.76), which diffuses
through the plasma membrane of yeast [18]. Inside the cell, the acid dissoci-
ates and releases a proton, making it necessary to use ATP to reach the cell
equilibrium required for essential biological functions and prevent cytosolic
acidification. The use of ATP causes a decrease in yeast growth during the first
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FIGURE 4.4 Transcriptional regulatory network visualization of Saccharomyces cerevisiae.

[Abbreviations: YAP: AP-1-like transcription factor; STB: protein STB, DNA-binding
transcription factor; DAL: transcriptional activator protein; GZF3: transcriptional regulator,
DNA-binding transcription factor; LEU: regulatory protein; PUT: proline utilization trans-
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activator; WAR: weak acid resistance protein; YRM: zing finger transcription factor; YRR:
zing finger transcription factor; PDR1: transcription factor, positive regulator of proteins
involved in permeability; CAD: YAP2, AP-1-like transcription factor; CIN: YAP4, AP-1-
like transcription factor; SMP: transcription factor, involved in osmostress response; RPN:
transcriptional activator protein; PDR: transcription factor; HSF: heat shock factor protein].
The classification of transcription factors in biological processes was carried out utilizing
PANTHER and the networks using YEASTRACT (http://yeastract.com/). Visualization of the
transcriptional regulatory network makes it possible to correlate transcriptional associations
such as gene activation or deactivation of transcription factors, green (positive), red (negative),
positive, and negative (brown).
hours of contact; this condition remains until the yeast manages its genetic
machinery to adapt to this adverse condition. The acetic acid concentration
can be higher than 5 g/l in lignocellulosic residue hydrolysates, increasing
the inhibition problem caused by acetic acid. At a high concentration of
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acetic acid, the physiological ability of yeasts to carry out fermentation is

decreased compared to that in normal conditions. Therefore, tolerance to
acetic acid at low culture pH is a key goal in yeast strain development for 2G
ethanol production. Meijnen et al. discovered that tolerance to acetic acid is
the result of a polygenic response of yeast, making it difficult for a targeted

genetic change to generate resistant yeast [19]. One of the most important
factors in resistance to acetic acid stress is Haa1p [20]. Haa1p is related
directly or indirectly to the regulation of the transcription of approximately
80% of the genes encoding protein kinases, multidrug resistance transporters,
proteins involved in lipid metabolism and in the processing of nucleic acids,
and proteins of unknown function, suggesting that this factor is one of the
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most important in the response of yeast to acetic acid. Other important
factors in resistance to acetic acid stress are Rim101p and Msn2p, which are
also displayed under other stress types, such as ethanol, high concentrations
of glucose and oxidative stress [21–23].
Furfural is well known to be transformed into furfuryl alcohol by
nicotinamide adenine dinucleotide-dependent dehydrogenases (NADH)
under anaerobic conditions. Therefore, enzymes that participate in the
reduction of aldehyde groups are essential to reduce the concentration of
inhibitors in lignocellulosic residues. The genes reported to participate in
the detoxification of furfural and HMF are ADH6, ADH7, ALD4, GRE3 [9];
ADH1 [24]; ARI1 [25]; and GRE2 [26]; in addition, the Y62 and Y76 genes
participate only in the detoxification of furfural, but no evidence has been
found that they also participate in HMF. Studies developed to evaluate the
capacity of ADH6 and ADH7 in the reduction of furfural and HMF show that
these enzymes have a high capacity to reduce aldehydes, as much as 100 times
greater than their oxidation capacity [27]. The synergistic activity between the
aldehyde dehydrogenase enzymes Ald5, Pad1 decarboxylase, and the alcohol
acetyltransferases Atf1 and Atf2 has been determined to provide resistance
to phenolic inhibitors [28]; thus, it is likely that they participate actively in
the transformation of furans present in lignocellulosic residues. Ma and Liu
demonstrated that transcriptional genes such as YAP1, PDR1, PDR3, RPN4
and HSF1 actively participate in the adaptation of S. cerevisiae to HMF [29].
In recent years, the existence of short-chain aldehyde reductase enzymes
known as SDR has been evidenced; SDR enzymes participate in the
transformation of aldehydes within yeast cells, including Ykl107wp. This
enzyme can reduce acetaldehyde, glycolaldehyde, furfural, formaldehyde,
HMF, and propionaldehyde, but it was not observed to reduce the six ketones
corresponding to the same compounds [30]. Aldehyde reductase enzymes
are highly diverse. SDRs have particular substrate preferences and are found
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in different places within the cell. In addition, considering that aldehydes can
cross mitochondrial, nuclear, and cytoplasmic membranes, the presence of
specific aldehyde reductases in each zone guarantees a decrease in cellular
damage [31, 32].
Although it is possible to direct the genetic modification of yeasts to

increase the production of TFs and increase resistance to acetic acid, furfural,
and HMF, it should be considered that several transcriptional factors are
activated during detoxification, so it is possible that affecting only one of
these factors will not have an overall impact. Likewise, the great diversity
of enzymes necessary to reduce cellular damage in each organelle should
be considered. Therefore, evolutionary adaptation is an interesting strategy
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because, during evolution, strains can undergo the necessary changes to allow
higher tolerance to the inhibitors present in the lignocellulosic hydrolysates
and even increase ethanol production and productivity [33–35].
4.2.1.4 METABOLOMICS ANALYSIS TO IMPROVE SACCHAROMYCES

CEREVISIAE STRESS TOLERANCE
The production of low-molecular-weight molecules or metabolites during

ethanol production is one of the main results of gene regulation in yeasts.
The complete collection of these metabolites integrates the metabolome
and is closely related to phenotype. The molecules generated in the cell
under specific culture conditions are directly associated with cellular
maintenance, growth, and function; this association has been used to analyze
the contribution of metabolic pathways to the cellular lifespan. However,
multiple metabolic pathways are affected by external stimuli, and it is
necessary to characterize the whole metabolome through metabolic analysis.
In recent years, metabolomic analysis has been utilized as a promising tool
to understand the molecular mechanism that drives the stress response and to
improve stress tolerance in yeasts [36–38].
Metabolomics analyzes have demonstrated the association between
ethanol stress tolerance and the production of specific metabolites in S.
cerevisiae. Li et al. demonstrated that the levels of metabolites produced
from intermediary molecules of the Embden-Meyerhof-Parnas (EMP)
pathway, glycine, serine, threonine, glycerol, alanine, isoleucine, and valine,
increase under ethanol stress, which indicates the inhibition of glycolysis.
Similarly, the levels of fatty acids are altered by chemical stress; lower levels
of palmitelaidic acid but higher levels of hexadecenoic and octadecanoic
acids were detected in S. cerevisiae under ethanol stress [36]. The evaluation
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of membrane phospholipids in S. cerevisiae under furan aldehyde stress

revealed an increase in phosphatidylethanolamine and its association with
the loss of membrane stability [11].
Regarding metabolites related to ethanol tolerance, metabolomic studies
have shown that intracellular accumulation of trehalose, proline, acetyl-CoA,

fumaric acid, aspartic acid, and TCA cycle-related metabolites leads to
improved ethanol tolerance [39]. These results have made it possible to establish
strategies to improve tolerance in S. cerevisiae by deleting selected genes to
increase (simultaneous deletion of LEU4 and LEU9) or reduce (simultaneous
deletion of INM1 and INM2) the concentration of metabolites such as valine
and inositol, which significantly increase ethanol tolerance [39].
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Despite all the successful results obtained through the years in under-
standing stress response mechanisms in S. cerevisiae, it is evident that a
rational strategy is necessary to improve yeast tolerance of ethanol or inhibi-
tory compounds. Such a strategy should combine omic analyzes (genomics,
transcriptomics, proteomics, metabolomics, and fluxomics) with predictive
computational modeling or simulation to design tolerant yeasts with better
metabolic properties for industrial ethanol production.
4.2.2 KLUYVEROMYCES MARXIANUS AS A POTENTIAL YEAST IN

ETHANOL PRODUCTION
Kluyveromyces marxianus is an unconventional yeast of great interest for use

in biotechnological processes aimed at the production of various metabolites,
such as aromatic molecules, higher alcohols, and lytic enzymes [10, 40–42].
Wild-type strains of K. marxianus have been widely studied, demonstrating
their ability and high potential to consume different carbon sources [43]. K.
marxianus has also been used as a model in various systems and strategies
of alcoholic fermentation to evaluate factors such as temperature, carbon
source aeration, enzyme expression, and the detoxification processes of
compounds present in hydrolysates. These tests showed ethanol yields from
agro-industrial residues and defined culture media (Table 4.1).
The use of K. marxianus in ethanol production has increased in the last
decade because it has several biological characteristics that give it interesting
properties for use in numerous fermentation processes. Thermotolerance
characteristics and robustness to chemical agents (organic acids, phenols,
furans, and aldehydes) present in lignocellulosic hydrolysates make K.
marxianus a model yeast suitable for use in improving ethanol yields in the
presence of toxic compounds [56, 57].
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TABLE 4.1 Ethanol Production by Kluyveromyces marxianus Using Different Carbon Sources
Raw Material Strain Fermentation Process Ethanol (g/l h) References
Barley straw K. marxianus CECT 10875 SSF 0.40 [44]
Sugar cane juice K. marxianus DMKU 3-1042 Batch 1.30 [45]
Wheat straw slurry K. marxianus CECT 10875 Batch 1.69 [46]
Wheat straw slurry K. marxianus CECT 10875 Batch 0.36 [47]
YPD K. marxianus BUNL‑21 Batch 0.06 [48]
Sweet sorghum juice K. marxianus DBKKUY-103 Batch 1.42 [49]
Wheat straw K. marxianus DBTIOC-35 SSF 0.86 [50]
YPDX K. marxianus CBS712 Batch 1.18 [51]
Kanlow switchgrass K. marxianus IMB4 SSF 0.23 [52]
Wheat straw K. marxianus CECT 10875 SSF 0.50 [53]
Corncob residue K. marxianus NBRC1777 Batch 1.05 [54]
Sugar cane bagasse K. marxianus NRRL Y-50883 (SLP1) Batch 0.29 [55]
Mineral medium K. marxianus NRRL Y-50883 (SLP1) Batch 0.30 [10]
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4.2.2.1 GENETIC REGULATION OF KLUYVEROMYCES MARXIANUS

DURING ETHANOL PRODUCTION UNDER STRESS CONDITIONS
Genetic regulation is one of the main biological mechanisms employed by yeast

at all stages of the cell cycle. However, several extracellular factors contribute
to the use of regulatory mechanisms associated with stress responses and cell
repair. The participation of some TFs, the overexpression and inactivation
of genes, and the synthesis and degradation of some metabolites have
been demonstrated when K. marxianus is employed in ethanol production
processes under certain fermentation conditions in the presence of different
carbon sources, nutrients, and toxic molecules [10, 42, 56].
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Ethanol production by K. marxianus has been evaluated mainly with
the use of agro-industrial residues and in defined culture media. Therefore,
understanding some of the molecular regulatory mechanisms employed by
K. marxianus during ethanol production is of great interest. In addition to
the carbon source used and its subsequent conversion to ethanol, several
biochemical and metabolic biological events disrupt ethanol conversion
pathways and mechanisms. Environmental or chemical effects provoke
biological responses by yeasts and consequently intervene in gene regulation
during the ethanol production process, inhibiting or decreasing the genera-
tion of ethanol [56, 58].
The presence of toxic molecules such as furfural and organic acids plays
an important role in gene regulation, biochemically affecting ethanol produc-
tion due to the overexpression of proteins and genes, affecting metabolic
pathways such as glycolysis, the pentose phosphate pathway (PPP), gluta-
thione, and butanoate metabolism, fatty acid metabolism, and the biosyn-
thesis of many amino acids such as Ala, Arg, Asp, Glu, His, Ile, Leu, Pro,
Trp, Tyr, and Val, in addition to MAPK signaling pathways. Some metabolic
pathways, such as those involving secondary metabolites and the synthesis,
and degradation of amino acids, TCA, and glyoxylate, act as intermediaries
in regulatory processes related to defense mechanisms and homeostasis,
maintaining the ideal intracellular balance for ethanol production [56, 59].
4.2.2.2 MOLECULAR STRATEGIES FOR THE GENETIC IMPROVEMENT

OF KLUYVEROMYCES MARXIANUS
The molecular techniques mostly used to improve K. marxianus for in ethanol

production include homologous recombination (HR) and CRISPR-Cas.
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These techniques have allowed the integration or overexpression of genes

that potentiate biological processes and reactions that participate as interme-
diaries in ethanol production [60].
Schabort et al. performed a metabolic regulation analysis using a tran-
scriptomics study on K. marxianus UFS-Y-2791 during glucose and xylose

consumption [61]. They concluded that gene regulation levels play an impor-
tant role in the regulation of metabolic fluxes in central carbon metabolism,
glycolysis reactions, and xylose adaptation. Gao et al. overexpressed thiore-
doxin TPX1 in K. marxianus Y179, achieving an increase in the maximum
rate of glucose consumption and the rate of ethanol generation by the strain,
a significant improvement compared to the control [62].
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Studies focused on ethanol production from xylose as a carbon source
have also achieved improvement by overexpressing enzymes through HR.
For example, the overexpression of enzymes such as xylose reductase
heterologous (XYL1) and xylitol dehydrogenase (XYL2) have increased the
participation of the phosphate pentose pathway and the flow into ethanol
production [63]. The increase in ethanol yields has been achieved through
the overexpression of native xylokinase (XYL3), L-ribulose-5-phosphate
4-epimerase (RPE1), ribose-5-phosphate isomerase (RKI1), transcetolase
(TKL1), and transaldolase (TAL1) genes as well as pyruvate decarboxylase
(PDC1) and ADH2. The XYL1 and XYL2 heterologous genes were selected
because of their preference for NADP(H) over NAD(H), thus helping to
rectify an imbalance in cofactors during growth on xylose [64].
4.2.2.3 KEY GENES ASSOCIATED WITH ETHANOL PRODUCTION IN

KLUYVEROMYCES MARXIANUS
Several genes and their association with the ethanol production process
have been reported to be involved in posttranslational regulation. In ethanol
production under certain stress conditions (temperature, ethanol, and
chemical agents), protein folding and the expression or repression of genes
coding for carbon source carriers and defense mechanisms have been found
to affect oxidation-reduction processes and intracellular transport, in addi-
tion to genes involved in the inhibition of routes and signaling pathways
associated with regulatory processes in ethanol production (Figure 4.5).
Assays performed in K. marxianus demonstrated the biological importance
of genes that code for intermediary enzymes in the ethanol production
process, mainly enzymes involved in carbon metabolism for both xylose and
glucose, enzymes involved in transport processes, mitochondrial enzymes,
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and mediators of cellular homeostasis processes under stress conditions

during ethanol production, such as MDH2, TDHs, and ADHs (Figure 4.5).
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FIGURE 4.5 Interactome of genes involved in ethanol production in Kluyveromyces
marxianus. [Abbreviations: DAK: dihydroxyacetone kinase; GUT: glycerol-3 phosphate
dehydrogenase; HXK: hexokinase; TSA: peroxiredoxin; SDH: succinate dehydrogenase;
ICL: isocitrate lyase; FBA: fructose-biphosphate aldolase; TDH: glyceraldehyde-3-phosphate
dehydrogenase; FBP: fructose-1,6-biphosphatase; KGD: 2-oxoglutarate dehydrogenase
complex; PGI: glucose-6-phosphate isomerase; TPI: triosephosphate isomerase; ADH: alcohol
dehydrogenase; GPM: phosphoglycerate mutase; CIT: citrate synthase; PFK: ATP-dependent
6-phosphofructokinase; PGK: phosphoglycerate kinase; MDH: malate dehydrogenase;
CDC: pyruvate kinase; ENO: enolase; LAT: dihydrolipoyllysine-resiude acetyltransferase
component of pyruvate dehydrogenase complex; ACO: aconitate hydratase; IDH: isocitrate
dehydrogenase; MLS: malate synthase; PCK: phosphoenolpyruvate carboxykinase;
ALD: aldehyde dehydrogenase; MAE: NAD-dependent malic enzyme; GDH: glutamate
dehydrogenase; GND: 6-phosphogluconate dehydrogenase]. The interactome was based on
S. cerevisiae using STRING: functional protein association networks (https://string-db.org/)].
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Seven ADHs (KmADH1 to KmADH7) have been identified in K.

marxianus; however, the function of KmADH5 to KmADH7 has been little
studied. Similar to S. cerevisiae, KmADH1 and KmADH2 are associated
with ethanol production, and both are expressed at high levels in aerobic
and anaerobic conditions. The function of KmADH3 is to use nonferment-

able carbon sources, and it is upregulated during the stationary phase. The
KmADH4 isozyme is related to ethanol detoxification, and its expression
also increases in the stationary phase. Expression analysis of KmADH5
suggests that this isozyme is expressed at the end of fermentation under
aerobic conditions; however, its expression in anaerobic conditions was
constant throughout fermentation. The function of the KmADH6 isozyme is
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associated with cellular processes carried out in the stationary phase under
anaerobic conditions. The metabolic function of KmADH7 is associated
with the oxidation of hemiacetal as an alternative route for the synthesis of
ethyl acetate, and its expression increases in the stationary phase [65].
Multiple sequence alignment analysis of the ADHs of K. marxianus
shows the similarities and differences between the main four KmADHs. The
similarity percentages of KmADH2 to KmADH4 with respect to KmADH1
are 82, 79, and 78% (Figure 4.6). Similar to that in S. cerevisiae, the amino
acid sequence reveals the metal binding site (zinc catalytic site) and the
NAD-binding site (according to NADH or NADPH dependence). Expres-
sion analysis demonstrated that the expression of KmADHs depends on the
growth phase and carbon source [65].
4.2.2.4 TRANSCRIPTION FACTORS (TFS), INTENSIFIERS, AND SILENCERS

INVOLVED IN ETHANOL PRODUCTION IN KLUYVEROMYCES MARXIANUS
In the process of regulating gene expression, the participation of expression

modulating sequences plays a very important role. These sequences can
intensify or silence the transcription process. Enhancers are sequences that
stimulate transcription and whose location can be thousands of nucleotides
away from the promoter. Silencers are sequences that inhibit transcription
and can also be located far away from the promoter. For K. marxianus,
few activating or repressor proteins have been reported that interact with
enhancer sequences or silencers. For example, Spt15 is a protein that intensi-
fies ethanol yields [66]. In contrast, the participation of TFs such as HSF1
and MSN2 confers resistance to chemicals such as furfural, phenol, and
acetic acid and increases lignocellulosic ethanol production [60].
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Apple Academic Press Genetic Regulation of Principal Microorganisms
FIGURE 4.6 Multiple sequence alignment analysis of the alcohol dehydrogenases (KmADH1 to KmADH4) identified in Kluyveromyces
marxianus. The conserved residues are highlighted in purple. Residues boxed in red correspond to the catalytic zinc-binding motif. Residues
boxed in orange belong to the binding site with NADH/NADPH. The multiple sequence alignment was obtained with the MUSCLE algorithm
(https://www.ebi.ac.uk/Tools/msa/muscle/), using the ADHs sequences of Kluyveromyces marxianus (strain ATCC 12424 for KmADH1-2, and
strain DMKU 3-1042 for KmADH3-4). Alignments were edited with Jalview software (https://www.jalview.org/).
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71
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Similar to S. cerevisiae and other yeasts, gene regulation in K. marxianus

is a complex regulatory network that affects different biological processes
(Figure 4.7). Although K. marxianus is a very interesting yeast for producing
2G ethanol, information about the activation of TFs during its stress response
is limited. However, the expression of several TFs by K. marxianus has been

demonstrated during heat stress (HSF1, MSN2, SFP1, and RPN4), oxidative
stress (MSN2, SNF2, GCNA), osmotic stress (MSN2), and stress by nutrient
starvation (SFP1) [56, 60].
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FIGURE 4.7 Transcriptional regulatory network visualization of Kluyveromyces marxianus.

[Abbreviation: HFS: heat shock factor protein; MSN: zinc finger protein; MSN: zinc finger
protein; OAF: oleate-activated transcription factor; MTF: mitochondrial transcription factor;
HCM: forkhead transcription factor; YNG: chromatin modification-related protein; MET:
transcriptional regulator; SNF: transcriptional regulatory protein; GCR: glycolytic genes
transcriptional activator; MED: mediator of RNA polymerase II; SFP: transcription factor;
RPN: transcriptional activator protein RPN; GCN: general control protein]. The visualization
of the transcriptional regulatory network for K. marxianus represents an approximation of
the transcriptional association between transcription factors, green (positive), red (negative),
positive, and negative (brown). For its construction, S. cerevisiae was used as a reference.
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4.2.2.5 METABOLOMIC ANALYSIS IN KLUYVEROMYCES MARXIANUS
Metabolomic analysis is a useful tool to increase knowledge about the

stress response mechanisms in K. marxianus. Similar to S. cerevisiae,
stress provoked by inhibitory compounds (ethanol, furan aldehydes, weak

acids, among others) inhibited the glycolysis pathway and, consequently,
the growth of K. marxianus. Several metabolites, such as amino acids,
sugars, and lipids, increased in concentration when K. marxianus was under
ethanol stress. Arginine, phenylalanine, glutamate, proline, and tryptophan
are related to the ethanol adaptation process, as the concentrations of these
amino acids increase when the yeast is exposed to ethanol stress. Trehalose
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is another metabolite that is recognized as a protein and plasma membrane
protector [67]. Flores-Cosío et al. observed an increase in the concentration
of phosphatidylethanolamine and a decrease in phosphatidylcholine when
K. marxianus was stressed by furan aldehydes, illuminating the effect of
these inhibitors on the plasma membrane and the capacity of K. marxianus to
reorganize its membrane [11]. Despite the potential of metabolomic analyzes
as tools to study the effect of inhibitory compounds on cellular metabolism
and physiology, this omic technique has been rarely used in K. marxianus, so
there are few studies that help to understand the stress response in this yeast
through its metabolome.
Recent studies have demonstrated that K. marxianus is a promising yeast
for ethanol production and other interesting metabolites, such as enzymes,
flavor, and fragrance compounds, and xylitol. K. marxianus has advantages
over other yeasts because it can assimilate several carbon sources (glucose,
xylose, arabinose, sucrose, inulin, fructans, among others). In addition, K.
marxianus is a thermotolerant yeast with a high growth rate and high resistance
to inhibitory compounds, and it can quickly adapt its metabolic machinery
to survive toxic environments. The main obstacle to its development at an
industrial level has been the limited knowledge of its genetics and physiology,
but this is rapidly changing thanks to the new omic technologies, making K.
marxianus a promising yeast for ethanol production.
4.3 GENETIC REGULATION OF ETHANOLOGENIC BACTERIA
Bacteria, similar to yeasts, are exposed to changing environments in which

biotic and abiotic factors can radically alter their metabolism. Bacteria
respond to such variations by regulating their gene expression; thus, they can
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adjust the metabolic pathways depending on the carbon source, pH, tempera-
ture, toxic compounds, and other nutrients available. Different mechanisms
regulate genes in bacteria and yeasts; bacterial genes are organized into
operons or clusters of coregulated genes. In addition to being physically
close in the genome, these genes are regulated to turn on or off together. This
characteristic of grouping related genes under a common control mechanism
allows bacteria to adapt rapidly to changes in the environment.
Transcription has three steps in bacteria: first, RNA polymerase binds to a
promoter site on DNA to form a closed complex; then, RNA polymerase starts
transcription by opening the DNA duplex to form a transcription bubble. In
the second stage, termed elongation, the transcription bubble moves along
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DNA, and the RNA chain is extended by adding nucleotides in the 5’ to 3’
direction. Finally, transcription stops, and the DNA duplex reforms when
RNA polymerase dissociates at a terminator site. In bacteria, each gene or
operon is flanked by a promoter and a terminator. The promoter is a specific
nucleotide sequence site where the RNA polymerase binds to DNA and starts
making RNA (mRNA). The terminator is a similar instruction in the DNA
where the RNA polymerase stops transcribing mRNA and dissociates from
the DNA. This mechanism is the purest form of gene expression regulation
in bacteria. Essential components of transcription are sigma factors (σ),
which are subunits of all bacterial RNA polymerases. They are responsible
for determining the specificity of promoter DNA binding and efficiently
control transcription initiation. In conclusion, the first step in bacterial gene
expression and the step most often controlled is transcription. Regulatory
factors usually determine whether a specific gene is transcribed by RNA
polymerase or not under specific environmental conditions.
4.3.1 ZYMOMONAS MOBILIS
Zymomonas mobilis is a gram-negative anaerobic bacterium that was first

isolated from palm wine and pulque in Mexico. This microorganism has
many desirable industrial characteristics due to its greater suitability than
yeasts in ethanol production [68]. Such attributes include a faster production
rate per cell, a lack of requirement for controlled oxygen during fermen-
tation, a higher sugar absorption rate, a wide tolerance to ethanol, and an
efficient growth rate across a broad pH range (3.5–7.5) [69, 70]. One of
the most interesting physiological characteristics of Z. mobilis is its carbo-
hydrate catabolism via the Entner-Doudoroff (ED) pathway [71]. The ED
pathway is almost entirely restricted to aerobic gram-negative bacteria due
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to low ATP production compared with the EMP pathway for glycolysis, but
it is also present in anaerobic bacteria such as E. coli and Z. mobilis, and it
produces one ATP molecule per glucose consumed [69]. In this pathway,
two key enzymes participate, PDC and two ADHs, which convert pyruvate
to acetaldehyde and then to ethanol [70]. Lignocellulose-derived inhibitors

have negative effects on the ethanol fermentation capacity of Z. mobilis
and other microorganisms. Such inhibitors include acetic and formic acid,
furfural, HMF, and phenols [72]. High temperatures, oxygen, and the ethanol
produced in fermentation itself can also act as growth inhibitors.
Different metabolic engineering experiments were performed to improve
bioethanol production through the use of genetic tools such as heterologous
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expression systems, the silencing of specific genes, and mutagenesis, among
others [73]. One example is a strain of Klebsiella oxytoca that was modified by
metabolic engineering by inserting the PDC gene from Z. mobilis to increase
its capacity for pyruvate decarboxylation, a key enzyme in the homoethanol
pathway of Z. mobilis [74]. In recent years, next-generation sequencing
(NGS) technologies have allowed the characterization of complete genomes
from different microorganisms of industrial interest, including Z. mobilis.
Knowledge of the genetic structure of these bacteria has allowed us to deeply
study the production of metabolites with biotechnological applications and
the adaptation mechanisms to environmental factors. The first genome of Z.
mobilis was sequenced in 2005, with a circular chromosome with a length
of 2,056,416 bp and 5 plasmids, and it was further annotated in 2009 [75,
76]. Subsequently, other genomes were also sequenced with an approximate
length of 2.01 to 2.22 Mb including 2 to 6 plasmids [73].
4.3.1.1 GENETIC REGULATION OF ZYMOMONAS MOBILIS UNDER

TEMPERATURE AND OXYGEN STRESS
To date, several differential gene expression studies have been performed to

understand the cellular dynamics of Z. mobilis under heat stress, under aerobic
and anaerobic conditions, and in the presence of chemical inhibitors, including
phenolic aldehydes, furfural, and ethanol. The objective of these studies is to
characterize the metabolic phenomena that occur during cellular adaptation
to biotic and abiotic agents and how this affects the expression dynamics and
regulation of key genes, leading to a decrease or increase ethanol production.
A study of the heat stress adaptation of Z. mobilis ZM4 by RT-qPCR
allowed the identification of overexpressed genes that are related to oxida-
tive stress and heat shock proteins (HSPs), which increase cell viability
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(Table 4.2) [77, 78]. The gene expression dynamics in Z. mobilis ZM4 were
also evaluated under aerobic and anaerobic conditions. In the absence of
oxygen, several overexpressed genes participating in the ED pathway that
optimize glucose metabolism were identified. Additionally, overexpressed
genes involved in ribosome-mediated polypeptide synthesis and amino acid

and cofactor biosynthetic genes were identified (Table 4.2). The growth rate
of Z. mobilis under aerobic conditions negatively influences fermentation
performance, since it increases the concentration of toxic compounds such
as aldehydes and decreases ethanol production due to the overexpression of
genes involved in stress response, transcriptional regulation, the metabolism
of sulfur compounds, and apoptosis and chemotaxis [79].
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TABLE 4.2 Genes of Zymomonas mobilis Z4 Differentially Expressed Under Different
Stress Conditions (Temperature and Aeration)#
Heat Stress
Gene ID Cellular Process References
sod, cat, ZMO1573, ZZ6-0186, ahpc Oxidative stress [77]*
dnaKJ, hsp20, clpB, clpA, clpS HSP genes [78]*
[75]*
Aeration conditions
Anaerobic conditions
glk, zwf, pgl, pgk, eno, pdc, adhB ED pathway [79]
rars1 Ribosome-mediated polypeptide
synthesis
leuC, trpB, argC, ilvI, ilvC, thrC, thiC, Amino acid and co-factor
and ribC biosynthetic genes
Aerobic conditions
ZMO0084, ZMO0641, ZMO0651 Chemotaxis [79]
ZMO1022, ZMO1460, NT01ZM1467 Metabolism of sulfur compounds
ZMO1121, ZMO1216, ZMO1387, Transcriptional regulators
ZMO1063
nadE Nicotinamide adenine dinucleotide
de novo biosynthesis
atpA, atpB ATPase alpha/beta chains family
tdsD, nifF Flavoprotein transcripts
ZMO1097, ZMO1830, ZMO1732, Stress response
ZMO0279, ZMO1118, ZMO0749
#
Genes highlighted in green are up regulated and those selected in red are down regulated. The
experiments highlighted with (*) were performed by RT-qPCR.
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INHIBITORY COMPOUND STRESS
Chemical inhibitors are commonly present during Z. mobilis fermentation

because these products may be present in LCB residues or may be derived

from the fermentation itself [80]. The study of the expression profile of Z.
mobilis with diverse inhibitors helps to characterize the metabolic pathways
involved in cell detoxification processes. Changes in the cell growth and
ethanol yield of Z. mobilis ZM4 in the presence of inhibitors such as phenolic
aldehydes, furfural, and ethanol have been evaluated. Phenolic aldehydes are
formed in the pretreatment of LCB used as raw material for the production of
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biofuels and have been reported as toxic agents that can affect cell growth and
fermentation [81]. Yi et al. evaluated the genomic response of Z. mobilis ZM4
in the presence of the inhibitors 4-hydroxybenzaldehyde, syringaldehyde,
and vanillin, identifying overexpressed genes from the respiratory chain and
transporter genes (Table 4.3) that help reduce inhibitors to their corresponding
phenolic alcohols and maintain ethanol production [82].
TABLE 4.3 Genes of Zymomonas mobilis Z4 Differentially Expressed Under Inhibitory

Compounds Stress*
Phenolic Aldehydes
Gene ID Cellular Process Reference
ZMO116, ZMO1696, ZMO1885 Respiratory chain [82]
ZMO0282, ZMO0283, ZMO0799, ZMO0800 Transporter genes
Furfural
flhA, fliE, fliG, flgH, flgL, ZMO0619, ZMO0285,
Cell motility [83]
ZMO0780, ZMO1525, oprM, ZMO0307, ZMO0779, and cell wall
ZMO0064, ZMO0835, ZMO0197, ZMO01331 membrane
ZMO0629, ZMO0356, ZMO0996, ZMO0216, ZMO1174, biogenesis
ZMO1311, ZMO0291
rpsD, rpsF, rplI, rbsR, frr, rbfA, proS, alaS, leuS, glyS, pheT, Protein synthesis
valS, gnlA, trpA, trpB, argG, gltB, ilvE, glnB, serA, serC
leuC
ZMO0351, addA, addB, rnhB, ung, ZMO1185, ZMO1652 DNA replication,
ZMO1930, ZMO1356, ZMO1426, ZMO1588, ZMO0362, recombination,
ZMO1231, ZMO1584, ZMO0354, ZMO1193 and repair
ZMO1336, ZMO0050, ZMO0774, ZMO0471, ZMO1738, Transcriptional

ZMO1944, ZMO0281, ZMO1283, ZMO1623 regulation
*
Genes highlighted in green are up regulated and those selected in red are down regulated.
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Another inhibitor derived from LCB is furfural. This toxic compound is

widely studied in yeasts due to growth inhibition and low ethanol production.
Microarray analysis was conducted to evaluate the transcriptional response
of Z. mobilis ZM4 under furfural stress [83]. The genes involved in furfural
tolerance were identified as both up- and downregulated and are related to cell
motility and cell wall membrane biogenesis: Several downregulated genes
are implicated in protein synthesis and DNA replication, recombination,
and repair, and upregulated genes are related to transcriptional regulation
in response to DNA damage, which has been extensively studied in other
bacteria, e.g., Escherichia coli and fermentative yeasts [84, 85].
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ETHANOL STRESS
Similar to other inhibitors, the intracellular and extracellular accumulation

of ethanol is generally toxic to Z. mobilis. To evaluate the gene expression
dynamics related to ethanol tolerance, a microarray analysis of Z. mobilis
ZM4 was performed, identifying low expression levels in different genes
involved in carbohydrate metabolism by the ED pathway, in addition to the
ldhA gene, which is involved in ethanol formation [86]. Up- and downregu-
lated genes related to cell motility and cell wall membrane biogenesis were
also identified. Remarkably, the fliE, fliG, and ZMO01311 genes were also
found to be downregulated in the presence of furfural [83].
Genes involved in the respiratory chain are also up- and downregulated:
in particular, ZMO1885 is upregulated in the presence of ethanol and
downregulated in the presence of phenolic aldehydes. Transcriptional
regulation is a cellular process involved in the adaptation process of Z.
mobilis, with the genes ZMO281 and ZMO0774 upregulated in the presence
of ethanol and phenolic aldehydes. Upregulation of genes implicated in
DNA replication, recombination, and repair also plays an important role
in the adaptation process, and the genes ZMO1193, ZMO1356, ZMO1426
and ZMO1584 have high expression levels in both ethanol and phenolic
aldehyde adaptation. Many genes in Z. mobilis are upregulated in relation to
stress tolerance, and the ZMO1623 gene is also present in phenolic aldehyde
stress (Table 4.4).
The study of the biology of Z. mobilis for more than 6 decades has
revealed its exceptional metabolic characteristics and its great potential as
an ethanol producer, combined with few growth cultivation restrictions. To
date, we have information about its metabolism and physiology, in addition to
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genomic, transcriptomic, and metabolomic data. This bacterium has recently

been studied through systems biology, which is a multidisciplinary approach
involving biological sciences, mathematical models and computer science
that aims to study the relationships that connect the components of a network
and the components themselves. With this approach, we can develop predic-
tive mathematical models of the biological processes of Z. mobilis.
TABLE 4.4 Genes of Zymomonas mobilis Z4 Differentially Expressed Under Ethanol Stress*
Ethanol
Gene ID Cellular Process References
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gnl ED pathway [86]
gntk Pyruvate biosynthesis
ldhA Ethanol formation
flhAB, fliDEFGHIKLMNPQRS, ZMO0613, Cell motility and
ZMO0614, ZMO0604, ZMO605, ZMO607, cell wall membrane
ZMO608, ZMO609, ZMO610, ZMO611, ZMO612, biogenesis
ZMO619, ZMO624, ZMO632, ZMO634, ZMO635,
ZMO642, ZMO643, ZMO648, ZMO649, ZMO651,
ZMO652
ZMO1311
ZMO0022, ZMO1571, ZMO1572, ZMO1032, Respiratory chain
ZMO1255, ZMO1256, ZMO1189, ZMO1669,
ZMO0678, ZMO1812, ZMO1813, ZMO1814, rnfAB
ZMO1844, ZMO0957, ZMO0958, ZMO1252,
ZMO1253, ZMO1254, ZMO1479, ZMO1480,
ZMO1113, ZMO1885, ZMO1809, ZMO1810,
ZMO1811, ZMO0569
ZMO1404, ZMO1623, ZMO0274, ZMO0626 Stress response
ZMO1356, ZMO1426, ZMO1484, ZMO1417, DNA replication,
ZMO1648, ZMO1193, ZMO1401, ZMO086, recombination, and
ZMO0598, ZMO1907, ZMO1187, ZMO1054, repair
ZMO1584, ZMO812
ZMO0054, ZMO2033, ZMO1697, ZMO0281, Transcriptional
ZMO1547, ZMO0774, ZMO0190 regulation
ZMO1107, ZMO0347
ZMO1180, ZMO2018, ZMO1395, ZMO1804, Transport systems
ZMO1025, ZMO1855, ZMO1522, ZMO1425,
ZMO1647, ZMO1262, ZMO0546
ZMO1649, ZMO1757, ZMO0899
*
Genes highlighted in green are up regulated and those selected in red are down regulated.
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4.3.2 CLOSTRIDIUM THERMOCELLUM
Clostridium thermocellum is an anaerobic thermophilic bacterium with a

high growth rate on cellulose; this characteristic is due to its highly efficient
extracellular free and multienzyme complex termed the cellulosome and its
accessory enzymes. A model of the multienzymatic systems is represented
in Figure 4.8; some proteins were renamed as follows: CipA (ScaA), OlpB
(ScaB), Orf2p (ScaC), OlpA (ScaD), SdbA (ScaF), and OlpC (ScaG) [87].
This enzymatic complex gives C. thermocellum the ability to solubilize the
cellulose contained in LCB and rapidly ferment it to produce ethanol. The
metabolic ability of C. thermocellum to produce ethanol directly from LCB
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makes it the main candidate microorganism for bioethanol production via
consolidated bioprocessing (CBP). Despite the biotechnological potential
of C. thermocellum, the industrial application of this bacterium is relegated
because of its disadvantages compared to other microorganisms, such as
mixed acid fermentation, low ethanol productivity and titer, low ethanol
tolerance, and low hemicellulose utilization [88–90]. To solve these biotech-
nological limitations, several strategies have been carried out to improve
ethanol production by C. thermocellum, from technological strategies such
as cocultivation with other bacteria to genomic strategies such as metabolic
engineering. In parallel, several genetic and transcriptomic studies have been
performed to understand the gene regulation involved in biomass degradation,
as well as the ethanol tolerance mechanism used by C. thermocellum [91].
4.3.2.1 GENETIC REGULATION OF THE CELLULOSOME
The main attribute that makes C. thermocellum a promising bacterium for

the production lignocellulosic ethanol is the ability to produce cellulosomes;
as a consequence, the regulation of cellulosome expression has been widely
studied. C. thermocellum, similar to other microorganisms, regulates gene
expression in response to the environment to adapt and synchronize its
metabolic reactions to new conditions. Genetic and metabolic analysis of
C. thermocellum showed that this bacterium employs more than 100 genes
for biomass degradation, including more than 70 genes that encode various
cellulosomal enzymes.
One of the most studied regulation systems in C. thermocellum is the homol-
ogous LacI transcriptional regulatory network for the celC operon [93–96]. The
celC operon is formed by a noncellulosomal GH5 endoglucanase gene (celC),
the glyR3 gene (GlyR3 protein) and the endo-1,3-β-d-glucosidase gene (licA);
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this operon is negatively autoregulated by the binding of the GlyR3 protein to

the celC promotor region [93]. The repression of the operon celC is relieved
by laminaribiose, which impedes the binding of GlyR3 to the celC promoter.
An extended model for the regulation of the six-gene cluster celC-glyR3-licA-
orf4-manB-celT was proposed by Choi et al.; in this model, the protein GlyR3
coregulates the expression of the celC and manB genes [96]. In the extended
model, the expression of the cellulosomal family 26 glycoside hydrolase ManB
is repressed by a high concentration of the protein GlyR3 in the presence of
laminaribiose. In contrast, at a basal concentration of GlyR3 (in the absence
of laminaribiose), the manB gene is expressed (Figure 4.9). The mechanism of
regulation of the celT gene that encodes a cellulosomal family 9 endoglucanase
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is unknown. In the same way, the mechanism regulating the orf4 gene and the
function of the protein it encodes are unknown.
FIGURE 4.8 Model of Clostridium thermocellum cellulosome systems.

Source: Adapted from: Ref. [92].
On the other hand, several regulatory mechanisms have been proposed

to be involved in the genetic regulation of the expression of the cellulosome,
including carbon catabolite repression and alternative σ factors. It is well
known that extracellular polysaccharides affect cellulosome composition by
regulating which enzymes and structural components are expressed [97]. The
expression of the individual components of the cellulosome in C. thermo-
cellum is probably regulated by a cluster of at least 6 paralogous alternative
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FIGURE 4.9 Expanded model of GlyR3 regulation in Clostridium thermocellum. Red and green indicate genetic repression and expression,
respectively.
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σI factors and their cognate membrane-associated anti-σ [97, 98]. Several

investigations have revealed that the main structural component of the cellu-
losome in C. thermocellum is CipA, a protein scaffold that includes nine type
I cohesin modules, a type II dockerin module, and a family III carbohydrate
binding module known for a strong affinity for cellulose (Figure 4.8).
Ortiz de Ora et al. demonstrated that 5 σI factors (identified as σI1, σI2,
σI3, σI4 and σI6) regulate the expression of 17 genes encoding different cellu-
losomal proteins [98]. They could relate the σI1–σI6 factors to the genetic
regulation of two of the most important components of the cellulosome,
the primary scaffoldin (CipA) and the most abundant enzyme (Cel48S). In
the same way, the regulons of the σI alternative factors were identified by
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bioinformatic analysis in conjunction with classical microbiology genetic
tools and the application of the heterologous B. subtilis host system [98].
All σI factor genes (sigl1-sigl6 and sig24C) are upregulated in the presence
of extracellular polysaccharides. In this context, the proposed mechanism to
activate alternate σI implied an extracellular carbohydrate-active module and
an intracellular anti-σ peptide domain [97] (Table 4.5).
Studies performed to understand the mechanisms of genetic regulation
of cellulosome production in C. thermocellum demonstrated that growth
conditions regulate the expression of cellulosomal components and that this
regulation is a response to the polysaccharides present in the culture media.
Nataf et al. identified five sugar ABC transport systems: four are specific
for β-1,4-linked glucose oligomers (cellodextrins), and one is specific for
β-1,3-linked glucose dimer (laminaribiose) [99]. The sugar transporters and
their substrate specificities demonstrated that C. thermocellum prefers to
assimilate cellodextrins rather than cellobiose or glucose. Genome analysis
also suggests that the bacterium lacks any other sugar ABC transporters, in
agreement that this strain can grow only on β-glucans. Consequently, the
sugars present in the culture media determine the composition of the cellulo-
some, which that subsequently influences the overall ability of the bacteria
to degrade lignocellulosic substrates and produce ethanol.
4.3.2.2 GENETIC REGULATION OF CLOSTRIDIUM THERMOCELLUM

DURING ETHANOL PRODUCTION
The regulation of genes involved in ethanol production is affected by the

substrate and growth conditions, similar to the regulation of cellulosome
expression. Although C. thermocellum is a promising bacterium for ethanol
production, mixed acid fermentation and low ethanol tolerance are the major
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TABLE 4.5 Regulatory Networks of σI Factors in Clostridium thermocellum

σI – anti-σ Regulon Gen Product C-Terminal Sensing of Anti-σ
Factors Factor/Activator Polysaccharides
σI1-Rsgl1 sigl1, cel8A, cel48S, sdbA, σI1, GH8-DocI, GH48-DocI, Cohll-X-3(SLH), GH74-DocI CBM3/cellulose
xgh74A
σI2-Rsgl2 sigl2, Clo 1313_0420, Clo σI2, DocI-UNK, Abf-DocI-GH43 CBM3/cellulose
1313_2216
σI3-Rsgl3 sigl3, cel48S, cipA, rga12A, σI3, GH48-DocI, 2(CohI)-CBM3-6(CohI)-X-DocII, PA14 dyad/pectin
rgl11A Rga-DocI-CMB35-Rga, Doc-CBM35-Rgl
σI4-Rsgl4 sigl4, cel8A, cel48S, cipA, σI4, GH8-DocI, GH48-DocI, 2(CohI)-CBM3-6(CohI)-X- CBM3/cellulose
Clo1313_1436 DocII, HP
σI5-Rsgl5 sigl5 – CBM42/arabinoxylan
σI6-Rsgl6 sigl1, sigl6, cel9V, cel48S, σI1, σI6, GH9-2-(CBM3)-DocI, GH48-DocI, 2(CohI)- GH10/xylans, cellulose
cipA, cseP, rsgl5, xyn11B, CBM3-6(CohI)-X-DocII, CotH-DocI, anti-σI5 (or Rsgl5),
xyn10D, xyn10Y, xyn10Z GH11-CBM6-DocI, CBM22-GH10-DocI, CBM22-GH10-
CBM22-DocI-CE1, CE1-CBM6-DocI-GH10
σ24C-Rsi24C sig24C – GH5/cellulose
Abbreviations: GH: glycoside hydrolase; Doc: dockerin; CBM: carbohydrate-binding module; Coh: cohesin; X, X-module (module of
unknown function); CotH: spore coat protein H; UNK: unknown sequence; HP: hypothetical protein; Abf: Alpha-L-arabinofuranosidase; Rga:
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rhamnogalacturan acetylesterase; Rgl: rhamnogalacturonan lyase; SLH: S-layer homology domain; CE: carbohydrate esterase.
Source: Adapted from: Refs. [97, 98].
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obstacles to its commercial application. However, to overcome the limitations

that slow down the use of C. thermocellum to produce ethanol industrially,
some approaches have been pursued to understand the mechanism of inhibi-
tion by ethanol, as well as approaches focused on increasing ethanol yield,
including metabolic engineering efforts to delete pathways for carbon flux to

lactate and acetate [4, 91, 100, 101]. Wild-type C. thermocellum is inhibited
by low ethanol concentrations (approximately 10 g/l) and is completely
unable to grow at 20 g/l ethanol. However, wild-type C. thermocellum had
to be adapted to tolerate 50–70 g/l ethanol [102, 103]. C. thermocellum uses
the EMP glycolysis pathway to generate pyruvate from sugars (cellobiose
or glucose); however, some differences from the traditional pathway are
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present [104]. The final steps of the EMP pathway involve the successive
reduction of acetyl-CoA and acetaldehyde with electrons provided by NADH
(i.e., the ALDH and ADH reactions); these two reactions are both catalyzed
by the bifunctional alcohol dehydrogenase (ADHE) enzyme. This enzyme
is related to the ethanol tolerance of C. thermocellum, as mutations in the
bifunctional ADHE result in increased ethanol tolerance [101]. Previous
work demonstrated that the expression of ADH genes, pyruvate ferredoxin
oxidoreductase genes (Cthe_2390, Cthe_2391, Cthe_2392 and Cthe_0340)
and other genes related to ethanol production were affected by the carbon
source (cellulose or cellobiose) and the growth rate [105, 106].
Recently, global transcriptomic analysis of C. thermocellum ATCC 27405
growing in dilute acid-pretreated Populus and switchgrass showed overexpres-
sion in genes related to nitrogen uptake and metabolism at the end of fermenta-
tion when populus was used; this overexpression coincided with increases in
ethanol concentration [107]. Similar results were obtained when C. thermo-
cellum was grown on crystalline cellulose [108]. These results demonstrated
that the expression of genes related to ethanol production is influenced by the
carbon source and the fermentation time, as well as the growth rate.
Although several studies have been carried out to understand the mecha-
nism of genetic regulation in one of the most promising bacteria to produce
ethanol directly to LCB, C. thermocellum, substantial further effort is
required to fully elucidate all the genetic regulatory mechanisms that control
the expression of proteins involved in biomass degradation and ethanol
production. The application of new omics technologies (genomics, tran-
scriptomics, proteomics, and metabolomics) allowed us to reach a complete
understanding of metabolism and its connection with the genetic regulation
of C. thermocellum. An understanding of the genetic regulatory mechanisms
in C. thermocellum can help improve this industrially relevant strain and
promote the use of cellulosome-producing bacteria in the CBP of biomass.
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4.3.3 OTHER ETHANOLOGENIC BACTERIA
Fermentation is a crucial process in bioethanol production, where ethanol

is produced from the metabolic activity of a microorganism, either bacteria
or yeast. We previously reviewed the characteristics of several bacteria,

such as Z. mobilis and cellulosic thermophiles, including C. thermocellum.
Another interesting alternative is extreme thermophilic anaerobic bacteria,
which are good candidates for bioethanol production due to their ability to
ferment an extensive variety of substrates that include hexoses, pentoses,
and disaccharides to produce ethanol, in addition to the relatively low
contamination risk by other microorganisms due to high fermentation
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temperatures [109].
Extreme thermophilic anaerobic bacteria have been isolated from
different environments, including geothermal areas, volcanic mud, and
canned products [110–112]. These bacteria are facultatively anaerobic
and tolerate extreme pH and salt concentrations during fermentation with
minimal nutrient supplementation [113]. Despite the numerous advantages
of employing thermophilic bacteria in fermentation for bioethanol produc-
tion, it is known that yeasts and other bacteria such as Z. mobilis can tolerate
a higher ethanol concentration than extremophiles due to the fatty acid
composition of the cell membrane [114].
Several studies have been performed with hemicellulolytic thermophiles
of the genera Thermoanaerobacter and Thermoanaerobacterium to increase
the ethanol yield and ethanol tolerance. These strategies include the suppres-
sion of other fermentation products through lactate and acetate metabolic
pathway knockout in T. saccharolyticum JW/SL-YS485 and the overexpres-
sion of enzymes such as NAD(P)H-dependent ADH in T. mathranii, which
is directly related to increased ethanol production [109, 115]. The expression
dynamics of the ADH enzymes ADHA, ADHB, and ADHE, which have
a key role in ethanol formation in T. ethanolicus, were evaluated, and the
expression of these enzymes was observed to be affected at high ethanol
concentrations [116].
In conclusion, the search for more efficient and resistant microorgan-
isms to produce ethanol continues. Microorganisms such as S. cerevisiae
and Z. mobilis have been extensively studied due to their ability to produce
ethanol; knowledge of their genomes and the application of omic techniques
have considerably illuminated their mechanisms of gene regulation during
ethanol production. Emerging microorganisms such as K. marxianus and C.
thermocellum have also aroused interest for use in ethanol production due
to their outstanding metabolic and physiological characteristics. However,
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knowledge about their gene regulation during ethanol production is still

limited, so at present, several studies are focused on understanding why these
microorganisms are promising for the industrial production of ethanol. The
development of new omics techniques will allow a complete understanding
of the regulatory mechanisms that govern the genetic expression of these

bacteria and yeasts in the short and medium term. Consequently, we will be
able to develop new strains capable of producing ethanol efficiently in highly
stressful conditions.
KEYWORDS
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• Clostridium thermocellum
• ethanol production
• genetic regulation
• Kluyveromyces marxianus
• Saccharomyces cerevisiae
• Zymomonas mobilis
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CHAPTER 5
Metabolic Engineering of Yeast,

Zymomonas mobilis, and Clostridium
thermocellum to Increase Yield of
Bioethanol
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S. SÁNCHEZ-MUÑOZ,1 M. J. CASTRO-ALONSO,1 F. G. BARBOSA,1
E. MIER-ALBA,1 T. R. BALBINO,1 D. RUBIO-RIBEAUX,1
I. O. HERNÁNDEZ-DE LIRA,2 J. C. SANTOS,3 C. N. AGUILAR,4 and
S. S. DA SILVA1
1
Bioprocesses and Sustainable Products Laboratory,
Department of Biotechnology, Engineering School of Lorena,
University of São Paulo (EEL-USP), Lorena–12.602.810, SP, Brazil,
E-mail: silviosilverio@gmail.com (S. S. Da Silva)
2
Bioremediation Laboratory, Biological Science Faculty,
Autonomous University of Coahuila (UAdeC), Torreón Campus,
Coahuila–27276, México
Bioprocesses, Biopolymers, Simulation, and Modeling Laboratory,
3

University of São Paulo (EEL-USP), Lorena–12.602.810, SP, Brazil
4
Bioprocesses and Bioproducts Group, Food Research Department,
School of Chemistry, Autonomous University of Coahuila (UAdeC),
Saltillo Campus, Coahuila–25280, Mexico
ABSTRACT
The price fluctuation of petroleum-based fuels makes biofuels one of the most
promising alternative energies for global economies. Since the biological
production of fuels, from vegetal biomass, offers sustainable and economically
attractive options compared to petroleum-based production. However, the
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complexity of vegetal biomass does not allow the process to be simple.

Furthermore, there are still many important biological and technological
barriers for the processing of biofuels being competitive. Besides, bioprocesses
normally required multiple steps of feedstock pretreatment and subsequent
conversion to fuel. Those steps are being consolidated into single microbial
processes using metabolically engineered species (e.g., Saccharomyces
cerevisiae, Zimmomona mobilis, and Clostridium thermocellum). This chapter
will review the advance in metabolic engineering and synthetic biology on
the main ethanol producers’ microorganisms, which allow the development
of new engineered systems aiming for a better transition from fossil fuels to
biofuels.
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5.1 INTRODUCTION
Economical fluctuations and CO2 emissions of oil-based fuels are increasing

day by day. These factors control the equilibrium of developing economies
and produce asymmetric responses in social and economic growth, also
harming the environment [1, 2]. Thus, biofuels production as alternative
energy gained attention and potentialized efforts in many research areas [3].
However, the possibility of a sustainable power source has been around for
a long time but did not receive considerable governmental attention mainly
because of low-cost oil since last 50 years. Furthermore, industrial 2G
bioprocesses are not well integrated to achieve cost-effective bioproducts in
established biorefineries [4, 5].
To compete with oil-based energies, the biofuels process can take
advantage of 2G biomass and must be enhanced in relevant steps, mainly
in the synthesis of biomolecules by microorganisms. Although microbes
have the inherent metabolic pathways for generation of these valuable
molecules, the natural biofuel synthesis is significantly low and limits
its production and commercialization at the industrial level [6]. Thus,
for achieving those goals, different strategies in synthetic biology and
metabolic engineering fields have been studied. Also, those areas could offer
the sustainability factor and the possibility of producing new molecules to
become a profitable bioprocess [7, 8]. These disciplines have been applied
to improve the microbial production level of advanced biofuels through the
over-expression of specific regulators and target enzymes, heterologous
gene expression, orthogonal pathway construction, protein engineering,
co-factor balancing, blocking of competitor pathways and down-regulating
genes, among others [6, 8–10].
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Metabolic Engineering of Yeast, Zymomonas mobilis 99
Established microbial industrial hosts like the bacterium Escherichia

coli and the yeast Saccharomyces cerevisiae are preferred for metabolic
engineering systems, because of their well-known genetic handling with
available ‘omics’ (genomic, proteomics, transcriptomics, or metabolomics)
databases, growth speed, low incubation costs, and their now established use
in industrial bioprocesses [6, 11, 12]. However, other microorganisms such
as the bacterium Zymomonas mobilis and the fungus Clostridium thermo-
cellum are of great interest in research field, because of their versatility to
produce several biomolecules under industrial conditions, that made them
excellent alternatives for biotechnology [13, 14].
In this chapter, we have addressed the current state of metabolic engineering
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in biofuel production and described the recent progress made in producing
bioethanol from the main genetically modified microorganisms.
5.2 METABOLIC ENGINEERING TO ENHANCE BIOPROCESSES
Biological organisms, enzymes, and their genetic modification formed the

basis of a growing collection of techniques grounded in molecular biology
and cell biology. As a result, biotechnology, and bioprocesses are knowledge
areas that industries and researchers have worked on, looking up to develop
and commercialize the building blocks of life to offer products and services
[15, 16]. For this purpose, metabolic engineering is the science that makes
possible the correct performance of the biotechnological field, rewriting the
metabolism of cells for different aims in each bioprocess [7].
Bioprocesses are used to produce pharmaceuticals, enzymes, vaccines,
foods, flavors, pigments, polymers, amino acids, fuels, and other important
chemical molecules [17, 18]. Briefly, a bioprocess usually consists of
feedstock preparation and pretreatment (upstream process), fermentation
or biocatalysis (core process), and separation for product recovery and
purification (downstream process) [19, 20]. Also, it could involve genetic
engineering for the manipulation of plants, animals, and microorganisms
such as yeasts, bacteria, and fungi, since those microorganisms may not be
able or well-performed for the production of our desired product [19, 21,
22]. Furthermore, the metabolic characteristics of living organisms often
impose challenges on bioprocessing, thus a wide study of cells is always an
important prerequisite for successful engineering design [17].
Due to diverse research developments, several synthetic gene constructs
and circuits have been designed within a wide number of host microorganisms
leading to a positive impact on different areas [23]. Nevertheless, metabolic
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engineered tools applied to industrial strains to generate high yield in biopro-

cess still have important challenges to consider them as promising alternatives
in most research areas [24].
There are some examples that can be listed to show the potential of
metabolic engineering in different steps of bioprocesses. One of them is the

conversion of lignocellulosic biomass (LCB) (upstream process) into mono-
saccharides using heterologous cellulases, a main critical step in biorefineries,
as its enhancement can help in cost saving achievements [25, 26]. Another
important step is fermentation (core process), and at this process level, several
strategies could be applied. For example, deletion of genes or sequences
that interrupt strategic cell responses, such as, autophagy, that is a recycling
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process of cellular components; in some cases, its interruption enhance the
biomass growth rate (ex. yeast) and consequently, a positive influence in the
production of some products like ethanol could occur [27–29]. Fermentation
has been well-studied for the application of several molecular tools. The most
used one could be the overexpression of specific enzymes. For example, in
citric acid production, pyruvate carboxylase is a key enzyme in the reduction
of the tricarboxylic acid cycle, and it is closely related to the formation of this
important biomolecule, thus the overexpression of this enzyme could lead to
a higher production of this organic acid [30, 31]. Other examples of metabolic
engineering tools applied to microorganisms for enhancing the production of
biomolecules are shown in Table 5.1.
5.3 ROLE OF GENETIC ENGINEERING IN SUSTAINABLE BIOFUELS/

ETHANOL PRODUCTION: BOTTLENECKS AND MODEL STRAINS
The progress in metabolic engineering allows the reconstruction and

development of “microbial cell factories” as a promising strategy for large
scale production of ecofriendly fuels [38]. The fermentation processes
through wild-type microorganisms produce low titer of biofuels since
the characteristics of end-product and conditions of processes generate
several stresses, making the microbial adaptability to large-scale industrial
processes very difficult [39]. Thereby, industrial biofuel production requires
microorganisms with ability to tolerate several stressing conditions during
bioprocesses, such as osmotolerance, shearing forces, organic acids, and
high temperature. The physiological responses related to those stress condi-
tions can be improved by genetic manipulation of a single or a few genes
[8]. Hence, several conventional genetic engineering tools and synthetic
biology techniques, such as genetic transformation, gene targeting, use
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TABLE 5.1 Metabolic Engineering Strategies Applied to Several Microorganisms for Enhancing the Production of Trending Industrial Compounds
(B: bacteria; Y: yeast; F: fungus)
Product Strain Genetic Modification Enhancement* Application References
Clavulanic acid Streptomyces clavuligerus Co-transcription of the late-stage gene 33% Antibiotic [24]
F613-1 (B) claR with the reporter gene neo
Coenzyme B12 Pseudomonas denitrificans Removal of riboswitches and the 2-fold Essential cofactor in many [32]
ATCC 13867 (B) replacement of promoters in operons biological rearrangement
of cluster I reactions
Glargine Escherichia coli JM109 Heterologous expression of the – Diabetes mellites [12]
(insulin analog) (B) prepeptide glargine insulin
Ethanol Saccharomyces cerevisiae Replacement of the regulatory gene 5.3% Fuel [33]
(Y) PHO4
Citric acid Yarrowia lipolytica (Y) Over expression of pyruvate 98.5% Additive for several [34]
carboxylase gene products
Metabolic Engineering of Yeast, Zymomonas mobilis
Ethanol Saccharomyces cerevisiae Disruption of ATG32 region 2.76% Beverages (sake) [29]
(Y)
Cellulases/ Trichoderma reesei RUT Construction of transcriptional 50–80% Saccharification of [35]
Xylanases C30 (F) activation linked to a domain of herpes lignocellulosic biomass
simplex virus protein VP16
Pullulan Aureobasidium pullulans Homologous expression of UGPase 1.7-fold Blood plasma substitutes, [36]
NRRL Y-12974 (F) food preservation,
adhesive, and others
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60
Gibberellin Fusarium fujikuroi (F) Co γ-ray radiation combined with 21-fold Growth hormones for [37]
(GA3) lithium chloride treatment to generate plants
mutants
*
Enhancement of production of modified strain compared with wild strain.
101
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of different promoters, clustered regularly interspaced short palindromic

repeats (CRISPR) and RNA-interference (RNAi) among others, had been
widely used to address bottlenecks to achieve large-scale production of
biofuels [40–42]. The major advances of conventional and modern genetic
tools to improve industrial biofuels production are discussed below.
5.3.1 GENETIC ENGINEERING TO ENHANCING SOLVENT

TOLERANCE OF BIOFUELS
One of the major challenges for achieve high levels of biofuels production
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is that fermentation performance is affected by toxicity of end products [43].
The toxicity effects of biofuels, particularly organic solvents, are related
to the logarithm of its octanol-water partition coefficient (logP). Organic
solvents with logP values between a range of 0.8 and 5 cause disruption
of cellular membranes and interferes with several vital metabolic functions,
such as membrane transport and energy generation. Moreover, solvents
generate damage to biological macromolecules, such as DNA and RNA [44].
Several genetic engineering strategies had been employed to attenuate the
toxicity of organic solvents and biofuels, and to elucidate stress responses of
microorganism through the overexpression of heat shock protein genes [45],
redirection of carbon metabolic flux [46], regulation extrusion of solvents
efflux pumps genes [47] among others.
HSP are involved in the synthesis, transport, and folding of proteins closely
related to metabolic activities [43, 48]. The role of HSP in enhancement of
biofuel tolerance was showed for the first time in Clostridrium acetobutylicum.
In that study, the overexpression of chaperone GroESL improved tolerance to
n-butanol (85%) [45]. Another work from this research group demonstrated
that the overexpression of GroESL, also increased the expression of other
HSP involved in solvent tolerance, including dnaKJ, hsp18, hsp90 [49]. In
other studies, with isobutanol, the microbial transcriptional profiles revealed
main genes related to heat shock stress and protein misfolding, including
rpoH, dnaJ, htpG, and ibpAB [50, 51]. The same strategy was applied in
Escherichia coli, Lactobacillus plantarum and Zymomonas mobilis and
showed significant improvement in survival and viability of cells in presence
of ethanol, 1-butanol, and 2,3-butanediol by the overexpression of GroESL,
ClpB chaperones and SecB multi-tasking chaperone [44, 52–55]. On the
other hand, Zhang et al. [56] showed that genes involved in central carbon
flux and TCA cycle (gapA and sdhB) are closed related to end product
tolerance. Genes gapA (glyceraldehyde-3-phosphate dehydrogenase A) and
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sdhB (FeS subunit of succinate dehydrogenase) provides high energy storage

to regulate the solvent stress.
Redirection of carbon metabolic flux can be performed by CRISPR
systems to improve the tolerance and production of biofuels. Wang et al. [57]
applied CRISPR interference in Klebsiella pneumoniae and found that amino

acid synthesis pathways interfere with n-butanol synthesis. The repression of
the main genes that encode valine, leucine, isoleucine, threonine, and alanine
pathways increased n-butanol tolerance and led to higher production (154%)
of this fuel. Similarly, Otoupal and Chatterjee [58], using the CRISPR
perturbations system in E. coli, identified the unknown genes yjjZ and yehS
with strong potential to improve tolerance to n-butanol and n-hexane. At
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the moment, it is known that yjjZ gene plays a regulatory role in cellular
metabolism as small RNA (sRNA) [58–60]. In another work, Huang and
Geng [61] developed a novel replicative CRISPR/Cas9 plasmids system
(pdC99 and pPC9) to integrate the 2,3-butanediol pathway in Saccharomyces
cerevisiae that redirected carbon metabolic flux to improve tolerance and
lead to higher production of 2,3 butanediol (50.5 g/L).
Efflux pumps are membrane transporters responsible for recognizing
and exporting toxic compounds from the cytoplasm to out of cell. Those
transporters are comprised by three types of proteins: inner membrane
proteins, periplasmic linkers, and outer membrane channels [43]. Several
studies observed that overexpression of exogenous efflux pumps and the
cloning of their operons are effective strategies to enhance the tolerance
to biofuels. Early studies reported that genes encoding the efflux pump
SrpABC of Pseudomonas putida from the RND family were expressed in
the presence of n-butanol [62, 63]. Since the elucidation of this mechanism,
other studies revealed that the overexpression of efflux pump srpABC or
the srpB subunit from P. putida in E. coli enhances n-butanol tolerance
(20–40%) [64, 65]. Similarly, the co-overexpression of the membrane ATP
binding cassette ABC and the efflux pumps Snp2 and Pdr5 in S. cerevisiae
increased the tolerance and cellular growth when it was underexposed to
exogenous n-decane [66]. Reyes et al. [47], found that overexpression of
another membrane transporter associated to the genes ygfO, setA, mdtA, and
pgsA improve n-butanol tolerance in E. coli. Furthermore, this study also
exhibited that underexposure to n-butanol can lead to adaptive structural
changes of the membrane lipid structure, since pgsA gene is involved in
cardiolipin biosynthesis. In a recent work, a negative feedback network
was introduced to E. coli strain using promoter PgntK, which regulated the
expression of the butanol efflux pump AcrB and controls membrane protein
expression to optimize growth. That study reported an increase in tolerance
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(40%) and production (35%) of n-butanol [67]. Other studies introduced

efflux pump AcrB in E. coli through directed evolution strategy and showed
increase significant tolerance to n-butanol (>25%), α-pinene (400%) and
n-octane (47%) [68, 69]. In contrast, He et al. [70] found that deletion of
acrB gene in E. coli also raised tolerance of n-butanol in terms of cell density,
which was improved by 82.8%.
5.3.2 GENETIC ENGINEERING FOR IMPROVED INHIBITOR

TOLERANCE
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Inhibitors derived from fermentation process due to the pre-processing of
lignocellulosic material limits the metabolism of strains in terms of micro-
bial growth and biofuel production [71]. These inhibitors can be categorized
into three major types: (i) furan derivatives, (ii) short-chain aliphatic acids
and (iii) phenolic compounds, as reviewed by Palmqvist and Hahn-Hägerdal
[72]. Among these compounds, furan derivatives such as furfural and
5-hydroxymethylfurfural (HMF) are some of the most toxic substances
since they induced serious damages to cellular metabolism. Furfural and
HMF inhibits glycolytic and fermentative enzymes (pyruvate, acetaldehyde,
and alcohol dehydrogenases (ADHs)) and cause breaks in double-stranded
DNA [73]. Thus, furan derivates also generate reduction in concentration
of intracellular ATP and NAD(P)H because the energy flux is redirected to
pentose phosphate pathway (PPP) for repairing the induced damages [74,
75]. Moreover, furfural also affect mitochondria and vacuole membranes, as
it induces the formation of intracellular ROS [76]. All the cellular damages
mentioned above result in prolongation of lag phase of microbial growth and
decrease specific growth rate, which lead to low yield of biofuel produc-
tion [46]. In addition, furfural also increase the toxicity of acetic acid and
phenolic compounds [77, 78].
Extensive studies have been performed to improved microbial tolerance to
these inhibitors through classical and novel mutagenesis strategies. Miller et
al. [75] reported that silencing of NADPH-dependent oxidoreductases (yqhD
and dkgA) increased furfural tolerance in E. coli. In contrast, several recent
studies showed that overexpression of oxidoreductases and proteins involved
in oxidative stresses improved inhibitor tolerance. Co-overexpression of
oxidoreductases (yqhD and FucO) showed increased tolerance to furfural,
improved glucose utilization, and enhance the production of isobutanol
(110%) [79]. Similarly, Song et al. [80] employed the strategy of increasing
NAD(P)H pool through overexpression of pncB and nadE genes. These genes
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are involved in nicotine amide salvage pathway that led to reduce furfural
toxicity and improved the production of isobutanol (2.5-fold higher). Oh et
al. [81] found that overexpression RCK1 gene encoding for a protein kinase
involved in oxidative stress improved acetic acid tolerance under glucose
and xylose conditions and reduce 40% of intracellular ROS levels. More
recently, Liu et al. [82] showed that the manipulation of intracellular redox
potential of genes encoding cofactors related oxidoreductases in Z. mobilis
is a promising novel strategy to improve tolerance to acetic acid, furfural,
and phenolic compounds in industrial biofuels production. In other studies,
glycerol supplementation results in other promising strategy to furfural
detoxification [83, 84]. Agu et al. [84] demonstrated that overexpression
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of two glycerol dehydrogenases dhaD1 and gldA1 reduced 68% of furfural
toxicity in Clostridium beijerinckii.
5.3.3 GENETIC ENGINEERING TO DEVELOPMENT OF

THERMOSTABILITY
The temperature of industrial fermentation process should be up to 40°C,

which prevents contamination and reduces cooling costs by decreasing water
and energy consumption. Moreover, high operating temperature improves
productivities in simultaneous saccharification and fermentation (SSF)
process because of the optimal temperature for enzymes that catalyze the
saccharification of biomass at ≥50°C [85–87]. However, this temperature
is higher in comparison with optimal growth temperatures of mesophilic
microorganisms, typically used in industrial processes of biofuels production,
e.g., E. coli.
Higher temperature causes serious cellular damages such as degradation
of cytoskeleton proteins, morphological abnormalities, and inhibition of cell
division and growth [86–88]. Thus, development of thermotolerant microbial
strains is other major challenge for the first and second-generation biofuels
production [8]. Adaptation laboratory evolution, random mutagenesis, and
CRISPR had been employed to elucidate mechanisms involved in microbial
thermostability. Several early genetic studies revealed that microorganism
acquired thermotolerance to ≥50°C through accumulation of trehalose
and overexpression of heat stress proteins such as chaperones, ubiquitin,
among others [89–91]. In other studies, single amino acid modification in
the pyruvate kinase, C-5 sterol desaturase and NADH dehydrogenase led
to increase the thermotolerance and biofuel production in S. cerevisiae and
Z. mobilis [85, 92]. Likewise, Nasution et al. [93] showed that the deletion
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of Dfg5 gene encoding glycosyl phosphatidylinositol-anchored membrane

protein improved thermotolerance and decreased level of ROS.
In last years, researches had been developed artificial systems using
synthetic biology to improve the thermostability of microorganisms through
the incorporation of systems constituted by heat shock and antioxidant

proteins. Jia et al. [94] developed an intelligent microbial heat-regulating
engine (IMHeRE) to improve the thermo-robustness of E. coli through the
integration of a thermotolerant system and a quorum-regulating system. At
cellular level, the thermotolerant system composed of different HSP and
RNA thermometers hierarchically led to increase the optimum temperature
by sensing heat changes. At community level, the quorum-regulating system
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dynamically regulates the altruistic sacrifice of individual cells to reduce
metabolic heat release by detection of temperature and cell density. This
study showed that the synthetic IMHeRE system improves cell growth (10%)
at >40°C. Xu et al. [95] engineered an artificial antioxidant defense system
to improve thermo-tolerance of yeast. They introduced several antioxidant
genes from S. cerevisiae and Thermus thermophiles HB8 to construct “Angel
yeast.” This synthetic yeast showed an increase of thermotolerance in terms
of cell density (65.2%) at >40°C. Li et al. [87] used CRISPR/Cas-based
gene activation screening in S. cerevisiae and identified the essential role
of delta-9 desaturase gene OLE1 in increasing fatty acid unsaturation and
reduction of lipid peroxidation caused by heat stress at 42°C. Similarly, Li
et al. [96] elucidated the role of 4-Hydroxy-2,2,6,6-tetramethylpiperidin-
1-oxyl (TEMPOL) as a stress-alleviating agent in S. cerevisiae. TEMPOL is
a redox-cycling nitroxide and membrane-permeable antioxidant escorted by
dual redox potential forms. This system enhanced expression of important
genes and the activity of enzymatic antioxidant defense system, altering the
ratio of cofactors to improve the non-enzymatic antioxidant defense system,
or by directly degrading ROS with the help of NADH. Recently, Tao et al.
[97] used ‘Cas9 nickase-based genome editing’ and found that the inactiva-
tion of the gene MspI improved the thermotolerance of C. cellulolyticum.
Despite the MspI gene belongs to the restriction-modification system, this
study revealed that the disruption of MspI gene can influence the expression
of other thermotolerant genes.
5.3.4 MOLECULAR TOOLS APPLIED TO VEGETABLE BIOMASS
Agricultural wastes and byproducts are complex substrates mainly

composed of cellulose, hemicellulose, and lignin, which proportions depend
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on the source and species of plants [98]. Cellulose and hemicellulose are
polymers composed of hexoses (glucose, mannose, and galactose), pentoses
(arabinose and xylose), and sugar acids (uronic acids). Lignin is an amor-
phous macromolecule, composed by phenylpropanoids (guacyl and siringyl
units). Lignin together with cellulose and hemicellulose forms a closed

structure that confers biological, chemical, and mechanical resistance to
cell walls [99]. These complex structures are one of the main bottlenecks
in biorefinery, which affect the production cost of biofuels. Lignin and
cellulose also limit the accessibility of enzymes to sugar polymers, thus
restricting their hydrolysis. Hence, lignocellulosic pretreatments are a key
step in bioprocessing.
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Hydrolysates obtained from sugar fractions of biomass are a mixture of
hexoses, pentoses, and products derived from lignin. The products derived
from lignin generate seriously microbial damages that inhibit fermentation
performance. The artificial modification and genetic engineering of plants
that result in low content of lignin are potential strategies to enhance plant
biomass hydrolysis to obtain maximum sugar releases [100]. Furthermore,
genetic strategies have more advantages than physical and chemical pretreat-
ments considering that they do not require additional energy or chemicals
input and result in lower environmental pollution [101].
The sense, antisense, or RNAi approaches has been used to upregula-
tion or downregulation of genes involved in lignin biosynthesis pathway to
alter their content, location, and type (Syringyl: Guaiacyl ratio) [102, 103].
Early studies revealed that the expression of p-coumaroyl-CoA 3-hydroxy-
lase (C3′H) gene is related to lignin synthesis [104–107]. Subsequently,
Coleman et al. [108] reported that RNAi suppression of C3′H expression in
hybrid poplar led to significant reduction of lignin content (56–59%). Simi-
larly, Chen and Dixon [109] reported that downregulation of the hydroxy-
cinnamoyl transferase (HCT) and caffeic acid 3-O-methyltransferase
(COMT) genes, reduced lignin content (40%) and increase sugar releases
(166%) in alfalfa. Shafrin et al. [110] introduced hpRNA-based vectors for
downregulation of Cinnamate 4-hydroxylase (C4H) and COMT genes in
jute, which showed significant reduction of soluble lignin (13–23%) and
fiber lignin (13–17%). RNAi downregulation of COMT gene also reduces
the Syringyl:Guaiacyl (S:G) ratio, improved the fermentable sugars yield
(>34%) and ethanol production yield (≥ 38%) in switchgrass and sugarcane
bagasse, respectively [111, 228]. In contrast, other studies showed that
reduction of S:G lignin ratio had no correlation with the reduction of biomass
recalcitrance [112, 113]. More recently, Zhang et al. [114] demonstrated
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that the overexpression of PdPFD2.2 gene encoding a prefoldin (PFD)

protein, resulted in the increase of lignin S:G ratio and sugar releases
(7.6%) in transgenic poplar. Likewise, Fan et al. [225] identify a novel
miRNA (miR6443) that modules syringyl lignin biosynthesis by specially
regulating F5H2 in Paulownia tomentosa. This strategy showed that the

improve of saccharification efficiency (24.5%) was positively correlated
with the increase of S:G ratio. Also, Sakamoto et al. [115] discover two
bacterial genes encoding coniferaldehyde dehydrogenase calB and couA
can function as novel genetic tools for lignin manipulation. In this study,
glucose releases increased significantly after modifications of CalB, CouA,
and F5H, by 21%, 55%, and 31%, respectively.
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5.3.5 GENETIC ENGINEERING STRATEGIES TO IMPROVE
BIOETHANOL PRODUCTION
Among biofuels, bioethanol is the most produced and commercialized in the

word. Industrial bioethanol plants depend on sucrose and starch-based raw
materials such as sugarcane in Brazil, corn in USA, and wheat, sugar beet,
and barley in Europe. Bioethanol generated from these feedstocks is known
as first-generation. First-generation bioethanol has several advantages like
low production cost and energy efficient production methods that result
in lower fossil fuel consumption [229]. However, first-generation biofuels
production generates substantial economic and environmental concerns
related to the increasing demand for crops competing for cultivable land.
Thus, researches have been interested in the development of commercial
production of second-generation bioethanol, which is produced from non-
food lignocellulosic materials such as agricultural wastes and industrial
byproducts as feedstocks. However, bioethanol generation from lignocel-
lulosic materials still confronts challenges in its commercial production, due
to the complexity of biomass chemical structure, metabolic characteristics of
microorganisms, and fermentation conditions [229, 230].
At present, the most widely studied microorganisms for the first- and
second-generation bioethanol production include yeast, bacteria, and fungi,
such as Saccharomyces cerevisiae, Zymomonas mobilis, and Clostridium
thermocellum (Figure 5.1) [230, 231]. The metabolic performance of these
microorganisms has been improved by genetic strategies in several aspects,
such as substrate conversion, development of substrate assimilation, and
improved inhibitor tolerance, as discussed in the following sections.
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Apple Academic Press Metabolic Engineering of Yeast, Zymomonas mobilis
FIGURE 5.1 Bioethanol production from 1st and 2nd generation biomass: metabolic routes integration. Genes in the dotted box are the
most manipulated to improve metabolic performance for bioethanol production in Saccharomyces cerevisiae (•), Zymomonas mobilis (•) and
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Clostridium thermocellum (•).
Source: Own authorship.
109
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5.4 MAIN METABOLIC ENGINEERED MICROORGANISMS FOR

ETHANOL PRODUCTION
5.4.1 YEAST: SACCHAROMYCES CEREVISIAE

Yeasts, especially Saccharomyces cerevisiae, when compared to other micro-

organisms, have advantageous characteristics that make them suitable for
bioethanol production. Features such as the ability to ferment high concentration
of sugars, high productivity of end products, and high tolerance to ethanol and
inhibitor compounds, are desirable aspects observed in this yeast [116–118].
These microorganisms are already commonly used for industrial
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bioethanol production refineries [119, 120]. During fermentation, some
stress conditions, such as high temperatures, ethanol concentration and the
presence of sugars (e.g., xylose), can limit the process. These factors lead
to the inhibition of the production process and consequently, the reduction
of the ethanol yield [117, 121, 122]. Thus, recombinant DNA techniques
and genetic modifications are constantly carried out in yeasts, in order to
improve the performance of this microorganism [120, 122].
5.4.1.1 STRESS DUE TO HIGH CONCENTRATION OF ETHANOL
In industrial process (e.g., Sake), the ethanol concentration reaches values

approximately 20% in the culture medium [123, 124]. Despite some yeasts
are tolerant to high concentrations of ethanol, its accumulation inhibits cell
growth and metabolic activities that limits the production yield [125–127].
In addition, negative tolerance to ethanol promotes the reduction of the
synthesis and activity of RNA and enzymes, which could result in punctual
mutations [120, 128].
As the concentration of ethanol in fermentation medium increases, the
permeability of the membrane is altered. Ethanol molecules interact with
the hydrophilic portion of the lipid bilayer, promoting the loss of membrane
integrity and increasing its permeability, which induces an increase in the
influx of protons, decreasing the pH to toxic levels [126, 129, 130]. In
addition to membrane alterations, high concentrations of ethanol promote the
denaturation of cellular proteins and the consequent loss of their functions,
for example, enzymes involved in specific pathways (e.g., glycolysis) could
be affected, which influences in central carbon metabolism [129, 131, 132].
Ethanol-tolerant strains can be obtained by genetic engineering techniques.
Studies have carried out analysis of microarrays to determine the transcriptional
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response to ethanol to generate mutations that increase end product tolerance,

from identified sensitive genes [133, 134]. Hirasawa et al. [133] also performed
microarray analysis followed by gene knockout and showed that the overex-
pression of genes related to tryptophan biosynthesis can confer tolerance to
ethanol stress for yeast cells. Techniques such as global transcription machinery
engineering (gTME), are also tools for obtaining ethanol tolerant strains. In
this case, genes of mutant transcription factors (TFs) that control the genes of
cellular metabolism are introduced. Studies have shown that a recombinant
S. cerevisiae with a mutation in the SPT15 gene (gene encoding the TATA
binding factor) can exhibit a new pattern of gene expression for several genes.
This recombinant isolate, with a completely different gene expression pattern,
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was able to tolerate high levels of ethanol [134].
5.4.1.2 STRESS DUE TO TEMPERATURE
As well as the high concentration of ethanol, high temperatures influence yeast

and other microorganisms, causing an imbalance of the plasma membrane,
increasing the permeability of the membrane and the flux of protons, making
it difficult to regulate the intracellular pH [117, 123, 135].
Tolerance to temperature stress is controlled by many genes involved
in protecting cells. Overexpression of genes involved in tolerance to high
temperatures is a commonly used tool. Studies found that the RSP5 gene in
Saccharomyces cerevisiae is responsible for the thermotolerant phenotype,
when it undergoes a mutation process in the promoter region, the RSP5-C
allele causes an increase in the transcription of the RSP5 gene, making it
thermotolerant [136, 137].
High temperature conditions, as well as stress due to ethanol tolerance,
are answered by two main cellular mechanisms: the accumulation of HSP
(described in Section 5.3.3) and carbohydrates (trehalose) [129, 132, 138].
In these stress conditions, the HSP and trehalose are responsible for folding
proteins and reducing membrane permeability [139–141]. Additionally,
the accumulation of trehalose makes cells able to tolerate high levels of
ethanol and high temperatures, exchanging it with water and stabilizing
the membrane and proteins [142]. Some genetic engineering tools aimed to
reduce trehalose degradation. One of the enzymes responsible for trehalose
degradation is vacuolar acid trehalase (ATH1). Performing a Null mutation
of this gene caused high survival rates under various stress conditions, and
the inhibition of ATH1 gene activity by antisense RNA decreased trehalose
degradation and increased ethanol tolerance [143, 144].
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5.4.1.3 FERMENTATION OF 5-CARBON SUGARS
Currently, many feedstocks used in the production of bioethanol have

5-carbon carbohydrates, such as xylose, in their composition [145, 146].
However, the presence of these carbohydrates is often a challenge in

bioethanol fermentation because of some yeasts, such as S. cerevisiae, do
not have the ability to assimilate pentose sugars [147–149]. Some species
such as Pichia, Schizosaccharomyces, and Pachysolen have been described
as good producers of ethanol from pentose sugars [150]. Furthermore, other
yeasts (e.g., Kluyveromyces marxianus) have the ability to co-ferment
hexose and pentose sugars [151].
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Pentose fermentation difficulties have been solved using genetically modi-
fied organisms or co-culture of two yeast strains [152, 153]. To metabolize
xylose, the microorganism must be able to incorporate pentoses into the cell
via membrane transporters. Subsequently, two different pathways for xylose
isomerization in xylulose can be followed: the balanced redox oxidoreductase
and the isomerase pathway. The first occurs when the enzyme xylose reductase
(XR) converts D-xylose to xylitol, then, the enzyme xylitol dehydrogenase
(XD) transforms xylitol into D-xylulose. Later, xylose is isomerized to xylulose
by the enzyme Xylose Isomerase (XI) without the need for a cofactor [149,
154]. Finally, this D-xylulose is further phosphorylated by a xylulokinase in
D-xylulose-5-P, which can enter the PPP [232].
Studies have been successful in the production of ethanol from xylose
using recombinant S. cerevisiae. These strains are constructed by inserting
genes that encode the heterologous metabolic enzymes XR and xylitol
dehydrogenase (XDH) from Scheffersomyces stipitis, Candida intermedia or
other strains [155, 227, 233]. In addition to the construction of recombinant
yeasts, studies carried out genetic modifications to increase xylose consump-
tion. For example, the overexpression of the non-oxidative enzymes of the
PPP, endogenous XKS1 gene (encoding xylulokinase) and the deletion of
GRE3 (encoding reductase enzyme capable of converting xylose to xylitol
using NADPH) in S. cerevisiae, resulted in higher growth rate during xylose
fermentation [156]. Examples of other genetic strategies of yeasts to improve
bioethanol production are shown in Table 5.2.
5.4.2 ZYMOMONAS MOBILIS
In addition to yeasts, other microorganisms have been gaining attention for

ethanol production, like the gram-negative bacteria Zymomonas mobilis. The
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TABLE 5.2
Metabolic Engineering Strategies in S. cerevisiae to Enhance Bioethanol Production
Challenge Strategy Genetic Alteration Enhancement in Mutants Ethanol References
Yield (-fold)
Tolerance to Deletion of Deletion of ACE2 encoding a transcription Higher tolerance between 1.37 [157]
ethanol non-essential genes factor. 5–15% (v/v)
Xylose Transformation Insertion of an endogenous gene that Ability to consume xylose 2.0 [158]
assimilation assimilates xylose with the overexpression
of the ADH1 (alcohol dehydrogenase) gene
Xylose Transformation and Overexpression of GLN1 with knockout of Increased consumption of 1.2 [159]
assimilation overexpression FPS1 and GPD2 xylose and reduction in glycerol
production.
Xylose Construction of Construction of a recombinant strain for Co-assimilation of glucose and 1.86 [160]
assimilation recombinant yeast co-fermentation of glucose and xylose. With xylose.
strain insertion of the PirXylA and OrpXylA genes
Cellulose Transformation Insertion of gene encoding heterogeneous Assimilation of cellulose 57.86 [161]
assimilation cellulase system (sestc) in the S. cerevisiae

genome
Xylose Transformation, Overexpression of the xylulose kinase gene Increased capacity to 1.48 [162]
assimilation superexpression co-ferment glucose and xylose
from lignocellulosic biomass
Tolerance to Transcriptomic Expression of the genes involved in the Enhanced yeast tolerance to 1.17 [163]
stress analysis and degradation of ROS and acetic acid species stress due to elevated reactive
transformation oxygen species (ROS) and
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acetic acid
Xylose Gene deletion Deletion of PDE1 and PDE2 genes The recombinant strain showed 2.0 [33]
assimilation involved in the PKA activity increased consumption of
xylose by 50% in relation to the
113
mutant strain (pde1Δ pde2Δ)
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TABLE 5.2
(Continued) 114
Yield (-fold)
Tolerance to Phenotypic analysis Simultaneous knockout of LEU4 and LEU9 Increased ethanol tolerance – [164]
ethanol and knockout (leading to the accumulation of valine) or
INM1 and INM2 (leading to the reduction
of inositol)
Xylose overexpression, Insertion of genes involved in the Increased capacity to ~2.0 [165]
assimilation evolutionary consumption of xylose (XYL1; XYL2; co-ferment glucose and xylose
engineering XKS1; TAL1; PYK1; MGT05196)
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Z. mobilis has desirable characteristics such as a generally recognized as safe

(GRAS) status, high glucose uptake and tolerance (up to 400 g/L), ethanol
tolerance (16% v/v), and grows in a broad pH range (3.5 to 7.5) [13, 166]. To
produce ethanol, this bacterium uses only Entner-Doudoroff (ED) pathway,
in consequence, low ATP and cellular biomass is produced, resulting in high

conversion efficiency (0.51 g ethanol/g glucose) [167, 168]. Besides ED
pathway, the high expression of pyruvate decarboxylase (PDC) and ethanol
dehydrogenase also makes Z. mobilis a good ethanol-producing microor-
ganism [169, 170]. Despite of these advantages, there are some difficulties to
use Z. mobilis in biotechnological processes, for example, the use of pentose
sugars (e.g., xylose, and arabinose) [171], the toxicity of ethanol, and inhibi-
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tors from lignocellulosic hydrolysates [13, 172]. In face of these limitations,
researchers have been focused on the development of genetic strategies in Z.
mobilis for improving ethanol production, as shown in Table 5.3.
As described before, some of the problems with a strong impact in ethanol
production by Z. mobilis are the inability of these bacteria to grow in presence
of inhibitors formed during the biomass pretreatment, such as acetic acid and
furfural [173]. The acetic acid, generated by hemicellulose and lignin deacety-
lation, can cross the cytoplasmic membrane of microorganisms by diffusion
when it is present in its undissociated form [174]. Inside the cell, acetic acid
is dissociated into proton and corresponding ion, resulting in a decrease of
intracellular pH, higher necessity of ATP to pumping protons out of the cells,
and growth inhibition [175, 176]. In an attempt to solve the problem caused
by the presence of acetic acid, Liu et al. [177] obtained acid-tolerant mutants
through chemical mutation and adaptative evolution. Similarly, Wu et al.
[178] studied acetic acid-tolerant mutant strains obtained via a multi-round
atmospheric and room temperature plasma (mARTP) mutagenesis.
On the other hand, furfural is formed during lignocellulose pretreat-
ment due to the dehydration of pentose sugars [166, 179]. Even in small
concentrations, this inhibitor can demonstrate a negative effect on terpenoid
biosynthesis and mRNAs related to the ED pathway, alterations of the central
carbon metabolism, membrane perturbation and decrease of NADH and ATP
concentrations [180, 181]. Regards to furfural inhibition, Z. mobilis mutants
were constructed by the error-prone PCR-based whole genome shuffling to
improve the tolerance to furfural [168]. The authors hypothesized that the
enhanced NADH-dependent furfural reductase activity detected during the
early log phase may be related to an increase of furfural tolerance of the
mutants, allowing an accelerated furfural detoxification process. Interest-
ingly, Wang et al. [182] considered both acetic acid and furfural inhibition
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TABLE 5.3
Metabolic Engineering Strategies in Z. mobilis to Enhanced Bioethanol Production 116
Yield (-fold)
Sodium ion Transformation Overexpression of ZMO0119 gene (Na+/H+ Higher glucose uptake, cell 2.09 [193]
inhibition antiporter) growth and Na+ tolerance
with 150 mM Na+ or hydro-
lysate added 90 mM Na+
Salt inhibition EZ-Tn5-based Gene ZMO1122 (himA) fractured, higher Higher tolerance under 1.5 1.2 [194]
transposon insertion expression of pdc and adh genes under 2% and 2% NaCl exposure
mutagenesis system NaCl
Ethanol Directed adaptative Alterations in clpP (ATP-dependent Higher tolerance in different – [185]
inhibition evolution protease), spoT/relA, ((p)ppGpp synthetase/ ethanol concentration, grow
hydrolase), and clpB (ATP-dependent in up to 100 g/L of ethanol,
chaperone) improvement in cell viability
Ethanol Random mutagenesis Global transcription factor RpoD protein Higher ethanol tolerance 0.78 [186]
inhibition (σ70), higher pdc gene expression and lower (10% v/v) and glucose
adh gene expression under 9% (v/v) ethanol consumption
exposure
Acetic acid Chemical mutagenesis Deletion in terminator region of ZMO0117 Higher tolerance to acetate 19.26 [177]
inhibition and adaptative (hydroxylamine reductase) and upstream of (244 mM), Higher YP/S and
evolution ZMO0119 increase in the conversion
rate
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Acetic acid Genome shuffling Single nucleotides inserted between ZMO_ Higher tolerance and 2.32 [182]
and furfural mediated by protoplast RS04290, (monofunctional biosynthetic ethanol productivity under
inhibition electrofusion peptidoglycan transglucosylase) and 7 g/L acetic acid and 3 g/L
ZMO04295 (cytochrome c) furfural exposure
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TABLE 5.3
(Continued)

Yield (-fold)
Acetic acid Multiplex atmospheric Single nucleotides variations inserted Higher acetic acid tolerance 44.7 [178]
inhibition and room temperature in regions between genes ZMO0952 (5–8 g/L)
plasma mutagenesis (tRNA methyltransferase)-ZMO0956
(ubiquinol-cytochrome C reductase);
ZMO0152 (pyruvate kinase)-ZMO0153
(DNA binding transcriptional factor);
ZMO0373 (hypothetical protein)-ZMO0374
(levansucrase)
Furfural Error-prone Higher NADH-dependent furfural reductase Higher tolerance to furfural 1.13 [168]
inhibition PCR-based whole activity (3 g/L) and higher concen-
genome shuffling tration of glucose uptake
Phenolic Transformation Expression of NAD+-dependent aldehyde Increase of the conversion 1.77 [189]
aldehydes dehydratase rate of furfural, hydroxy-
bioconversion benzaldehyde, and vanillin
to less-toxic compounds
Use of biogas Atmospheric and room Nucleotides deletion in CDS of gene Ethanol was produced from 1.62 [195]
slurry such as temperature plasma ZMO_RS07255 (carbamoyl-phosphate biogas slurry to replace
substrate mutagenesis combined synthase large subunit) and between gene water and nutrients, under
with adaptive ZMO_RS06410 (FUSC family protein) sterilized and unsterilized
laboratory evolution and ZMO_RS06415 (DNA polymerase III condition.
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subunit delta)
117
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to obtain Z. mobilis tolerant mutants through genome shuffling mediated by

protoplast electrofusion.
As well documented, high concentration of ethanol can also influence
the growth rate and cause inhibition of intracellular metabolites, damage of
peptidoglycan cell wall, and consequently reducing the potential of pumping

protons across membrane [183, 184]. Regarding this problem, Carreón-
Rodríguez et al. [185] obtained and characterized two ethanol-tolerant
mutant strains, through the cultivation of Z. mobilis ZM4 in medium by
increasing ethanol concentrations in consecutive steps. They observed that
overexpression of the spoT/relA gene increased the synthesis of (p) ppGpp
alarmone, that influenced the increase expression of genes related to amino
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acid synthesis mutation. Despite the improvement in ethanol tolerance, most
of the ethanol-tolerant mutant strains did not increase ethanol production.
However, Tan et al. [186] obtains ethanol-tolerant mutants and enhance
ethanol production yield by utilizing random mutagenesis of the sigma factor
RpoD protein (σ70).
Phenolic aldehydes are also produced during lignocellulose pretreatment
by the degradation of lignin components and can pass across cell membranes
due to its low molecular weight [166]. These inhibitors can cause damage to
internal structures and DNA, leading to the inhibition of RNA and protein
synthesis. Besides, it causes an increase of the cell membrane fluidity,
decrease of the intracellular potassium levels and cell growth, and cell
morphology abnormalities [187]. An alternative to reduce toxic effects of
these inhibitors is the direct conversion of phenolic aldehydes to less-toxic
compounds. However, the phenolic aldehydes are recalcitrant molecules
with low water solubility, which disturbs their bioconversion [188]. In this
context, Yi et al. [189] constructed an intracellular oxidative pathway to
enable simultaneous biodetoxification of phenolic aldehydes and fermen-
tation in Z. mobilis. Experiments in vivo demonstrated a strong oxidative
capacity of aldehyde dehydrogenase and upregulation of key genes of the
ED pathway and the oxidative phosphorylation.
Z. mobilis is a promising candidate to be used in the industrial process
with economically viable production [173]. Comparing to the model yeast
S. cerevisiae, this bacterium presents higher ethanol productivity (about
four-fold faster) [190, 191]. Moreover, the possibility to use low pH and
non-sterile conditions to grow Z. mobilis reduces the cost process and the
chance of contamination [192]. Therefore, the characteristics of Z. mobilis
added to studies of metabolic engineering that promote enhancement in
bioproduction, allow large-scale commercial production of bioethanol with
a positive economic balance for industries.
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5.4.3 CLOSTRIDIUM THERMOCELLUM
In addition to the powerful metabolic machinery available in Saccharomyces

cerevisiae and Zimomonas mobilis, other microorganisms are gaining attention
in the industrial field due to their potential for bioethanol production from a
wide range of carbohydrates (e.g., lignocellulosic biomass (LCB)) [196–199].
On this matter, Clostridium thermocellum is a gram-positive anaerobic
bacterium that grows at temperatures above 50°C and can metabolize different
substrates such as lignocellulose, cellobiose, xylose, and hemicelluloses [14,
200]. In the case of the lignocellulosic material, the hydrolysis occurs through
the cellulosome, which is a hydrolytic enzyme complex with a synergistic
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action bound to a backbone scaffold protein and attached on the surface
of the cell wall of the microorganism [14]. Owing to C. thermocellum can
solubilize lignocellulose and produce ethanol as an additional fermentation
product, its application is considered as a low-cost consolidated bioprocess
with many advantages in one single step [201]. However, C. thermocellum is
inhibited in high concentrations of ethanol. This fact is related to alterations
in the membrane composition observed through the accumulation of long-
chain branched fatty acids during its culture [202]. Consequently, this ethanol
inhibition difficulties its widespread industrial adoption. In this sense,
metabolic engineering has been crucial to enhance the bioethanol production
capacities of C. thermocellum (Table 5.4). Hence, to turn this microorganism
a better ethanol producer, different protocols of transformation and deletion
have been widely tested by researchers [203–205]. For instance, deletion
experiments are routinely performed in strain DSM 1313 due to high
transformation efficiency [206, 207]. On the other hand, the development
of counter selectable markers such as tdk, hpt, and pyrF make possible an
easier manipulation of the C. thermocellum chromosome [203, 208, 209].
Furthermore, genetic tools such as gene deletion and targeted mutagenesis
have contributed with additional modifications as an attempt to meet the
industry demands [210, 211].
Among genetic tools, gene overexpression has provided substantial infor-
mation about the complexity of the clostridial metabolism and the possible
ways for increasing bioethanol production [212]. Similarly, heterologous
expression of bioethanol production pathways has been performed in native
strains as a potential solution to the commercialization of this biofuel [213,
214]. In addition, the carbon flux and metabolic pathways affected in C.
thermocellum by the inactivation or complete removal of specific genes have
been another alternative selected by researchers [215]. Since bioethanol
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TABLE 5.4
Metabolic Engineering Strategies in Clostridium thermocellum to Improve Bioethanol Production 120
Yield (-fold)
Increasing of Growth-based Deletion of the genes responsible for Growth in batch culture for 2,000 4.2 [216]
ethanol yield evolutionary organic acid formation ldh and pta h, without pyruvate production
engineering and increased the ethanol titer
4-fold relative to the wild type
Increasing of Gene deletions from Deletion of the genes responsible for Ethanol selectivity of 40:1 1.5
ethanol yield markers inserted and organic acid formation ldh and pta relative to organic acids
removed in the host
chromosome
Conversion of Transformation Heterologous expression of H2 production reduced by 55%, 3.25 [215]
phosphoenolpyruvate kinase (Tsac_1363) from T. carbon recovery of 97%, grow in
pyruvate (PEP) to saccharolyticum, with modification up to 35 g/L of ethanol
pyruvate of the star codon GTG of
phosphoenolpyruvate carboxykinase
Conversion of Transformation Gene for malic enzyme and part of H2 production reduced by 65%, 3
phosphoenol- malate dehydrogenase deleted carbon recovery of 94.2%,
pyruvate (PEP) to grow in up to 35 g/L of ethanol,
pyruvate no NADH-dependent activity
detected
Increasing of Transformation Deletion of deletion of the gene initi- No phosphate acetyltransferase 1.7 [220]
ethanol yield ation regulator, spo0A (Cthe_0812) specific activity detected;
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and phosphotransacetylase (pta, pyruvate concentrations two folds
Cthe_1029) by removing base pairs higher
+174 to +1,074 of the 1,077 bp gene
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TABLE 5.4
(Continued)

Yield (-fold)
Increasing the Transformation Deletion of hydrogenase maturase Maximum ethanol titer of 73 mM 3.19 [221]
flux to ethanol (hydG), lactate dehydrogenase at 20 g/L Avicel, growth in the
by removing side (Clo1313_1160; ldh), pyruvate- absence of pH control
product formation formate lyase (Clo1313_1717;
pflB), pfl-activating enzyme
(Clo1313_1716; pflA), phosphotrans-
acetylase (Clo1313_1185; pta) and
acetate kinase (Clo1313_1186; ack)
Redirection Cloning and Deletion of genes encoding Formate production eliminated, – [217]
of carbon and transformation pyruvate: formate lyase (pflB) and acetate production decreased by
electron flux PFL-activating enzyme (pflA) 50% on both complex and defined
medium
Increasing Cloning and Heterologous expression of pyruvate Two-fold increase in pyruvate 2.03 [222]
the affinity of transformation decarboxylase gene (pNW33N-pdc) carboxylase activity
the pyruvate from Zymomonas mobilis
ferredoxin
oxidoreductase
Improving ethanol Cloning and Heterologous expression of T. Hydrogen production decreased 1.5 [214]
yield transformation saccharolyticum ethanol production
operon (adhA, nfnAB, and adhEG544D)
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Improving ethanol Cloning and Deletion of hydrogen production Maximum ethanol titer of 280 1.2
yield transformation mM (12.90 g/L) within 75 h
121
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production demands the reduction of metabolic power for increasing its

yield, this has been a key point to achieve large-scale processes. Thus, to
enhance the NADPH pool, the most frequently engineered metabolic route
is PPP [216, 217]. In this sense, due to the abundance of pentose sugars in
LCB, and the lack of xylose consumption by C. thermocellum, studies have

been developed to enhance the assimilation of this economic substrate [218,
219]. For instance, Verbeke et al. [218] reported that the deletion of the ATP-
dependent transporter (CbpD) in C. thermocellum partially alleviated xylose
inhibition. Moreover, the authors observed a decrease in the total and molar
yields (mol xylitol: mol cellobiose consumed) of ~41% and ~46% respec-
tively, when deleted a putative XD, encoded by Clo1313_0076. Banerjee et
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al. [122] also analyzed the heterologous expression in C. thermocellum of the
genes xylA (xylose isomerase) and xylB (xylulokinase) from a thermophilic
anaerobic bacterium Thermoanaerobacter ethanolicus. The results from
this study suggested that the combined activity of these enzymes converted
xylose to xylulose-5-phosphate, which was then converted to ethanol
through the incorporation to PPP, glycolysis, and malate shunt pathway.
Searching for new metabolic reactions through enzyme engineering is one of
the expanding areas to improve actual scenario in the industrial sector [198].
5.5 CURRENT STATUS AND FUTURE PERSPECTIVES OF

ENGINEERED MICROORGANISM FOR ETHANOL PRODUCTION:
INDUSTRY
Fossil fuels can potentially be replaced by biofuels in our daily lives, albeit
much work must develop to make the current biofuels production technology
economically feasible. Several considerations have to take in count for cost
reduction, such as maximum theorical yields in energy terms and cheaper
biomass feedstock fermentation. The transition from conventional fuels
to bioenergy systems requires the design and discovery of new metabolic
pathways, as well of new enzymes, new approaches in the bioprocess
engineering and development of microbial consortium capable of tolerate
high ethanol concentrations [223].
As described in sections above, recent advances and future perspectives
in microbial genetic engineering due to the fusion of biological approaches
derived from data of transcriptomics, proteomics, and metabolomics have
led to design novel synthetic circuits that might allow us to cross the gap
between the laboratory scale to the industrial marketplace [8].
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Additionally, to researches presented before, other novel genetic strat-

egies (e.g., CRISPR/Cas) continue to be developed to simplify the use of
model microorganisms (S. cerevisiae, Z. mobilis and C. thermocellum)
in biorefineries. For example, a recent study utilized a novel strategy
(SHPERM-bCGHR) to replace the PHO4 gene in S. cerevisiae, without the

introduction of antibiotic resistance genes and giving stability for long-term
industrial ethanol production [33]. Furthermore, numerous efforts have been
started to boost ethanol production by suppressing glycerol formation. Liu et
al. [207] developed a strategy in S. cerevisiae based on CRISPR/Cas9 novel
system, which optimize the ethanol metabolic pathway by the disruption and
combinatorically of three genes (ADH, GPD, and ALD). Results showed
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an increase of ethanol production and yield by 40% and 22% respectively.
In addition, Xue et al. [224] used CRISPR/Cas9 technology to disrupt the
alcohol dehydrogenase 2 (ADH2) gene via complete deletion of the gen in S.
cerevisiae. Their results demonstrated an improvement up to 75% in ethanol
yield compared with the native strain.
5.6 CONCLUSIONS
Demanding on fuels cost reduction have occasioned in increased alterna-

tives to replace fossil fuels. In addition, several novel strategies have been
implemented to improve biofuel production by the microbial metabolism
engineering in order to look forward for better yields and improvement in
industrial biofuels production. Data recollected in this chapter indicates
that the field of metabolic engineering and synthetic biology has been
expanding rapidly in the case of yeast. This has made possible the construc-
tion of complex metabolic pathways, well-regulated metabolic networks,
synthetic strains, useful recombinant vectors, specific genetic manipulation,
and high-capacity systems. Well-established metabolic engineering and
synthetic biology provides not only a novel biochemical approaches insight
into the lignocellulosic biofuel industry, but also an additional insight into
deciphering the pathways behind bioethanol production by different micro-
organisms. Thus, the main improvements to scale up bioethanol production
process, must be related with the development of high-performance strains,
such as high ethanol concentration tolerance, thermostability, and detoxifica-
tion of inhibitors. Finally, economical scenarios must be assessed to deter-
mine the true commercial value of bioethanol production through metabolic
engineered microorganisms.
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KEYWORDS
• bioethanol
• biofuels
• global transcription machinery engineering
• metabolic engineering
• prefoldin
• synthetic biology
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CHAPTER 6
Increasing Ethanol Tolerance in

Industrially Important Ethanol
Fermenting Organisms
KALYANASUNDARAM GEETHA THANUJA1 and
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SUBBURAMU KARTHIKEYAN2
1
Department of Agricultural Microbiology,
Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India
Department of Renewable Energy Engineering, Tamil Nadu Agricultural
2
University, Coimbatore, Tamil Nadu, India, E-mail: skarthy@tnau.ac.in
ABSTRACT
The growing population and increased urbanization have set great demand for
energy. With the deprivation of fossil fuels, interest has focused on renewable
sources. Among them, bioethanol is an attractive and green source being used
as a promising alternative to reduce environmental pollution. In the process of
ethanol production, varied ranges of feedstocks are being converted through
microbial fermentation. Widespread industrial biocatalysts are exploited in
bioethanol production like bacteria, yeast, fungi, and algae according to the type
of raw material, environmental conditions, and resources available. However,
the organisms leading the ethanol industries are Saccharomyces cerevisiae,
Pichia stipites, Zymomonas mobilis, and Clostridium thermocellum. Their
capacity to yield higher ethanol, ability to ferment sugars, growth in simple
and inexpensive media, resistance to inhibitors, and contaminants makes
them potential candidates in the fermentation process. While the maintenance
of growth and metabolic efficiency of these microbes with resistance to
various stresses is the desirable factor, several challenges predominate in the
microbial fermentation inhibiting the overall ethanol production in which
ethanol toxicity is a significant concern. The key advantages of microbial
strain employed for industrial ethanol production are, ethanol tolerance
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limits and substrate concentration, ultimately decreasing productivity and

increasing the costs of bioreactor. Ethanol toxicity is the complex and multi
loci trait, affecting various cell functions and inhibits key glycolytic enzymes
by denaturation mechanisms. The process renders the microbial cell to
undergo reprogramming in cellular and metabolic activities with increased

repair functions. However, it is crucial to understand the consequences
of ethanol stress defense mechanism for ethanol tolerance improvement
strategies. Various approaches have been attempted to circumvent the ethanol
stress and enhance ethanol tolerance from optimizing its media to rewiring
of genetic setup. Towards the establishment of ethanol tolerant phenotypes,
integrative system, and the interplay of genes involving complex network is
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essential. With the assistance of molecular tools, the strain with improved
ethanol tolerance can be achieved.
6.1 INTRODUCTION
The energy consumption across the globe is ever-widening for improved

living standards. The demand for energy hunts mainly towards fossil fuels
as the major resource which sets step for environmental pollution, global
warming (GW), depleted natural resources, etc. With large depletion of the
world’s petroleum reserves and growing costs, interest vested on the produc-
tion of ethanol. Ethanol has been widely acknowledged as an alternate fuel
for transport which can be readily produced from various lignocellulosic
feedstocks through microbial fermentation. Generation of high yield ethanol
can be achieved through the implementation of consolidated bioprocessing
(CBP). The feasibility of CBP gets hindered by various stress factors causing
the industrial fermentation troublesome.
Various fermentative microbes, namely S. cerevisiae, Z. mobilis, and C.
thermocellum, have been exploited successfully in the industrial production
of alcohol-related products owing to their unique characteristics (Table 6.1).
Although the organisms involved in the industrial processes have great
sturdiness, they often lack tolerance to certain stress factors.
To ensure the viability of the industrial process, the end product has to
accumulate with high yield and titer which becomes the near-universal stress
factor. The end product of fermentation, ethanol interrupts with glycolytic
enzymes harming the cell membrane of fermentative bacteria. To maintain
the pace with escalating demand for ethanol, the pressing prerequisite is to
overcome the barriers in the production and recovery process. This chapter
provides an overview of different strategies to develop ethanol tolerant
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Increasing Ethanol Tolerance in Fermenting Organisms 143
strains of the above-mentioned organisms and highlight specific strategies

unique to individual organisms.
TABLE 6.1 Comparison of Physiological Characteristics of S. cerevisiae, Z. mobilis, C.

thermocellum
Categories S. cerevisiae Z. mobilis C. thermocellum
Growth condition Facultative Facultative anaerobe Thermophilic anaerobe
aerobe
Taxonomy Eukaryotic Gram-negative Gram-positive bacterium
bacterium
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pH preference 2.0–6.5 3.5–7.5 6.0–8.5
Metabolic pathway EMP ED ABE fermentation
Theoretical ethanol yield >90% >90% 2.5%
Ethanol tolerance (w/v) 15% 16% 1–2%
Safety GRAS GRAS GRAS
The brewer’s yeast, Saccharomyces cerevisiae is promising for industrial

alcoholic beverage production and bioethanol fermentation. Ethanol begins
to accumulate in the early stage of fermentation, and due to the depletion of
essential nutrients, the quantity of ethanol outstretches to toxic levels towards
the end of the process. Although, they have maximum tolerance between 115
and 200 gL–1, their accumulation higher than 150 g.L–1 in the culture broth
limits yield and productivity. Ethanol is found to damage the mitochondrial
DNA of yeast cells and inactivates key enzymes like dehydrogenase and
hexokinase. Beyond the threshold limit, ethanol stress significantly influences
lipid production, ion homeostasis, up-regulation of several genes, trehalose
metabolism, and altered transcription behavior. Several strains of yeast have
adapted mechanisms to tolerate a higher concentration of ethanol than most
other organisms, yet their mechanism remains complex to elucidate. Ethanol
tolerance in yeast is a complex phenotype influenced by various genetic,
physiological, physical, and environmental factors.
Z. mobilis is an ethanologenic, gram-negative, and obligate fermentative
bacterium recruiting unique metabolism to produce ethanol by the EntnerD-
oudoroff pathway. They are well recognized for ethanol and glucose tolerance
and exhibit rapid fermentation than S. cerevisiae. Further, they own desirable
industrial biocatalyst criteria such as increased specific-productivity, wide pH
for production (pH 3.5–7.5), reduced cost for sophisticated aeration control,
and safe status of production [1]. With more than 7% initial concentration
of ethanol and at ≥35°C, the cell mass and ethanol production get disrupted.
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The fermentation capacity of various Z. mobilis strains was assessed and

optimized parameters for ethanol production [2] Compared with S. cerevi-
siae, the studies on the physiological and genetic basis of ethanol tolerance
in Z. mobilis is relatively lower. Some ethanol tolerant strains have been
developed by ethanol-adaption, mutagenesis, and genetic engineering [3–5].

Ethanol adaption (EA) in wild strains of C. thermocellum revealed a greater
percentage of fatty acids and an increase in the percentage of plasmalogens
significantly contribute to membrane rigidity counteracting the effects of
ethanol [6]. The mechanism of ethanol tolerance was explored by systematic
metabolomics to analyze the phenotypes of ethanol tolerant and wild type
strains. The results revealed an accumulation of cellodextrin in the ethanol
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tolerant strain depicting its potential mechanism for stress resistance [7].
6.2 EFFECTS OF ETHANOL ON YEAST, Zymomonas mobilis AND

During the fermentation of ethanol, microbial cells experience various stresses

like increasing ethanol concentration, high temperature, feedback inhibition,
inappropriate substrate concentration, osmotic pressure, etc. The small size of
ethanol and the functional group renders solubility to both aqueous and lipid
environments. It easily passes through the plasma membrane and enhances
the fluidity and permeability and causes a series of consequences at the
cellular level [8]. Recent studies evidence that ethanol tolerance is a poly-
genic phenotype trait governed by multiple alleles with complex interaction
and varies between the strains. As a response mechanism, saturated lipids,
transmembrane lipids, sterols, and hopanoid gets enhanced in the membrane
of Clostridium, Zymomonas, and Saccharomyces [9, 10]. In general, for
the recovery of ethanol from classical downstream, Saccharomyces should
possess the ability to grow and produce ethanol in 4% (v/v) ethanol [11]. It
involves cellular dysfunctions, lowered respiratory rates and ATP, the forma-
tion of reactive oxygen species (ROS) which then induces DNA damage and
oxidative stress. The flux of protons gets deregulated ultimately resulting in
decreased proton motive force compromising the nutrient uptake. Among
them, a high level of ethanol interrupts not only the physiology of the cell
affecting growth, viability, and fermentation but also overall ethanol produc-
tion. Table 6.2, briefs the effect of ethanol on microorganisms. Microarray
and several expression tools revealed the genes and their complex network
associated with ethanol tolerance disclosing its enigmatic nature. There
are many challenges faced in developing strategies for enhancing ethanol
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tolerance. This necessitates the need for ethanol tolerant strains which could
be achieved through several reprogramming strategies.
TABLE 6.2 Effect of Ethanol on the Physiology and Metabolism of Fermenting Microorganisms
Effect References
S. cerevisiae At low concentration: [17]
Inhibits cell division, growth rate and decreases cell volume
At high concentration:
Decreases cell vitality and causes death
Function of ribosome is inhibited [18]
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Governs vitality and viability of the cells, thereby terminating [19]
fermentation; Inhibits glucose and amino acids transport
systems, denaturation key glycolytic enzymes
Membrane permeabilization, cytosolic, and vacuolar [20]
acidification
Dissipation of membrane integrity and increase in [21]
membrane permeability due to intercalation of lipid bilayer
Z. mobilis Decreased cell mass and ethanol production, Inhibition of [22]
fermentation process
Decreased rate of substrate conversion [23]
Inactivation of enzymes involved in ethanol production [24]
C. thermocellum Imbalance in NADPH [3]
Interruption in proton motive force and ATP production [25]
Few studies revealed the genes involved in ethanol stress guiding the
rational strategies to progress the process performance. The genome sequence
of Z. mobilisZM4 revealed the sigma factor (σE, ZMO4104) plays a crucial
role in against ethanol stress [12]. Alcohol dehydrogenase II (ADH2) protein,
encoded by the adhB gene was found to be the major protein involved in
ethanol stress [13]. Further Pallach et al. [14] have proposed the exopoly-
saccharides (EPS) components of Z. mobilis contributes to its high ethanol
tolerance mechanism.
Ethanol fermentation using thermophilic bacteria has been suggested as
one of the novel systems. However, the membrane composition of S. cerevi-
siae and Z. mobilis provides higher tolerance to ethanol. C. thermocellum is
a gram-positive, thermophilic anaerobe that serves as an efficient candidate
in CBP due to its ability to rapidly fermenting cellulosic biomass. One of
the challenges in the industrial application hindering their potential is low
ethanol yield, titer, and tolerance producing acetate, lactate, H2, formate as
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further fermentation products [15]. The complexity of ethanol tolerance in C.

thermocellum was elucidated by eliminating the H2 production by redirecting
carbon flux towards only ethanol production, thereby rendering the electrons
available for reduction of acetyl CoA to ethanol [16].
6.3 METHODS FOR IMPROVING ETHANOL TOLERANCE
Various tolerance mechanisms rendered by the organisms are depicted

in Figure 6.1. Ethanol on passing into the plasma membrane increases
membrane fluidity which in turn deteriorates membrane integrity through
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partitioning into a lipid bilayer. The aliphatic chains of ethanol get deeply
inserted into the hydrophobic interior of a lipid bilayer and favor a high
degree of permeability. As a tolerance mechanism, the cell wall lipid
composition is increased with polyunsaturated fatty acids, ergosterol, and
phosphatidylcholine. The cell wall architecture gets remodeled to attain
robust nature through various signaling pathways [26]. The increased
membrane permeability also results in cytosolic and vacuolar acidification
for which vacuolar H+-ATPase (V-ATPase) plays a crucial role in the
maintenance of intracellular pH homeostasis. The intracellular transport of
H+ into vacuoles by H+ V-ATPase assists in translocation across the vacuolar
membrane through hydrolysis of ATP, thereby counteracts the ethanol stress.
Further several genes would up regulate upon ethanol stress leading to an
alteration in the transcription machinery which renders protection to the
organism by various physiological changes. Therefore, the design of the
ethanol tolerant organisms can be concentrated on the above-mentioned
background with numerous techniques as follows. The creation of strains
with augmented stress tolerance is however attainable through a range of
approaches (Figure 6.2), combining with recent technology could limit time
and tedious multistep. Traditional approaches for the development of ethanol
tolerant phenotype include rational selection approaches for modification
followed by probability-driven processes like adaptive evolution or random
mutagenesis. The strain development for ethanol tolerance has certain
gold standards like mutagenesis and selection traditionally in the industry.
The advent of recombinant tools gives rise to more sophisticated alternate
strategies to ameliorate alcohol toxicity. The advanced omics technologies
such as genomics, proteomics, transcriptomics, and metabolomics have
aided to understand the complex alcohol toxicity and tolerating mechanism.
Exploring the molecular basis of ethanol tolerance en routes the development
of rational approaches. The metabolomic analyzes of E. coli on the responses
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to ethanol and butanol tolerance elucidated several amino acids like valine,
glycine, isoleucine, glutamic acid and osmoprotectants like trehalose. Most
transcriptomic analyzes have shown the role of small molecular weight
compounds on alcohol tolerance.
FIGURE 6.1 Ethanol tolerance mechanism of mentioned microbes. (a) change in membrane Author Copy
composition; (b) maintenance of intracellular pH by H+ ATPase pump; (c and d) production of
stress-responsive proteins/genes; (e) vacuole acidification/vacuole mediated transport.
6.3.1 GENES INVOLVED
High ethanol concentration associates several 100 genes concerning cell

wall and membrane organization, amino acid biosynthesis, lipids, and fatty
acid metabolism, and few genes are presented in Table 6.3. The studies on
comprehensive gene expression, regulatory networks, and pathway-based
and integrated resistance have revealed numerous genes [27]. To explore
the molecular mechanism beneath the tolerance of yeast cells, the cell wall
integrity (CWI) pathway, and high-osmolarity glycerol (HOG) pathway was
studied along with their genes involved. Upon ethanol exposure, CWI in
collaboration with HOG triggers the transcription regulation of cell wall
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biosynthesis genes FKS2, CRH1, and PIR3 thereby causes remodeling of the
cell wall [28]. The mutants lacking CWI genes were found to be sensitive to
ethanol.
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FIGURE 6.2 Various approaches are available to develop ethanol tolerant strains.
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Msn2p is the transcription factor, whose activation is controlled by protein

kinase A (PKA), which regulates the interconversion of glucose 1-phosphate
and glucose-6-phosphate. Msn2p binds to the stress response elements,
inducing up-regulation of more than 200 genes. The protein sequencing by in
silico analysis of msn2 discovered, serine residue at position 625 as an effec-

tive target of PKA. Replacement of serine residue with alanine augmented the
cell susceptibility to ethanol indicating its crucial role [29]. The transcriptomic
and proteomic studies of S. cerevisiae 131 under ethanol stress revealed 937
differentially expressed genes and 457 differentially expressed proteins [18].
These expressions also revealed 10% (v/v) ethanol could induce sexual
reproduction, signal transduction such as G-protein, silent information regulator
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(sir) proteins, and aromatic amino acids (AAA) helping in the survival of cells.
TABLE 6.3 Genes Involved in Ethanol Tolerance Mechanism

Genes Function Alteration in Gene References
tps1 Catalyze trehalose Increased accumulation of [30]
synthesis intracellular trehalose by
over expression of tps1
CRH1 Cell wall biosynthesis Transcriptional activation [28]
triggers remodeling of cell
wall
SNF1 Develop cell resistance and Overexpression of SNF1 [31]
consumption of glucose
under stress conditions
Rice metallothionein Protection against Heterologous expression in [32]
isoform OsMTI-1b oxidative stress yeast conferred tolerance
YAP1 and SOD2 Key stress responsible Ensures signaling function [33]
elements desired for ethanol tolerance
ERG2, ERG3. ERG4 Ergosterol biosynthesis Upregulation under ethanol [34]
Hsp40, Hsp82, Heat shock proteins stress
Hsp104, KAR2
TH14, TH15, and Biosynthesis of thiamine
TH13
PRO1 Proline biosynthesis Downregulation under
ethanol stress
6.3.2 NUTRITIONAL STRATEGY
Wide exploration of the mechanism is mandated to produce advanced yeast

variants or mutants. The conventional methods of improving strains towards
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higher productivity have had partial accomplishment beyond the identifica-

tion of medium optimization and various chemical protectants (Table 6.4).
TABLE 6.4 Nutritional Strategy for Enhancing Ethanol Tolerance

Optimized Conditions Effects References
Addition of 0.02 g/l zinc sulfate Enhanced cell viability in ethanol shock [35]
treatment
Supplementation of zinc with other Maximum viability, ethanol, and biomass [36]
metal ions production
Addition of KCl Increased ergosterol and ATP content [37]
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with improved biomass and cell viability
Supplementation of Mg2+ in the 50% cells remained viable in exposure of [38]
culture media 20% (v/v) ethanol for 9 h
Exponential feeding of vitamins Increase in final ethanol titer and ethanol [39]
tolerance
Supplementation of CaCl2 and Protection against toxic effect of ethanol [40]
MgCl2
Non-limiting oxygen condition 23% increase in viable cell mass [41]
Supplementation of 10–20 mM Reduction in cell mortality [22]
magnesium
6.3.3 EVOLUTIONARY ENGINEERING
It follows the engineering principle by variation and selection in genetic

diversity. Random mutations in the genome induced by several stress factors
are known to be adaptive evolution (domestication). They serve as a tool
to develop strains through the continuous passage of strains and effectively
change a few characteristics of physiological/phenotypic origin. They are
also regarded as a capable approach for the non-recombinant alteration of
industrial yeast strains. The existence of stress maintains the yeast cells
under selective pressure and genotypes which survive are preserved. Step-
wise increase of ethanol concentration in culture medium was reported to
choose ethanol tolerant strains of the S. cerevisiae [42–44].
Change in the DNA sequence of six independent populations of geneti-
cally identical yeast upon exposure to gradually increasing concentration of
ethanol was monitored over two years. The novel computational analyzes
analyzed the mutational dynamics and molecular mechanisms of ethanol
tolerance are achieved through complex and different mutational pathways
[45]. Evolved populations exhibited intricate interaction of de novo single
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nucleotide mutations, copy number variation, ploidy level, and clonal

interference significantly contributing to ethanol tolerance. Liquid nitrogen
incubation of cell culture for 30 min followed by thawing at 30°C for 20
min (Freeze-thaw treatment) was found to enhance ethanol tolerance [46].
The mutant developed upon freeze-thaw treatment had a profound change

in the dynamic structure of membrane lipids and capable of accumulating
trehalose thereby contributing to ethanol tolerance. Further aneuploidy of
chromosome III is attaining interest in industrial strains of S. cerevisiae as
an adaptive trait [47]. Strains tolerating 12% (v/v) ethanol were developed
by Turanli-Yildiz et al. [48] by in vivo evolutionary engineering and also
hypothesized the link between ethanol tolerance and diploidization of yeast
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strain. Selective pressure of inhibitory concentration of hexanol evolved
tolerant S. cerevisiae BY4741 [49]. The sequential transfer method with
increasing ethanol concentration in Z. mobilis ZM4 mutant ER7ap exhibited
better performance than wild type [50].
6.3.4 GENETIC APPROACHES
6.3.4.1 RANDOM MUTAGENESIS
The classical strain improvement of industrial microorganisms is using

random mutagenesis and screening. Their well-established history of efficient
application to make genetically diverse phenotype is an engrossing tool in the
post- “omics” era. Although they have been used successfully in the strain
improvement program, random methods and global techniques have to be
combined to generate complex phenotypes. This method is often coupled
with an adaptive laboratory evolution strategy since classical mutagenesis
demands immense time for continuous screening and rarely yields elite
strains. Furthermore, ethanol tolerance as a complex phenotype requires
synergistic functions of a variety of genes thus making it complex to genetic
engineering. For improving these complex phenotypes, random mutagenesis
of TATA-binding protein is the strategy for transcription machinery engi-
neering. Sufficient selection followed by screening is essential in industrial
applications. Mathematical modeling serves as an adroit tool to achieve this
selection. It was attempted with the help of transmission electron micros-
copy (TEM) in probiotic yeast S. cerevisiae var. bouldardii CNCM I-745 by
Baranyi and Roberts model [51]. Hoponaid (hpn) transposon mutation, at
elevated ethanol concentration the head group composition and abundance of
hopanoids prevents membrane dissolution in Z. mobilis [52].
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6.3.4.2 GENOME SHUFFLING
The combinatorial method which combines the primacy of multi-parental

crossing enabled by DNA shuffling with the recombination of whole genomes
generally connected with conventional breeding. This strain improvement

technology however originated from protoplast fusion/electrofusion and
greatly relies on them, it markedly differs in the recombination giving rise
to stable and high-performance strain (Table 6.5). Genome shuffling is an
evolutionary engineering technology that accelerates the strain development
process by a sequence of three processes. First, the genetically divergent
population is creation followed by inducing mutation. Second, the popula-
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tion is screened to identify the best performing isolate. Third, the genomes
of supreme cells are shuffled either by sexual or asexual means following
protoplast fusion or sporulation, respectively, in single strain recursive
protoplast fusion between multi-parent strains.
TABLE 6.5 Different Genome Shuffling Techniques

Strain Method Profound Effects References
S. cerevisiae Recursive protoplast fusion Tolerates 25% (v/v) ethanol [53]
SM-3
S. cerevisiae Recursive protoplast fusion Resulted in hybrid strain [54]
NCIM 3090 followed by Random (SP2-18) with increased cell
amplified polymorphic DNA viability than the parent strain
S. cerevisiae EMS mutagenesis and Increased ethanol yield and [55]
S310 recursive sporulation of tolerance after three rounds
diploid cells of genome shuffling
S. cerevisiae Random mating and selection Increased ethanol tolerance [56]
and fermentation capacity
S. cerevisiae Q Inactive protoplast fusion Higher ethanol tolerance in [57]
and L beer production-14%(v/v)
6.3.4.3 GENOME EDITING
In the past years, novel genome editing tools like transcription activator-like
effector nuclease (TALEN's), zinc finger nuclease (ZFN) have been used to
engineer strains with desirable traits in industrial settings. However, there are
only a few studies applying the gene-editing tools for ethanol tolerance. Zhang
et al. [58] have demonstrated TALEN assisted multiplex editing (TAME) for
improving ethanol tolerance in yeast by multiple genetic perturbations in the
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genome. Mitsui, Yamada, and Ogino [59] have achieved higher cell viability
in low pH and high ethanol concentration in S. cerevisiae using Clustered
regularly interspaced short palindromic repeats (CRISPR)-CRISPR associ-
ated protein (Cas). The large-scale rearrangement of genomic DNA can be a
promising strategy for the improvement of ethanol tolerant strains.
6.3.4.4 OVER-EXPRESSION
The iron-sulfur clusters (ISC) are the prosthetic groups responsible for
catalytic and regulatory functions found in both prokaryotes and eukaryotes.
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The components of the mitochondrial ISC in S. cerevisiae were reported to
be related in iron homeostasis due to ethanol tolerance and are encoded by
the genes NFS1, ISU1, ISU2, ISA1, ISA2, JAC1, SSQ1, YAH1, GRX5, and
IBA5. The over-expression of Jac 1p and Isu 1p in S. cerevisiae UMArn3 was
evaluated and revealed their counteracting ability of metabolic unbalance
caused by an increase in ROS generation [60]. Further Snf complex of S.
cerevisiae, a member of AMP protein kinase family engages in a series of
cellular functions and responds to environmental stresses. The effect of Snf
over-expression revealed a 39% increase in cell survival rate over parental
strain at 8% ethanol accompanied with altered expression of genes involved
in glucose transport and accumulation of fatty acids [31].
6.3.4.5 GLOBAL TRANSCRIPTION MACHINERY (gTME)-MANIPULATION

OF MULTIPLE GENES
In recent times, engineering the global transcription machinery has been

investigated to reprogram the microbial transcription profile. It is regarded
as an efficient tool in bringing a high degree of pleiotropy. The method of
reprogramming transcription to elicit desired cellular phenotypes for indus-
trial and technical applications has spurred interest in yeast engineering
for stress tolerance. The transcriptional machinery of eukaryotes is quite
complex with a substantial set of general and specific transcription factors
(TFs). It modifies the key protein to synchronize the global transcription by
error-prone polymerase chain reaction epPCR.
Engineering or mutagenesis in TF to modify DNA binding specificity and
RNA polymerase II to induce perturbations have been implied successfully
to develop ethanol tolerant strains of S. cerevisiae. In their transcription, two
major complement complexes sharing TATA box-binding protein-associated
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factors (TBP), namely RNA polymerase II transcription factor D (TFIID)

and SAGA (Spt-Ada-Gcn-5 acetyltransferase) are involved. Both SAGA
and TFIID work together almost mRNA genes, however, all yeast mRNA
strongly relies on TFIID [61]. Reprogramming global regulator RpoD via
epPCR improved glucose consumption and ethanol yield under ethanol stress
in Z. mobilis [54]. Further, incorporation of gene modules increased fatty acid
production which served a crucial criterion for ethanol tolerance [62].
Beyond yeasts being physiologically ideal, the complex array of stress
factors exhibited have to be encountered cooperatively. Conventional breeding
techniques bestow bounded up-gradation of strain robustness. However,
their painstaking standards assist “omics” technologies in recent research.
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Exploring genetic elements required to maintain ethanol tolerance would be
a better approach rather than genetic modifications. On the other hand, altera-
tion in the gene for ethanol tolerance could compromise several fermentation
parameters. The ever-expanding knowledge on yeast genome and functions
makes an obscure platform for commercial-scale ethanol tolerant strains.
Recent precise technology for genome editing enabled quick engineering of
yeast strains which would develop strains for numerous applications.
6.3.5 SMALL RNA (sRNA) ENGINEERING
sRNA is non-coding functional RNA with 50–300 nt capable of blocking

translation or altering RNA’s stability by various mechanisms thereby
regulating protein expression. Hence, they represent strong tools in governing
central pathways to engineer complex phenotypes [63]. The Discovery of
novel sRNA candidates in Z. mobilis was attempted by Cho [64] using a union
of computational approaches. The differential expression of selected sRNA
under different ethanol concentrations was analyzed, followed by the effect of
ethanol stress on the expression of sRNA. Their results revealed three sRNA
namely Zms2, Zms6, and Zms18 were differentially expressed, suggesting
their prime role in the regulatory mechanism of ethanol tolerance. Further,
Zms4 and Zms6 were found to coordinate a large network of gene regulation
including sRNA-sRNA interaction and the absence of identified sRNA made
the cells ethanol sensitive [65]. Advancement in identifying novel sRNA
using prevalent “omics” datasets with the bioinformatics pipeline, RNA
seq examiner for phenotype-informed network engineering (REFINE) was
proposed in Z. mobilis. REFINE approach combines existing computational
tools with the new pipeline (sRNA phenoscore), to identify differentially
expressed RNAs from different growth conditions REFINE sRNA prediction
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relies on scores assigned to differentially expressed intergenic regions

suggesting their possible regulation relevant to the phenotype [66].
6.3.6 AdhE MUTATION
AdhE, a gene encodes for aldehyde dehydrogenase, the bifunctional and

bidirectional enzyme. It is responsible for catalyzing two-terminal steps in
the formation of ethanol: acetyl-CoA reduction to acetaldehyde by aldehyde
dehydrogenase (ALDH) and reduction of acetaldehyde to ethanol by alcohol
dehydrogenase (ADH) with two reduced electron donors, NADH, and
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NADPH. In C. thermocellum, the role of adhE is crucial and their deletion
strain showed >90% activity loss of ALDH, ADH, and >95% reduction of
ethanol production [67]. Point mutation resulted in the replacing certain
amino acids at particular sites of ADH [3].
The genome of six isolates with n-butanol tolerance was re-sequenced and
the mutations in the coding sequence of Clo1313_0853 and Clo1313_1798
were studied [5]. The gene Clo1313_0853 catalyzes the hydrolysis of
phosphatidylcholine and phospholipids to produce phosphatidic acid (PA).
The mutation in the gene truncates the protein by frameshift and protects
the membrane in the presence of n-butanol/ethanol. Mutation in the gene
Clo1313_1798, encoding alcohol dehydrogenase (adhE) increases its
ability to use NADPH as a co-factor yielding increased ethanol and titer.
The increased NADPH activities are associated with AdhE mutant, where
the reverse flux through ADH reaction affects NADH/NAD+ ratio and the
NADPH/NADP+ ratio. Deletion of adhE strain (LL1111) increases tolerance
and inhibits several primary alcohols by reverse flux through AdhE [68].
6.4 CONCLUSION
Ethanol tolerance in fermenting organisms is not a one-way approach and

includes the interplay of events. It requires numerous pathways and networks
to be recognized. Developing ethanol tolerant strains is often arduous due
to a lack of in-depth perceptive of tolerance mechanism. Further ethanol
tolerance research involves various strains of yeasts and other ethanol
fermenting microorganisms under varied conditions that make difficulty in
interpreting the results across the studies. In this regard, rational engineering
and evolutionary engineering are crucial to identify genetic targets. Conven-
tional methods of gene expression and evolutionary engineering can be used
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resourcefully with the aid of synthetic biology and omics techniques. The
ethanol-related industries may take advantage of these findings to lower the
cost of ethanol production.
KEYWORDS
• bioethanol
• Clostridium thermocellum
• ethanol tolerance
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• exopolysaccharides
• yeast
• Zymomonas mobilis
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Inactive Protoplast Fusion in Saccharomyces cerevisiae. Biomed Res Int., 1979318.
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the ethanol tolerance and yield by superoxide and iron homeostasis mechanism in an
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61. Warfield, L., Ramachandran, S., Baptista, T., Devys, D., Tora, L., & Hahn, S., (2017).
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CHAPTER 7
Challenges in Developing Sustainable

Fermentable Substrate for Bioethanol
Production
CHARU SARAF and KAKOLI DUTT
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Department of Bioscience and Biotechnology, Banasthali Vidyapith,
Rajasthan–304022, India, Tel.: 9910331924,
E-mail: charusaraf17@gmail.com (C. Saraf), Tel.: 9887398384,
E-mail: kakoli_dutt@rediffmail.com (K. Dutt)
ABSTRACT
The ability to harness fire from burning wood was the first application of fuel-
based energy. For a long time, wood and other derived products were the sources
of energy to support various activities like cooking, metallurgy, pottery, etc.
The discovery of fossil fuels and the development of the steam engine opened
a new age of applications. But in this modern world, overuse of fossil fuels to
sustain various technologies has started to take a heavy toll in the form of envi-
ronmental pollution. Thus, the new search for alternative energy sources which
can efficiently replace the fossil fuels and have minimal environmental issues
has become mandatory, with several biofuels such as bioethanol and biodiesel
gaining prominence. Ethanol production, a massive global commercial venture
shows continuous evolution in the production process for yield enhancement.
The developments observed in the characterization of different biomass as raw
material for ethanol production has led to their categorization as first, second,
third, and fourth-generation fuels. Numerous reviews and research articles
account for the advantages and disadvantages of these substrates against each
other. Additionally, the pretreatment and the fermentation processes play a pivotal
role in the final yield obtained. Thus, in this chapter, an attempt has been made
to present a comprehensive summary of ethanol production with emphasis on
the different substrates, pretreatment, and fermentation processes. In the chapter,
bottleneck of the bioethanol commercialization has also been briefly discussed.
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7.1 INTRODUCTION
Over six decades ago, petroleum products were inarguably the source of energy
that kept the world functional and expanding. Nevertheless, in the beginning
of the 1970s, proclamation to control the production by the members of

the Organization of Arab Petroleum Exporting Countries (OPEC) hiked the
petroleum price, causing an energy crisis [1]. Additionally, climatic changes
and greenhouse gas emissions (GHG) awakened the interest in alternative
energy sources to achieve environmental sustainability [2, 3]. Biofuels
(bioethanol and biodiesel) were welcomed as environmentally sustainable
alternative energy sources [4]. Alcohol obtained from fermentation route
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quickly became a part of fuel industry as blended fuel ‘gasohol’ [5].
“Fuel for future” a term coined by Henry Ford for ethanol is a major
contender among several key alternative energy sources. Obtained either
from biomass through microbial fermentation or as a byproduct of several
industrial processes [6], ethanol shows valid promise not only as feedstock
for chemical industries but also as biofuel (Figure 7.1). The oxygenated
nature of alcohol enhances its combustion efficiency which added to the
properties of eco-friendly, less toxicity, higher heat of vaporization and
flexibility of sugar substrate for alcohol fermentation increases its appeal
[7–10]. Ethanol has higher octane number (106–110) than gasoline (91–96),
resulting in enhancement of gasoline performance on blending [11–13].
Although, literature reported shows unlimited benefits of bioethanol with
respect to environment and economy, its global development as a fuel and
chemical feedstock is facing a serious bottle neck of usable and fermentable
substrates [14, 15]. Significant researches are going on which are globally
supported by private conglomerates, think tanks, NGOs, and Government
funded projects. This chapter provides an overview of the evolution of
ethanol as a fuel, fermentable substrates, technological approaches, and
fermentation conditions for bioethanol formation with a brief outline on the
hurdles in bioethanol commercialization.
7.2 BRIEF HISTORY OF ETHANOL
Uses of ethanol as fuel was first reported in 1826 in America and subse-
quently after 50 decades by Nikolaus Otto in 1876, who used ethanol to
power an early internal combustion engine (Figure 7.2). Before the American
Civil War, ethanol was used for lighting but this was severely curtailed due
to liquor tax imposition. After the tax was repealed, ethanol resumed its
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Challenges in Developing Sustainable Fermentable Substrate 163
function as fuel. In fact, Henry Ford's Model T in 1908 was designed to run
on either gasoline or pure alcohol (adapted from: www.ott.doe.gov/biofuels/
history.html).
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FIGURE 7.1 Various applications of ethanol.
FIGURE 7.2 The engine made by Nicolaus Otto in 1876.

Source: Public domain.
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Prior to the Second World War, industrial use ethanol was produced by
fermenting blackstrap molasses, a cheap, easy to handle, most economical
and easily available substrate. After the war, the low-priced easy avail-
ability of petroleum-based fuel and natural gas curbed the interest in use of
agricultural crops for fuel production which wiped out the economic incen-
tives for production of liquid fuels from crops leading to the generation of
a significant lacuna. Since, Government interest was lost, many wartime
distillers were dismantled and others were converted to beverage alcohol
plants [16].
The revival of alcohol-based fuel started in 1970s, when the supply of
oil started being disrupted due to instability in the Middle East. The major
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oil companies of America began to market ethanol as a gasoline extender
and octane booster (adapted from: www.ott.doe.gov/biofuels/history.html).
Gasohol is a mixture of 9 volumes of gasoline and only 1 volume of ethanol.
The total ethanol production in the USA has increased from 0.175 billion
gallons in 1980 to 15.8 billion gallons in 2017 (adapted from: www.afdc.
energy.gov/data/). In 2015–2016, Brazil had produced 8 billion gallons of
ethanol and sold gasoline with a blend of 18 to 27.5% ethanol [17]. Among
the global players, the United States, Brazil, and European Union (EU) are
the major producers of ethanol followed by China among the developing
countries, see Figure 7.3.
FIGURE 7.3 Global bioethanol production.

Source: Adapted from www.afdc.energy.gov/data/.
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7.3 ORGANIC FEEDSTOCKS FOR ETHANOL PRODUCTION
Sugar rich substrates are required to produce alcohol and can be categorized
into four generations [18–22]. Since alcohol is produced via the breakdown
of hexoses and pentoses, the efficiency is related to the accessible amount of

these monosaccharides. In the overall fermentative reaction using a mono-
hexose (glucose) and a monopentose (xylose) represented, shows that for
every 1 mole of glucose and 3 moles of xylose, 2 and 5 moles of ethanol may
be obtained, respectively. The stoichiometry, neglecting NAD(P)H balance,
is shown in Eqns. (1) and (2) [23].
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1GLUCOSE + 2ATP 2 ETHANOL + 2CO2 + 2ADP (1)
3XYLOSE + 5ADP + 5Pi 5ETHANOL + 5CO2 + 5ATP + 5H2O (2)
7.3.1 FIRST GENERATION BIOETHANOL
Cash crops like cereals and sugar crops were identified as readily ferment-
able substrates for first-generation ethanol (Table 7.1). It has carbon dioxide
(CO2) benefits and is commercially available even today. Sugar crops like
sugarcane and sugar beet contribute 40% of the ethanol production in the
tropical areas like India, Brazil, and Colombia, and the remaining 60% is
contributed by the starch crops which is used by the United States, EU, and
China [27, 50–52]. High corn and sugarcane harvests lead to an increase in
global biofuel production by 9% in 2014, accounting for 74% (of the total)
of ethanol followed by 23% biodiesel (adapted from: http://www.ren21.net/
wpcontent/uploads/2015/07/REN12-GSR2015_Onli nebook_low1.pdf).
7.3.1.1 SUGAR BIOMASS
Before 1750, sugar production was limited to sugar cane (Saccharum offici-
narum) grown in the tropical and subtropical regions and shipped globally
[53]. During 1880, a German chemist, Andreas Marggraf extracted sugar
from beets (Beta vulgaris) and sugar beet soon replaced sugar cane as the
main source of sugar in continental Europe as it was found that sugar beet
contains enough amounts (16–20%) of sucrose than sugar cane [54, 55]. The
other sources of sugar production are included in Table 7.2.
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TABLE 7.1
Different Sources for Ethanol Production 166
SL. Generation Source of Examples References Advantages Disadvantages
No. of Fuel Ethanol
1. First- Sugar sources Sugarcane (Saccharum officinarum) [24, 25] • High sugar yield • Seasonal availability
generation Sugar beet (Beta vulgaris) [13, 24] • Low conversion costs • Threat to food
Biofuels security
Sweet sorghum (Sorghum bicolor) [13] • Require less energy
Starch sources Corn (Zea mays) [24, 26, 27] • Availability across the • Energy-intensive to
Sorghum (grains) (Sorghum bicolor) [28] world produce
Wheat (Triticum aestivum) [24, 29] • Ease of conversion • rise in price of the
• Storage capability for crops
Cassava (Monihot esculenta) [24, 30]
a longer period
Potatoes (Solanum tuberosum) [31, 32]
• High ethanol yield
Sweet potatoes (Ipomoea batatas) [33, 34]
2. Second- Lignocellulosic Perennial grasses (switchgrass, [35, 36] • Abundant • Costly
generation sources miscanthus, reed canarygrass, giant reed) • Non-food biomass into • Difficult to make
Biofuels Aquatic plants (Eichhornia crassipes) [37, 38] fuel • More
Forest material [39–41] energy-intensive
Agricultural residues [2, 26, 43, 44]
Organic portion of municipal wastes [45–47]
3. Third- Algae Microalgae (Pleurochrysis carterae) [24, 48] • Renewable source for • Costly
generation Cyanobacteria (Chlamydomonas sp., [49] biofuels • Chance of
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biofuels Cynaothece sp., Spirulina platensis) • High yield contamination
• Easy to obtain large
biomass
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TABLE 7.2 Other Sources of Sugar Substrates [56]

Sugar Plants Producing Countries
Date sugar Date palm Algeria, Iraq
Palm sugar Palm species like Palmyra, saga or India, Sri Lanka, Malaysia,
Toddy palm, coconut, and nipapalm Philippines, etc.

Maple sugar Maple tree (Acer saccharum) North America (the USA and
Canada), Japan
The sugar industry promotes solutions aiming at higher yield and no

waste process for ethanol production from sugar beet as well as from inter-
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mediates and byproducts like molasses. Another byproduct of the industry,
i.e., sugar beet pulp (SBP), is a potential feedstock for biofuels. It contains
20–25% cellulose, 25–36% hemicellulose, 20–25% pectin, 10–15% protein,
and 1–2% lignin content on a dry weight basis [55, 57].
Sugar derived from plant biomass is a mixture of both hexoses and pentoses
represented mainly by glucose and xylose, respectively. Since the wild-type
strains of Saccharomyces cerevisiae do not metabolize xylose, researchers
have developed two approaches to increase the fermentation yields of ethanol
derived from sugar biomass. The first approach via genetic engineering is to
add pentose metabolic pathways in yeast and other natural ethanologens. The
second approach is to improve the ethanol production by modifying microor-
ganisms having the ability to ferment both hexoses and pentoses [58, 59]. A
general procedure for ethanol production from sugar biomass is illustrated in
Figure 7.4. After fermentation, distillation is done which generate is vinasse
as a byproduct, which can be used as an animal feed or as a fertilizer [56].
7.3.1.2 STARCH BIOMASS
Grain crops (corn, barley, wheat, or grain sorghum) and tuber crops (cassava,
potato, sweet potato, Jerusalem artichoke, cactus, or arrowroot) contain large
quantities of starch [60]. Isolated native starch from different sources can be
used for further conversion into bio-based products or bioethanol production.
The residue from starch isolation contains proteins and fiber, which has great
potential for application in food and feed production (adapted from: http://
www.bmbf.de/pub/Roadmap_Biorefineries_eng.pdf.). USA is the biggest
corn starch producing countries with 80% of the worldwide market. There,
95% of ethanol is produced from corn, and the rest from barley, wheat, whey,
and beverage residues [61]. Cassava in Thailand was also under investiga-
tion for bioethanol production. Cassava tubers contain by mass 80% starch
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and less than 1.5% proteins. Pretreatment of cassava tubers include cleaning,
peeling, chipping, and drying. The dried cassava chips are used for bioethanol
production [62]. There are two established procedures for processing starch
biomass summarized in Table 7.3; Figures 7.5 and 7.6.
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FIGURE 7.4 Diagram of ethanol production from sugar biomass [56].
TABLE 7.3 Dry-Grinded and Wet-Mill Method [63–65]

Dry-Grinded Wet-Mill Method
Uses whole grains or tubers part of the Involves separation of different components
starch crops from the raw materials and only starch is used
It can be operated at a small-scale level It requires extensive equipment and high capital
with fewer requirements of equipment investments for producing large amount of
and capital investment ethanol and a variety of co-products
It generates two major products: ethanol The various high value products like corn gluten
and distiller’s dried grains with soluble meal (CGM) and corn gluten feed (CGF) are
(DDGS). also produced, which can add to the commercial
feasibility of the process.
The ethanol yield is 10.6 L/bushel The ethanol yield is 9.5 L/bushel
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FIGURE 7.5 Diagram for dry-grind ethanol production from starch crops [65].
FIGURE 7.6 Diagram of wet-mill ethanol production from starch biomass [64].
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The first-generation biofuels possessed two major issues, firstly, fuel vs. food
trade-off, and secondly, biofuels potential of CO2 reduction (biofuels produc-
tion could release more carbon than CO2 sequestration in the feedback growth
process) [66]. This trade-off situation would affect the producers, distributors,
related markets, and finally, the regional and national economies [67–69].
7.3.2 SECOND GENERATION ETHANOL
The use of food crops for ethanol production became debatable due to multiple
reasons; the lignocellulosic biomass (LCB) (Figure 7.7 and Table 7.1) came
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into prominence as feedstock for the second-generation bioethanol [71–73].
Biomass of various agricultural wastes like rice husks, corn stalks, wheat, and
barley straws, etc., and forest biomass such as grasses, wood, etc., are placed in
this category [74, 75]. Some industrial wastes such as brewer's spent grains and
spent grains from distillers, municipal solid wastes such as food waste, kraft
paper and paper sludge containing cellulose were also considered [76–79].
They are inexpensive, abundant and non-competitive with the food crops
[80]. However, an additional pretreatment step apart from the conventional
three-step strategy (Figure 7.8), is required to break down the lignin present in
the lignocellulosic material for subsequent hydrolysis and fermentation [81].
By using a suitable pretreatment method [82], the biomass is degraded into
cellulose, hemicelluloses, and lignin polymer which can be further degraded
into byproducts like furfurals, furans, acetic acid, etc. (Figure 7.9).
In general, recognition of new trending knowledge of energy-based
biofuels (compared to fossil fuels) spurred the doubt of economic efficiency
of biofuels [84]. As instance, cellulosic biofuel production is highly energy-
intensive, which means the energy contained in this type of biofuels is lower
than the energy required for its production [85]. Researchers in the past
decades have attempted many experiments with limited success to lower
the cellulosic ethanol production costs [66]. Study using microbial or fungal
systems for more effective and faster cellulose breakdown and fermentation
process has also been attempted [86, 87]. However, no wide-scale commer-
cial solution has been found and the development in this field is still ongoing.
7.3.3 THIRD GENERATION ETHANOL
First and second-generation biofuels sources require large amount of arable

land and are seasonally dependent, making their availability discontinuous
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for production. The three generations of biofuels have been compared with
the petroleum products in Table 7.4. In a report by Kim and Day [89], the
Louisiana sugar mills in the USA operate only quarterly as sugar cane avail-
ability is only from October to December, making capital investments for
facility upgrade and maintenance very difficult [89]. Comparatively, micro-

algal cultivation will be a continuous process with less land requirement.
Also, non-arable land can be used which provides a higher incentive [90–93].
Thus, a need for a fermentable substrate with all year availability emerges
which if realized can change the entire economic of the industry (Table 7.5).
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FIGURE 7.7 Types of lignocellulosic biomass and their composition [70].
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TABLE 7.4
Comparison of Three Generations Biofuels with Petroleum Products [88] 172
Petroleum Products First Generation Second Generation Third Generation
Technology Petroleum refinery Microbial fermentation, Pretreatment, hydrolysis, Metabolic engineering for direct
chemical, and enzymatic and fermentation, synthesis, fractionation of algal
transesterification transesterification biomass
Feedstock Crude petroleum Vegetable oils and corn Non-food, cheap, and abun- Algae
sugar feedstocks dant plant waste biomass
(agricultural and forest
residue, etc.)
Products CNG, LPG, diesel, petrol, Biodiesel, corn ethanol, Hydrotreating oil, bio-oil, Biodiesel, bioethanol, biohydrogen
kerosene, and jet-fuel sugar alcohol FT oil, lignocellulose
ethanol, butanol, mixed
alcohols
Benefits • High energy density: high • Environmental friendly • Environmental friendly • Environmental friendly
compact portable source of • Economic and society • Not competing with food • Not competing with food and
energy used for most forms of security agricultural land
mechanical transportation
• Oil productivity is very high when
compared with all other biomass
• Algae is the most promising
non-food source of biofuels
• A rapid reproduction rate
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• Algae can grow in saltwater and
harsh conditions
• Algae thrive on CO2 from gas and
coal-fired power plants
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TABLE 7.4
(Continued)
Petroleum Products First Generation Second Generation Third Generation

• Algae biofuel contains no
sulfur, is non-toxic and highly
biodegradable.
Problems • Depletion • Limited feedstock • Agricultural land • Low product yield at large scale
• Declining of petroleum reserve • Food vs. fuel consumption • Less biomass production
• Environmental pollution competition • Complicated processes
• Economic and ecological • Blended partially with

problems conventional fuel
Challenges in Developing Sustainable Fermentable Substrate
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173
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TABLE 7.5 Carbohydrate Content and Ethanol Yield in Different Feedstocks

SL. Sources Carbohydrate Ethanol Yield References
No. Content (%) (gallons/acre)*
1. Corn 70–72 370–430 [63]
2. Sorghum 68–70.7 326–435 [28]

3. Wheat 65.3–76 277 [29]
4. Sugar beet 8–12 536–714 [95]
5. Microalgae (Chlorella vulgaris) 37–55 5,000–15,000 [74]
*
Ref. [93].
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Microalgae are the oldest living organisms on Earth [96] which can grow
100-fold faster than terrestrial and can double their biomass in less than one
day time period [97]. It is beneficial as microalgae can grow faster and fixes
the CO2 at a higher rate than terrestrial plants. Lignin is absent in microalgae
making it easier to convert their starch and cellulose to monosaccharides
[24, 98]. These microalgae can be used for ethanol production via various
hydrolysis strategies and fermentation processes [24, 93, 99] (Tables
7.1 and 7.6; Figure 7.10). Nahak et al. [105] reported that seaweed and
marine algae such as Eneteromorpha sp. contain 70% carbohydrate (dry
weight basis), which can be explored for bioethanol production [105]. The
reported microalgal species which are employed as a fermentable substrate
for bioethanol production are listed in Table 7.6. The microalgal cells
store carbohydrates in the outer layer of cell wall: pectin, agar, alginate;
inner layer of the cell wall: cellulose, hemicelluloses; and inside the cell:
starch [91]. These carbohydrates are hydrolyzed to fermentable sugars like
glucose for the bioethanol production via microbial fermentation [48, 72].
The procedure includes cultivation of microalgae followed by the recovery
of microalgal biomass from the medium, cell disruption for the release
of biomolecules, saccharification (hydrolysis), fermentation, and finally
separation by distillation [51, 91]. Chemical hydrolysis method facilitates
the solvent extraction of lipids, thereby recovering both fermentable sugars
and lipid from the microalgal biomass, where fermentable sugar can be
converted to ethanol and lipid into biodiesel, increasing the commercial
potential by two folds [106].
Algae-based fuel industries are still searching for innovative ways to
reduce the production cost via reducing the costs of systems infrastructure
and integration, algae biomass production process, harvesting, and dewa-
tering techniques, extraction, and fractionation, and finally biofuels conver-
sion process [107] (Figures 7.8–7.10).
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TABLE 7.6 Carbohydrate Concentration and Microalgal Biomass Conversion to Ethanol

Microalgae Carbohydrate Concentration (%w/w) Ethanol Yield References
Total Starch Glucose (gethanol g–1 biomass)
Chlamydomonas 59.7 43.6 44.7 0.235 [100]

reinhardtii
Chlorococcum 32.5 11.3 15.2 0.520 [101, 102]
humicola
Chlorococcum sp. – – – 0.383 [101, 102]
Chlorococcum 32.5 11.3 15.2 0.261 [101, 102]
infusionum
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Chlorella vulgaris 50.9 – 48.0 0.209 [24]
Chlorella vulgaris – – – 0.400 [103]
Dunaliella sp. – – – 0.011* [104]
*
Theoretical conversion.
FIGURE 7.8 Ethanol production from lignocellulosic materials [27].
7.3.4 FOURTH GENERATION BIOETHANOL
This generation biofuel is under development and experimental stages. Thus,

they combine a variety of technology, processing, and feedstock level [66].
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The main feedstock for the fourth-generation biofuels production is geneti-

cally engineered, high biomass with low lignin and cellulose contents. This
effectively eliminates the hurdles of the second-generation substrates. Another
strong contender is metabolically engineered algae with high oil yield contents,
increased carbon entrapment ability, and improved cultivation, harvesting, and

fermentation processes, which is a vast improvement to the third-generation
substrate [108].
FIGURE 7.9
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Degradation products occurring during hydrolysis of lignocellulosic material [83].
In order to enhance the ethanol production and tolerance towards it,

certain microorganisms have been modified, for example, E. coli. This
microorganism is extremely well studied, takes up genetic changes well,
and additionally, it can grow three times faster than yeast and 100 times
faster than most agricultural microbes [15]. Beside the strong efforts, the
researchers have not reported a single commercially available CBP organism.
However, Brethauer and Studer [109] have reported a microbial consortium
which could be used instead of using a single microbe. The proposed model
utilizes Trichoderma reesei, S. cerevisiae and Scheffersomyces stipites.
The former organism secretes enzyme for hydrolysis; the middle and later
microbes ferment hexoses and pentoses, respectively [109]. But the major
obstacle in this approach is to control the consortium and it is also difficult
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to find microorganisms having identical fermentation conditions. Geneti-

cally modified microorganisms reported for ethanol production are listed in
Table 7.7.
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FIGURE 7.10 Ethanol production from microalgae [51].
7.4 MICROORGANISM USED FOR FERMENTATION
Fermentation is based on the discipline of chemistry, biochemistry, and

microbiology, in which the fermentable sugars are converted to ethanol by
microorganisms [90]. In this, Saccharomyces cerevisiae and Zymomonas
mobilis are the most common and well-studied microorganisms with appli-
cations in the food and beverage industry, molecular genetic research, and
biotechnological ethanol production [111, 112]. The theoretical values of
ethanol production are 87–95% and 94–97%, respectively [113–117], with
the difference in the metabolic pathways opted for the sugar catalysis as
mentioned in Figures 7.11 and 7.12. Other microorganisms reported for
ethanol production are Aerobacter, Bacillus, Klebsiella, Thermoanerobacter,
Aeromonas, Escherichia coli, S. bayanus, Candida sp., Pichia stipitis,
P. angophorae, Pachysolen tannophilus, Fusarium, Mucor, Neurospora,
Monilia, and Rhizopus [90, 117–123].
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TABLE 7.7 List of Genetically Modified Microorganisms Used in Ethanol Production [110]
Microorganisms Strain Features
Yeast Candida shehatae NCL-3501 Co-ferment xylose and glucose
Saccharomyces cerevisiae D5a Improvement in yield of ethanol

Saccharomyces cerevisiae 590E1 Ferment glucose and cellobiose
Saccharomyces cerevisiae RWB217 Ferment glucose and xylose
Saccharomyces cerevisiae RWB218
Bacteria Zymomonas mobilis ZM4 Ferment xylose and glucose
Pichia stipitis BCC15191
Zymomonas mobilis AX101 Ferment arabinose, glucose, and
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xylose
Thermoanaerobacterium Improved ethanol yield, have
saccharolyticum ALK2 ability to ferment arabinose,
glucose, xylose, and mannose
Thermoanaerobacter mathranii Improved ethanol yield
BG1L1
DSM1313 and YD01
Escherichia coli KO11 Ferment xylose and glucose
Escherichia coli FBR5 Ferment xylose and arabinose
Pichia stipites A Adapted at hydrolysate increased
Pichia stipitis NRRL Y-7124 concentration
7.5 PRETREATMENT
Pretreatment of biomass is needed to remove or modify the surrounding matrix

of lignin and hemicellulose prior to hydrolysis of the polysaccharides like
cellulose and hemicellulose [124]. The main aim of pretreatment is to increase
the enzyme accessibility improving digestibility of cellulose. Different
pretreatment methods have a different effect on the cellulose, hemicellulose,
and lignin fraction, thus forming the basic criteria of method selection [80].
Here, the pretreatment methods are discussed according to the biomass used.
7.5.1 LIGNOCELLULOSIC MATERIALS
Pretreatment of lignocellulosic material is done to fractionate, solubilize,

hydrolyze, and separate cellulose, lignin, and hemicellulose [70, 125].
It includes various categories: physical, chemical, physicochemical, and
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FIGURE 7.11 Ethanol biosynthesis pathway in Saccharomyces cerevisiae [111].
FIGURE 7.12 Entner-Doudoroff (ED) pathway in Z. mobilis [114].
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biological [126]. These methods differ from each other in context to mode of
action, reaction conditions and overall outcomes.
Physical method includes uncatalyzed steam explosion (SE), liquid
hot water (LHW) pretreatment, mechanical comminution and high energy
radiation [124, 127]. The methods increase the surface area and pore volume,
decreases the degree of polymerization (DP) of cellulose and lignin [128].
By using uncatalyzed SE method, Grous et al. [129] achieved the enzymatic
digestibility of untreated poplar chips from 15% to 90% after treatment
[129]. Perez et al. [130] used LHW to pretreat wheat straw and obtained
maximum hemicellulose-derived sugar recovery of 53% and enzymatic
hydrolysis yield of 96% [130].
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Although, the physical pretreatment method has some disadvantages like
energy consuming, ecologically unhealthy, and non-viable for commercial
process. Chemical pretreatments were initially exploited for the production
of high-quality paper products. The improvement in biodegradability of
cellulose by removing lignin and hemicellulose was the primary goal [124].
It includes techniques like alkali (e.g., NaOH, KOH, NH4OH and Ca(OH)2),
acid (H2SO4, HCl, HNO3), organic acids (fumaric and maleic acids) and
several cellulose solvents such as alkaline H2O2, ozone, glycerol, dioxane,
phenol, and ethylene glycol [124, 131, 132]. Millet et al. [133] reported an
increase in digestibility of NaOH-treated hardwood from 14% to 55% with a
decrease of lignin content from 24%–55% to 20% [133].
Physicochemical pretreatment is a combination of physical conditions
and chemicals. It includes SE, ammonia fiber explosion (AFEX), soaking
aqueous ammonia (SAA), ammonia recycling percolation (ARP), wet oxida-
tion and CO2 explosion. It increases the accessible surface area, decreases
cellulose crystallinity, and removes hemicellulose and lignin from the ligno-
cellulosic materials. The yields of enzymatic hydrolysis of AFEX-pretreated
newspaper (18–30% lignin) and aspen chips (25% lignin) were reported by
McMillan [23] as only 40% and below 50%, respectively [23].
Biological pretreatment is a friendly method used for lignin removal
[22]. It is carried out using microorganisms, particularly fungi, which are
white rot, brown rot, and soft rot fungi [134]. White and soft rot fungi
attack on both cellulose and lignin, whereas, brown rot fungi attack only
on cellulose. The biological pretreatment was studied by Hwang et al. [135]
using four different white-rot fungi for 30 days [135]. The team found that
glucose yield of pretreated wood by Trametes versicolor MrP 1 reached
45% by enzymatic hydrolysis while 35% solid was converted to glucose
during fungi incubation. Beside this, the lower hydrolysis rate and longer
incubation period as compared to the other pretreatment methods are some
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disadvantages of the biological pretreatment method. However, the benefits

of using biological pretreatment method, such as are low energy requirement
and mild environmental conditions, are unignorable [136].
7.5.2 MICROALGAL BIOMASS
To release carbohydrate trapped within microalgal cells, either chemical

or enzymatic methods may be used to disrupt the microalgal cell wall.
Microalgae has a simple cellular structure and lignin is also absent in them,
thus mild reaction conditions are sufficient [24, 137]. This method is fast and
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inexpensive than the enzymatic method. However, the extreme conditions
like high temperature and pressure can degrade carbohydrate into furfural,
acetic acid, gypsum, vanillin, and aldehydes which are potential fermentation
inhibitors leading to yield reduction [91, 132].
The enzymatic hydrolysis of the carbohydrate, particularly starch, by
alpha-amylase (liquefaction) in a random manner generates oligosaccharides
with three or more ɑ-(1→4)-linked D-glucose units as an intermediate
product. Subsequently, starch saccharifying enzyme (amyloglucosidase) is
introduced to the liquefied starch and simple reducing sugars are generated
[83, 100]. This method has advantages over the chemical method such as
higher conversion yield, minimal byproduct formation, mild operating condi-
tion, and low energy input [138].
7.6 FERMENTATION PROCESS
Both six-carbon (hexoses) and five-carbon (pentoses) sugars (from cellulose

and hemicellulose hydrolysis) derived from the biomass can be used as a
substrate for the fermentation process [82]. The fermentation parameters are:
temperature range, pH range, alcohol tolerance, growth rate, productivity,
osmotic tolerance, specificity, yield, genetic stability, and inhibitor tolerance.
The selection of microorganisms is also dependent upon their compatibility to
co-exist with products, processes, and equipment for bioethanol production.
Though, Saccharomyces cerevisiae and Zymomonas mobilis are widely used
for bioethanol fermentation, they show inability to efficiently convert xylose
[23, 139, 140]. Thus, natural xylose-fermenting yeasts like Pichia stipites,
Candida shehatae and C. parapsilosis are also used [23, 141]. Depending
on the process design, the combination of the above-mentioned parameters
may vary, being consecutive or simultaneous [142]. The most commonly
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used bioconversion processes are: separate hydrolysis and fermentation

(SHF), simultaneous saccharification and fermentation (SSF), simultaneous
saccharification and co-fermentation (SSCF) and consolidated bioconver-
sion process (CBP) (Figure 7.13).
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FIGURE 7.13 Types of fermentation processes [140].
7.6.1 SEPARATE HYDROLYSIS AND FERMENTATION (SHF)
Two different reactors used for the hydrolysis and fermentation process
[142]. This has the advantage that both the process can be carried out under
different conditions like temperature, pH, and time [101, 102, 142]. Thus,
more substrate could be produced during the hydrolysis process by using the
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acid, alkali, or enzyme. However, neutralizing the acid or alkali will be an

additional step. On the other hand, enzymatic hydrolysis leads to accumula-
tion of glucose and cellobiose, which will inhibit the cellulases and thus
reduce the hydrolysis rate and the overall yield of the process [142, 143].
7.6.2 SIMULTANEOUS SACCHARIFICATION AND FERMENTATION (SSF)
In a single reactor, both hydrolysis and fermentation process take place [144].
Since, yeast will directly convert the glucose and cellobiose into bioethanol,
these compounds could not cause the inhibition [142]. SSF has advantages
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over the SHF that it requires lower enzyme load, higher bioethanol yield,
shorter fermentation time, and reduced risk of contamination by external
microflora [145]. However, compromises in the operating conditions are
seen during the SSF process [146]. Additionally, it is difficult to recycle the
enzymes and yeast, which makes the process challenging for the scaling up
for commercialization purposes [147].
7.6.3 CONSOLIDATED BIOCONVERSION PROCESS
In the process, the same microorganisms carry out both saccharification and
fermentation of biomass in a single stage [148]. CBP is also known as direct
microbial conversion (DMC). CBP uses a single microorganism community
for cellulase production, cellulose hydrolysis, and fermentation in a single step,
has two major benefits: (i) avoid the cost of cellulase production, (ii) saves the
energy [149, 150]. Clostridium thermohydrosulfuricum, Thermoanaerobacter
ethanolicus, Thermoanaerobacter mathranii, Thermoanaerobacter brockii
strain, etc., are some thermophilic cellulolytic anaerobic bacteria that have
been studied for their potential as bioethanol producers [13].
7.6.4 SIMULTANEOUS SACCHARIFICATION AND CO-FERMENTATION

(SSCF)
In this fermentation, microorganism can completely assimilate all the sugars

released during the pretreatment and hydrolyze LCB. Sanchez and Cardona
[151] reported that the mixed cultures of yeasts can assimilate both hexoses
and pentoses [151]. However, hexose-utilizing microorganisms grow faster
than pentoses-utilizing microorganisms and thus, hexoses conversion to
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ethanol gets elevated. In nature, there is an existence of microorganisms

which can assimilate both hexoses and pentoses for ethanol production.
This conversion efficiency can be increased by genetic modification of the
microorganisms [81].
7.7 BOTTLENECKS IN COMMERCIALIZATION OF ETHANOL
The commercialization of the bioethanol is dependent on the economics of

the process. Simultaneously, the ease of implementation is another important
factor for the success of a new bioprocess technology. There are many other
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factors which also influence the commercialization of ethanol production
including the availability of feedstock, cost of raw material and ethanol
recovery cost. The prices of feedstock depend on the location, seasons, local
state of the supply-demand conditions and transportation needed [152].
Studies are done which state that biomass feedstock made up 40% of the
ethanol production cost [139, 152]. The main challenge is to economize the
operating costs of biomass conversion processes that include pretreatment
and enzymatic hydrolysis, toward a full-scale commercialization. Extensive
studies are done with aim of developing a process which reduce bioconver-
sion time, use of cellulose during fermentation and enhance the total ethanol
yield [152, 153]. Another cost adding factor is the separation and recovery of
ethanol from the fermented liquor of lignocelluloses hydrolysates [136, 151].
Although microalgae are rich in carbohydrates and proteins, limited reports
are available on its use as a source for fermentation for ethanol production
[42]. Thus, despite being a fully functional industry, ethanol production
still has lacunae that need to be addressed to improve the efficiency of this
commercial endeavor and its economics.
7.8 CONCLUSION
With the sharp increase in population and vehicles, only bioethanol will not
be sufficient to match the energy demand. However, with each generation,
the bioprocess technology is undergoing massive changes, through which
we are slowly but contently increasing production potential. The changes
initiate from the nature of substrate to the microorganisms and the process
involved. Dealing with the current scenario of possible feedstock with their
advantages and disadvantages, it becomes necessary to evolve better and
sustainable methodologies for higher yield outputs. Feedstock generated
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from forest and agricultural wastes, algal biomass and engineered organisms
could lead to the development of efficient technologies, thereby reducing the
ethanol price and environmental pollution, and also preserving the natural
resources. The fourth-generation biofuels are a new concept and could have
a significant potential in the near future.
ACKNOWLEDGMENTS
We are thankful to Professor A. Shastri, Vice-Chancellor of Banasthali

Vidyapith, Rajasthan, for kindly extending the facilities of “Banasthali
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Center of Education” for research in basic sciences sanctioned under
CURIE program at the Department of Science and Technology.
CONFLICTS OF INTEREST
The authors declare that there is no conflict of interest to disclose.
KEYWORDS
• bioethanol
• feedstock
• fermentation
• pretreatment
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CHAPTER 8
Emerging Strategies for Ethanol

Purification
ALAN D. PEREZ,1 JAVIER QUINTERO,2 and JUAN A. LEÓN3
Sustainable Process Technology (SPT), University of Twente, Enschede,
1
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Overijssel, The Netherland, E-mail: a.d.perezavila@utwente.nl
Department of Chemical Engineering, National University of Colombia,
2
Manizales, Caldas, Colombia, E-mail: jaquinteroj@unal.edu.co

Laboratory of Process Intensification and Hybrid System (LIPSH),
3
Department of Chemical Engineering, National University of Colombia,

Manizales, Caldas, Colombia, E-mail: jaleonma@unal.edu.co
ABSTRACT
This chapter is focused on the separation technologies mainly applied for

ethanol recovery from fermentation broths and aqueous solutions during the
process of fuel alcohol production. Conventional technologies to separate
ethanol such as distillation, azeotropic distillation, extractive distillation
(ED), and adsorption using molecular sieves are described, in order to present
the current separation equipment used in biorefineries and distilleries. On
the other hand, novel and alternative technologies to recover, concentrate,
and dehydrate ethanol, either from the treated wine or directly from the
fermenter are presented. These alternative technologies are able to increase
the performance of a fermentation, reduce the number of equipment, reduce
the operation and capital costs, associating a higher energy efficiency, as
compared to the conventional separation process to recover ethanol. Although
there are several separation technologies proposed to recover ethanol, in the
following chapter, only the most representative and researched technologies
are described.
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8.1 INTRODUCTION
The ethanol purification is an important stage in the production process, since

the ethanol quality defines the use and price of this component (commodity)
in the market. Although ethanol can be commercialized at its azeotropic

composition, the ethanol is highly demanded in the energy market (fuel
alcohol). As a result, ethanol must be dehydrated and its azeotrope must be
broken, in order to achieve optimum condition to blend it to gasoline.
The processes commonly used for the ethanol purification are distilla-
tion, azeotropic distillation, and ED. These latter are mature, stablished, and
applied technologies to recover ethanol in the current biorefineries. However,
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the thermodynamic limitations in distillation, the use of entrainer/solvents,
and the need of several number of columns in the distillation trains make
these technologies not very efficient to recover ethanol, especially to use it
as fuel alcohol.
In addition, adsorption has been successfully applied in several biorefineries
to recover ethanol from aqueous solutions. Extractive or azeotropic distillation
columns can be replaced by adsorption to overcome the azeotrope (ethanol
dehydration). For instance, the use of molecular sieves (from potassium
aluminosilicates) has been applied in industrial cases, achieving to replace
the azeotropic distillation [1]. Although adsorption with molecular sieves
can associate high capital costs, it is a technology with a high separation
performance with a lower operation cost as compared to traditional distillation
schemes.
On the other hand, pressure-swing distillation has been extensively studied
to separate ethanol by reducing the operation pressure inside a column in the
separation train. At vacuum condition, the separation by distillation leads
to the disappearance of the azeotrope. However, it is necessary the use of
columns composed of a large number of plates, and high reflux ratios to
achieve an ethanol with a high purity. Also, high capital and operation costs
can be associated to maintain the vacuum condition for a large volume [2].
Moreover, several efforts have been made in order to propose separation
technologies with a higher performance than those previously mentioned.
For instance, the use of non-conventional and non-volatile solvents to
increase the ethanol volatility, and consequently, to overcome the azeotropic
condition. In this chapter, ED using dissolved salts, ionic liquid and hyper-
branched polymers (HyPols) are presented.
Also, ethanol recovery through membrane technology and its integration
to distillation is described. Pervaporation (PV) has been demonstrated to
be able to separate ethanol without thermodynamic limitations, which is a
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Emerging Strategies for Ethanol Purification 197
significant advantage as compared to conventional distillation. However, the

capital cost associated to PV are substantially high. The integration of PV
to distillation has been approached in order to compensate the distillation
economy with the PV performance, achieving successful industrial cases of
a separation stage composed of hybrid PV-distillation systems.

On the other hand, ethanol is the primary metabolite produced in the
fermentation process. However, it is the main inhibitor of the yeast cells as
well, compromising the yeast cell activity at high ethanol concentration in
the fermentation broths. Therefore, a simultaneous ethanol removal during
its formation can improve the fermentation performance. In this chapter, it is
described a pervaporative fermentation and a liquid-liquid extraction (LLX)-
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fermentation as alternative to increase the fermentation performance.
8.2 CONVENTIONAL SEPARATION TECHNOLOGY FOR ETHANOL

RECOVERY
8.2.1 CONVENTIONAL DISTILLATION
In the first generation of alcoholic fermentation, ethanol is diluted in the

fermentation broths (5–12 wt.%). The fermenter downstream is mainly
composed of water [3, 4]. The free-biomass alcoholic solution (filtered
fermentation broth), called “wine or beer” is processed to recover the ethanol
produced in the fermentation stage.
Due to the high volatility of ethanol in aqueous solutions, distillation is
a versatile separation technology to recover the ethanol from the wine [5].
Although ethanol separation by distillation is an energy-intensive process, it
is an effective and profitable separation to recover ethanol from a bioprocess.
At low ethanol composition in aqueous solutions, the constant in Henry’s law
presents values significantly higher than 1, allowing a high ethanol separation
as a function of the liquid-vapor equilibrium [6]. A large capacity of processing
is able in distillation columns since low reflux ratios are required.
On the other hand, ethanol promotes the formation of a minimum
boiling azeotrope in an aqueous solution. As a result, the maximum ethanol
composition attainable by conventional distillation (at 1.013 bar) is close to
95.6 wt.%, which is the azeotropic composition. Commonly, the ethanol-
water mixture close to the azeotropic composition is called hydrated ethanol
in distilleries. The distillation process to obtain hydrated ethanol is presented
in Figure 8.1.
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FIGURE 8.1 Distillation process to obtain hydrated ethanol from sugarcane molasses with
thermal integration in the column condenser.
Source: Adapted from: Dias et al. [7].
In general, the ethanol composition in the hydrated ethanol in distilleries

is in the range of 92.5–94.6 wt.%, and is produced by two-column arrange-
ment, as Figure 8.1 shows [7]. The first column is used to recover the ethanol
and a fraction of water from the wines (some organic components in very
low compositions are separated as well). This column is divided in three
sections. Section D is located at the top of the column and is commonly
composed of 6 stages. Section A1, where the pre-heated wine is fed, is an
intermediate section constituted by 8 stages. Section A at the bottom of the
column, which can be composed of 16 to 24 stages. The distillation product
of this column is a second alcohol stream (91 wt.%) produced at the top of
Section A, and the stillage at the bottom of the column. In addition, from
Section D and A are produced liquid and vapor Phlegma streams with an
ethanol composition about to 30 wt.%, respectively.
Both phlegma streams are fed to a second distillation column B, which is
composed of 40–50 stages (Figure 8.1). In this column, the mixture is recti-
fied to completely recover the ethanol at the top of the column, achieving
a distillate close to the azeotropic composition (hydrated ethanol). On the
other hand, the excess of water in the phlegma streams is eliminated at the
bottom of column B.
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In almost all distilleries, the wine is pre-heated close to the bubble

temperature of the mixture to reduce the vapor condensation, improving the
performance and energy efficiency of the distillation column. An integrated
heating system, involving the stillage stream and top product of column B is
usually applied (Figure 8.1). The wine is used as a cooling service stream in
the condenser C2 at the top of column B, producing a partial condensation
of the hydrated ethanol, followed by a total condensation in condenser C3
with water. The wine leaves C2 at 60°C. The heated wine is used to reduce
the stillage temperature as well. The wine achieves a temperature of 90°C,
before being fed to the first distillation column.
The produced ethanol in biorefineries is mainly used as fuel alcohol.
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Therefore, hydrated ethanol is not still applicable for blending in gasoline in
flex-fuel engines, which are the most common technology using fuel alcohol.
Azeotropic ethanol must be dehydrated. At least an ethanol composition of
99.3 wt.% or higher must be produced to be added in gasoline [8]. Since it is
not possible to overcome the azeotropic composition by conventional distil-
lation, azeotropic distillation (AD) is used to produce anhydrous ethanol.
AD is an economical technology widely applied in Brazilian distilleries [9].
8.2.2 AZEOTROPIC DISTILLATION (AD)
The separation of an ethanol-water mixture by azeotropic distillation (AD),

involves adding a third volatile component denominated entrainer. This is
made in order to form a new azeotrope with the components to be separated
[10]. The relative volatilities are modified as compared to a binary distilla-
tion of ethanol-water mixture, allowing achieving an ethanol composition
further than the azeotropic composition [11]. Due to an entrainer is involved
in an AD, additional columns are necessary to recover individually each
component, including the entrainer.
According to the thermodynamic and molecular structure of the entrainer,
two types of AD can be considered. In a homogenous azeotropic distillation
(HAD), a polar entrainer is used. Then, there is not a formation of a second
liquid phase at the bubble temperature of the mixture. In a heterogeneous
azeotropic distillation (HTAD), a second liquid phase is formed. This is due
to a nonpolar entrainer is applied [11]. In addition, a decanter to split the
organic and aqueous phases is required in a HTAD. Therefore, a liquid-liquid
and liquid-liquid-vapor equilibria must be considered in a mixture separation
by HTAD.
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Figure 8.2 is presented the flow sheet of a HTAD for the ethanol dehy-
dration. The hydrated ethanol and the entrainer are mixed and fed to the
azeotropic distillation column (1), where anhydrous alcohol is produced
at the bottom of the same column. The product at the top of the column
(1) is mostly composed of the entrainer, a fraction of water, and a little

of ethanol. Inside the decanter (2), the organic and aqueous phases are
split. The organic phase (reflux stream) is recycled to the column (1). The
aqueous phase (distillate stream) is fed to the entrainer recovery column
(3). Water, as the heavy component, accompanied by ethanol traces, is
separated at the bottom of the column. The entrainer and a small amount
of ethanol from the top of the column (3) are recycled to the column (1)
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again.
FIGURE 8.2 General scheme of azeotropic distillation for the separation of ethanol-water
mixtures: (1) azeotropic distillation column; (2) decanter; and (3) distillation column for the
entrainer recovery [12].
Benzene was widely used as an entrainer for the ethanol dehydration

by HTAD in the past [12]. Currently, the use of benzene has been replaced
for other entrainers, due to its carcinogenic effects. Cyclohexane is one of
the most used entrainer for the ethanol dehydration [13]. Although this is
widely applied in distilleries, this component is highly flammable. In addi-
tion, toluene can be used as entrainer as well, producing a similar separation
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behavior of benzene [11]. The entrainer is not necessarily restricted to a pure

component, a mixture of hydrocarbon (e.g., hexane-isooctane-cyclohexane,
gasoline) are potential candidates as entrainer in an HTAD for the ethanol
dehydration [14].
8.2.3 DEHYDRATION PROCESS BY MOLECULAR SIZE DIFFERENCE:

ADSORPTION WITH MOLECULAR SIEVES
Molecular sieves are cylindrical or spherical granular substances called

zeolites, which can be natural or made from potassium aluminosilicates.
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They are identified according to their nominal size of the internal pores
whose diameter is generally measured in angstroms. Molecular sieves are
materials that are characterized by their excellent capacity to retain on their
surface defined types of chemical species. These species are usually solvents
(water most of the cases), which are desired to be removed from a mixture to
obtain a final product with its given specifications [15].
Adsorption has been applied as an alternative process for ethanol
dehydration. Recently, new distilleries plants have opted to use adsorption
rather than AD to produce anhydrous ethanol. Adsorption on molecular
sieves is an established technology for the ethanol dehydration as an
energy-efficient process. The anhydrous ethanol production by a pressure
swing adsorption (PSA) process is shown in Figure 8.3. Commonly, a
vapor-phase adsorption (vapor feed) is used to process directly the output
stream from the conventional distillation stage (hydrated ethanol) [16].
Two zeolite adsorption units (A and B) in parallel arrangement are applied
for a continuous ethanol dehydration process. While in one bed (e.g., A) is
operating for dehydrating of superheated ethanol vapor at high pressure,
the other bed (e.g., B) is being regenerated at vacuum conditions by
recirculating (splitter valve C) a small portion of the anhydrous ethanol
through the saturated sieves [17].
Adsorption using molecular sieve for ethanol dehydration can present
a lower energy consumption as compared to azeotropic distillation, since
the stream to be processed only must be vaporized one time. On the other
hand, zeolite is a highly selective material. Water is strongly adsorbed,
requiring high temperatures or a low operating pressure to regenerate the
zeolite bed. In addition, the constant use of superheated vapor for the
regeneration of the sieve bed, accelerates the sieve deterioration due to
thermal shock [18].
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FIGURE 8.3 Schematic diagram of ethanol dehydration with molecular sieves.

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8.3 NONCONVENTIONAL TECHNOLOGIES TO RECOVER ETHANOL
8.3.1 EXTRACTIVE DISTILLATION (ED) WITH NONCONVENTIONAL

SOLVENTS
Similar to AD, in an ED a third heavy component (solvent) is added to the

ethanol-water mixture. Although the solvent can modify the relative volatility
of the mixture, there is not an azeotrope formation as AD. Also, the solvent is
commonly fed to the column in a different location than the hydrated ethanol
feed. This is made in order to allow an extraction section in the extractive
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column. In addition, in ED some topological aspects differ from AD and can

be found somewhere else [19].
8.3.1.1 ED WITH DISSOLVED SALTS
A dissolved salt can be used as solvent in an ED for the ethanol-water

separation. The “salt effect” significantly alters the volatility of the binary
mixture, overcoming the azeotropic composition inside the column. In the
case of ethanol aqueous solutions, the liquid-vapor equilibrium is enhanced
for ethanol. It means that the constant of Henry's law is increased, allowing
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a vapor with a higher ethanol composition as compared to a conventional
distillation. In the case of water, its volatility is reduced by the salt effect.
This effect is very effective for the separation of azeotropic mixtures, as in
the case of ethanol-water mixture, promoting a vapor composition of ethanol
higher than the azeotrope.
However, the use of salts as an extraction agent in ED has not reached a
successful application due to the technical problems. The recrystallization of
the salt (recovery step), its dissolution, and the special needs of the construc-
tion materials to avoid corrosion problems have limited the applicability of
dissolved salts as a solvent in ED in the industry [20]. Although the limitation
by technical issues in this technology promotes little interest in the industry,
dissolved salts are a potential alternative to produce anhydrous ethanol at a
low cost.
In Figure 8.4 is shown a simplified scheme of ED with dissolved salts
for ethanol separation from aqueous solutions. The ethanol-water mixture is
fed to the ED column (1), where the separation by distillation is governed
by the modified liquid-vapor equilibrium due to the salt effect. The salt feed
is located immediately after the splitter at the top of the column, where
it is dissolved (2) in the reflux stream [22]. Since the salt is not a volatile
component, it only remains inside the liquid phase and flows downward along
the column. Then, the salt can be recovered at the bottom of the column and
recycled by evaporation or drying, rather than by distillation.
The most common salts used in ED for ethanol dehydration are
CH3COONa, CH3COOK, CaCl2, Ca(NO)2, KI, and NaI [22–24]. In addition,
a mixture of these latter salts can be employed. For instance, a salt with
a ratio 70/30 of CH3COOK/CH3COONa is able to produce ethanol with a
composition of 99.8 wt.%. The energy consumption and capital costs are
lower as compared to AD using benzene as entrainer and conventional
ED with ethylene glycol as solvent [22]. On the other hand, a pilot plant
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for producing anhydrous ethanol using CaCl2 was performed [21]. It was
possible to reduce the energy consumption by more than 50% as compared
to conventional ED and vacuum distillation. In other work, different
salts (KCl, CaCl2, NaCl, and KI) were theoretically evaluated to produce
anhydrous ethanol [25]. In all cases, an ethanol composition higher than 99

wt.% was achieved. However, CaCl2 is the most effective saline agent with
the lowest energy consumption, even lower than a conventional ED with
ethylene glycol.
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FIGURE 8.4 Typical separation of ethanol-water mixture by extractive distillation using
dissolved salts. Extractive distillation column (1); and salt dissolver (2).
8.3.1.2 ED USING IONIC LIQUIDS (ILS) AS SOLVENTS
An ED with ionic liquids (ILs) is similar to an ED with conventional solvents.

However, the top products can be solvent-free streams due to the properties
of ILs. Furthermore, this process presents several operative advantages as
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compared to ED with dissolved salts, since crystallization and an accelerated

corrosion are avoided with ILs [26].
Typically, ILs are composed of an inorganic polyatomic anion and large
organic cations. Further, at room temperature, the ILs are at liquid state.
Therefore, as a separation agent, ILs presents the advantage of conventional

solvents and the high separation ability of dissolved salts. Several combina-
tions of anions and cations can be made, based on the separating agent
structures and the separation performance. Also, several commercial ILs
such as, 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM]+[BF4]−),
1-ethyl-3-methylimidazolium tetrafluoroborate ([EMIM]+[BF4]−) and 1-butyl-
3-methylimidazolium chloride ([BMIM]+[Cl]−) can be found in the market [5].
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In general, the ED with ILs presents a similar configuration of an ED
using conventional solvents. According to Figure 8.5, hydrated ethanol
(azeotropic) and the solvent (ILs) are fed to the ED column (1). The IL is fed
at the top of the column to increase the separation performance. A solvent-
free anhydrous ethanol is produced at the top of the ED column, while the
bottom stream (solvent with water) is partially split by a flash evaporation
(2). The solvent is recovered in the regeneration column (3), where water
is produced at the top of the column, while the recovered solvent from the
bottoms is recycled again to column (1).
FIGURE 8.5 General scheme of an extractive distillation using ionic liquids as solvent.
Extractive distillation column (1); flash drum (2); and solvent regeneration column (3).
Source: Adapted from: Seiler et al. [27].
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Several studies have shown the potential of using IL for the ethanol
dehydration. For instance, the liquid-vapor equilibrium for ethanol-water-
solvent (IL) mixture was evaluated close to the azeotropic region [28]. The
ILs used were 1-butyl-3-methylimidazolium bromide ([BMIM][Br]) and
1-ethyl-3 methylimidazolium bromide ([EMIM][Br]). In both cases, the

ethanol composition in the vapor phase overcame the azeotrope, reaching
a composition higher than 99 wt.%. Also, low energy demand for ethanol
dehydration using 1-Ethyl-3-methylimidazolium tetrafluoroborate [EMIM]
[BF4] was achieved in other work [29]. The results showed a thermal energy
reduction of the 14.6% and 28.6% as compared to glycerol and gasoline,
respectively. Moreover, a cost reduction of 21%, 18%, and 59% in columns,
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reboilers, and cooler can be achieved using ILs as solvents in comparison to
a conventional solvent ED [30].
8.3.1.3 ED USING HYPERBRANCHED POLYMERS
A hyperbranched polymer (HyPol) is a tridimensional macromolecule with a

tree-based arrangement. The HyPols are characterized by presenting several
random branched points with functional groups. These macromolecules can
be manufactured with a low cost and can be designed with specific physical
and chemical properties, which is favorable for industrial applications.
Currently, different companies such as the Perstorp Group (Sweden), DSM
Fine Chemicals (The Netherlands) and Hyperpolymers (Germany) are manu-
facturing HyPols on an industrial scale [31].
Since the HyPols can be designed with different polarity features by
adjusting the functional group at the end of the polymer branch, selective
solvents can be produced using HyPol technology. Also, properties as low
viscosity, high thermal stability, and no volatility can be presented in HyPols.
Based on these latter properties, HyPols have been attracting as solvents in
ED. However, the works associated to the use of HyPols are mostly focused
on the impact of the liquid-vapor equilibrium rather than the distillation
itself, since there is not still much information about experimental data of
the HyPol-mixture interaction.
Seiler et al. [32] tested poly(ethylene glycol) and hyperbranched
polyester as solvents to identify the effect on the liquid-vapor equilibrium of
the ethanol-water mixture. The relative volatility of ethanol can be increased
from 1 to 1.1 and 1.25 at the azeotropic composition for hyperbranched
polyester and poly(ethylene glycol), respectively. In other work, HyPol
polyglycerol and ethanediol (conventional solvent) were compared in an
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ED application [33]. The ethanol relative volatility is increased up to 1.8

at the azeotropic composition using polyglycerol, which is a similar value
reached by ethanediol. However, the advantage of the non-volatile HyPol
polyglycerol lies in the variety of separation and regeneration methods
to recover the solvent. In addition, using HyPol polyglycerol as a solvent

could produce a reduction of the energy requirement of 19% as compared to
conventional ED.
8.3.2 ETHANOL SEPARATION USING MEMBRANE TECHNOLOGY
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8.3.2.1 PERVAPORATION (PV)
A pervaporation (PV) process is an operation to separate liquid mixtures

through a selective layer, called membrane. Unlike distillation, PV is
not limited by the mixture thermodynamic. Therefore, PV is a potential
technology to separate azeotropic mixtures as ethanol-water case. In addition,
the thermal energy requirement in PV can be significantly lower as compared
to distillation. The separation by PV depends on the intrinsic properties of
the selective layer material. Considering that the components in the mixture
present different affinities to the membrane material, the combination of their
different permeation rate through the membrane produces the separation.
In Figure 8.6 is illustrated a PV process. A heated liquid mixture is fed to
a PV unit, being contacted on one side of the membrane. The components of
the mixture are transported through the membrane wall until the other side
of the membrane is reached, the permeate side. The component transferred
across the membrane wall is recovered as a vapor stream. The mass transfer
through the membrane is driven by the difference of the vapor pressure
between the feed and permeate. In order to maintain the driving force as
high as possible, vacuum pressure is usually applied on the permeate side.
Although the vacuum pressure is commonly generated by a pump in a lab-
scale PV unit, in the industry, this is generated by the rapid condensation of
the permeate itself [34].
The performance of PV membranes can be defined according to the
selective component removed from the feed. The membrane selectivity is
a measure of the permeability ratio for the components associated with the
mixture, which can be calculated as the ratio of binary permeability. In the
case of the ethanol-water mixture, the membrane selectivity can be deter-
mined as follows [35]:
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PEtOH
θEtOH / w = (1)
Pw
In Eqn. (1), P is the permeability coefficient of the components. P is a
mass transfer parameter, in which is condensed the mass transfer mechanisms

(sorption and diffusion) through a PV membrane based on the solution-
diffusion theory [36]. The membrane selectivity is an independent parameter
of the feed conditions, it depends exclusively on the membrane material [37].
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FIGURE 8.6 Sketch of a pervaporation process.
Source: Adapted from: Baker [34].
Two types of membranes can be identified for the ethanol-water mixture

separation. Organophilic membranes (ethanol selective membranes), where
θEtOH/w is higher than 1. It means that the ethanol affinity in the membrane
material is higher than water affinity, producing a permeate enriched in
ethanol (PEtOH > Pw). Otherwise, water is selectively removed in hydrophilic
PV membranes. The permeate is enriched with water, which means that
PEtOH > Pw.
8.3.2.1.1 Organophilic Membranes for Ethanol Recovery
According to the vapor-liquid equilibrium for the ethanol-water mixture,

the highest constant in Henry’s law is reached in the ethanol composition
from 0 to 20 wt.%. The highest PV performance for ethanol recovery can be
achieved in this composition range. Consequently, PV is highly applicable
to recover ethanol from the fermentation downstream. Besides, the energy
consumption in PV is lower as compared to distillation, due to only a fraction
of the feed is evaporated on the permeate side [38].
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Hereto, polydimethylsiloxane (PDMS) is the most researched membrane

material for ethanol recovery from aqueous solutions. It is highly applicable
to separate ethanol at low composition, due to the high hydrophobic nature
of this material. Different companies manufacture commercial PDMS
membranes, such as SolSep BV (Apeldoorn, Netherlands), Pervatech BV

(Enter, Netherlands), Sulzer Chemtech (Neunkirchen, Germany), and
Celanese Corp. (NC, United States) [39]. In general, PDMS membranes can
present permeate fluxes in the range of 0.001–1 kg m–2 h–1 and a selectivity
from 1.8 to 4 [35, 40]. In many cases, the ethanol composition in the permeate
is located under the liquid-vapor equilibrium curve for the ethanol-water
mixture. Therefore, it is necessary a cascade arrangement of PV modules to
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achieve a similar separation duty of a conventional distillation. It involves a
large membrane area, a meaningful increase in capital cost.
Also, poly(1-trimethylsily-1-propyne) (PTMSP) membranes have shown
potential characteristics to recover ethanol from aqueous solution. In the case
of a PV with PTMSP membranes, the permeate flux and the ethanol selectivity
can be 3 and 2 times higher as compared to PDMS membranes, respectively
[41]. Although PTMSP membranes initially showed a high performance,
these are hardly applied for practical separation due to their instability. For
instance, in a separation of an ethanol aqueous solution (50 wt.%) by PTMSP
membranes, the permeate flux and ethanol selectivity can be reduced up to
60% and 22% after 200 h of operation [42]. PTMSP membranes have been
submitted to different studies in order to improve the stability by modifying
the polymer synthesis, as grafting copolymer to the selective layer. The
synthesis of PTMSP using NbCl5 and TaCl5/Al(i-Bu)3 as catalyst allowed a
stable membrane, even with a low pH PV [43]. In a hybrid PTMSP/PDMS
membranes, a higher permeate flux and ethanol selectivity were achieved as
compared to a solely PTMSP membranes [44]. No membrane instability was
reported. In addition, a permeate with an ethanol composition up to 70 wt.%
was reached from a feed with 7 wt.% of ethanol.
Although PDMS has been the preferred choice to recover ethanol,
inorganic membranes present a substantial improvement in the separation of
ethanol from aqueous solution. Membranes based on hydrophobic zeolites
have shown a higher performance for recovering ethanol in comparison to
PDMS membranes [39]. A special characteristic of zeolite membranes is
a lower swelling as compared to unmodified polymeric membranes, as a
result, a high selectivity can be achieved in PV at high temperatures [45].
MFI membranes (silicate-1 and ZSM-5) are the representative zeolite-based
membranes. It is possible to perform a PV at high temperature with MFI
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membranes, which cannot be made using polymeric membranes due to

membrane swelling. The permeate flux and ethanol selectivity reached by a PV
with an MFI membrane can be 2 and 5 times higher than a PDMS membrane
[46]. However, inorganic membranes (especially zeolite-based membranes)
can be up to 50 times more expensive than polymeric membranes [47]. In

addition, it is difficult to manufacture this type of membranes commercially.
8.3.2.1.2 Hydrophilic Membranes for Ethanol Dehydration
In the field of ethanol dehydration, PV presents a significant advantage
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as compared to azeotropic distillation and other conventional distillation
schemes. A small amount of water is needed to be separated. Consequently,
the operation costs for the dehydration by PV can be significantly lower than
an azeotropic distillation. An installed ethanol dehydration process by PV
was compared to commercial dehydration with AD [48]. The ethanol losses
in PV were neglected, and the alcohol efficiency in PV was about to 99.7%.
On the other hand, the ethanol quality in the AD was lower as compared to
PV. The permeate had not any impurity of the entrainer. Besides, the dehy-
dration by PV presented a reduction of operation costs around 30%. This
reduction was associated with low energy consumption (thermal utilities) in
PV, and the entrainer make up in AD. This latter represented about 10% of
the total operation cost.
For the application of PV and membrane selection in the ethanol dehy-
dration, the liquid-vapor equilibrium and PV performance can be used. In
Figure 8.7 is presented the comparison of the separations between distillation
and PV with different membrane material. A proper dehydration membrane
should be under the 45° line in the liquid-vapor equilibrium of the ethanol-
water mixture. For this case, only the membrane selectivity is considered.
In general, the three membrane types are able to remove water from the
mixture selectively. For hydrated ethanol (≈ 93 wt.%), PVA membranes are
the most appropriate to produce anhydrous ethanol. In the range of 85–95
wt.%, the highest water selectivity is achieved by the PVA membrane. This
reason makes that PVA membrane be widely used in the industry to dehy-
drate ethanol [48].
1. Organic Membranes: As it was mentioned above, PVA-based
membranes are one of the most studied and applied PV membranes
for alcohol dehydration. As a polymeric membrane, PVA is able to be
swelled in ethanol solutions, decreasing its selectivity. However, due
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to its highly hydrophilic character, superior abrasion resistance, and

cost, PVA is an effective material to be applied in the water removal
from hydrated ethanol. In general, the PVA membrane fluxes can
be in the range of 0.06–6.33 kg m–2 h–1 and the water selectivity can
vary from 43 to 890 for a maximum water content of 10 wt.% [50].

Besides, at low water content as the hydrated ethanol composition,
PVA membrane can become glassy [51]. The water flux and selectivity
can be significantly reduced. Therefore, recent works have been
focused on improving the mechanical and physical properties of PVA
membranes. This is made by crafting other copolymers and additives
in the PVA matrix or preparing composite membranes [52–55].
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Chitosan (CS) membranes are well-known hydrophilic membranes,
which have been widely applied for ethanol dehydration. However, the
performance of CS membranes can be decreased in aqueous solution
due to its uncontrollable swelling [56]. Similar to PVA, CS membranes
have been researched in order to improve their physical properties.
For instance, a hybrid CS/PVA membrane was synthesized for an
alcohol-water mixture (10 wt.% water) separation at low temperature
[57]. A permeate flux of 0.113 kg m–2 h–1 was achieved and, the water
selectivity was up to 17,000. In other work, a PV with hybrid CS/poly
(sodium vinyl-sulfonate) was performed to dehydrate an ethanol solu-
tion at the azeotropic composition [58]. The experiments were carried
out during 120 h, and the membrane presented a stable performance
with a permeate flux of 1.98 kg m–2 h–1 and a water composition of
99.5 wt.% on the permeate side.
2. Inorganic Membranes: An advantage of inorganic membranes, for
example, ceramic membranes, is the stability and mechanical prop-
erties in PV at high temperatures. Although the membrane selectivity
is naturally given by the membrane material, the permeate flux and
permeate composition are dependent on the vapor pressure of the
component. In the case of a dehydration membrane, the water pres-
sure and, consequently, the water flux can be enhanced by increasing
the feed temperature. Since inorganic membranes do not present
thermal instability, a wider operation range for PV can be presented
as compared to organic membranes.
Microporous silica membrane is one of the most representative inorganic
membranes for alcohol dehydration. A PV with silica membranes for an ethanol-
water mixture (6 wt.% water) can achieve a permeate flux of 0.417 kg m–2 h–1
and a water selectivity of 207 [59]. Moreover, this type of membrane is widely
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manufactured and commercialized by several companies, such as, ECN, TNO,

PervaTech, and SMS [60].
FIGURE 8.7 Comparison for the separation of ethanol-water mixtures by distillation based on
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the liquid-vapor equilibrium (—) and by three pervaporation membranes: (---) cellulose triacetate
(CTA), an anionic polyelectrolyte membrane (– • –) and poly(vinyl alcohol) (PVA) (…).
Another highly hydrophilic material is titanium dioxide (TiO2). This

material has been attracting the attention of researchers since it can be poten-
tially applied in PV for alcohol dehydration. TiO2 is commonly used as a
filler in PV membranes, using nanoparticle of this, improving the mechanical
and physical properties of organic membranes [61, 62]. A permeate flux of
0.340 kg m−2 h−1, and a membrane selectivity of 196 can be reached for the
separation of ethanol at 90 wt.% in an aqueous solution at 80°C. Similar
to TiO2, zeolites have been used as a filler in organic [63] and inorganic
membranes [64].
Although PV presents significant advantages as compared to distilla-
tion, it is difficult to replace an entire distillery for a PV system completely.
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Considering the high amount of material to process in a biorefinery, it would

require a large membrane area to take the ethanol composition in the wine
to the azeotropic composition. Currently, the membrane prices are consider-
ably high, even for polymeric membranes. Although distillation involves
high-energy consumptions, producing hydrated ethanol is more economi-

cally feasible by distillation than PV. Therefore, PV for ethanol recovery in
biorefineries is mainly focused on using it as an assistant operation. It means
that PV can be coupled to a main unit of the process to improve it. This is
especially observed in hybrid processes as fermentation or distillation.
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8.3.2.2 HYBRID DISTILLATION-PERVAPORATION (PV) COLUMNS
As it was previously mentioned, PV is a proper technology to produce

anhydrous ethanol. In order to compensate the high capital costs of PV
membranes, distillation, and PV can be coupled in a hybrid separation.
Therefore, ethanol can be concentrated close to the azeotropic composition
by distillation, while it is simultaneously dehydrated by PV. In this case, PV
replaces the dehydration by AD or adsorption using molecular sieve driers.
In Figure 8.8 is shown the hybrid distillation-PV column for ethanol
dehydration. The ethanol is concentrated in the first distillation column,
similarly to the conventional distillation process (Figure 8.1). Liquid and
vapor phlegma streams are produced with an ethanol composition about
30 wt.%. Unlike to the conventional distillation stage, a PV membrane is
externally connected to the column B. The hydrated ethanol in the distillate
stream produced by the column B is continuously fed to the PV unit, where
the water removal takes place. In order to increase the driving force in the
separation by PV, the feed temperature and pressure are increased to 130°C
and 4 bar, respectively [34]. A retentate with an ethanol composition higher
than 99.3 wt.% can be achieved. A permeate with a low amount of ethanol
is recycled to the column B, specifically, to the stripping section where the
water composition is high.
Although in Figure 8.8 is shown a single-stage PV, in the industry, three
or four stages are required for ethanol dehydration. It is due to temperature
drop during the PV. Therefore, inter-heating stages are necessary to compen-
sate for the temperature drop inside each PV module. In some way, this
allows maintaining relatively constant the PV temperature. In many cases,
the thermal energy for PV is supplied by integrating the column condenser,
stillage, and bottom stream with the PV feed stream.
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FIGURE 8.8 Hybrid distillation-pervaporation column to dehydrate ethanol from the
phlegma streams generated in the first column in a conventional distillery.
In the case of large biorefinery, a different design of a hybrid distillation

should be proposed. Vapor permeation is applied to improve the thermal inte-
gration in the process since the energy costs are significantly high. These have
to be considered in the design. The main difference between a PV and vapor
permeation is the physical state of the feed. In PV, a liquid feed is consid-
ered. While in vapor permeation, the feed is vaporized before entering to the
membrane unit. In both cases, the driving force is the difference in the vapor
pressure between the sides of the membrane. According to this, it is possible
to reach higher permeate fluxes in a vapor permeation than PV. However, the
vapor condensation inside the membrane module should be considered.
A hybrid distillation-vapor permeation process for ethanol separation
and dehydration is shown in Figure 8.9. Initially, the ethanol concentration
from the wine is concentrated in a vacuum stripper column (A) at 0.5 bar.
The top and bottom products in column A are an overhead vapor (~65 wt.%
of ethanol) and a liquid stream mostly composed of water (~0.1 wt.% of
ethanol), respectively. The product of the top is compressed up to 3 bars
(B), resulting in a temperature increase of the vapor. This vapor can supply
a part of the energy required for the evaporation in the column A. The water
content in the compressed vapor is reduced around 75% inside the vapor
permeation unit 1 (C), producing a permeate with an ethanol composition of
7 wt.%. The permeate is recycled directly to the column A, taking advantage
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of the latent heat of this stream. Anhydrous ethanol is produced in the vapor
permeation unit 2 (D). Since the ethanol composition is significantly high
in the feed of D, the permeate generated in this unit (57 wt.% of ethanol) is
condensed and mixed with the wine fed to the separation system. Finally, the
anhydrous ethanol (vapor) stream is thermally integrated with the column

A reboiler for its condensation (transfer of the latent heat). Approximately
30% of the thermal energy in the column reboiler is supplied by the retentate
condensation leaving from D. Using the arrangement presented in Figure
8.9, it is able to reduce the energy consumption by up to 50% as compared to
the hybrid distillation-PV system.
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FIGURE 8.9 Hybrid distillation-vapor permeation process for ethanol-water separation in

large-scale biorefineries [65]. Vacuum stripper column (A); compressor (B); pervaporation
unit 1 (C); and pervaporation unit 2 (D).
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Different works have presented the advantage of the hybrid distillation-PV

system to recover ethanol. It was demonstrated that a hybrid system, using NaA
zeolite membranes requires 52% less energy as compared to the azeotropic
distillation, producing an anhydrous ethanol with a composition of 99.4 wt.%
[66]. Besides, the ethanol dehydration by PV can be more energetic efficient

than using molecular sieves in industrial cases. PV requires just 23% of the
energy used in an adsorption process with molecular sieves [67]. In other
work, a hybrid vapor stripping-vapor permeation process, termed membrane
assisted vapor stripping (MAVS) was applied to separate alcohols in aqueous
solutions [68]. Using MAVS technology allows a 65% more energy efficient
separation of ethanol as compared to conventional distillation.
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8.3.3 IN SITU ETHANOL RECOVERY: HYBRID FERMENTATION
TECHNOLOGY
The conventional process for the production of ethanol by batch fermentation

of sugars by yeast has dealt with several drawbacks such as the high freshwater
consumption, the high energy consumption, and the inhibition by substrate or
product [5, 69]. In order to avoid the product and substrate inhibition, low
substrate concentration is used. Consequently, low reaction rates are achieved
with slow cell growth, which hinders the downstream processing, increasing
the energy consumption in the separation stage, and the total cost of the
process [69–72].
Strains genetically modified with high tolerance to substrate and ethanol
within the fermentation broth have been developed in order to overcome
the inhibition drawback [69, 70, 73]. However, this approach to improve
the conventional process for production of ethanol still is an uneconomic
process as compared to fossil fuels.
In situ product recovery (ISPR) is a potential strategy to improve ethanol
production [69]. ISPR involves ethanol removal while it is produced within
the bioreactor, maintaining the ethanol concentration under the inhibi-
tion limit (90 kg m–3). It provides several benefits to the process, such as,
decreasing of the water usage, increase the reaction rate, and increase ethanol
productivity, allowing a continuous fermentation process [69, 74].
Several separation methods for its integration to the ethanol fermentative
process, such as PV, solvent extraction, and adsorption, have arisen as poten-
tial technologies for ISPR and overcome the abovementioned drawbacks of
the conventional ethanol production.
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8.3.3.1 INTEGRATED LIQUID-LIQUID EXTRACTION (LLX) AND

ETHANOL FERMENTATION PROCESS
Liquid-liquid extraction (LLX), also called solvent extraction, was proposed

as a separation method for in situ ethanol removal from fermentation broth

in the 1980’s, and remains as a potential separation process to be coupled to
ethanol fermentation. The key considerations for the integration of the LLX
to the ethanol fermentation involve the selection of the solvent and strategy
of operation for the integrated LLX-fermentation system.
The main criteria for selection of the solvent to be used in a coupled
LLX-fermentation system may be summarized in the distribution coefficient,
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selectivity for the solute, and biocompatibility with the yeast. It is desirable
the solvent be low cost, low viscosity, completely immiscible with the
aqueous phase, high selectivity and distribution coefficient for ethanol, and
simple regeneration (for recovery of the product from the solvent phase, in
this case, the ethanol).
Thermodynamically, the distribution coefficient at equilibrium condi-
tion in a liquid-liquid system (equality of chemical potential between both
phases) is defined as the ratio between the equilibrium concentrations of
solute in the organic phase and the aqueous phase [75]. A high distribution
coefficient is convenient because it results in a decrease in the number of
stages of the LLX process and reduces the amount of required solvent for
removal of the desired solute [76, 77]. The polarity of the solvent influences
on the solubility of ethanol. The distribution coefficient of polar solvents
increases as the number of polar groups in the structure of the solvent
increases. The distribution coefficient of ethanol (KD,EtOH) in several solvents
has been widely tested, and in Table 8.1 are shown the highest distribution
coefficient values achieved.
The separation factor, defined as the ratio of the distribution coefficient
of ethanol to water (α = KD,EtOH/KD,water), is used to define the ability of the
solvent to remove the ethanol from water. Values of the separation factor
higher than the unity result in a selective extraction of ethanol from water.
For the selection of the solvent, it is also important to know the selectivity
on other components that are usually within the fermentation broths, specifi-
cally sugars and by-products that may achieve considerable concentrations
during fermentation. Alcohols and esters have shown a high separation factor
for ethanol [78]. Furthermore, the branched-chain solvents have higher
values of the separation factor as compared to its counterpart, the linear-
chain solvents [76]. In many cases, the solvent has shown high selectivity
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but not a high enough distribution coefficient. Looking for a compromise

between selectivity and distribution coefficient, a mixture of solvents has
been proposed [76]. It is usually used in the LLX of organic acids from
fermentation broths [79, 80], where the mixture of solvents not only provides
reasonable distribution coefficient and selectivity but also provide of a kind

of tuning of the physicochemical properties of the solvent mixture, such as
density, viscosity, and interfacial tension [79].
TABLE 8.1 Solvents with High Values of Distribution Coefficient (around 1 or higher
than 1) for Ethanol Extraction
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Solvent KD
1-Hexanol 1.0–1.2
3-Methylcyclo-hexanol 0.93
3-Methyl-3-pentanol 1.3
4-Methyl-2-pebtanol 1.1
2-Ethyl-butanol 0.69–1.03
3-Ethyl-3-pentanol 1.1
Phenol 2.15
o-Isopropylphenol 1.4
o-ter-butylphenol 1.4
Valeric acid 1.3
Hexanoic acid 0.944–1.1
Methyl acetate 0.91
Ethyl propionate 2.53
50% Hexan-1-ol + 50% 2-ethyl-1-ol (w/w) 1.03
On the other hand, microorganisms are very sensitive to the conditions of

their environment. These conditions refer to the pH, temperature, agitation,
and composition of the fermentation broths [81]. During the fermentation,
the optimal conditions for cell growth are set to avoid the stress on the cells.
The presence of an organic solvent on the fermentation broths produces
some stress on the cells, a toxic effect. The toxicity of organic solvents on
the microorganism of the fermentation may arise in two ways. Direct contact
between the microorganism with the solvent into the aqueous/organic inter-
face (phase toxicity) and by the soluble portion of the organic solvent within
the aqueous phase (molecular toxicity) [79, 82].
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The stress of the organic solvents on yeast cells affects their morphology
and physiological activity [81, 83]. The accumulation of organic solvents
on the membrane cells modifies their fluidity and permeability [81], which
involves hindrance of nutrients transport, leakage of metabolites, and in the
most severe cases, lysis of the cell [76, 83, 84]. On the other hand, organic
solvent within the cells may disturb the metabolic reactions decreasing the
activity of enzymes because it blocks the enzymatic active sites [81, 83,
84]. As a response to the presence of organic solvents, both the membrane
cell and into the cell, cells may change its lipid and protein composition in
the membrane to modify the plasma membrane properties, such as rigidity,
permeability, and fluidity [84]. However, any response of the cells to the
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organic solvent is energy demanding, which means that a portion of the
consumed substrate must be used to supply this energetic demand [79, 83],
decreasing both the cell growth and the metabolite production [76, 79].
Nonpolar solvents are nontoxic to cells [83]. However, nonpolar solvents
provide of low distribution coefficient to ethanol. In general, solvents with a
high distribution coefficient are toxic to cells [76, 79]. For instance, n-alcohols
with high molecular weight have a low distribution coefficient for ethanol,
while n-alcohols with low molecular weight have high toxicity and solubility
in water [70]. According to several works, the efficiency on the in-situ ethanol
removal and toxicity by using yeast cells follows [85, 86]: carboxylic acid >
alcohols > esters > amines > ketones > ethers > hydrocarbons. However, the
branch or length of the linear structure may affect [86].
Natural solvents, for instance, vegetable oils, which are very lipophilic,
are nontoxic to the whole cells [83]. Solvents, such as n-amyl alcohol,
1-octanol, and 1-docecanol have been tested for in situ ethanol removal from
the fermentation broth, resulting n-amyl alcohol the solvent with the highest
recovery percentage, providing the highest ethanol productivity, despite its
toxic effect [72]. Also, n-dodecanol has probed as an efficient solvent for
removal of ethanol by LLX in a continuous fermentation [87, 88].
The toxicity of solvents on the yeast may be reduced and even avoided,
by immobilizing the cells [71, 89]. Several compounds have been tested
for immobilization of cells, such as alginates, collagen, carrageenan, agar,
agarose, glutaraldehyde, and some materials as support such as CS, cellu-
losic material, natural zeolite, and γ-alumina [90, 91]. Calcium-alginate
gel entrapping is the most used method due to it is low cost, provides high
enzymatic activity and simple preparation [90]. Immobilized yeast has
been tested in LLX-fermentation systems for ethanol production, achieving
higher ethanol productivities and yields that its counterpart, the conventional
ethanol fermentation process [72, 76, 88].
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In addition, a proper LLX-fermentation configuration is able to reduce the

toxicity of solvents, providing an efficient ethanol extraction with a minimum
contact between yeast cells and solvent [71, 76]. Several configurations for
continuous LLX-fermentation, considering direct and indirect contact of the
yeast cell with the solvent are shown in Figure 8.10.
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FIGURE 8.10 LLX-fermentation arrangements: (a) solvent directly mixed with the fermen-
tation broths inside the fermenter; (b) solvent directly mixed with the fermentation broths in
an external LLX unit; (c) solid porous support contactor inside the fermenter; (d) solid porous
support contactor in an external LLX unit; (e) solvent directly mixed with the fermentation
broths in an external LLX unit with yeast cells filtration; and (f) solid porous support contactor
in an external LLX unit with yeast cells filtration.
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In Figure 8.10(a), the ethanol fermentation and LLX are simultaneously

carried out within the bioreactor. An output stream (B1) from the bioreactor
is processed in a decanter, splitting the aqueous (B2) and organic (S1) phases.
Therefore, B2 is the fermentation broth depleted in ethanol, and S1 is the
solvent highly concentrated in ethanol. The B2 stream is recycled and mixed

with the fermenter feed (F). The stream S1 is processed in a subsequent step
for regeneration of solvent and recovery of the product. In the regenera-
tion stage, the regenerated solvent (S2) is recycled to the bioreactor. P is an
output stream from the regeneration stage, which is concentrated in ethanol.
Worth noting that for this configuration, immobilized cells are recommended
to reduce any toxic effect due to a direct contact yeast-solvent.
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The ethanol fermentation also may carry out simultaneously with LLX,
using separate units as Figures 8.10(b, d–f) show. In Figure 8.10(b), the
fermentation broths (B1) from the bioreactor are fed to LLX column. Here,
the solvent phase is in intimate contact with the fermentation broths (ethanol
is transferred from fermentation broth to solvent phase), and both phases are
split in a subsequent unit, the decanter. The aqueous phase from the decanter
(B2) is returned to the bioreactor once is enriched of the substrate with the
fermenter feed (F), while the solvent phase (S1) is fed in the unit for its
regeneration. The clean solvent (S2) and the concentrated ethanol (P) are
produced in the regeneration unit. Finally, the clean solvent (S2) is returned
to the LLX column to perform a new extraction cycle. In this scheme,
immobilization of cells is required in order to avoid the direct contact of the
cells with the solvent phase.
An alternative scheme with a direct yeast-solvent contact is shown in
Figure 8.10(c). However, the mechanism of contacting the fermentation
broths and solvent is significantly different as compared to Figure 8.10(a).
In this case, the yeast cells are directly contacted to the solvent by using
a solid porous support submerged in the fermentation broths. The solid
surface of the support spatially separates the fermentation broths from the
solvent, while the porous, which are filled by the solvent, are exposed to the
fermentation medium. Then, the solid porous support works as contact media
(contactor) between fermentation broths and the solvent phase, providing a
high interfacial area between the phases.
The configuration shown in Figure 8.10(d) is a modification of the
scheme shown in Figure 8.10(c), since the LLX is externally performed
using a contactor (LLX-C). An output stream from the bioreactor is fed
to the LLX-C. In the LLX-C unit, the fermentation broths and solvent are
partially separated by a contactor (solid porous support). Both phases are
contacted through the filled porous of the contactor. Similar to the case of
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Figure 8.10(c), the contractor provides a high interfacial area between the
phases, but also, applying an external LLX unit.
The schemes presented in Figure 8.10(e) and f are variants to those previ-
ously showed in Figure 8.10(b) and (d), respectively. The difference lies in
the use of a parallel arrangement of filters before the extraction unit, in order
to avoid direct contact of the yeast cells with the solvent. It is an alternative
for fermentation processes with non-immobilized yeast cells. Due to the
filters suffer from fouling by the yeast cells and solids in the fermentation
broths, two filters must be implemented (F1 and F2). While F1 is operating,
F2 is in a cleaning stage or vice versa.
For all schemes shown in Figure 8.10, the molecular toxicity may be
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avoided if there is no recirculation of the fermentation broths (B2). However,
recirculation of the fermentation broth reduces the use of fresh water in the
process significantly, providing a positive impact on the cost of the process
and in the environment. Immobilization of cells works to overcome this
molecular toxicity drawback. Another option is finding a suitable solvent
for ethanol removal, which be nontoxic on the yeast cells. Nowadays, the
seeking of solvents for several processes still is an important field for the
design of new reactive and separation processes. Ionic liquids (ILs), deep
eutectic solvents (DESs) and aqueous two-phase systems (ATPS) are
emerging as potential solvents to be applied in several processes. ILs and
DESs have shown high efficiency for the extraction of several organic acids
[76, 77, 92, 93]. These novel solvents have great potential for the removal
of several metabolites of fermentations. However, it still requires research
on the applicability of these solvents in the ISPR for ethanol fermentations.
Once the solvent is used to remove the ethanol from fermentation broth, it
requires a regeneration step, as was shown in all configurations of the LLX-
fermentation process. There are several choices for the regeneration of the
solvent, such as vacuum flash vaporization, distillation, gas stripping, PV,
and back extraction [76, 77]. Use one or another technique is highly related
to the physicochemical properties of the solvent and ethanol, for example, the
difference in density or differences in volatilities. However, the most used
technique for the regeneration of the solvent rich in ethanol is vacuum flash.
8.3.3.2 PERVAPORATIVE FERMENTATION (PV-FERMENTATION)
PV is a promising technology to be applied in fermentation processes to

recover ethanol from the fermentation broths. According to previous sections,
organophilic membranes can be used to remove ethanol, while this is produced
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during the fermentation process. In this case, PV is not an invasive method.

Unlike to other types of hybrid fermentation, where the fermentation medium
can be contacted to solvent or, subjected to vacuum pressure, a solid membrane
does not affect the fermentation medium. It maintains the ethanol under the
inhibition concentration.
The general scheme of a PV-fermentation is shown in Figure 8.11. In a
hybrid fermentation-PV system, the membrane (C) can be located inside or
outside the fermenter (A). However, an internal membrane is more susceptible
to be fouled due to the high solid concentration (mainly for yeast cells) in
the fermentation broths. As a result, the operating time and membrane perfor-
mance can be rapidly reduced. In order to minimize the membrane fouling due
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to the suspended solid, a fraction of the fermentation broths is withdrawn and
passed through a rotatory filter (B). Around 99% of the yeast cell is retained
in B, and recycled to the fermenter again [95]. In the case of a continuous
fermentation, around 85% of retained yeast cells is feedback to the fermenter
again [96]. In the membrane module, ethanol is continuously recovered by a
PV at the fermentation conditions. The retentate is recycled to the fermenter.
FIGURE 8.11 Pervaporative fermentation system with an ethanol selective membrane

composed of (A) fermenter; (B) rotatory filter; and (C) external pervaporation membrane.
Source: Adapted from: Léon et al. [94].
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Commonly, PDMS membranes are applied to recover ethanol from

the fermentation broths due to their high stability [97]. For a fermentation
process (29–35°C) is not significantly important the thermal stability of the
membrane. However, PDMS membrane is swelled during the fermentation.
Several studies have reported improvements in the fermentation process by

coupling it to PDMS membranes. For instance, in a continuous membrane
fermenter separator (CMFS) the ethanol production was increased up to
20% as compared to a conventional fermentation [98]. In other work, it
was possible to reestablish high substrate consumption rates and ethanol
productivity after the inhibition by ethanol was achieved in a continuous
and closed-circulating fermentation system (CCCF) with PDMS membranes
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[99]. In Figure 8.12 is shown this latter impact of PV on the fermentation
performance. Once PV is initiated, the cell activity arises, increasing the
substrate consumption. Consequently, the ethanol productivity is increased,
allowing an overall ethanol concentration up to 609.8 kg m–3. This is almost
6-fold higher than a conventional fermentation. The reestablishment of the
fermentation performance after the ethanol inhibition condition is reached, is
a general effect of PV on the fermentation process [94, 100, 101].
FIGURE 8.12 Ethanol productivity (—) and glucose consumption rate (…) in a continuous
and closed-circulating fermentation system (CCCF) with PDMS membranes.
Source: Adapted from: Fan et al. [99].
On the other hand, the components in the fermentation broths influence

the PV performance. As it was previously mentioned, yeast cells can cover
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the membrane surface, reducing the permeate flux by PV. In addition, a

maximum glucose concentration of 100 kg m–3 increases the ethanol
selectivity [102]. However, the permeate flux is reduced. In the case of a
high concentration of dissolved salts (e.g., NaCl), the ethanol flux can be
increased due to the increase of its vapor pressure [97]. Furthermore, the
reduction of the membrane selectivity can occur by the presence of the
main fermentation products at a high concentration [97]. Ethanol, acetone,
n-butanol, and 2-propanol at 100 kg m–3, 10 kg m–3, 20 kg m–3 and 2–5 kg
m–3, respectively, reduce the PDMS membrane selectively. Also, the pH
can modify the hydrophobicity of PDMS membranes [103]. At low pH, the
water permeability is increased. The ethanol selectivity is reduced.
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KEYWORDS
• chitosan
• extractive distillation
• hyperbranched polymer
• liquid-liquid extraction
• membrane assisted vapor stripping
• polydimethylsiloxane
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CHAPTER 9
Design and Engineering Parameters of

Bioreactors for Production of Bioethanol
DAVID FRANCISCO LAFUENTE-RINCÓN,1
INTY OMAR HERNÁNDEZ-DE LIRA,2 HÉCTOR HERNÁNDEZ-ESCOTO,3
Author Copy
MARÍA ALEJANDRA SÁNCHEZ-MUÑOZ,4
HÉCTOR HUGO MOLINA CORREA,4
CRISTIAN EMANUEL GÁMEZ-ALVARADO,4
PERLA ARACELI MELÉNDEZ-HERNÁNDEZ,3 and
JAVIER ULISES HERNÁNDEZ-BELTRÁN4
1
Bioprocesses and Bioprospecting Laboratory, Biological Sciences Faculty,
Autonomous University of Coahuila, Torreón–27000, Coahuila, México
Faculty of Mechanical and Electrical Engineering, Autonomous
2
University of Coahuila, Torreón–27000, Coahuila, México

Chemical Engineering Department, University of Guanajuato,
3
Noria Alta s/n, Guanajuato, 36050, Mexico

4
Bioremediation Laboratory, Biological Sciences Faculty,
Autonomous University of Coahuila, Torreón–27000, Coahuila, México,
Tel.: +52-871-7571-785, E-mail: ulises.hernandez@uadec.edu.mx
(J. U. Hernández-Beltrán)
ABSTRACT
As an effort to find renewable sources of energy, the ethanol production

from lignocellulosic biomass (LCB) represents an environmentally friendly
alternative to fossil fuels. However, the recalcitrant nature of lignin on
holocellulose tackles the biomass use as feedstock and forces the use of
pretreatment methods in a first step to switch the raw substrate into digestible
cellulosic solids. In a second step, enzymatic hydrolysis process transforms
the holocellulose into sugars, which in turn are converted to bioethanol by
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using yeast or bacteria in the final fermentation step. In these three steps,
there are many challenges to face as the conversion of a high concentration
of raw materials, saving chemical and biological reagents, and the reduction
of the processing time. Therefore, it brings into play several engineering
tasks such as the configuring of bioreactors in the aspects of geometry and

equipment to achieve effective transfer of mass and heat, and the setting of
process conditions. In this context, this chapter reviews the main engineering
parameters and features to be considered in the design of bioreactors for the
steps of pretreatment, enzymatic hydrolysis and fermentation.
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9.1 INTRODUCTION
The fast growth of the human population has increased the demand for
energy and consequently bringing into play concerns as global warming
(GW) and reduction of natural sources [1]. One of the main aspects that the
world faces is the high consumptions of fossil fuels which represent around
80% of the global energy usage [2]. Thus, the R&D in renewable sources of
energy has become a priority. Biofuels are produced from biomass and have
been evaluated as an alternative to reduce both the dependence of fossil fuels
and environmental pollution [3]. In recent decades, they are emerged as an
effective strategy to deal with the matter related to the decreasing of fossil
fuels and the increasing of greenhouse gas (GHG) emissions [4]. Biofuels
can be classified in terms of generation according to the raw material used.
First generation biofuels are derived from food crops which unfortunately
creates ethical concerns and the increase of food prices [5]. To solve these
issues, the second-generation biofuels are produced from inedible LCB
such as agro-residues, forestry wastes, herbaceous biomass, wood wastes or
municipal wastes [6]. Among the most promising biofuels, the bioethanol of
second generation is considered as a fuel substitute for road transportation
due to it is an eco-friendly product with a higher octane number and emits
fewer concentrations of GHGs than gasoline [7].
The LCB is converted into bioethanol through the steps of pretreatment,
enzymatic hydrolysis, and fermentation that usually are carried out in a
bioreactor which is a controlled reaction system where an optimum environ-
ment is provided to enable the highest possible yield [8]. Inherently, different
configurations and operation modes of bioreactors have been explored,
by studying the impact of different process conditions involved such as
substrate concentration, catalyst concentration, temperature, pH, reaction
volume, mixing, among others [9]. A bioreactor commonly used is the stirred
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Design and Engineering Parameters of Bioreactors 235
tank reactor (STR) that can be operated under the different modes batch,
fed-batch or continuous, and other innovative bioreactor designs have been
proposed to process lignocellulosic materials [10]. It draws the convenience
to consider the design and engineering parameters involved in the bioreactors
for bioethanol production in the steps of pretreatment, enzymatic hydrolysis,

and fermentation. Thus, in this book chapter, aspect of geometric configura-
tion in the bioreactors of each step and operation conditions to perform the
processes are discussed.
9.1.1 LIGNOCELLULOSIC BIOMASS (LCB)
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Lignocellulosic biomass is an attractive renewable feedstock for bioethanol
production due to its huge abundance, low cost and sustainable supply [11].
Lignocellulose is composed by three main biopolymers such as cellulose,
hemicellulose, and lignin [12]. Table 9.1 shows the chemical composition for
different LCB. In general, the fractions of cellulose, hemicellulose, and lignin
corresponds to 25–55%, hemicellulose 5–35% and 5–30%, respectively.
TABLE 9.1 Chemical Composition of Different Lignocellulosic Biomass According to the

Fraction of Cellulose, Hemicellulose, and Lignin
Type of Lignocellulosic Chemical Composition (% w w–1) References
Biomass Cellulose Hemicellulose Lignin
Bamboo residues 38.4 ± n.a. 21.3 ± n.a. 29.6 ± n.a. [112]
Barley straw 45.7 ± 0.2 32.6 ± 0.5 5.2 ± 0.0 [11]
Oak sawdust 44.7 ± 0.3 14.8 ± 1.3 26.7 ± 0.8 [13]
Plantain pseudostem 34.7 ± 0.5 10.1 ± 0.1 30.8 ± 0.3 [14]
Rapeseed straw 30.0 ± 2.2 12.7 ± 0.7 21.1 ± 0.2 [113]
Rice straw 36.3 ± 1.2 27.9 ± 1.3 14.11 ± 0.5 [114]
Sorghum straw 26.9 ± 1.2 32.5 ± 1.9 10.1 ± 1.8 [15]
Spruce sawdust 55.4 ± 1.3 4.2 ± 0.4 28.7 ± 1.0 [13]
Sugarcane bagasse 54.2 ± 2.2 27.9 ± 3.6 10.3 ± 0.1 [16]
Wheat straw 39.25 ± 1.1 33.65 ± 1.1 7.35 ± 0.3 [115]
Cellulose is a linear polymer that is composed of D-Glucose subunits

linked by β-1,4 glycosidic bonds, which form the cellobiose dimer. This long
chain is linked together by hydrogen bonds and van der Waals forces [17].
Cellulose is generally present in crystalline form (80%) which is hardly to
hydrolyze and the rest in amorphous form being more susceptible to enzymatic
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degradation [18]. On the other hand, hemicellulose is a polysaccharide

with a lower molecular weight than cellulose and is composed of different
sugars forming shorter and branched chains. These sugars can be divided
into different groups such as pentoses (D-xylose, L-arabinose), hexoses
(D-glucose, D-mannose, D-galactose), hexuronic acids (D-glucuronic acid,

4-O-methyl-glucuronic and D-galacturonic) and deoxyhexoses (rhamnose
and fucose). The main chain of a hemicellulose consists of a single unit
(homopolymer), such as xylan, or two or more units (heteropolymer), such
as glucomannan. Sugars are linked by β-1,4 glycosidic bonds and sometimes
by β-1,3 glycosidic bonds. Hemicellulose is amorphous, therefore, the
differences in the composition of sugars, the presence of shorter chains and
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the ramifications of the main chains make the hemicellulose structure easier
to hydrolyze than cellulose [19].
Lignin is the most abundant phenolic biopolymer in nature and is the
third major component in lignocellulosic materials. Lignin is related to both
hemicellulose and cellulose, forming a physical seal that is an impenetrable
barrier in the plant cell wall. It is present in the cell wall to provide
structural support, impermeability, and resistance against microbial attack
and oxidative stress. Lignin is an optically inactive, non-water soluble,
amorphous heteropolymer that is formed from phenylpropane bound
through non-hydrolysable bonds. This polymer is generally synthesized by
three different phenylpropanoid derivatives: coniferyl (guaiacyl propanol),
coumaric (p-hydroxyphenyl propanol) and sinapyl (syringyl propanol)
alcohols; synthesized from phenylalanine through various derivatives of
cinnamic acid. Phenylpropanoid alcohols are bound in a polymer by the
action of enzymes that generate intermediates in the form of free radicals.
This heterogeneous structure is linked by C-C and aryl-ether bonds linked to
β-aryl-aryl-glycerol ether, these being the predominant structures [20–22].
9.1.2 PROCESS OUTLINE OF BIOETHANOL PRODUCTION
Figure 9.1 shows the general process for bioethanol production from LCB, in
which three main steps appear: (1) pretreatment methods have been identified
as the first barrier in is used to break the lignin wall and disrupt the crystallinity
of cellulose to allow the accessibility of the enzymes; (2) enzymatic
hydrolysis process consists in the conversion of holocellulose (cellulose plus
hemicellulose) to reducing sugars through the action of enzymes on pretreated
biomass; (3) fermentation is the step in which the reducing sugars like glucose
is converted to ethanol by using yeast or bacteria [23].
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FIGURE 9.1 Outline of bioethanol production process.
9.1.2.1 PRETREATMENT
The conversion of LCB into added value bioproducts represent a prominent

strategy to look forward in the circular bioeconomy. However, the pretreatment
of LCB still be the most complex step in the conversion to biofuels from the
economic point of view. LCB due to its chemical composition are consider as
a recalcitrant biomass, resistant to the breakdown by biological, physical, and
chemical process [24]. Various strategies have been explored to overcome
these restrictions, including novel methods of pretreatments and the utilization
of innovative bioreactors configuration. Several pretreatments’ technics for
biomass have been studied, which can be divided into, mechanical processes
(particle size reduction), chemical processes (use of diluted acids or alkalis),
physicochemical processes (steam explosion (SE) and hot water), biological
processes (microbial consortium or enzymes) or the combination among
those [25]. Albeit these techniques enhance significant cost to the process at
commercial scale.
The most used physical method is a mechanical one which consists
in the reduction of the particle size and it represents the first action in the
processing of LCB because it has been established as an essential to increase
the accessible surface area and the porosity of the particles, in addition to
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reducing the crystallinity of the cellulose [26]. However, there is no universal

optimum particle size and before to start the processing of every LCB
this issue should be addressed [6]. After that, chemical, physicochemical,
biological pretreatment, or combination of them must be followed. Chemical
and physicochemical pretreatment methods use the reagents at low

concentrations, i.e., a common practice is the use of dilute acid like sulfuric
acid on LCB and carry out at high temperature in an autoclave for several
minutes [27]. As well, the combination of reagents results in a most effective
way, i.e., an alkali reagent like sodium hydroxide combined with hydrogen
peroxide as oxidative reagent which not only improves the depolymerization
of lignin and the digestibility of lignocellulosic materials at a low operating
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cost but also maximizes the use of cellulose and hemicellulose [28]. Alkaline-
oxidative is an environment-friendly pretreatment that can be carried out
at mild temperature and pressure, which leads to the formation of minor
inhibitors [16]. Regardless of the type of LCB, if ethanol is the final product,
the pretreatment must guarantee a maximum recovery of holocellulose to
achieve high concentration of reducing sugars. Furthermore, pretreatment
has been identified as the crucial step, both technically and economically, in
the conversion of global LCB due to its strong influences to restrict the other
steps in the chain of bioethanol production.
9.1.2.2 ENZYMATIC HYDROLYSIS
Although lignocellulosic materials can be hydrolyzed using acids or

enzymes, the process must consider the use of environmentally friendly and
economically viable technologies. Enzyme-based hydrolysis is presented as
the best option due to its high conversion efficiency and minimal production of
by-products like inhibitors which negatively affect the performance of further
fermentation process [29]. In addition, the operating conditions are moderate,
i.e., temperatures used in enzymatic hydrolysis are between 45–60°C. It is
not corrosive for the bioreactor used and the process requires a lower energy
consumption. In contrast, acid hydrolysis requires a higher temperature like
120°C or more and produces furfurals and hydroxymethylfurfural as inhibitors
for subsequent fermentation process [15].
Polysaccharide hydrolysis involves at least three different enzymes
which are simultaneously working to degrade the polymer to its monomer.
Enzymatic hydrolysis of cellulose is performed by cellulases or cellulolytic
enzymes that catalyzes the cleavage of β-1,4 glycosidic bonds, present in
the insoluble cellulose, to produce glucose. Cellulase enzymes comprise
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endo-1,4-β-D-glucanases, exo-1,4-β-D-glucanases (cellobiohydrolases) and

β-glucosidases. The main reaction mechanism of cellulases is depicted in
Figure 9.2. First, endoglucanases start breaking β-1,4 glycosidic bonds by
the addition of a water molecule, releasing cellodextrin with a reducing and
a non-reducing end from the amorphous regions of the cellulose backbone.

Then, exoglucanases hydrolyzes cellodextrins, from its reducing and a non-
reducing end, to produce cellobiose (disaccharide of two glucose units).
Finally, β-glucosidases catalyzes the cleavage of cellobiose producing two
soluble monomers of glucose [30–33]. Although enzymes are biological
catalysts that perform chemical reactions efficiently, its activity is strongly
regulated for different factors as temperature and pH, as well as, adsorption,
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mixing conditions, and solid-liquid ratio that will be further discussed.
FIGURE 9.2 Action mechanism of cellulase enzymes on cellulose fraction.
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On the other hand, enzymatic hydrolysis can be influenced by the

concentrations of substrate and final product, the enzymatic activity, and the
reaction conditions [33]. Recently, one of the main strategies to reach a high
concentration of reducing sugars is by using a high concentration of substrate,
however, this aspect affects directly to the mixing of the reaction and simul-
taneously to the process conditions such as pH and temperature cannot be
constant along the time, even the adjustment of these parameters is difficult
[34]. Thus, there is no possible to ensure the best performance considering
that the mixing trouble begin from 7% w v–1 of substrate concentration [14].
Therefore, the research activities in enzymatic hydrolysis have been focused
to reach high solids concentration of substrate between 20–40% w v–1 using
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engineering strategies as fed-batch or semi-continuous configurations for the
reaction instead of a batch one and to deeply study the relationship of the
several parameters which affect the enzymatic hydrolysis process to increase
the profitability of fermentable sugars production [35].
9.1.2.3 FERMENTATION
In contrast to conventional ethanol production processes from starch and

sugar raw materials, lignocellulosic sugars become a highly challenging
environment due the fermentable broth may contain several compounds
released in pretreatment and enzymatic hydrolysis steps which could affect
the performance on fermentation process [36]. This fact requires robust
fermenting microorganisms with high tolerance to inhibitors derived from
biomass, ethanol concentration, and mechanical and osmotic stress [37].
Saccharomyces cerevisiae yeast is the most used microorganism for industrial
alcoholic fermentation [38]. The most attractive characteristics of S. cerevi-
siae are: (i) it is generally recognized as safe (GRAS); (ii) it can consume all
types of hexoses; (iii) it achieves ethanol yields close to theoretical; and (iv)
it can produce ethanol at concentrations as high as 13% (v v–1). Furthermore,
the resistance of S. cerevisiae to lignocellulose-derived inhibitors is high,
and therefore, it is the preferred microorganism for lignocellulosic ethanol
production [39]. Despite showing all these interesting characteristics, the
main drawback of S. cerevisiae is its inability to ferment xylose, which is the
second most important sugar after glucose in lignocellulose. The challenge
in the fermentation process to make large-scale production is the obtention
of bioethanol broths of at least 4% w v–1 due from that concentration value,
and the purification process starts to be feasible from an economical point of
view. This means that fermentation process must be loaded with a broth of
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at least 80 g L–1 of six-carbon reducing sugar, considering the stoichiometric

conversion of glucose to ethanol is 0.51 g g–1 [15].
9.2 BIOREACTORS FOR PRETREATMENT PROCESS
The reactor configuration, process conditions, and the operation mode during
the pretreatment conversion process are essential to gap the high yields and
productivity in the biomass conversion at industrial levels [40]. More recently,
different studies have been conducted in several bioreactor configurations
with the aim to obtain higher yields of glucose in the pretreatment stage [8].
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The pretreatment step has many methods for its development and each
one present their own phenomenology into the reaction. The mechanical
method is the most used among the physical pretreatments [41] but does
not require a bioreactor for reduction of particle size but rather a milling
equipment, however, the reduction of particle size is a process condition that
can limit the performance of the whole bioethanol production chain [42].
For this reason, a study of the optimum particle size for each LCB is a key
issue [26]. Other common physical pretreatments are the microwave and
hydrothermal methods. Microwave reactors (MWR) have noticed interest
for lignocellulosic pretreatment. Microwaves are a kind of electromagnetic
radiation that is shaped like energy propagating in a vacuum in the absence
of any material in motion. Microwaves are originated by the separation of
two charges positive and negative of equal magnitude which are separated by
a fixed distance. MWR are mainly composed by a generator, a cavity section,
a waveguide, and an advanced control monitoring system. The principle
of these reactors is based on Maxwell’s equation [43]. Hermiati et al. [44]
studied microwave-assisted acid pretreatment of sugarcane trash, with a
maximum work temperature of 180°C and biomass capacity of 100 mL. The
sugar production from the pretreated LCB was 20.2 g L–1 of xylose, which
offers a good yield scale under these conditions. In addition, Peng et al. [45]
designed a pilot-scale continuous microwave irradiation reactor (MWIR).
The temperature range of operation is between 0 to 300C, with a maximum
power of 6 kW. MWIR ethanol yield indicated a value of 31.2 g 100 g–1 of
pretreated corn straw. Table 9.2 describes the main characteristics of reactors
system for conversion of LCB considering the reaction system, including the
agitation method, reaction volume, reactor configuration, and the reducing
sugars or ethanol yield. On the other hand, an emerging technology to convert
wet biomass into valuable products due to the action of temperature (200–
370°C) and high pressure (4–20 MPa) is hydrothermal liquefaction reactors
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TABLE 9.2 Main Characteristics of Reactors System for Conversion of Lignocellulosic Biomass
Biomass Reactor System Agitation Tank Configuration Sugarsa or Ethanolb References
Volume Process Concentration Yield
b
Agave bagasse Tubular reactor Rushton-type stirred 1.0 L Batch SSF 37.6 g L–1 [116]
blades
Sugarcane bagasse Hydrodynamic cavitation Recirculation pump 2.0 L Fed-batch SSF b28.44 g L–1 [48]
b –1
Dry corn Hydrodynamic cavitation Low-shear impeller 379 L Batch SSF 35.8% w w [49]
b
Reed grass Hydrodynamic cavitation Recirculation pump 150 ml Batch SSF 25.9 g L–1 [50]
a –1
Apple pomace Bubble column bioreactor Air bubbles – Batch 51.8 g L fermentable sugar [117]
a
Agave bagasse High pressure stirred reactor Anchor impeller 1.0 L Batch 3.7 g L–1 xylose [8]
a
Sugarcane bagasse Plasma in liquid reactor N/A 1.0 L Batch 51.3% glucose [118]
b –1
Sargassum horneri Stirred tank bioreactor Rushton impeller 2.0 L Batch S-SSF 2.89 g L [119]
a
Sugarcane bagasse Supercritical CO2 reactor CO2 bubbles 100 ml Batch 1.17 g L–1 fermentable sugars [120]
b
Tobacco stalk Rotating reactor Rotation 2L Batch 27.5 g Kg–1 TS [121]
b
Sugarcane bagasse Fluidized bed reactor Air bubbles 2L Batch SSCF 0.34 g g–1 [122]
b –1
Birch biomass Steam explosión react N/A 4L Batch SSF 80 g L [123]
a
Wheat Straw High pressure reactor Rotation 1L Batch 72.7% glucose [21]
a –1
Sugarcane trash Microwave reactor N/A 100 ml Batch 20.2 g L xylose [44]
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3 b –1
Corn straw Microwave irradiation reactor N/A 4m Batch SSF 31.2 g 100 g LCB [45]
Lignin-rich stream Hydrothermal liquefaction N/A 43 ml Batch – [47]
reactor
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(HTLR) [46]. Several studies have revealed the potential of HTLR for bagasse
pretreatment to produce a liquid product, known as bio-oil. Miliotti et al. [47]
evaluated the valorization of lignin-rich stream from lignocellulosic ethanol
production at industrial scale using hydrothermal liquefaction. The stainless-
steel reactor was operated in batch mode with a volume capacity of 27 mL.
It has been observed that reactors configuration and operation mode, in order
to improve the LCB pretreatments, should consider the development of new
technologies and novel methods to overcome the lab-scale into industrial
level. Special attention might be focused on the energy reduction process and
the improvement of sugars-bioethanol yields.
The design of bioreactors in chemical and physicochemical pretreat-
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ments differs in the temperature parameter. If the temperature is moderate,
between 25–70°C, stirred tank bioreactors (STBR) are normally used. If the
temperature to be used is higher, for example, more than 100°C, the bioreac-
tors used also tend to withstand high pressures. The substrate concentration
plays an important role to determine the type of stirring and can vary from a
moderate concentration of 5% to a high concentration of >12% (w v–1) [51].
This entails to the use of mechanical mixing and the increasing of speed
of the stirring. The more concentration, the faster the stirring speed, which
leads to higher energy requirement. For this reason, the engineering strate-
gies used for processing high concentrations of substrate in chemical and
physicochemical pretreatments are fed-batch or semi-batch and continuous
modes of operation [10]. Besides, bioreactors must withstand to acid and
alkaline values of, i.e., the value of pH using an acid reagent like sulfuric
acid is <1 or by using an alkaline reagent like sodium hydroxide is >11 [27].
Another physical pretreatment but also can be performed as physio-
chemical one is hydro-dynamic cavitation. It involves the generation of
cavitation in freely flowing liquid which is constricted with a venture tube
or orifice plate [52]. Cavitation phenomenon can be defined as the formation
and subsequent growing and collapse of microbubbles due to the changes in
pressure at constant temperature. Bernoulli's equation is the main principle in
this technique, where the drop in pressure at the constriction below the vapor
pressure of the flowing liquid generates bubbles or hydrodynamic cavities
[53]. Hydrodynamic cavitation reactors (HCR) have been effectively applied
for delignification of several LCB, such as reed grass [50], sugarcane bagasse
[48], dry mill corn [49] among others. The great advantage of HCR is its much
lower energy consumption compared with ultrasonic reactor, as well it has
been observed that this pretreatment affects both delignification and crystal-
linity of cellulose to increase yields in bioethanol production. Terán et al. [48]
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proposed hydrodynamic cavitation (HC)-assisted alkaline hydrogen peroxide

pretreatment in a continuous process, the system contained two devices to
generate cavitation. HCR consisted in a 2 L stainless steel reactor, provided by
two centrifugal pumps of 1.3 hp.
Also, in the system was included two tanks for lignocellulosic biomass
preparation and for the cavitation devices consisted in a plate of 8 to 24
orifices at different configuration. On the other hand, Ramirez-Cadavid et
al. [49] built a pilot scale HCR. The system included a 379 L heat-jacketed
vessel able to maintain temperatures from 82°C to 94°C. The agitation was
provided by a low shear impeller mixer.
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9.3 BIOREACTORS FOR ENZYMATIC HYDROLYSIS PROCESS
9.3.1 OPERATION MODE AND GEOMETRIC CONFIGURATION
In case of the saccharification bioreactor, operation mode and geometric

configuration must be carefully selected in order to optimize the maximal
lignocellulosic bioconversion into added-value products. Hence, the
bioreactor must accomplish specific requirements to ensure the efficient use
of the substrate and to fulfill the enzyme conditions which involves the prior
study of the biological system. When the process works is optimized, it is
expected to obtain high rates of glucose yield per volume of reactor by means
of controlling certain parameters mainly temperature and pH [54]. However,
enzyme-substrate ratio has to be guaranteed, as well as the agitation mode
to ensure optimal interaction among cellulases and cellulose in order to
increase the hydrolysis efficiency [55].
There are three operation modes according to the substrate feeding process:
1) discontinuous, semi-continuous, and continuous mode. Discontinuous or
batch mode is a closed process where substrate is initially added without
additional feed or removal of secondary products. This mode is commonly
applied in many industries for bioethanol production [56]. Semi-continuous
or fed-batch mode involves the intermittent addition of fresh nutrients at a
certain period during the hydrolysis stage. This mode has several advantages
as promoting a higher bioconversion process than batch mode, the constant
mixing process prevents the rising of substrate viscosity, it decreases the
enzymatic inhibition rates by reducing the unspecific enzyme binding and
it provides a longer time to hydrolyze the biomass into fermentable sugars
[57–59]. Continuous mode consists in the continuously feeding of the reactor
with the concomitant removal of by-products from the reaction vessel, thus
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the liquid volume of the reactor keeps constant. However, this process can be
affected by substrate inhibition [60].
Different types of configurations in the bioreactor fields had been employed
including well-known configurations as STBR, which is the most commonly
used for enzymatic processes, and the last advances on STRB which focus
on the conversion of energy crops and residual materials into sugars and
ethanol include separate hydrolysis and fermentation (SHF), simultaneous
saccharification and fermentation (SSF), simultaneous saccharification and
co-fermentation (SSCF) and consolidated bioprocessing (CBP) (Figure 9.3).
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FIGURE 9.3 Different types of bioreactor configurations for sugars and bioethanol production.
[Abbreviations: SHF: separate hydrolysis and fermentation; SSF: simultaneous saccharification
and fermentation; SSCF: simultaneous saccharification and co-fermentation; CPB: consolidated
bioprocessing].
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STBR consist in cylindrical vessels with one or more impellers assembled

with an external motor. This configuration is widely used in a batch mode
enhancing the mixture homogeneity where biochemical reactions occur.
These types of bioreactors are equipped with baffles to prevent vortexes,
heating-cooling systems, or thermal jackets, and can be built, depending on

the size, of glass (laboratory scale), stainless steel or carbonate [61].
SHF carries out the enzymatic hydrolysis and fermentation in two
independent reactors for saccharification and fermentation processes. This
process can be subject to different conditions since the reactors are separate
which allows to enzymatic hydrolases work at optimal temperature, which
is higher than fermentation, and to reduce the quantity of saccharifying
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enzymes than other simultaneous processes [62, 63].
SSF combines enzymatic hydrolysis and fermentation to obtain value
added products in a single step, which can reduce significatively the time
and cost of the process. Another advantage is the risk reduction of inhibitory
compounds of saccharification. Hence, SSF has been widely used for produc-
tion of biofuels from lignocellulosic materials. However, in comparison with
SHF pH and temperature parameters cannot be individually set and an equi-
librium point might be necessary for the process to work properly [64, 65].
SSCF is referred to the process where enzymatic hydrolysis can be
conducted simultaneously with the co-fermentation of glucose and xylose
in a single process. This process take advantage of the biochemical affinities
due glucose and xylose compete for the same transport systems in the
co-fermentation of xylose. Thus, SSCF is promising for ethanol production
[66]. SSCF have some advantages over SHF which includes the removal of
hydrolysis end-products, short processing time and low contamination risk.
Besides, it has been proved its high efficiency and rates of ethanol yield [67].
CBP is a system where the enzyme production, substrate hydrolysis and
fermentation are performed in one step by lignocellulolytic microorganisms.
The production costs by using this process are lower for biofuel obtention,
due implies lower energy inputs and higher conversion efficiencies in
comparison to SHF. However, CBP has certain disadvantages, mainly
involved with enzyme production because monocultures have shown some
limitations. While co-cultures might have different enzyme production
requirements due bacteria can ferment LCB at mesophilic or thermophilic
temperatures and thus, different rates of cellulose hydrolysis and biofuel
production can be observed [68, 69].
Jacket or coil for heating/cooling system is one of the most important
instruments for laboratory-scale bioreactors, the vessel can be positioned
into a thermal incubator or can be adapted to an electrical heater resistance,
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also the external wall of the vessel can be equipped with a thermal jacket in
order to maintain the optimal temperature and equilibrium for the biological
system. Otherwise, large-scale bioreactors possess an internal coil heating-
cooling system.
On the other hand, as seen before, STRB is one of the most common
systems for lignocellulosic bioconversion and in order to heat transfer and
homogenization be adequate different types of agitation have been studied.
It is worth to mention that agitation promotes the equal distribution and
interaction among all the biological matter and the mixing efficiency will
result in an optimal product yield. As seen in Figure 9.4, there are different
types of impellers that can achieve different mixing patterns as axial or
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radial, depending on the movement direction of the components.
Three blade elephant ear impellers (Figure 9.4(A)) are commonly used
in STRB. It has been showed that a configuration of two impellers of this
type have two lowered energy consumption, diminished mixing time and
enhanced homogenization of the medium. At the same time, this configuration
has produced higher glucose conversion by diminishing the inhibition rate of
soluble by-products [70]. Peg mixer (Figure 9.4(B)) has been recognized for
being more convenient on enzymatic saccharification at high solid loadings
where viscosity is challenging, due it promotes the enzymatic binding on the
cellulose substrate and overcomes mass transfer limitations [71].
On the other hand, helical ribbon impellers (Figure 9.4(C)) are used for non-
Newtonian fluids and substrates with high viscosity that present challenges for
the mixing process. This impeller design allows to provide and efficient mixing
not only for this type of fluids but an alternative for enzymatic hydrolysis at
high solid loadings [72]. Besides, it has been demonstrated that this impeller
enhances glucose yields and ethanol production by using less energy [73].
Finally, Rushton turbines are commonly present in most of the vessels
which promotes a radial flow mixing pattern (Figure 9.4(D)). These impellers
have been recently combined with other configurations as the elephant ear
impeller due combines the axial and radial flow overcoming the disadvantage
of Rushton turbines, which creates a staged mixing patters that produces an
irregular enzyme-substrate interaction [70].
9.3.2 PROCESS CONDITIONS
The main parameters that must be under tight control and play a crucial
role in the effectiveness of enzymatic catalysis are substrate concentration,
enzyme concentration, temperature, pH, and mixing velocity.
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FIGURE 9.4 Different impeller designs for bioreactor systems. (A) Elephant ear impeller;
(B) peg mixer; (C) helical ribbon impeller; and (D) Rushton turbine.
First, the enzymatic hydrolysis development depends on the adsorption

of cellulases on the surface of the lignocellulosic material, most endogluca-
nases and exoglucanases have a carbohydrate-binding module that enhances
the binding process. This process takes about 10 to 15 min, which is related
to the reaction rate [74]. It is also known that cellulolytic enzymes activities
are optimal at a range from 45 to 55°C and pH in a range of 4–5 [60, 75].
Besides, mixing conditions and solid-liquid ratio are essential in saccharifi-
cation of polysaccharides due it is necessary to mass and heat transfer and to
enhance the interaction among substrate-enzyme [76].
It is well-known that during the hydrolysis process where glucose is
available to further fermentation, there is some risk of feedback inhibition by
the end-product of fermentation. This problem has been overcome by SHF
configuration reactors. However, enzyme and substrate concentrations also
play an important role due to excessive substrate can cause an end-product
inhibition of glucose that accumulates in the hydrolysis step [77]. Nonethe-
less, there are multiple studies that are based on the optimization process to
reach the maximal enzymatic activities and obtain high yields, modifying
several parameters during enzymatic lab-scale approaches, as well as large
scale studies. Table 9.3 summarizes some studies related to some conditions
for enzymatic hydrolysis of agricultural residues where high hydrolysis
yields of product were reached.
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TABLE 9.3 Conditions of Conversion of Agricultural Residues with High Hydrolysis Yield
Configuration mode/ Biomass Concentration Culture/Enzyme Concentration Hydrolysis Hydrolysis References
Bioreactor System Conditions Yield (%)
Corn Stover
3L fed-batch steam- Corn stover 30% Trichoderma reesei, 30 FPU g–1 cellulose 50°C, pH 4.8, 85.1% [124]
explosion reactor (w v–1) 150 rpm.
Continuous pilot-scale Corn stover 12–15% 10.7 FPU g–1 cellulose 45°C, pH 4.8, 400 rpm 80% [59]
reactor
1L-batch reactor Corn stover and dry 15 FPU/g cellulose þ 64 pNPGU g–1 50°C, pH 4.8, >95% [125]
distiller’s grain and cellulose 200 rpm.
solubles 18%
Straw
Design and Engineering Parameters of Bioreactors
2L-batch reactor Barley straw 15% 7.5 FPU g–1 solids plus 13 CBU g–1 solids 50°C, pH 5, 57 rpm. 81% [126]
–1
250 ml-SSF Barley straw 15% 7 FPU g solids plus 8.4 IU β-glucosidase 50°C, pH 5.5, 150 59% [127]
g–1 solids plus 72 U xylanase g–1 solids rpm.
250 ml batch reactor Wheat straw 20% 7 FPU g–1 DM plus β-glucosidase 50°C, pH 4.8, 6.6 rpm 60% [128]
–1 –1
Batch reactor Rye straw 17% 13 FPU g cellulose plus 35 CBU g 45°C, pH 5, 120 rpm 65% [129]
cellulose
Bagasse
150 ml batch reactor Sugarcane bagasse C. acetobutylicum ATCC 824, 9.6 FPU g–1 50°C, pH 6.7, 120 rpm 55% [130]
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9+8+7+6% (w v–1) solids
1L-fed batch Sweet sorghum bagasse 30 FPU g–1 cellulose 50°C, pH 4.8, 100 rpm 60% [131]
hydrolytic reactor 20%
249
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9.4 BIOREACTORS FOR FERMENTATION PROCESS
The most common bioreactor used for fermentation experiments is a stirred

tank operated in batch mode and the ideality is to achieve the continuous mode.
Batch configuration is a discontinuous process where the bioreactor is initially

loaded with a specific reaction volume that remains constant throughout the
fermentation. The batch reactor is a simple but efficient from an operational
point of view with low-cost, compared with other more sophisticated, usually
the main components in batch reactors are the tank, the stirred and mixing
system, and several controllers of variables as shown in Figure 9.5, i.e., in
the industry prefer to the steel and concrete to avoid the corrosion issues that
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are materials expect a long lifetime [78]. Additionally, the batch system is
the simplest operation mode which implies the least risk of contamination.
Meanwhile, the continuous mode fresh culture medium is continuously added
and extracted from the bioreactor [79]. The volume of the bioreactor is kept
constant because both the inflow and outflow are equal. Normally for this
kind of process is used a membrane for filtration of the ethanol [80]. In this
way is easy to recover a higher amount of ethanol avoiding the inhibition of
the ethanol that limits the growth of the microorganism [81].
9.4.1 GEOMETRIC CONFIGURATION AND INSTRUMENTATION
The configuration of the fermentation system is directly related to the

sugar conversion process, where the separate processes of saccharification
and fermentation and the simultaneous processes of saccharification and
co-fermentation are the most common system [82]. Co-fermentation is based
on the transformation of hexoses and pentoses contained in the fermenta-
tion broth. For which reason several strains of yeasts and bacteria have
been studied with the aim of developing an efficient process of hydrolysis
and co-fermentation of xylose and glucose. The advantage of developing a
process to carry out SSF is the reduction in the number of vessels [83], in
addition, a high ethanol yield can be obtained due to the relief of glucose
inhibition in cellulase activities for a more complete cellulose hydrolysis
[84], but the main drawback is finding a fermentative micro-organism that
has operating conditions similar to those required by the enzymatic complex
used during saccharification [83].
The design of the reactor structure and their components to reach the
established process, in this case for second-generation bioethanol, some acces-
sories in a STR operated under batch mode, are jackets or thermal blanket
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for temperature regulation, stirred system composed with propeller of type

marine, rushton or helical, sensor for pH adjustment, and other components
to maintaining a controlled environment (Figure 9.5). The heating-up control
is an important operation in the industry; the jackets are considered the best
way to control the temperature with practically no variation of temperature

in comparison with other systems like thermal blankets. Jacketed reactors are
extensively used for fermentation and other different industrial processes like
crystallization. Nowadays, the use of new materials for the equipment in the
industry becomes more versatile with the use of polymeric complementation
with metallic parts in the structure of the reactor [85].
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FIGURE 9.5 Design of stirred tank bioreactor detailing the process conditions involved in
fermentation process.
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By having a completely homogeneous stirred tank bioreactor, it can

fulfill the characteristics of a chemostat in which the growth of the micro-
organism is kept in a steady state by controlling the concentration of the
substrate. This can be achieved by maintaining a continuous inflow and
outflow where fresh medium is added and part of the inhibitor products is
removed [86].
For the stirring system for the mixing of the substrate, we can identify
several options of mixing from propeller to plates, and as well the baffles
on the reactor walls. Other accessories on the bioreactor that support the
stirring are the air diffusers, but also its combination with mechanical
stirring is another strategy that provides a homogeneous in reaction when
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the broth presents resistance to mixing due to a saturation of substrate
or huge growth of microorganism [87]. Stirred systems with successful
results is using a blade stirrer for radial flow, it gives easiness of mixing
from powders to fluids [88, 89]. Meanwhile, the air diffusers are most
used in columns bioreactors in combination with continuous stirred tank
reactors (CSTR).
The measurement of cells in fermentation is usually the cheaper analysis
for the cell counting in Neubauer chamber via Microscopium. However,
the disadvantage is still the no on-line measurement of cells [90, 91]. More
sophisticated instrument is the use of fluorescent in situ hybridization
(FISH) however is expensive. Isaka et al. [92] compared both analyzes, the
cell counting by Neubauer chamber still more versatile for simplicity and
low cost. In new development, several works apply different techniques
to counting cells. One of them is the on-chip as high-speed camera with
high optical resolution and flow technology record to analyze in real-time
the flows [93]. Sobahi and Han [94] fabricated an on-chip system of two
layers; a crystal substrate with electrode detection and the other microfluid
channel layer on top and used Saccharomyces cerevisiae at 0.8×106 cell
mL–1 and 1×106 cell mL–1 concentration diluted in peptone and dextrose
to test.
The temperature measurement plays one of the key roles to keep high
performance in fermentation, moreover, it needs to adjust flows on jackets
and homogeneous stirring in the reactor. Temperature sensors commonly are
added into a middle place of the reactor or close to the bottom lower, but
the instrument sensor does not remain immersed inside the volume reac-
tion, usually are external in the reactor [95]. For lab reactors also is used
an electric heater, Roukas and Kotzekidou [96] used an electric heater in a
batch reactor to adjust to 30°C a fermentation by Saccharomyces cerevisiae.
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The monitoring of pH is crucial for optimum ethanol production because any

variation in pH could make operational issues and inhibit the fermentation.
Claros et al. [97] used a pH sensor in CSTR in real-time. Meanwhile, De
Vleeschauwer et al. [98] reported the use of pH sensors JUMO® BlackLine
to monitoring a sequencing batch reactor (SBR) wastewater could be applied

in a fermentation reactor.
On the other hand, another design aspect to consider is the sterilization
issue to keep an aseptic environment that through the sterilization of the
bioreactor, medium, air, and auxiliary and accessories. The sterilization
process can be carried out by thermal, physical, chemical, and radiation
process being the first two the most used. The sterilization of industrial
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reactors is normally carried out by passing steam through pipes and a jacket
for a certain time, for the sterilization of substrates the most common is
to sterilize in the same way, passing steam through pipes and a jacket at
a pressure of 0.1 mPa, or sterilize the substrate and solutions in industrial
autoclaves and subsequently being injected through peristaltic pumps into
the reactor, however, the sterilization potential can be reduced if the cleaning
of the reactor is not efficient, if there are sediment residues from previous
processes, the risk of contamination increases dramatically. However, some
accessories cannot be sterilized many times or in the same way, i.e., if the
method used is by moist heat or dry heat sterilization. Although the bioreactor
design allows high temperatures for its sterilization, also with the use of
several times, accessories like pH sensor can be suffer a sensitivity loss. The
alternatives to not apply high temperatures are the use of gamma rays, ozone
gas, or oxygen radicals [99, 100].
9.4.2 PROCESS CONDITIONS
The sugar contained in the hydrolysate are converted to ethanol by

microbial fermentation [101]. For the industrial production of bioethanol,
the microorganisms used must be resistant to environmental stress such as
that generated by an acidic pH, high levels of sugars at the beginning of
fermentation (which causes hyperosmotic stress) and can grow rapidly on
various lignocellulosic substrates [83]. The most frequently microorganism
used is the yeast Saccharomyces cerevisiae because it is a microorganism
recognized as safe, capable of consuming all types of hexoses and reaching
high theoretical ethanol yields, in addition to being able to produce ethanol
concentrations of up to 18% (v v–1) [101]. Other microorganisms also
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used are Zymomonas mobilis, Pichia stipitis, Candida shehatae, Candida

tropicalis, Pachysolan tannophilus and Kluyveromyces marixianus
(thermotolerant). Sometimes a co-culture of microorganisms is used to
ferment the hexoses and pentoses contained in the fermentation broth.
Several process conditions affect the performance of the microorganism into

convert the reducing sugars into ethanol. In a first instance, the concentration
of cells can be at moderate or high concentration, i.e., a concentration of
2×107 cell/mL is considered a moderate cell density, meanwhile 8×107 cell
mL–1 is a high one. The fermentation process should be optimized to find
the effective cell concentration taking into account the performance and
reaction time. Phukoetphim, Salakkam, Laopaiboon, and Laopaiboon [132]
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tested the viability of microorganism at different ethanol concentration
(v v–1) produced in the fermentation process, by using a concentration of
5×107 cell mL–1 to ferment a broth of 100 g L–1 of reducing sugars and in
a batch reactor system. In comparison with other bioreactor system such
as, fed-batch reactor with the same concentration of cells obtained a high
production of ethanol. Another alternative is the use of pellets with a certain
volume of cells, Ju and Ho [102] reported a 0.66 g g–1 in cell pellet, with a
cell density of 2.57 × 103.
The stoichiometric conversion of glucose to ethanol is 0.51 g g–1. Achieving
this conversion efficiency is difficult because microorganisms use a certain
part of the carbon consumed into cellular metabolism and growth. The yeast
S. cerevisiae is one of the microorganisms that manages to produce ethanol
from high loads of substrate, being able to ferment in media containing up
to 400 g L–1 of glucose. It is important to consider this parameter because
microorganisms can be inhibited by high concentrations of substrate as well
[103]. The concentration of reducing sugars depending on the resistance of
S. cerevisiae, the concentration of ethanol could reach the 11–13% (w v–1)
[104, 105]. Pereira et al. [106] used cellulosic hydrolysates for alcoholic
fermentation and it is carried out in 250 mL Erlenmeyer flasks. They use
anhydrous glucose to reach the concentration of 100 g L–1. These experiments
were conducted for 8 h. Ariyajaroenwong et al. [107] used immobilized cells
of S. cerevisiae in sweet sorghum stalk for the fermentation of sweet sorghum
juice that had a concentration of between 120 and 280 g L–1 of total sugars. In
this case, no agitation was used to prevent the detachment of the immobilized
cells from the carriers.
Several substrates have different nutrient ratios, in the case of the C:N
ratio, the relation of the carbon fermentable of lignocellulose substrates are
interesting in the election for the facility to add less amount of nutrients
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to enhance the substrate. The supplementation of lignocellulose residues

provides an acceptable range of carbon-nitrogen ratios, for the right
development of yeast, the gain of energy, and the production of primary
metabolites. Several biomasses increase their fermentable carbon after
pretreatment, to become more available, as were mentioned in his chapter,

different pretreatments are used to break the recalcitrant structure of lignin.
Usually, the media for ethanol fermentation is supplemented with a limiting
nutrient nitrogen nutrient as urea, extract yeast, peptone, and salts to enhance
the fermentation potential of the lignocellulose residues.
On the other hand, the ethanol yield is linked to parameters like tempera-
ture and pH. An average operational temperature is 28–34° C using Saccha-
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romyces cerevisiae [108, 109], although the temperature ratio can vary by
the aim of growth of yeast, Lip et al. [110] reported the growth of yeast
in several temperature conditions, where the maximum growth rate was
between 30–34°C. Most of the fermenting broth used for bioethanol produc-
tion has pH in the range of 4.5–5.5 tested in different sugars concentrations.
The pH could improve de cell growth and ethanol production or can inhibit
the microorganisms in the acid or alkali medium; however, several studies
demonstrated an optimum range for pH conditions between 4–6 [111].
Table 9.4 shows yeast strains used in bioethanol production at different
process conditions of agitation and temperature and as well the concentra-
tion of ethanol obtained from certain sugar concentration.
9.5 CONCLUSIONS
The literature reviewed in this chapter indicates that the field of novel reac-
tors designs and operation process has been intensifying in pretreatment,
enzymatic hydrolysis, and fermentation processes. This has made viable the
use of a great range of LCB and cross the gap of lab-scale to biorefinery
concept. Properly established bioreactor configuration and complex process
conditions provides an important approach insight into the lignocellulosic
bioethanol industry. The main enhancements to scale up biofuels production
process, might be related to develop novel reactors and integrated process
capable to reduce net energy consumption, as well as combined technologies
for better yields in sugars and bioethanol concentration. Finally, tecno-
economical scenarios of the design of new bioreactors configuration must
be considered to evaluate the commercial value of bioethanol production
through LCB since commercial point of view.
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TABLE 9.4 Yeast Strains Used in Bioethanol Production at Different Process Conditions
Biomass Inoculated Yeast Cells Sugar Concentration Ethanol Yield T (°C) Agitation References
(g L–1) (%) (rpm)
Sugarcane bagasse 25 g L–1 (dry basis) S. cerevisiae 100 (glucose) 54.69 31 100 [106]
–1
Sugarcane tops 25 g L (dry basis) S. cerevisiae 100 (glucose) 65.52 31 100
Sugarcane straw 25 g L–1 (dry basis) S. cerevisiae 100 (glucose) 74.70 31 100
–1
Sweet sorghum 0.38 g L S. cerevisiae NP 01 228 87.72 30 – [107]
7 –1
Sugarcane bagasse 7.8×10 cell mL S. cerevisiae AR5 78.6 (glucose) 95 33 120 [16]
Spent coffee grounds 1 g/L S. cerevisiae RL-11 52.5 26 30 200 [133]
Sorghum stover S. cerevisiae MTCC 173 200 34 30 120 [134]
Giant reed S. stipitis CBS 6054 33.4 33 30 150 [135]
Corn stover S. cerevisiae ZU-10 66.9 41.6 30 120 [136]
Ipomea carnea S. cerevisiae RPRT90 72.1 46.1 30 150 [137]
Wood S. cerevisiae 37.47 49 30 150 [138]
Reed S. cerevisiae ATCC 123 93 38 150 [139]
Water hyacinth Kluyveromyces marxianus K213 23.3 16 42 – [140]
Wheat straw 1 g/L Kluyveromyces marxianus CECT 22.8 45 42 150 [141]
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Paper sludge S. cerevisiae GIM-2 27.8 68.34 31 60 [142]
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KEYWORDS
• bioethanol
• bioreactor design
• enzymatic hydrolysis
• fermentation
• pretreatment
• process parameters
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2021).
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CHAPTER 10
Integrated Production of Ethanol from

Starch and Sucrose
C. A. PRADO,1 S. SÁNCHEZ-MUÑOZ,2 R. T. TERÁN-HILARES,3
L. T. CARVALHO,2 L. G. DE ARRUDA,4 M. L. SILVA DA CUNHA,2
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P. ABDESHAHIAN,1 S. S. DA SILVA,1 N. BALAGURUSAMY,5 and
J. C. SANTOS2
Biopolymers, Bioprocesses, Process Simulation Laboratory,
1

2
Bioprocesses and Sustainable Products Laboratory, Department of
Biotechnology, Engineering School of Lorena, University of São Paulo
(EEL-USP), Lorena–12.602.810, SP, Brazil,
E-mail: jsant200@usp.br (J. C. Santos)
3
Material Laboratory, Catolic University of Santa Maria (UCSM),
Yanahuara–04013, AR, Perú
Biopolymers, Bioprocesses, Process Simulation Laboratory,
4

5
Bioremediation Laboratory, Biological Science Faculty,
Autonomous University of Coahuila (UAdeC), Torreón Campus,
Coahuila–27276, México
ABSTRACT
In recent years, several scientific and technological advances in bio-refinery

have greatly contributed to the evolution of the ethanol industry around
the world. These contributions have increased our view and knowledge
about technologies developed for first-generation (1G) and, more recently,
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second-generation (2G) ethanol. Nowadays, advanced technologies are

employed to generate this alcohol in top ethanol-producing countries. In
Brazil, for instance, a new innovation is the flex biorefinery in the first-
generation model, which has been considered as a primary concept for
the use of corn in the off-season of sugarcane, thus increasing the annual
production of bioethanol. In this concept, there is also the possibility of flex
biorefinery with 1G and 2G ethanol production. In fact, the conversion of
sugarcane biomass into the fermentable sugars for the production of second-
generation ethanol is a promising alternative to meet the future demands
for this biofuel and could be successfully integrated with sugarcane and
corn-based 1G facilities. However, in order to attain such success and to take
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advantage of those flexible industries, the construction of a bridge between
science and industry is essential. In this view, investments in research and
development with the transfer of new technologies from the academy to the
industry are required. Furthermore, the training of skilled labor to deal with
new technological challenges is essential.
10.1 INTRODUCTION
Petroleum-based energy sources are being exhausted, because of increasing

world energy demand and the non-renewable global crude oil reserves [1–3].
As a consequence of the depletion of this conventional energy source, oil
prices are unstable and directly bring about price fluctuation in the transpor-
tation fuel [4]. For these reasons, the change of the current model to the use
of biofuels as a sustainable option is highly necessary, as a matter of energy
security [5, 172] and it causes to mitigate the GHG emissions resulting from
the burning of fossil fuels [2, 173].
Considering the aforementioned issues, many attempts are being made
to use research and new technologies for the developments of traditional
industries of biofuels, particularly ethanol.
Indeed, although a number of cars have been designed to run on ethanol,
gasoline is still the primary fuel option for cars in many countries. In this
regard, because of the 1970’s oil crisis, the Brazilian government launched
the “Proalcool” program in 1975 with the aim of reducing the country’s
dependence on oil imports. Nowadays, many cars in Brazil are flex with the
possibility of using ethanol or gasoline. In this context, there are different
policies in many countries for increasing ethanol blended with commercial
gasoline [174, 175].
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Integrated Production of Ethanol from Starch and Sucrose 273
Currently, there are many projects for the production of bioethanol across
the world, which is known greatly in line with the corn biorefinery in USA
and sugarcane biorefinery in Brazil [176], while there are other countries
investing in the utilization of this biofuel. For example, in America, Mexico
has invested in studies focused on the first-generation biomass such as

sugarcane, agave, and sorghum [177–180]. Moreover, the expansion of the
corn crop in this country for bioethanol production was evaluated [181];
however, the current legislation prohibits the use of corn as an energy raw
material, due to its importance as the main source of food in the country.
The share of ethanol in the consumption of liquid fuels in bioethanol
production improved from 55% in 2012 to 75% in 2017 in the world [182].
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The global ethanol production in 2019 was 37.8 billion gallons in which
USA and Brazil had 83% and 26% of the total, respectively [183]. In the
United States, corn is the main raw material to produce ethanol so that in
2017 it represented 10% of the demand for vehicular fuel. In Brazil, on
the other hand, sugarcane (juice or molasses) is the main raw material for
ethanol production [176].
Raw material choice is the fundamental step for the ethanol industry,
which can represent up to 42% of the production costs [184, 185]. In addi-
tion, the commercial adoption of biofuels depends on the evaluation of the
broad levels of efficiency [185, 186] considering several economic, social,
environmental, and strategic criteria, such as national energy security. Hence,
in order to use a source of bioenergy, the chosen biomass needs to meet
these requirements. The ideal characteristics for using a biomass feedstock
as a source of energy include high agricultural production, favorable natural
cycles, low energy consumption in its cultivation, a low production cost, the
low levels of contaminants, and a low demand for nutrients [6]. Furthermore,
it is important that the selection of biomass can favor the carbon balance
when considering life cycle of the biofuel, thus it can take into account the
entire production and use in the production chain [175, 176, 184, 185].
Currently, ethanol can be classified as the first generation (1G) when it is
produced from food crops such as sucrose and starch. Ethanol is known as
the second generation (2G) when it is produced from biomass residues and
byproducts such as sugarcane bagasse and corn cob, while third-generation
(3G) of ethanol is referred to the ethanol produced from microbial biomass,
namely microalgae. In this context, some authors indicate ethanol as the
fourth generation (4G) of ethanol when it is produced from genetically
modified organism, e.g., cyanobacteria through a process named ‘photo-
fermentation’ (direct conversion of light and carbon dioxide (CO2) into
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ethanol) [7]. Moreover, ethanol has been called as 1.5G when it is produced
in the 1G industry, but additional sugars from cellulosic fibers is utilized, for
example, from corn grain [8].
These concepts are important to a systematic study of new options for
the integration of biorefineries and raw materials. In Brazil, the main raw
material for ethanol production is sugarcane juice that is produced only
for the alcohol, or as more commonly observed, sugarcane molasses used
in integrated sugar and ethanol industries [9]. However, Brazil is a great
producer of corn, mainly in some regions such as central west of the country.
Thus, sugarcane has an interseason period from December to April, which is
an interesting opportunity for 1G biorefineries using sugarcane with corn as
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raw materials in flex biorefineries [184].
Indeed, corn and sugarcane biorefineries are established processes
used in the world to produce ethanol [3, 4, 10] such as Brazil so that such
countries could integrate those raw materials and take advantage of their
characteristics. The aim of the present chapter is to discuss the possibility of
the integration of the use of both raw materials (corn and sugarcane) and to
give future perspectives of the integration in flex biorefineries. In this way, a
brief general overview is presented in the first sections.
10.2 MAIN CARBON-RICH RAW MATERIALS FOR ETHANOL

PRODUCTION IN DIFFERENT COUNTRIES
Several countries have evaluated the production of ethanol from biomass

feedstocks available in their region. There are a variety of renewable carbon
sources with the appropriate potential for bioethanol production in integrated
biorefineries [187]. Corn, for example, is produced in large quantities for
ethanol generation in the world compared to any other crop. Around 850
million tons of maize kernels are produced worldwide, indicating an average
productivity of 5.2 t/ha [11].
Vegetable biomass is classified as different carbon sources according to
the type of carbohydrate. They can be divided into starchy compounds such
as corn and cassava, sucrose-based substances (sugarcane juice) and lignocel-
lulosic materials such as sugarcane bagasse, corn stover and corn straw [12].
Table 10.1 shows a number of potential biomass for ethanol production
in the world. It is noteworthy that lignocellulosic biomass (LCB) represents
appropriate raw materials for the production of bioethanol. Agro-industrial
residues and woody by-products include residues derived from forest, the
paper and cellulose industries (sepilho, shavings, and declassified chips of
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eucalyptus and pine), sawmills (sawdust), and agricultural residues produced

from crops such as cereal straw, corn, wheat, corn cob, rice husks and oats
[188]. In this context, it has been estimated that more than 75.73 million
tons of dry biomass are generated worldwide from crops and agro-industrial
residues.
TABLE 10.1 Main World Carbon Sources (Starchy and Sucrose-based Ones) for Ethanol
Production
Countries Biomass Ethanol Production Biomass Residual References
Production (million gallons) in Biorefinery
(million tons) (million tons)
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United States 342 (corn) 149 75 (corn ethanol 68.25 (corn straw) [13, 14]
(data of 2018) (biomass from production)
corn for ethanol
production)
Brazil (data of 642.7 26 (sugarcane ethanol 96.3 (sugarcane [11, 15,
2019) (sugarcane) production) 2.5 (corn bagasse 189]
95.60 (corn) ethanol production lignocellulosic)
Europe Union 61.60 (corn) 13 (corn) 21 (beet) 64.28 (wheat [7, 14]
(data of 2019) 168.28 (wheat) straw)
35.82 (beet)
China (data of 215.00 (corn) 8.45 (corn) – [16]
2015)
Canada (data 203 (corn) 4.36 (corn) – [17]
of 2019)
India (data of 115.6 (rice) 2.23 (rice) 22.57 (rice straw) [18, 190]
2019) 85.72 (corn)
21.16 (wheat)
Considering the large quantity of raw materials, ethanol production

industries could be established as flexible, allowing the utilization of
different biomass feedstocks according to the seasonal availability. Indeed,
the seasonality of the crops is an important issue to be considered for the
production of biofuels. Sugarcane production, for example, has a well-
defined off-season, from December to April. In addition, as a new solution,
integrated biorefineries have been installed in the Central-West region of
Brazil, considering the production of alcohol from corn in those months
[187]. Therefore, ethanol production will cease, if no raw material is supplied
during December to April. This fact occurs due to the perishability of
sugarcane, which cannot be stored higher than 48 hours [191]. This problem
is not common for corn, and thus, it is a suitable alternative to sugarcane in
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the interseason period of sugarcane cultivation in Brazil [19, 192]. Thus, the
utilization of sugarcane and corn reveals an interesting example of a highly
flexible and productive biorefinery.
10.2.1 CORN
The raw materials used for the first-generation fuel ethanol are mainly crops
rich in starch or sucrose such as corn, cassava, and sugarcane [2, 20]. Corn
(Zea mays) is a cereal grain, which is widely grown all over the world. It is
widely used as a staple food for humans or as animal feed, due to its numerous
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nutritional properties. All studies suggest that its origin is Mexican since its
domestication started 7,500 to 12,000 years ago in the center of Mexico [21].
Maize contains facilities for its cultivation and therefore has a great potential
for production and adaptation to technology. It has a mechanized cultivation
that benefits greatly from modern planting and harvesting techniques [20].
The world production of corn was 850 million tons in 2019, which was more
than rice (678 million tons) and wheat (682 million tons) [184]. Corn is
grown in different regions of the world. The largest producers are the USA
and China, which have produced 37 and 21% of the total world production,
respectively [11, 22].
10.2.2 SUGARCANE
The sugarcane cycle plant has an average of six years, in which four or five
harvests occur. Sugarcane belongs to a group of tall perennial grass species
of the genus Saccharum. This plant is native in the tropical regions such as
the South of Asia and North of Brazil, and can be used for the production
of ethanol and sugar [193]. Its stems are robust, articulated, and fibrous.
Sugarcane height can be two to six meters. Researchers have been mixed
sugarcane species to develop complex hybrid for enhancing its performance
in different areas. Sugarcane belongs to economically important plants such
as corn, wheat, rice, and sorghum [194, 195]. Sucrose is the principal sugar
obtained and can be extracted and purified in biorefineries for further use as a
raw material in the food industry, or it would be fermented to produce ethanol
[196]. In 2019, sugarcane grew on about 26.0 million hectares of agricultural
lands in more than 90 countries, with a worldwide harvest of 174 million tons
[184]. Brazil is the largest producer of sugarcane in the world. Other largest
producers are Thailand, India, China, Pakistan, and Mexico [11].
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10.3 PROCESS IN BIOREFINERIES FOR ETHANOL PRODUCTION

FROM CORN AND SUGARCANE
The development of biorefineries leads to the efficient utilization of the LCB,

which can be used directly in the generation of bioenergy or serve as raw

materials for chemical, enzymatic or fermentative processes [23, 197]. Those
integrated facilities can work with different raw materials and technological
routes, aiming to simultaneous production of different valuable products.
The research on new methods for biorefinery integration in power plants
for production of biofuels from corn and sugarcane can reduce costs and
increase profits. For this purpose, the understanding of how each biorefinery
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works and what are their bottlenecks of production is crucial.
10.3.1 CORN TO ETHANOL BIOREFINERY
The production of ethanol from corn is an established process used in

American biorefineries [198]. Commercial ethanol fuel is obtained through
the following main steps including the conversion of starch into simple sugars
(fermentable sugars), the run of fermentation process and distillation process
[14, 24–26]. The process of breaking the starch polymer into fermentable
sugars consists of three sequential unit operations, namely milling, liquefac-
tion, and enzyme-based saccharification [14, 21, 27].
Milling is a physical process that aims to reduce particle size, in which
corn is processed through a hammer mill to produce corn flour. There are
two well-established methods to produce bioethanol from corn and the major
differences between them are related to this step. Wet-milling ethanol produc-
tion is a large-scale capital-intensive process that consists of steeping corn
grains in water to facilitate the separation of the various fractions of the corn
kernel (starch, fiber, germ), allowing them to be processed to obtain various
by-products. Dry milling is the most common process, corresponding to 90%
of the US operational plants [14, 20, 28, 199]. In this case, the compounds of
the grain are not separated and they are processed with the starchy fraction.
Proteins, oils, and other composts remain unchanged in fermentation and
will be separated further [21]. Since the latter is more common, it will be
more discussed in detail.
In the conventional dry-grind process, after grinding grains in hammer
mills, the cornflour is mixed with water to form corn slurry with 27–37%
of solid content [29]. Then, the pH value of the mixture is adjusted and
α-amylase, a thermostable enzyme, is added [24, 200]. The next step is called
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liquefaction or cooking, in which the slurry is heated to high temperatures

to soften the tightly bonded grain of starch that becomes more digestible.
The jet-cookers inject steam at high temperatures and high pressures. In this
step, the process time depends on how the high temperature is utilized. The
slurry can be cooked at 165°C for 3–5 minutes or at 90–105°C for 1–3 hours
[14, 27], resulting in a mixture with gelatinized starch that is converted to
dextrins and oligosaccharides by -amylase [20, 30].
The output from liquefaction is called corn mash which is rich in
short-chain saccharides, showing partial solubility with no fermentative
characteristic [30]. The corn mash is cooled approximately to 30°C [14,
21], and it proceeds to the saccharification process, in which the conversion
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of dextrins into fermentable sugars occurs. This hydrolysis happens in the
presence of glucoamylase (GA, also called amyloglucosidase or AMG) for
converting dextrins into glucose [10, 201]. In the past, the saccharification
was carried by acid hydrolysis in a hazardous operation that occurred in
extreme conditions of temperature and pH with sugar production yields of
85% [20]. The utilization of enzymes led to reduce the disadvantages and
raise the yield up to 95–97% [30].
The corn hydrolysate is subjected to ethanol fermentation in which yeast
Saccharomyces cerevisiae consumes glucose and produces ethanol and
CO2 under anaerobic conditions [1, 31, 200]. The yeast grows in seed tanks
and then is transferred to the sugary syrup in the fermentation tanks [21].
Fermentation is the main operation in a distillery plant. It runs for 42–55 h
using multiple fermenters as batch operation of the plant [32].
There are many different possible configurations for this process. The
most common modes are SHF (separate hydrolysis and fermentation) and
SSF (simultaneous saccharification and fermentation) [202]. The main
benefit of the SSF process is to avoid the osmotic shock to yeast due to
the high glucose concentrations. In this process, glucose is slowly released
through the saccharification process and immediately consumed by the yeast
to produce ethanol, preventing a glucose concentration that can cause inhibi-
tion [33, 203, 204]. The SSF technique can provide up to 8% more ethanol
than the SHF for the same amount of grain [24, 32].
The fermentation product is composed by a liquid portion containing
14–20% of ethanol [29, 199]. Thus, the distillation goal is to obtain a
product with higher ethanol purity (92–98%) [21, 34]. Ethanol is separated
from water and non-fermentable residues which form the stillage [201]. This
process can be separated in two stages. In the first column, unconverted
solids and heavy compounds are removed at the bottom, while the top outlet
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feeds the second column with a liquid portion containing 30–40% ethanol. In
the second column, ethanol is purified in which hydrous ethanol is obtained
[32]. Pure ethanol can be recovered by combining distillation and molecular
sieves which capture residual water to achieve anhydrous ethanol [35].
The stillage includes fiber, oil, and protein components of the grain,
as well as non-fermented starch. The stillage can be processed to obtain
byproducts [24]. It can be centrifuged to separate the solid and liquid
fractions and produce a variety of coproducts known as distiller’s grains
[36]. The most popular is distillers dried grains (DDGs) which is used as
animal feed [14]. In addition to animal feed, another coproduct obtained
from the dry grind operational plants is distillers corn oil. It is recovered from
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the concentrated stillage liquid portion obtained from the centrifugation of
whole stillage [10, 198].
The requirement of high temperatures is an unfavorable factor in bioethanol
industries, since it increases the energy demand, which adversely affects the
cost-effectiveness of the product, even though the elevated temperatures help
contamination control [37]. In the context of 1G corn ethanol, some of the
research’s address reducing operational costs such as the energy demand [38].
Other issues that also draw attention are crop improvement [5], new enzymes
[3], genetic engineering in yeast strains [39, 40] and coproduct recovery
(Figure 10.1) [41].
10.3.2 SUGARCANE TO ETHANOL BIOREFINERY
In Brazil, this biomass is largely used for 1st generation ethanol [6, 199].
The 1st generation ethanol is produced by processing sugarcane via
fermentation of sucrose-rich juice or molasses [42, 43]. In integrated sugar
and alcohol industries (the most common), there is a strict relation between
ethanol production and other main products (sugar and energy generated
by burning bagasse) which is influenced by market demand, within the
biorefinery concept [44, 45]. Figure 10.2 exemplifies a common industrial
processes of a sugarcane biorefinery (1st generation) with joint production of
ethanol, sugar, and energy.
At the reception, the harvested cane is aimed at cleaning tables and the
feeding system. Furthermore, in this stage the impurities, such as vegetable
and mineral residues are removed. If the harvested cane is chopped, a dry
wash is adopted to minimize the loss of sugars during the reception process.
Then, the clean cane is sent for preparation [46, 195].
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In the preparation stage, the sugarcane is crushed by a series of crushing

knives, allowing a uniform plant material, facilitating the next stage of juice
extraction. Before extracting the juice, the chopped cane is submitted to a
magnet treatment to remove metallic residues [189]. In the juice extraction
stage in a usual process, the chopped cane is crushed via mills (a set of three to
five cylindrical rolls), where the juice is separated from the vegetable fibrous
fraction (bagasse). They are usually sequential mill systems. The last set of
milled sugarcane is soaked into warm water, a way to increase the extraction
of residual sugars [200]. Despite the separation that has been already done,
a fibrous fraction still remains in the juice. In order to remove it, sieves are
used, and the solid material is sent for recirculation in the mill system. The
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juice from the first group of mills is often utilized for sugar production, due
to its great degree of purity and concentration of sugars. On the other hand,
the juice of the other groups of mills which are combined with molasses is
used for ethanol production [202–206].
FIGURE 10.1 General scheme for the production of 1G corn ethanol in biorefinery.
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FIGURE 10.2 General scheme for the production of 1G ethanol, sugar, and energy from
sugarcane in biorefinery.
The juice treatment is aimed at removing residual impurities in the juice. It

consists of a set of physical-chemical treatments, ranging from the separation
of fibers, sand, and particulate material with the use of sieves, heating of the
juice (30 to 70°C), the addition of lime and phosphoric acid, followed by a
second heating (105°C), and an addition of flocculating agent until clarifica-
tion of the juice [47] [184]. Depending on the manufacturing, this process
may be simpler once it is adapted according to the type of mill used and the
purity of the juice after extraction. In some cases, the impurities removed in
the juice treatment are still used for the recovery of sugars by filtration [6, 48].
The concentration of the treated juice is adjusted (juice concentration
stage) to an ideal value for fermentation [7]. Typically, a molasses mixture
obtained from the production of sugar is firstly combined with the treated
broth, and when necessary, it is secondly used by evaporators for concen-
tration adjustments [49]. Therefore, the juice is prepared for fermentation,
which consists of a biological conversion of sugars into ethanol by the yeast
Saccharomyces cerevisiae with the generation of CO2 and other by-products
(organic acids, higher alcohols, etc.), [48, 50]. The fermentation is conducted
in the batch mode and is fed by cell recycling from the previous fermentation.
The process is carried out in closed reactors continuously or discontinuously
at a temperature of 30 to 34°C for a few hours [6, 51].
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As a result, a liquid with the low ethanol content (maximum 10° GL)
is obtained, and CO2 which is still washed in adsorption columns is used
to recover transported ethanol. After fermentation, the juice extracted is
submitted to centrifugation, and the cells are removed in this process,
followed by treating with water and sulfuric acid solution as a method to

reduce future contamination [207, 208]. It is then stored and sent to the
following fermentation batches [13, 52].
The liquid proceeds to the distillation/rectification stage, where ethanol is
purified in distillation and rectification columns, resulting in hydrated ethanol.
This ethanol can also increase the degree of purity and it is dehydrated in
alternative purification systems such as azeotropic distillation with cyclohexane,
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extraction using monoethylene glycol, or adsorption in sieves [53, 209].
The first generation biorefineries are considered a consolidated tech-
nology with a low-cost production line. However, they are still dependent
on agricultural resources, arable land, climatic conditions, domestic supply
market and investment capital which should be implemented in new regions
or countries [184].
The industrial facilities for the production of 1G ethanol are also designed
for joint production of sugar and energy cogeneration. This has been resulted
from the burning of existing bagasse and straw, as well as the generation of
other products such as biogas obtained from fermented broth and proteins in
the form of yeast cells [13].
The production of sugar within the biorefinery follows common lines for
the production of ethanol. In this approach, the concentration of the treated
juice is adjusted so that heated processes of evaporation of the broth, crystal-
lization, and drying of the sugars occur (humidity between 0.5 and 2%) [52,
205]. Molasses obtained after crystallization can be used to produce ethanol.
In the energy cogeneration system, bagasse (for mechanized harvests) is
burned in a boiler and produced steam is driven to turbines, which in turn
are connected to electric generators [49, 54]. Thus, a part of the generated
electricity can be sold to distributing companies, and the exhausted steam
can also be used in various operations at process units that require thermal
energy [23, 55, 56, 208].
10.4 2G AND 1.5G ETHANOL FROM BY-PRODUCTS OF CORN AND

SUGARCANE
The first-generation bioethanol is already a well know and established tech-

nology [57]. However, the increasing ethanol demand results in the feedstock
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production, affecting the food and land usage concerns [19, 57]. In order to
better apply the feedstock instead of the focus on the increase of its production,
the second-generation biofuel has been a prominent technology since it relies
on lignocellulosic materials [58]. Lignocellulosic substances are variable and
available materials, which can be divided into agricultural residues, forest resi-
dues, municipal solid waste, and energy crops (herbaceous or woody plants)
[2]. The main components of this biomass are cellulose, hemicellulose, and
lignin with small quantities of extractives and ashes [59]. Some examples of
biomass applied for the second-generation bioethanol are sugarcane bagasse
[60], eucalyptus [61], hazelnut shell [62], corncob [63], banana crop [64], rice
straw [65], coconut husk [58], and sugar beet [210]. Each biomass presents
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specific quantities of cellulose, hemicellulose, and lignin so that sugarcane
bagasse, for example, is composed of 20–25% lignin, 40–50% cellulose and
30–35% hemicellulose, making it a potential feedstock for 2G biofuel [57,
66]. Another example of potential biomass is sugar beet which is also used
for bioethanol production and presents a high quantity of hemicelluloses
(24–32%), and cellulose (22–30%) with a very low lignin amount [210].
Even though the lignocellulosic residues are abundant and do not interfere
in food production, it is necessary to optimize the biomass bioconversion
in order to produce an economically viable second-generation bioethanol
[66]. Bioconversion of lignocellulosic materials consists of four main steps
including pretreatment, enzymatic hydrolysis, fermentation, and product
recovery [67]. In general, pretreatment contributes to the second-generation
bioethanol production by facilitating the subsequent hydrolysis through the
modification of the amorphous region and porosity of matrix with separating
cellulose from hemicellulose and the lignin [68]. Many technologies have
already been developed for lignocellulose pretreatment, and they can be
divided into physical, chemical, physical-chemical, and biological [211].
Each type of the pretreatment has demonstrated advantages and disadvan-
tages, acting in different methods to facilitate cellulose hydrolysis.
A large number of technologies have been focusing on the development
of an efficient and economic lignocellulosic pretreatment. Pretreatment is
one of the main macro-steps of the process in biorefineries and is closely
related to the subsequent stages of hydrolysis and fermentation. Therefore,
it is necessary to use a method that presents, in addition to high efficiency,
low energy consumption and reduced chemical catalysts to result in a high
recovery of carbohydrate fractions, low or no formation of fermentation
inhibitors and high simplicity for use in a larger scale.
Physical methods include milling [69], extrusion [70], and irradiation
[71]. Different methods of irradiation such as gamma rays and microwave
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have demonstrated to make significant changes in the lignocellulosic mate-

rial including the increase of surface area, expansion of pore volume and the
decrease of degree of polymerization (DP) [211].
Chemical pretreatment is also commonly applied to enhance delignifica-
tion and reduce cellulose crystallinity. Some examples of chemical pretreat-

ment are acid [72], alkaline [212], organosolv [73] and ionic liquids (ILs)
[74]. A combination of two different chemical pretreatment, such as acid and
alkali pretreatment can be applied as demonstrated by Gomes et al. [213].
The authors developed a the two-stages sugarcane bagasse pretreatment with
sulfuric acid followed by sodium hydroxide which was combined with high-
solid enzymatic hydrolysis, resulting in more than 150 g/L of glucose [213].
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Other examples of combinations are steam explosion (SE) [75], ammonia
recycle percolation [214], microwave-chemical [76], and hydrodynamic
cavitation (HC) [77]. For example, in an optimized condition of NaOH
pretreatment, a maximum glucose production equivalent to 85% of enzymatic
digestibility is observed. For the pretreatment of CH, it is possible that this
number reaches 96% digestibility. This is a new solution for the bottleneck
regarding 2 G produce ethanol [78]. Pretreatment based on HC has advantages
compared to other methods, such as the requirement of milder pretreatment
conditions and shorter process times [77]. Microorganisms such as fungi
and bacteria are also used in lignocellulosic pretreatment. They are capable
of modifying and degrading the complex structure into simpler substrates
by enzyme digestion. Some examples of microorganism are white-rot,
brown-rot, and soft-rot fungi. White-rot fungi stand out for providing
better sugar yields [215, 216]. However, microbiological pretreatment has
considerable disadvantages, especially the low hydrolysis rate caused by the
presence of inhibitors and the broth conditions [79].
After the pretreatment, it is necessary to convert the polymeric carbo-
hydrate obtained from lignocellulosic material into fermentable sugars
(Figure 10.3), due to the yeast inability to process carbohydrate polymers
[217]. Therefore, two hydrolysis methods can be applied in order to provide
second-generation bioethanol production, including acid and enzymatic
hydrolysis, which are chosen according to the biomass composition [67].
Acid hydrolysis can be performed by dilute acid or concentrated acid with
demanding different temperature and pressure conditions [17]. Sulfuric acid
(H2SO4) [80] is mostly applied for acid hydrolysis, however, researchers have
also developed different methods, including hydrochloric acid (HCl), nitric
acid (HNO3), and phosphoric acid (H3PO4) [81]. Enzymes can also be used to
hydrolyze both cellulose and hemicellulose components, in order to produce
fermentable sugars. Enzymatic hydrolysis (enzymatic saccharification) has
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Apple Academic Press Integrated Production of Ethanol from Starch and Sucrose
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FIGURE 10.3 Schematic process of ethanol conversion for 2nd generation of biomass (stages for 2G pretreatment, enzymatic hydrolysis, and
fermentation).
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high advantages such as the requirement of mild process conditions (low

temperature and atmospheric pressure) with avoiding acid corrosion [17, 82].
This type of hydrolysis enhances the soluble sugar production by cellulase,
xylanase or amylase [82]. In order to reduce the cost of 2G bioethanol and
make it large scale commercialization, it is necessary to improve the process

economics, for example, by decrease in the cost of cellulase production and
enzymatic hydrolysis [83].
Fermentation is the main process for bioethanol production. It converts
soluble sugars into the alcohol by the metabolic process of microorganisms.
A range of microorganisms can be applied for the fermentation process,
however, not all of them can process both pentoses (e.g., xylose and
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arabinose) and hexoses (e.g., glucose and mannose) [67, 218]. A co-culture
system has been reported in the literature, favoring both pentose and hexoses
fermentation. Farias and Filho [84] evaluated a co-culture fermentation using
hexoses-fermenting yeasts (such as Saccharomyces cerevisiae) and xylose-
fermenting yeasts (Scheffersomyces stipitis and Spathaspora passalidarum),
resulting in a maximum ethanol titer of 49.2 gL−1. Four main alternative
processes are commonly developed for bioethanol production, namely SSF,
SHF, SSCF, and consolidated bioprocessing (CBP) [7]. Each alternative
interferes in the process with advantages and disadvantages, giving the
lignocellulosic bioethanol production options according to a variety of the
processes, especially by consideration of the feedstock, microorganism, and
final product [41, 208]. Due to the many advantages of SSCF process, this
process was investigated for sugarcane bagasse pretreated with alkali assisted
HC. With the developed methodology, 62.33% of the total hydrolyzed carbo-
hydrate and 17.26 g/L of ethanol production were obtained, indicating 0.48
g of ethanol/g of glucose and xylose consumed by Scheffersomyces stipitis
NRRL-Y7124 [219].
Overall, the use of LCB for the second-generation bioethanol has
presented many advantages. The sustainable pathway, fossil fuel substitu-
tion, and the use of a variety of feedstock are some benefits of the second-
generation ethanol production [19]. Despite technological barriers, plants
of 2G bioethanol are already operating in the world [6]. Some examples
of the second-generation commercial ethanol plants are GranBio, Raízen,
Poet-DSM, BetaRenewables, Abengoa (plant bought by Synata Bio), and
DuPont [85]. Among them, two Brazilian companies, namely GranBio and
Raizen have reached commercial scale by predominantly using sugarcane
bagasse as the LCB [86]. In Nevada, Iowa, USA, the cellulosic ethanol plant
of DuPont has developed a projected production of 30 million US gallons of
bioethanol from corn stover as a lignocellulosic material [87].
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Considering a hybridization mode of the first and second-generation

technologies, the 1.5 generation technology uses the sugars remained in
the corn grain fiber in corn-based ethanol plants [88, 220]. The 1.5G is a
new ethanol production technology, which uses the same industrial facilities
for the production of the first-generation ethanol, in particular corn ethanol

facilities (the platform where this technology was first implemented) [89].
This technology was incorporated at the end of 2016, in corn ethanol
refineries in the United States (USA), due to the ethanol and corn oil produc-
tion increase and the environmental interest for using a cellulosic origin [14].
The 1.5 G technology was proven and tried in the pilot fermenters (15,000
gals) for production (585,000 gals) up to 1,000 hours [221]. The first 1.5 G
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biorefinery was built in March 2017. In this first plant, the ethanol produced
up to 5 million gallons of cellulosic ethanol per year was attained using this
technology [185]. In the corn ethanol refineries, this form of ethanol produc-
tion is combined with the 1G. Moreover, the 1.5G technology is still attractive
for the usage of cellulosic raw material, and it allows a reduction in GHG
emissions and indulges companies with producer titles. Therefore, renewable
products can receive technological and government incentives [14, 90].
The corn grain fiber, which is used in biorefineries for the production of
1.5 G ethanol, has some particular characteristics. Firstly, it includes its pure
and homogeneous composition formed by cellulose, hemicellulose, residual
starch, and little lignin, being a minimally recalcitrant source. Furthermore,
this material requires fewer pretreatment steps which support the diminishing
of the coast of ethanol production [220, 222].
Production of 1.5 G integrates a process that transforms corn fiber to
cellulosic ethanol with existing ethanol plants. In this way, the pathway to
cellulosic ethanol is accomplished by combining mechanical, chemical,
and biological processes. The fiber stream is submitted to an acid pretreat-
ment that deconstructs it; thus, it can access the cellulose. Additionally,
the cellulose stream is broken down into sugars with the enzyme cocktail
(Novozymes), and then the C5 and C6 sugars are transformed into ethanol
with other process detailed in the following parts [8].
The 1.5 G process was developed through the collaborations with the two
world-leading biotechnology companies. Nowadays, two different ways are
adopted for the bioconversion of the corn grain fiber into ethanol. One way is
to adopt the process separately, where the hydrolysis of starch and grain fiber
occurs separately. On the other hand, a second way is to adopt combined
systems of pretreatment of the grain (starch and fiber) before fermenta-
tion [14]. The companies that start this process have with ICM’s patented
technologies, selective milling technology (SMT), and fiber separation
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technology (FST). SMT selectively grinds corn slurry to make the starch and
oil more accessible in the entire process. Novozymes provided the enzyme
cocktail which converts the cellulose stream into accessible sugars. DSM has
developed yeast that ferment both the C5 and C6 sugars.
Fiber enzymatic hydrolyzes and fermentation processes can be carried

out separately or in the SSF process [223]. In both processes, enzymatic
cocktails are used in the low doses composed of cellulase for converting
cellulose, hemicellulose, and residual starch into sugars (C5 and C6), which
are used for fermentation [224]. Subsequently, the liquid obtained is sent to
distillation and dehydration, following the same processes as the production
of 1G corn ethanol. Protein and non-fermentable residues are removed and
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presented in the aqueous fraction after distillation/dehydration, which are
still utilized for animal feed [222].
The 1.5 G technology demonstrates a great potential for ethanol generation,
and its production process has a great potential to be adapted in a smoothie
way for the facilities already existing in a corn starch ethanol plant [14]. Some
future considerations can be suggested for the improvement of lignocellu-
losic bioethanol, such as process optimization to minimize water and energy
usage, the development of engineered microorganisms and the combination
of different microorganisms, which increases the fermentation capacity for
bioethanol production [91]. In addition to these strategies, a significant reduc-
tion in the enzymes and pretreatment costs is necessary to improve the yield
and productivity of the bioethanol for increasing its commercial scales around
the world [92, 93]. Therefore, integration, and consolidation to improve the
whole second-generation bioethanol production will make this a competitive
market with petroleum fuel in the biorefinery process.
10.5 INTEGRATED CORN AND SUGARCANE TO ETHANOL

BIOREFINERY (1ST GENERATION FLEX INDUSTRIES)
Even with the high performance of the ethanol production process, there is still
a way for top countries such as Brazil to increase its productivity even more.
This could occur due to the approach of several crops with higher productivity
in terms of ethanol per unit of area [94]. For example, in Brazil, sugarcane
can be produced between 6,000–7,500 L/ha, compared to sugar beet (EU)
5,500 L/ha and corn (USA) 3,800 L/ha [95–97]. This higher productivity of
sugarcane implies the lower ethanol production costs. However, even with
a harvest cycle higher than that of corn or sugar beet, there is an off-season,
in which sugarcane cannot be processed, since it is not possible to store it.
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Nevertheless, this is different from corn, which can be stored all year round
[96]. Both biomass has differences in relation to their derived products. For
example, in addition to ethanol, sugarcane gives a rise in electricity and
sugar. This underproduction has a strategic value for the sugarcane mills, as it
allows the capture of value in different markets [48]. In this context, corn can
raise ethanol and other food products, such as oil and protein for animal feed
(BBGDS). According to RFA [225], for each corn unit converted to ethanol,
a third part returns to the animal nutrition. However, corn bioprocess plant is
not self-sufficient in energy terms as the Brazilian one.
Therefore, con is the biggest gain for the Brazilian bioenergy process to
invest in new biorefinery models, such as the "flex" ones, which are capable
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of integrating two different feedstocks, namely corn and sugarcane, or the
use of their residues as an alternative feedstock [9]. In the last case, one
strategy for biorefinery model occurs when it integrates residues of second-
generation biomass in first-generation process, such as sugarcane bagasse
and straw, which could help the sugar-energy sector to overcome low levels
of profitability of ethanol [98].
Fuel ethanol production generally consists of five-stage process: (i) raw
material collection; (ii) pretreatment; (iii) hydrolysis; (iv) fermentation; (v)
separation and (vi) dehydration (downstream). However, there are some
differences when it is produced from different feedstocks [99]. In the case of
flex biorefineries, some steps are adapted and currently these are key points
of challenges to increase conversion yields. Some of these integrations and
flex modes are presented in Figure 10.4.
Moreover, the application of alternative biorefinery processes help to
reduce the momentum for investments in the construction of new plants in
the region. At the same time, this situation needs intense search for innova-
tions with the potential to increase the profitability levels of these young
companies. This incentive could increase the production of corn ethanol
in flex mills to 1.3 billion liters [226]. Other phenomenon that this process
integrates is fermentation of sugarcane juice (or molasses) and starchy
feedstocks, providing a number of advantages over conventional distilleries
[94]. This process has a faster fermentation (34–36 h) in comparison with
the traditional process (45–60 h) adopted by distilleries in the USA. In this
approach, less sugar is deviated for yeast multiplication and production of
cellular biomass [227]. Thus, this mode of “flex” biorefinery uses the same
distillation system applied for sugarcane and extends the period of ethanol
production to 345 days a year, reducing initial investments and fixed costs.
For this new process each corn ton allows to produce 415 L of ethanol and
250 kg of DDGS (dry distillation grain) [94, 96, 228].
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FIGURE 10.4 Biorefinery integration of 1st and 2nd generation of sugarcane and corn as feedstock. [Abbreviations: CS: corn stover; CC: corn cob;
SCS: sugarcane straw; SCB: sugarcane bagasse).
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Therefore, the integration of ethanol production with sugarcane and

corn has the potential to offer the most significant economic advantages
in comparison to standalone units, since important operations such as
inoculum, feedstock, laboratories, and technical personnel may be shared
between them [9]. Flex-fuel plants of corn and sugarcane have an energy
balance and reduction of GHG emissions that do not affect the fermenta-
tion performance. This process is economically viable in regions with corn
supply at low prices and high demand of DDGS for animal feed. Thus, it is
an opportunity for Brazilian biorefineries in corn producing regions [96].
In addition, ethanol production from starchy gives two feedstock options to
reduce the end problem of biofuels, and to enable the use of LCB-derived
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sugars for the other best production [23, 100, 227].
In the case of lignocellulosic materials, the cellulose and hemicellulose
content account for more than 60% of dry weight (ex. sugarcane bagasse)
and can be converted into fermentable sugars either by acid or enzymatic
hydrolysis [7, 100]. These sugars could provide substrate to produce not only
ethanol, but also other biofuels such as butanol and 1-methlypropanol. All
those strategies could make the possible development of a new economically
and energetically panorama for flex biorefineries [227].
For example, in Brazil, there are two industrial plants integrated
into operation for the production of second-generation ethanol that use a
semi-industrial process. These processes were originated from investment
programs of the Brazilian government in the second-generation ethanol
launched in 2011 [96, 101]. Nevertheless, the production costs of the second-
generation ethanol are still high, mainly concerning equipment handling at
bagasse pretreatment and the use of enzymes [96, 102].
Comparing characteristics between the different types of biomass,
researchers have found that sugarcane is currently the most promising
source for the production of biofuels [229]. However, the flex biorefinery
shows that it is possible to have one process more promising that sugarcane
biorefinery [94, 96]. Table 10.2 shows different situations corresponding to
the first- and second-generation plant scenarios dedicated exclusively to 1G
processing (situation 1), and corn processing (situation 2). This case shows
consolidated ethanol production in the world with different biomass. Situa-
tion 3 shows the lowest amounts of GHG compared to all other processes
studied in Table 10.2. Moreover, this situation could impact in a positive
way, when corn is integrated into a sugarcane flex plant. The main factor that
contributes to decrease GHG emissions is the inclusion of corn in integrated
biorefineries (situation 3 and 4) which in this process exists the higher ethanol
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TABLE 10.2 Examples of Different Processes of Ethanol Production in Biorefineries Flex with Two Different Biomass Feedstocks (Sugarcane 292
and Corn/1G and 2G)
Biorefinery Process Characteristics, Advantages, and Limitations References
1. Sugarcane ethanol 1G • Conventional soil preparation [98, 106, 107,
• Mechanized planting and harvesting 231]
• Without pre-harvest burning

• Requirement of a shorter planting area
• High yield of sugarcane ethanol production (77 ton/ha)
• Industry does not work in sugarcane crop off-season
2. Corn ethanol 1G • O straw collection [7, 14, 108]
• High mechanized farming and livestock
• Chemical pest control.
• Corn is a popular and highly successful agricultural crop available worldwide.
• It requires a larger planting area.
3. Flex corn and sugarcane • Flex plant with the processing of corn only in the off-season of sugarcane. [109, 110]
ethanol (Flex-Example 1) • Cell recycling.
• The inclusion of corn in the system increases the production of bioethanol and decreases
the pollution emission in the air.
• There is a loss of sugars in solids by removal operation before the fermentation step.
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4. Integrated biorefinery of • This biorefinery work with the corn and sugarcane in all 12 months of the year [7, 98, 104]
corn and sugarcane ethanol • In this process, there is a six-year cycle (planting + five cuts).
in Midwest Brazil (Flex-
Example 2) • Possibility for co-fermentation of the broth of the two raw materials during the sugarcane
harvest. It can solve probable operational problems (for example, stoppages).
• This biorefinery has reduced the impetus for investments in the construction of new plants
in the Midwest Brazilian region.
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TABLE 10.2
(Continued)
Biorefinery Process Characteristics, Advantages, and Limitations References
5. Corn and sugarcane ethanol • In this biorefinery, corn is stored and used together with sugarcane to produce ethanol for 8 [48, 96, 108]
integrated production months per year.
off-season (Flex-Example 3) • This type of biorefinery, with the integration of the sugarcane and corn culture, can
increase the production of Brazilian ethanol.
6. Flex sugarcane 1 G, 2 G • Cellulosic materials such as bagasse which are already present in the 1G ethanol production [7, 48, 103,
chain could be used as raw material for 2G production by conventional infrastructure. 105]
• High pretreatment price
• High enzyme cost
• Low pentose fermentation yield
Integrated Production of Ethanol from Starch and Sucrose
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293
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production and it is possible to achieve this yield because of the amount of

corn processed [9, 230].
Another possibility is the co-fermentation of sugarcane and corn broth
produced during harvesting, which makes it possible to solve operational prob-
lems (for example, stoppages at the sugarcane milling), incorporating gains in

the production system. In this context, the integration of the sugarcane culture
with the harvest of up to eight months, into other energy crops such as the corn
could increase the sustainability of ethanol as a bioenergy [9]. Other flex plants
are capable of processing sugarcane and bagasse from sugarcane (situation 5),
and they could help the sugar-energy sector to overcome the current adverse
environmental situation and decrease the levels of ethanol profitability [94, 227].
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Furthermore, the situation 6 could reduce the investments in the construc-
tion of new plants in the world, because of the use of 2 G biomass [103]. At
the same time, this situation sparks an intense research for innovations with
a potential to increase the profitability levels of these young sectors, and for
the investment in more flex bioprocess [104]. One disadvantage is that this
process needs an overwork on the logistics side, because freight and storage
is not a simple issue for industry [13]. In addition, the 1G + 2G configuration
has the greatest efficiency (44.4%) compared to 1G biorefinery configuration
[105]. Another problem for this situation is that investors demand a return of
5-fold investment. However, the most important factor in situation 6 is the low
investment (50 million to adapt an existing biorefinery) of these biorefineries,
which opens many possibilities for developing countries [14, 103].
10.6 TECHNOLOGICAL CHALLENGES AND PERSPECTIVES FOR

SUCROSE AND STARCH INTEGRATION INTO BIOREFINERY
(2nd GENERATION AND FLEX)
Currently, the demand for biofuels has increased due to environmental, social,
and technological care [13]. In this view, there is a great worldwide interest
in the use of biomass residues, such as cellulosic residues (for example,
sugarcane bagasse and corn straw) for the production of biofuels 2 G [7, 103].
In the context of scaling up production, there is the integrated biorefinery
configuration, called flex biorefinery, in which there is a possibility of integrating
different types of biomass for the production of 1G ethanol. For example, in
Brazil, there is a high rate of productivity in this biorefinery configuration during
a four-month period in which there is no ethanol production, due to the lack of
raw material and thus the plant goes through a long period of maintenance [104,
111]. This fact occurs because of the perishability of sugarcane [232]. As seen
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before, this problem is not common for corn, and the best option is the use of
both feedstocks for ethanol production [23, 105].
All biorefineries have the potential to meet the demand for the produc-
tion of bioethanol, but there are still many challenges in the integration of
1G-1G (different biomasses), and 1G and 2G biorefineries. For example,

economic problems, substrate approach, costs in main steps (pretreatment
and saccharification), low total sugar (C5 and C6 sugars) conversion yield in
wild type of microorganisms are prominent complications [13, 233].
The second generation has many challenges that hinder the integrated
process of 1G and 2G. In the case of 2G, it works with lignocellulosic
materials for biofuel production [234]. These feedstocks could contain
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little protein or other nutrients (corn) that fermentation microbes require,
however, not all feedstocks have these advantages, and it is important to add
some supplementation [98, 103].
Other challenges that need to be considered are harvest, transportation
costs and storage. For example, sugar crops (sugarcane) are especially difficult
to store without losses, and this might also be a problem with lignocellulosic
feedstocks [104]. Another challenge in the process is the stage of saccharifica-
tion, which is important because current enzymes for hydrolysis are more
expensive and less effective than corresponding enzymes used with starch
[111, 208]. Another alternative for saccharification process is to conduct SSF
of lignocellulose due to the feedback inhibition of the enzymes [23, 197].
Pretreatment is the most significant bottleneck in biorefinery process;
however, pretreatment is not needed for all sugar crops. It is used for
lignocellulosic materials where it is an overriding feedstock. However, the
lignocellulose pretreatments are often sufficiently intensive and generate
inhibitory compounds, and this is prejudicial to the total process. In addition,
it is difficult for the industrial process, since pretreatments have high costs
and do not have short process time options to be applied for the continuous
industrial process [98, 112, 113].
Another challenge is that microbial growth is limited due to the presence
of inhibitors in the fermentation process generated in previous stages
(pretreatment). In addition, different sugars (C5 and C6 sugars) in substrates
obtained from LCB are difficult to be metabolized by many wild type
microorganisms because it comes from pretreated biomass. In fermentation,
there are difficulties with the concentration of sugars and solids content in
the medium which are also unsatisfactory factors for the system [111, 236].
The cost of integrated 1G + 2G biorefineries requires two to four times
more investment than 2G ethanol prices to ensure competitiveness, which is
one of the main problems in flex production. However, this can be fixed when
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pretreatment and hydrolysis technologies are chosen [8, 23]. As a result, it is

necessary to investigate more developed schemes, particularly with staged
hydrolysis that can provide more competitiveness for the 1G alternatives
[114, 237–240]. On the other hand, the operational unit is important due
to its cost is the heat exchange process which can be reduced by adopting
developed schemes for evaporation and backward integrated evaporation
that is a new perspective for flex biorefinery [98, 241–244].
1G and 2G biorefineries have higher investments in costs than 1G+ 2G
+ electricity integration, due to the hydrolysis step with cogeneration which
presents a lower yield. The 1G + electricity schemes have provided better
results and it is superior to 1G+2G + electricity equivalents, mainly because
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of its highest investment costs [234, 241]. Other problems of sugarcane 1 G
and 2 G plants are related to enzyme parameter costs and reduced electricity
production. However, the advantage of this process lies in ethanol prices with
lower capital costs, presenting the greatest potential to increase the economic
utilization of 1G + 2G (flex) schemes. In this regard, the cost is lower than
2G biorefinery process and it has greater productivity than 1G [197].
Other option in the biorefinery integration is the development of plants with
corn and sugarcane as feedstocks that results in 1G and 2G processes. This
biorefinery could have high productivity which could help solving the global
demand of ethanol [112, 245]. Thus, sugarcane biomass is more versatile to be
used in this kind of biorefinery, because it could be integrated with different
biomasses (sugarcane and corn) in 1G or 2G integrated [98, 241, 246].
Energy integration in a sugarcane biorefinery can provide economical
advantage, environmental benefits, and increased ethanol production. The
last factor is related to the lower steam consumption in the plant due to
energy integration and consequently, and less bagasse needs to be burnt in
the electricity generation. Hence, its surplus can be made available for the
production of second-generation ethanol [51, 115, 234].
There are other integration processes with different advantages, such as,
1G, 2G and cogeneration system (1G+2G+COGEN), 1G and 2G from corn,
1G and 2G for sugarcane, 1G (biomass A) and 2G (biomass B) that could be
the future of fuels from biomass [111, 113]. These integrations could give
us positive results with 2G, which involves the production of 1G and 2G
ethanol in a combined distillery, enzymatic hydrolysis, and cogeneration
plant [112]. These kind of biorefineries offer many techno-economic and
environmental alternatives.
Total production cost calculations for these kinds of biorefineries can be
resulted in 74 settings, covering 5 fuel output types, 8 feedstock types, 12
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countries, and 8 combinations of agricultural management systems between

2010 and 2030 worldwide. This shows that many countries have interested
in this 1G and 2G flex technology [112]. However, this integration is not able
to meet the international demand and it is important to modify the production
of bioethanol according to the international market demand for these agri-

cultural products [13, 104]. Biorefinery integration modes could meet the
demand for biofuels and its use which will be a part of a big energy program
for each country by considering its necessities and approach to its resources.
10.7 CONCLUSION
Author Copy
First and second-generation bioethanol production from sugarcane and corn
are successful processes established in several countries, and it is currently
analyzed to find out the possibility for the integration of other biorefinery
modes to enhance some particular bottlenecks. Those bottlenecks are the
seasonal lack of the feedstocks and approach of second-generation sugars.
Several strategies, such as the alternation of feedstocks in off-seasons and
the integration of second-generation into the first-generation processes are
deeply evaluated. However, the integration of these modes in biorefinery is a
hard-analytical work to understand the role that plays each step-in bioethanol
production from two feedstocks with differences in structure, sugar content,
and processing. Even though the correct application of those strategies
could result in a successful integration from both biomass, the main steps in
bioprocesses such as pretreatments, saccharification, and fermentation need
more work to obtain a cost-effective competitive conversion.
KEYWORDS
• distillers dried grains
• hydrochloric acid
• saccharification and fermentation
• separate hydrolysis and fermentation
• simultaneous saccharification and co-fermentation
Non Commercial Use

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CHAPTER 11
Conversion of Sweet Sorghum Juice to

Bioethanol
IOSVANY LÓPEZ-SANDIN,1 FRANCISCO ZAVALA-GARCÍA,1
GUADALUPE GUTIÉRREZ-SOTO,1 and HÉCTOR RUIZ LEZA2
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1
University of Nuevo León, Faculty of Agronomy, Francisco Villa S/N Col.
Ex Hacienda El Canadá – 66415, General Escobedo, N. L., México,
E-mail: francisco.zavala.garcia@gmail.com (F. Zavala-García)
2
Biorefinery Group, Food Research Department,
Faculty of Chemistry Sciences, Autonomous University of Coahuila,
Saltillo–25280, Coahuila, Mexico
ABSTRACT
The continuous decrease in fossil fuel reserves and their impact on the
environment, lead the search for new alternative energy sources. In this
context, biofuels provide a viable substitute that contributes to the reduction
of polluting emissions into the atmosphere and increases the conventional
fuels efficiency. However, biofuels confront great challenges such as
obtaining raw material, requiring biomass sources to mitigate this problem.
In this sense, the sweet sorghum variety ROGER has shown potential in the
production of bioethanol, which depending on the production conditions can
have ethanol concentrations in the juice of 81.2 g/l. This chapter mentions
aspects related to the bioethanol production from sweet sorghum juice.
11.1 INTRODUCTION
The continuous use of fossil fuels has led to the reduction of their reserves and
environmental pollution because of the emission of considerable volumes
of GHG and other pollutants into the atmosphere [1]. However, over the
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years, actions aimed at reversing the energy and environmental paradigm

through the search for new renewable and eco-friendly energy sources have
increased [2]. In this sense, biofuels generate a significant reduction in GHG
emissions with respect to those derived from oil [3, 4].
The biofuels production faces four major challenges: 1) the raw material
must not compete with food production, 2) it must have a neutral balance of
polluting emissions (CO2), 3) its production must be continuous, ensuring the
supply of industrial facilities (extraction plants, biorefineries, fermenters),
and 4) extraction methods must be profitable from the energy and economic
point of view to compete in the long term with the price of other sources of
non-renewable energy [5].
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Bioenergy is the most extensively used energy source in the world,
mainly in solid form and to a minor extent in liquid and gaseous form. Thus,
the continuous technological advance has allowed the development of more
efficient biomass conversion technologies that make it possible to obtain
biofuels in their different physical states. However, most of these achieve-
ments have not been implemented in commercial facilities yet [6, 7].
With respect to liquid biofuels (bioethanol and biodiesel), they are used
as mixtures or substitutes for gasoline and diesel, being used mainly in
transportation and industry. According to the origin of the raw material and
the technology used to obtain it, they are classified as first, second, third, and
fourth generation, the latter still under development [5, 8], which depend
on sources of sugars, starch, and LCB. Where the conversion to bioethanol
depends on the nature of the raw material, mainly on its biochemical
composition [7].
In the case of first-generation bioethanol, it is a product that derives
mainly from the sugars fermentation contained in agricultural crops with a
high sucrose content, such as sugar cane, beet, and sweet sorghum, or from
the starch contained in the corn grain, wheat, barley, tubers (like potato) and
roots (like cassava), with the disadvantage of being crops intended for food,
so their use as energy material generates a food safety controversy. In addi-
tion to contributing to deforestation by eliminating large areas of primary
forests for their establishment [9]. However, sweet sorghum [Sorghum
bicolor (L.) Moench] has agronomic characteristics that allow it to supply
raw material for the production of biofuels, without affecting the diet and
agricultural areas [10, 11], since it is the food base of millions of people,
mainly in semi-arid regions around the world [12].
Sweet sorghum is a short-cycle plant with a high degree of tolerance
to biotic and abiotic stresses, which can achieve high biomass yields
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Conversion of Sweet Sorghum Juice to Bioethanol 317
and accumulate considerable volumes of fermentable sugars in the stem

parenchyma [13–15]. Sorghum has historically been used in the production
of syrup and molasses, but its bioenergetic potential makes it a viable raw
material for the manufacture of bioethanol and other products [16, 17].
The agronomic yields of sweet sorghum are related to the variety

and cultivation conditions, consolidating a strong line of research aimed
at determining the factors that can affect the productivity of agronomic
traits associated with obtaining bioethanol [94]. Hence, the importance of
studying the crop production process to identify (among other factors) the
best conditions for plant development in terms of energy and production,
considering agricultural activities such as soil preparation [18] and fertilization
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[19]. They have a high influence on the development of the crop, on the energy
consumptions of the system [20] and the definition of the optimal moment of
its harvest [17]. Although this would not be complete without the study of the
fermentation kinetics that defines the final production of bioethanol [21]. For
this reason, ethanol productivity varies between sweet sorghum genotypes,
due to its characteristics such as the amount of juice they can retain in their
stems and the concentration of sugars, which can influence the process of
conversion to bioethanol, where the temperature and the size of the yeast
inoculum play an important role. The latest has a great influence on the typical
biomass conversion processes, specifically in the sugar fermentation, where
one of the traditionally used yeasts is Saccharomyces cerevisiae [7].
Thus, the potential of sorghum in the bioethanol production is also
determined by the energy necessary to obtain it, energy balances being
necessary in order to reduce consumption and achieve high energy efficiency.
In this sense, an agricultural operation is efficient when the energy obtained
is greater than that used. However, the energy ratio depends on the crop,
the production system and the intensity of management. The last is mainly
associated with the optimal management of the resources used in the
production process such as water, fuels, fertilizers, pesticides, machinery,
electricity, and others [22].
In general, obtaining bioethanol is a complex process that must be
continuously improved, even more so by the introduction of new raw
materials. Therefore, the incorporation of a new plant material always requires
the study and evaluation of its potential application, as well as the study of
the sweet sorghum variety. This chapter give general aspects related to the
production of bioethanol from sweet sorghum juice, considering agronomic
and energetic parameters of the crop, besides of the conversion process to
bioethanol, using a new sweet sorghum variety developed in the Universidad
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Autónoma de Nuevo León, leaded by the research group focused in sorghum

plant breeding of the Agronomy Department.
11.2 BIOLOGY OF SWEET SORGHUM
Sorghum is an herbaceous plant of type C4 that belongs to the grass family

(Poaceae) with high ecological plasticity that allows its cultivation in tropical,
subtropical, temperate, and semi-arid regions. Thus, taking advantage of
soils with reduced natural fertility such as sandy ones and, due to its multiple
uses, it is divided into grain, broomcorn, sweet, and forage sorghum [23,
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24]. The morphological and physiological variations of sorghum plants have
provided the material for natural selection to act on them and accumulate
individual differences in a similar or artificial way. These differences allow
the different uses of cultivated sorghums, such as the production of ethanol,
grain, honey, brooms, etc. In such a way, that they are currently grouped
into various categories, based on their main products and uses, as well as the
distinctive or genetic characteristics of the plant [24, 25].
Taxonomically, sorghum was first described by Linnaeus in 1753 under
the name Holcus. He originally delineated several species of Holcus, some of
which were later moved to the Avenae tribe, where the generic name Holcus
now belongs. In 1794, Moench distinguished the genus Sorghum from
the genus Holcus [26]. The genus Sorgum is divided into five subgenera:
sorghum, chaetosorghum, heterosorghum, parasorghum, and stiposorghum.
Within the Sorghum subgenus, the wild species, S. bicolor, S. halepense, and
S. propinquum can be mentioned [27].
Sorghum exhibits several morphophysiological forms and great variation
for flower morphology, resulting in the classification of several basic and
intermediate races. Harlan and de Wet [96], classified Sorghum bicolor (L.)
Moench, subspp. bicolor in five basic races and ten hybrids as shown in the
following subsections.
11.2.1 BASIC RACES
• Race bicolor (B);

• Race guinea (G);
• Race caudatum (C);
• Race kafir (K);
• Race durra (D).
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11.2.2 INTERMEDIATE/HYBRID RACES
• Race guinea-bicolor (GB);

• Race caudatum-bicolor (CB);
• Race kafir-bicolor (KB);

• Race durra-bicolor (DB);
• Race guinea-caudatum (GC);
• Race guinea-kafir (GK);
• Race de guinea-durra (GD);
• Race kafir-caudatum (KC);
• Race durra-caudatum (DC);
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• Race kafir-durra (KD).
11.3 GLOBAL SORGHUM PRODUCTION
The different uses of sorghum and its high capacity for adaptation to
different climatic conditions have maintained its productive levels, as well
as the areas devoted to this crop. The global trend of sorghum production is
shown in Table 11.1. According to Mundia et al. [28], there are mainly 10
factors that significantly impact sorghum production: (1) climate change,
(2) population growth/economic development, (3) non-food demand, (4)
agricultural inputs, (5) demand for other crops, (6) scarcity of agricultural
resources, (7) biodiversity, (8) cultural influence, (9) prices, and (10) armed
conflicts. Furthermore, several of these factors can simultaneously affect
sorghum production and their degree of impact can be the combination of
several factors, and their magnitude differs geographically.
TABLE 11.1 Global Sorghum Production by Tonnage (in millions)

Region Year
2010 2012 2014 2016 2018
World 60.181 57.321 68.278 63.661 59.342
Africa 25.073 23.583 29.298 30.430 29.782
America 22.500 21.056 26.820 23.024 19.244
Asia 10.384 9.676 9.498 7.132 7.974
Europe 0.713 0.763 1.375 1.280 1.079
Oceania 1.512 2.243 1.287 1.796 1.262
Source: FAO [29], Food and Agriculture Organization of the United Nations.
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11.4 SWEET SORGHUM VARIETIES
In the production of first-generation bioethanol, many sweet sorghum geno-

types have been reported (Table 11.2). Some of these varieties stand out for
their high resistance and adaptation to different production conditions [30],

for example:
• Dale: Resistant to lodging and diseases, with approximately 120 days
to maturity.
• Della: Mid-season variety, disease-resistant, approximately 114 days
to maturity.
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• Sugar Drip: High productivity with late sowing, susceptible to most
sorghum diseases, with approximately 110 days to maturity (early
maturity).
• M81-E: Late maturing, resistant to lodging and diseases.
• Theis and Brandes: Late maturing, at least 2–3 weeks later than
Dale, resistant to lodging and disease.
TABLE 11.2 Agronomic Performance of Some Sweet Sorghum Varieties Used to Produce
Bioethanol
Varieties Juice (m3/ha) °Bx (%) Ethanol (m3/ha) References
Keller 18.9–20.8 16.5–18.5 1.487–1.544 [31, 32]
Dale 21.1–22.9 15.3–18.7 1.331–1.360 [31, 32]
Della 18.2–24.2 13.7–16.2 1.281–1.429 [31, 32]
M81-E 23.1–23.4 14.9–17.1 1.419–1.496 [31, 32]
Sugar Drip 9.7–13.8 14.4–16.3 0.704–0.842 [31, 32]
11.5 ADVANTAGES OF SWEET SORGHUM
Sweet sorghum is a subspecies of sorghum [Sorghum bicolor (L.) Moench]

that is characterized by its high sugar content rather than grain production
[33]. Its agronomic characteristics make it a viable source in the current
energy situation and the food conflicts generated using agricultural crops
in the production of fuels. In addition, it has considerable advantages [10,
34–36] such as:
• It does not compete for agricultural ground as it can to be cultivated
on marginal lands that are not optimal for food production;
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• It is tolerant to drought, high temperatures, floods, soil salinity,

acidity toxicity, allowing its development under different agroclimatic
conditions;
• Presents high productivity and efficiency in the use of solar radiation,
as well as nitrogen (N)-based fertilizers compared to other crops;

• It requires less inputs, chemical reactions and energy in the bioethanol
obtainment from stem juice with a minimum cost of cultivation;
• It has high concentrations of fermentable sugars in the juice of its
stems, producing more ethanol per unit area than other crops;
• Provides high volumes of raw material to produce second-generation
bioethanol.
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11.6 SWEET SORGHUM PRODUCTION
Sweet sorghum is one of the energy crops with high potential for the
bioethanol production, which can be obtained from the juice of the stems,
grains, bagasse, and straw. However, the cultivars with greater interest stand
out in certain agronomic traits such as: (i) high biomass yield; (ii) stems
rich in juices with high sugar content; (iii) high percentage of extractable
juice; (iv) stem geometry capable of resisting lodging; (v) a long period of
industrial use that prolongs the harvest season; and (vi) tolerance to different
production conditions [11, 30].
Compared to other plant materials with potential in the bioethanol produc-
tion such as sugar cane, sugar beet, and corn, sweet sorghum has higher
levels of directly fermentable reducing sugars in its stems and requires fewer
inputs per production unit of biomass. In addition, it has high adaptability to
adverse edaphoclimatic conditions and a greater efficiency in the use of solar
radiation and nitrogen fertilizers than corn and sugar cane [34, 37].
Although sweet sorghum is a crop with a great capacity to adapt to
various agroclimatic conditions, crop yield in any region is associated with
plant growth, which depends on climatic, biological, edaphic factors and
agronomic practices. Some of these factors are summarized below:
1. Precipitation and Temperatures: It adapts to a wide range of
annual precipitations (from 550 to 800 mm) and temperatures (15
to 45°C), under various climatic and soil conditions. Although the
optimum temperature for plant development is between 25 and 40°C
and requires a day duration between 10–14 h [38]. However, these
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annual differences (particularly low rainfall and low temperatures)

can influence biomass production, stem juice and sugar content,
affecting crop yields and consequently ethanol. Also, they limit the
response of the plant to the application of N and the magnitude of its
effects will depend on the growing season [17, 39].

2. Edaphic Conditions: The soil provides the nutrients and other
elements necessary for the plant development [40]. The case of sweet
sorghum can be cultivated in different types of soils and, therefore,
availability of nutrients. However, knowing the nutrient and water
requirements for the plant and the physical, chemical, and biological
properties of the soil. In most cases, it is necessary to modify mainly
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the initial soil conditions to favor the growth of the crop and increase
yield [41, 97]. Ramírez et al. [42] reported that in Mexico there are
more than 19 million hectares with high optimal conditions under
irrigation conditions, although, with the supply of water, it can be
cultivated throughout the country.
3. Seedtime: Sweet sorghum is an annual plant with a short life cycle
(approximately 4 months) that, depending mainly on the agrocli-
matic conditions and the genotype, can be planted two or three times
a year [14, 43]. According to Han et al. [44], sweet sorghum can
be planted from mid-March to early June with favorable results.
Nevertheless, planting sorghum in early. May and harvested at the
hard dough stage can substantially increase the fermentable sugar
content. Though, a late seedtime can decrease the efficiency of the
use of solar radiation and consequently the productivity of the crop
[45], which may be due to the gradual decrease of the foliar area and
the height of the plant as a consequence of sensitivity to photoperiod
or shorter days [46]. In addition, it delays the harvest exposing the
plants to the attack of pests and diseases that are predominant at the
end of the crop cycle [47]. In general, the sowing date can influence
the contents of sucrose, total sugar, pH, electrical conductivity of the
juice and the Brix degrees [48].
4. Sowing Density: It influences the growth parameters of sorghum and
its yield. For example, optimal plant density can increase biomass
yields, since they maintain a high and stable net assimilation rate,
besides a positive balance between photosynthesis and respiration
processes [40, 49, 50]. Since the plants have a greater vertical angle
of the leaf with respect to the main stem, increasing the capture of
light and consequently a higher photosynthetic rate [51, 52]. In this
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sense, it has been observed that a high planting density (≈16.7 plants/
m2) increases the juice, sugar, and biomass yields. However, the stem
diameter and the internodes number decrease contributing to a higher
lodging rate [49, 53].
5. Fertilization: The constant exploitation of agricultural soils has

decreased their nutrient reserves, requiring artificial sources to
compensate for the deficit. Which depends largely on the properties
of the soil and the type of crop to be established. For example, N,
and C deficiency limits plant growth and decreases their agronomic
yields [50, 54, 55]. For the sustainable production of sweet sorghum
as a raw material to produce bioethanol, it depends on agronomic
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practices that maintain the necessary levels of N and C in the soil
[56]. Thus, the physical-chemical analysis of the soil is necessary
to make up for its deficiencies. In this sense, a positive response of
sweet sorghum has been observed with applications of ≥40 kg/ha of
N, increasing the biomass, juice, and sugars yields [57–60].
6. Pests Management and Control, Diseases, and Weeds: Undesir-
able plants compete for nutrients, water, and light, they can release
substances through their roots and leaves that turn out to be toxic to
crops. In addition, they provide a favorable habitat for the proliferation
of other pests (arthropods, mites, pathogens, and others) by serving as
their hosts, which can affect the harvesting process and contaminate the
production obtained [61]. On the other hand, pest, and disease cause
considerable crop losses; for example, the yellow aphid Melanaphis
sacchari causes crop damage at all stages of sorghum development [62,
63]. This pest feeds on the sap of the plant, reducing its vigor and yield
[64], and even causing 100% of crop losses under severe attack [65].
7. Irrigation: It tries to supply rainwater when it is insufficient to
supply the water needs of the crop, maintaining productive soils with
optimal humidity levels for growth, in addition to contributing to
increased agricultural yields. Water helps plants in the absorption of
nutrients from the soil and to perform other physiological functions
[66]. Sweet sorghum is a drought-resistant crop that, compared to
other crops (such as sugar cane), requires a lower volume of irrigation
water (approximately 400 mm), which depends mainly on climatic
conditions and the variety [67, 68]. Thus, there is the possibility of
reducing irrigation water needs through sweet sorghum cultivars
with deep roots that intercept most rainwater and in some productive
environments no require irrigation [69].
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8. Harvest: The optimal harvest time is important in reducing produc-

tion costs and obtaining high-quality plant material (high concentra-
tions of fermentable sugars and biomass) destined for the bioethanol
production [70, 71]. In this sense, it has been reported that the sweet
sorghum harvest may be carried out from the soft mass stage of
the grain (the filling of the grains is complete and they begin to
harden) and extend to physiological maturity (the grains begin to
dry out), which would imply between 104 to 130 days after sowing.
Although, this time will depend on the variety and environmental
conditions [71–74].
Once harvested, sweet sorghum faces some challenges such as
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the rapid fermentation of sugars, generating an acceleration of its
decomposition in a few hours, indicating that it should not be stored
for long periods [70, 75]. Therefore, the rapid stem grinding and the
processing of the extracted juice (4 to 5 hours) is essential considering
the high content of fermentable sugars and the metabolic activity of
contaminating bacteria. However, to reduce this postharvest effect,
the juice may be refrigerated, or chemical preservatives can be added
[76, 77]. However, it could change the chemical composition of the
juice [78].
In general, the bioethanol production requires the continuous
supply of sweet sorghum, the transport and storage of high volumes
of plant material, as well as minimal post-harvest losses of ferment-
able sugars [34].
11.7 ENERGY USE IN THE SWEET SORGHUM PRODUCTION
The energy balance allows evaluating the efficiency of the crop production
methods, considering the energy inputs and outputs of the production system.
The energy balance differs widely between agricultural production systems
and life cycle analysis is commonly used to assess the energy efficiency and
environmental effect of bioethanol production. Therefore, energy demand
is classified according to the energy inputs of each production system
[79]. Accordingly, an efficient process requires that the energy invested in
producing a unit of biofuel, including the agricultural and industrial stages,
be less than the energy leaving the system.
In the sweet sorghum production, the inputs associated with agricultural
operations are decisive in energy consumption. Wherein production systems
with low input requirements are characterized by lower consumption and,
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hence, greater energy efficiency. In this sense, it has been reported that the
greatest contribution to energy consumption corresponds to the diesel used
to move agricultural machinery and in nitrogen fertilization [22, 80]. There-
upon, consumption of energy has a wide range of variation depending on the
production conditions of sweet sorghum. For example, Jankowski et al. [80]

obtained that the low input technology required between 14.9 and 15.8 GJ/
ha, with an energy gain of 170 GJ/ha, although high input technology had
higher energy gains with 226 GJ/ha.
On the other hand, Garofalo et al. [22] with conventional tillage and
the application of 150 kg/ha of N had an energy consumption in diesel of
4,508 MJ/ha, with an energy yield of 35.2 GJ/ha. Compared to the treatment
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without tillage and without fertilizer with an energy consumption of 2,260
MJ/ha. For the bioethanol production, an energy production of 22.6 to 70.5
GJ/ha has been obtained from fresh sweet sorghum biomass [81]. Likewise,
a net energy gain of 8.37 MJ/l of bioethanol and an energy efficiency of 1.56
have been reported [79].
11.8 CONVERSION OF SWEET SORGHUM JUICE TO BIOETHANOL
The juice of the sweet sorghum stalk has shown to be a raw material of
great potential for the biofuels production which, after being transformed
(Figure 11.1) into anhydrous ethanol, is mixed with gasoline and is used in
motors. During the bioethanol production, the stems are crushed to extract
the juice rich in fermentable sugars with an exact proportion that varies
between genotypes (53–85% sucrose, 9–33% glucose and 6–21% fructose)
and that can be converted directly into ethanol with high efficiency, mainly
by the yeast Saccharomyces cerevisiae. Which is a fast-acting microor-
ganism that tolerates high alcohol concentrations (up to 150 g/l) and shows
high bioethanol yields. Furthermore, it maintains high cell viability under
different fermentation conditions [30, 82, 83]. When used in sweet sorghum
juice in the bioethanol production, its growth curve shows three phases: the
exponential phase observed approximately in the first 18 h of incubation, the
slow phase observed approximately between 18 to 24 h and the stationary
phase observed approximately between 24 to 72 hours [95]. Likewise, the
kinetics of bioethanol production depend on several factors such as tempera-
ture, yeast load, pH, aeration time and rate, stirring conditions, nitrogen
source and initial sugar concentration. Therefore, in the bioethanol produc-
tion from sweet sorghum juice, a wide range of yield between 39.2 and 127.8
g/l has been reported [83–85].
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FIGURE 11.1 Conventional process diagram for obtaining first-generation bioethanol from
sweet sorghum juice.
11.9 THE SWEET SORGHUM VARIETY “ROGER” IN THE

PRODUCTION OF BIOETHANOL
The variety ROGER was registered under the same name and with
registration number SOG-261-050315 by the Universidad Autónoma de
Nuevo León in the National Catalog of Plant Varieties. This genotype
is characterized by an average of 75 days to flowering and a cultivation
cycle of 130 days. Depending on the production conditions, this crop have
approximately a fresh biomass yield of 51.66 t/ha, juice of 22.53 m3/ha and
°Bx of 16.04% [74].
ROGER was produced in the Marín experimental field of the Faculty
of Agronomy belonging to the Universidad Autónoma de Nuevo León
(UANL), located in the municipality of Marín, in the state of Nuevo Leon,
Mexico. This is geographically located at 25° 52´ 13.5´´ north latitude
and 100° 02´ 22.56´´ west longitude, with an altitude of 355 meters above
sea level. The climate corresponds to a BS1 (hˊ) hw (é), described as a dry
hot steppe with rains in summer, annual rainfall that fluctuates between
250–500 mm and average annual temperature of 22°C. The type of soil is
vertisols, thin, compacted, with high content of clay and calcium carbonate,
low content of organic matter and pH between 7.5–8.5. ROGER, like other
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varieties of sweet sorghum, has a high capacity to adapt to different agro-

climatic conditions. However, when these are not optimal for the efficient
growth of the crop, they need to be reestablished by carrying out different
agricultural operations.
11.9.1 AGRICULTURAL OPERATIONS
In one of our experiments, in the soil preparation, the traditional tillage

integrated by the operations of clearing, subsoil plowing (up to a depth of
0.75 m), plowing, and harrowing was used. The sowing was carried out at a
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depth of 0.05 m and distance between rows of 0.8 m, using an experimental
planter (Almaco® CTS EO, Nevada, Iowa, USA). Organic fertilization
(chicken manure) was applied 20 days before sowing with doses of 3 t/ha
(N-60 kg/ha, P-65 kg/ha and K-75 kg/ha) and 20 days after sowing thinning
was performed to obtain an average density of 18 plants/m2. For pest control,
a dose of 0.6 l/ha of the pesticide Pounce 340 CE® was applied, 28 and 59
days after sowing and in particular for the yellow aphid a dose of 0.3 l/ha of
Murralla® was applied at 32 and 81 days after sowing. For weed control, 1 l/
ha of Phyto Amina 40® was applied at 35 and 64 days after sowing. Irrigation
by band (superficial drip) was carried out before sowing and five auxiliary
irrigation after sowing at intervals of 14 days. The harvest was carried out
manually (130 days after sowing) at a cutting height of 0.03 m to 0.04 m
from the stem base. However, to determine the harvest moment with the
highest bioethanol yield, samples were taken at different plant physiological
stages (PS) described by Vanderlip and Reeves [86]. In the first sampling, the
grains showed soft mass (PS7). In the second sampling, the grains showed a
stage of dough (PS8). In the third sampling, the culture was at physiological
maturity (PS9).
11.9.2 ENERGY CONSUMPTION
In our study, the tillage and fertilization effect on energy consumption during
the establishment of the crop was evaluated. The tillage treatments consisted
of a minimum tillage system, conventional tillage and conventional tillage
with breaking of the plow layer; while the fertilization treatments consisted
of the use of organic and inorganic fertilizer and without fertilizer. For the
above, the methodology described by De las Cuevas et al. [87] and Paneque
and Sánchez [88], based on the proposals of Bridges and Smith [89] and
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supported by the information presented by Stout [90]. This methodology

determines the energy cost in mechanized agricultural operations consid-
ering the energy required in construction, manufacturing, and transportation
materials, fuel, lubricants/filters, maintenance/repair, labor, fertilizers,
pesticides, and seeds. As a result, it was obtained that the energy demand
varied depending on the production system used, where the systems with
low inputs required less energy supply. In this sense, minimum tillage and
no fertilization showed the lowest energy consumption values with 21.3 and
17.2 GJ/ha, respectively. Likewise, these systems had the highest energy
efficiency values with 15.1 and 18.7, respectively. On the other hand, the
highest net energy production was 340.3 and 351.4 GJ/ha, obtained with
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deep conventional tillage and organic fertilization, respectively. Where, the
highest contribution to energy consumption was using diesel used in agricul-
tural machinery in a range of 0.41 to 1.7 GJ/ha and in inorganic fertilization
with 8.4 GJ/ha [74].
11.9.3 BIOETHANOL PRODUCTION
To evaluate the potential of the variety ROGER in the obtaining bioethanol,

several tests were carried out at the laboratory level in which the effect
of stirring conditions, inoculum size, phenological stage of the plant
and freezing of the juice was evaluated. This was done using a standard
submerged culture methodology in 250 ml Erlenmeyer flasks, incubated at
30°C for 48 h after being inoculated with the yeast Saccharomyces cerevisiae
PE-2. To estimate the juice ethanol yield, 100 ml of the supernatants were
taken and placed in a rotavapor (IKA® RV 10 Digital, China) at 30°C, 132
mbar and 50 rpm for a period of 40 min. The distillate obtained from the
rotavapor was used to determine ethanol concentration using the potassium
dichromate method [91, 92].
Table 11.3 shows the most representative results of the effects of
inoculum, agitation, juice conditions (fresh and frozen) and the phenological
stage of the plants in the bioethanol production. The highest bioethanol yield
(78.1 g/l) was obtained with the juice of PS7 plants at 48 h of fermentation
under stirring conditions and with 2% inoculum. Although, there was no
significant difference between the phenological stages evaluated. Regarding
the freezing of the juices, it had no influence on the obtaining of bioethanol,
since in comparison with fresh juices and under the same fermentation
conditions, there was no significant difference between them. This allows
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the juice to be preserved for long periods without affecting its quality and
consequently the bioethanol yield.
TABLE 11.3 Bioethanol Production Conditions of with Juice of the Sweet Sorghum Variety
ROGER
Treatments Culture Conditions Bioethanol Yield (g/l) Time (h)
Fresh juice Agitation 150 rpm, 2% inoculum 78.1 48
Fresh juice Without agitation, 2% inoculum 34.7 24
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Frozen juice Agitation 150 rpm, 1% inoculum 47.3 24
Juice PS7*
Agitation 150 rpm, 2% inoculum 70.3 48
Juice PS8* 65.7 48
Juice PS9* 57.9 48
*
Phenological stage of the plant described by Vanderlip and Reeves [86]. All the tests were
carried out in triplicate, with a standard deviation of less than 5%.
In a test with 100 l of juice, the bioethanol production kinetic was deter-
mined. The juice was placed and sterilized in a stainless-steel tank with a
capacity of 200 l and the changes in pH, °Bx, reducing sugar concentra-
tion (RS), and bioethanol concentration were determined at 0, 24, 48 and
72 h of fermentation. The pH measurement was made with a potentiometer
(Corning® pH meter 430, USA). The °Bx were determined with a portable
digital refractometer (ATAGO® 3810, PAL-1, Atago USA Inc., Bellevue,
USA). The RS quantification was performed by the method described by
Miller [93], using glucose as standard.
As a result, the bioethanol production curve showed a logarithmic phase
up to 48 h, maintaining the stationary phase until 72 h (see Figure 11.2),
during which the maximum bioethanol concentration was reached (81.2 g/l).
The concentration of reducing sugars and °Bx showed a decrease of 64.5
and 59.1%, respectively at 72 h. However, the highest consumption rate was
observed at 48 h with 59.6 and 52.3%, respectively. It is worth mentioning
that the juice was not supplemented with other sources of carbon, nitrogen
or other types of nutrients, nor was the pH adjusted during the fermentation
process. Therefore, the behavior of the production kinetics suggests that the
juice obtained from the variety does not have levels of compound that inhibit
the development of yeast.
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Bioethanol production kinetic.
FIGURE 11.2
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11.10 CONCLUSIONS
Sweet sorghum is a low-input energy crop that has high adaptability to various
production conditions and great potential in bioethanol production. In this
sense, the sweet sorghum variety ROGER has potential in the first-generation
bioethanol production and can be cultivated in semi-arid climates, achieving
a high production of biomass, juice, and sugar. On the other hand, during
the crop establishment, energy consumption oscillates depending on the
production system used, with low-input systems being those with the lowest
energy consumption. The higher energy input was from the consumption of
diesel fuel (0.41 to 1.7 GJ/ha) and of chemical fertilizer (8.4 GJ/ha). Thus,
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the higher energy efficiency with values of 15.1 and 18.7 was obtained with
minimum tillage and without fertilizer application, respectively. Whereas for
the stem juice, it does not require supplementation or addition of nutrients
for its conversion to bioethanol. However, the yields fluctuate depending on
the fermentation conditions, where the maximum yield can be obtained after
72 h of fermentation (81.2 g/l).
KEYWORDS
• bioenergy
• biofuels
• fossil fuels
• greenhouse gases
• production systems
• variety ROGER
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CHAPTER 12
Biotechnology Development of
Bioethanol from Sweet Sorghum Bagasse
DANIEL TINÔCO
Biochemical Engineering Department, School of Chemistry,
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Federal University of Rio de Janeiro, Rio de Janeiro–21941909, RJ, Brazil,
E-mail: dneto@peq.coppe.ufrj.br
ABSTRACT
Sorghum is one of the most cultivated cereals in the world, with great
potential for energy and fuel production. Its lignocellulosic fraction can
be used for cellulosic ethanol production, after undergoing treatment steps
to make sugars available. This chapter presents the most recent trends in
the bioprocess for the ethanol production from sweet sorghum bagasse,
highlighting the main physical, chemical, physical-chemical, and biological
pretreatments used for cellulose digestibility such as acid-base, SE, ammonia
fiber expansion (AFEX), organic solvents, ILs, microwave, and combined
methods. Saccharification and fermentation approaches were also presented,
such as simultaneous (SSF), separate (SHF), and co-fermentation (SScF)
processes. Finally, a biotechnological evolution of sweet sorghum bagasse
was presented from the main scientific reports in the last 12 years on its use
as a raw material for the fuel cellulosic ethanol production.
12.1 INTRODUCTION
Sorghum is one of the most cultivated cereals in the world, along with
wheat, rice, corn, and barley [1], being composed on average of 50–60%
lignocellulosic biomass (LCB) [2]. Considered a viable energy crop for
alcohol fuel production, sorghum, especially the sweet variety, has great
potential for the generation of cellulose-based products such as ethanol,
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butanol, and wood-plastic composites [3]. In addition to the free fermentable

sugars present in the stem, sweet sorghum bagasse can be processed and
converted into fuel ethanol and energy, giving this biomass a multi-purpose
aspect, therefore being considered as a biorefinery crop [4].
The sweet sorghum bagasse processing for the bioethanol production

is characterized by an initial treatment step, followed by the cellulose
saccharification, and the fermentation of released pentoses and hexoses. After
harvesting, drying, and storage, sweet sorghum bagasse can be submitted
to physical, chemical, physical-chemical, and biological action, responsible
for preparing the LCB for the enzymatic hydrolysis. The main physical
treatments are drying, grinding, sieving, granulating, extruding, steam flaking,
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extraction, and decortication. Chemical methods are based on dilute acids
and alkalis, organic solvents, ILs, and oxidative agents. Physical-chemical
treatments combine mechanical and chemical processes, being the SE, liquid
hot water (LHW), supercritical CO2, AFEX, and microwave the most used
methods. Biological treatment can be combined with other methodologies,
being marked by the use of enzymes and microorganisms (bacteria and fungi)
capable of degrading the lignocellulosic material [5–7].
The next step is the enzymatic hydrolysis and fermentation. These
processes can be conducted in different approaches: simultaneously in the
same bioreactor or separately in different bioreactors [7]. SSF is generally
conducted at low temperatures due to the thermal tolerance of the microor-
ganisms used. However, this condition limits the fermentation efficiency,
requiring a pre-saccharification step under optimized conditions. Once the
sugars are released, the temperature can be readjusted to the ideal fermentation
condition and, then, the microorganism inoculation can be completed. This
process is classified as delayed simultaneous saccharification and fermenta-
tion (DSSF) [8]. The SHF has the advantage that the saccharification and
fermentation steps can be conducted under ideal individualized conditions.
Meanwhile, the ethanol yield and productivity, and the bioprocess economy
tend to be lower than those obtained with the SSF approach. Simultaneous
saccharification and co-fermentation (SScF) are similar to the SSF process,
with the additional fermentation of free sugars present in the sweet sorghum
stem. Therefore, the C5 and C6 sugars can be simultaneously converted to
ethanol [9].
This chapter aimed to present the main trends related to the cellulosic
ethanol biotechnology from sweet sorghum bagasse, highlighting the treatment
methodologies and the most used saccharification and fermentation approaches
in recent years, especially between 2009 and 2020. The biotechnological
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Biotechnology Development of Bioethano 341
evolution was also presented for the cellulosic ethanol production, highlighting
the most used combinations of pretreatment and sweet sorghum fermentation,
as well as the microorganisms and most relevant aspects involved in the sweet
sorghum ethanol bioprocess.
12.2 SORGHUM
12.2.1 WORLD PRODUCTION
Sorghum is the fifth most cultivated cereal in the world, with an expected
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production of 327 Mtons by 2027 [10]. The main producing regions are
Africa (40.1%) and the Americas (38.1%), where sorghum is used in human
food and for the alcoholic drinks and biofuels production. Next are Asia
(17.3%), Oceania (3.2%), and Europe (1.4%) (Figure 12.1).
FIGURE 12.1 World sorghum production [1].
According to the Food and Agriculture Organization of the United

Nations (FAOSTAT), among the main countries producing sorghum in the
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world between 1998 and 2018, four are African, three American, two Asian,
and one from Oceania (Figure 12.2). The United States is the largest sorghum
producer in the world, with an average production of 10.6 Mtons, followed
by Nigeria with around 7.4 Mtons, India with 6.65 Mtons, and Mexico with
6.1 Mtons. Sudan and Sudan (former) account for a production of 4.4 and 3.8
Mtons, respectively [1]. Argentina is a reference in South American sorghum
production with 3 Mtons, together with Brazil, which has widely cultivated
sorghum in association with sugarcane for bioethanol production, although
not among the 10 largest producers [11]. Since 1960, Ethiopia has increased
its sorghum production, with a recent amount of 2.9 Mtons. In addition to
India, China has stood out in Asia since the sorghum participation in its
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domestic market increased. China has imported large cereal quantities in the
past, about 3 Mtons in 2012 and 18 Mtons in 2014 [10]. Today, China has
an average production of 2.6 Mtons, being the ninth largest producer in the
world and contributing, together with India, with more than 85% of Asian
sorghum production [12]. In Oceania, the only main contributor is Australia,
with a production of 1.9 Mtons.
The average world sorghum production and the corresponding planted
area for the period 1998–2018 was 63.1 Mtons and 45 Mha, respectively,
which represents a world average yield of 1.4 t/ha1 (Figure 12.3).
FIGURE 12.2 Main sorghum producers-average 1998–2018 [1].
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FIGURE 12.3 Area harvested and production of sorghum in the world [1].
12.2.2 SWEET SORGHUM
Sorghum (Sorghum bicolor (L.) Moench) is a tropical crop from an African

source, classified into basic types: sweet, grain, forage, low-lignin, and
biomass [7]. The sweet type has great industrial and agricultural interest due
to its chemical characteristics, in particular, the composition in free ferment-
able sugars of its stem, which contains about 12 to 18% (w/v) sugar. The
sugars present in sweet sorghum juice correspond to 53–85% sucrose, 9–33%
glucose, and 6–21% fructose [4]. In addition to a sugar-rich composition,
sweet sorghum has the following agricultural characteristics: high produc-
tivity, high-stress tolerance (temperature, salinity, and water), and high adap-
tation to the cultivation and management infrastructure [4, 12]. Compared to
traditional biomasses such as sugarcane, sweet sorghum requires about 2 and
4-fold less fertilizer and water, respectively, being grown in up to 3 annual
cycles [2]. The most suitable soils for the sweet sorghum cultivation are the
red or black clay [4], and the average plant growth temperature ranges from
12°C to 37°C [12].
The most popular sweet sorghum varieties, identified with biofuel produc-
tion potential, were: Dale, Della, M81-E, Sugar Drip, Top 76–6, Umbrella,
Keller, Rio, Wray, and AFL Tcv27751. The Top 76–6 variety was considered
the most suitable for the bioethanol production in a study carried out with
six of these 10 varieties, as it presented higher productivity, resulting from
the soluble sugars found in its stem, and a greater nutritional value of its
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grains, with a low concentration of polyphenols and high-quality relative

protein [13]. Hybrid varieties have been investigated, such as Hybrid H8015,
which are capable of producing biomass with higher yields, in a shorter
growth cycle, and with guaranteed seed production, which contributes to
the bioethanol economy [4]. The varieties GK-coba, Mn-1054, Ramada,

Mn-4508, and SS-301 were also identified and analyzed for their productivity
and their sugar and fiber content. GK-coba and Mn-4508 presented high sugar
content in the stem, while Mn-1054 and Ramada had a large fiber amount.
The SS-301 variety had a high sugar and fiber content, which provided juice
and bagasse with high potential for ethanol production [14].
Sweet sorghum can be cultivated in tropical and temperate regions. In
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tropical countries, sweet sorghum is widely cultivated in the rainy seasons
from June to July, and dry seasons from September to October, while in
temperate countries, the planting is limited to once a year [4]. In Brazil, where
sugarcane is the main feedstock for bioethanol production, sweet sorghum
has been cultivated in the sugarcane off-season, between November and
May, allowing the operational integration of the commercial distilleries [15].
In the USA, with their corn bioethanol, sweet sorghum has been planted
between May and June, which has contributed to biomass yields of 26 to 29
t/ha, with an ethanol production of at least 14,500 L/ha [16].
12.2.3 BIOENERGY PRODUCTION
Sweet sorghum can be used in the production of solid (biochar), liquid

(bioethanol, biodiesel, and bio-oil) and gas (bio-hydrogen, biogas, and
synthesis gas) fuels [7], from use of all its constituent parts: grains (starch),
juice (sucrose) and biomass (lignocellulosic fraction). Sweet sorghum can be
also used for heat and power co-generation. Therefore, it is classified as an
important biorefinery crop [4].
Sweet sorghum is capable of producing integrated first and second-
generation ethanol, due to its rich saccharine juice and its high lignocellulosic
composition, with 50–60% bagasse [2]. Although promising, the bioethanol
production from sweet sorghum by biorefinery concept has challenges as
the biological conversion, which requires a high yield of the generated
bioproducts. The process implementation based on microorganisms and
technologies able to simultaneously hydrolyze cellulose, overcome the
lignin recalcitrance, and ferment different sugars like glucose, xylose,
and cellobiose, with minimal inhibitor release, are requirements for the
bioprocess success. The use of low-cost cellulolytic enzymes and its reuse
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also contributes to making biological conversion even more productive and

economically competitive [7].
12.2.4 SWEET SORGHUM BAGASSE
Bagasse, a fibrous lignocellulosic material, represents approximately

two-thirds of the sweet sorghum dry matter [17]. It consists of 27–44.6%
cellulose, 25–27% hemicellulose, 11–24.7% lignin [4], and other
compounds as: minerals (K, P, Mg, and Ca), nutrients (niacin, thiamine,
riboflavin, and B6), amino acids (proteins), dietary fibers (soluble and
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insoluble), fatty constituents (saturated and unsaturated), and vitamins [12].
Its calorific value (18.3 MJ/kg) is comparable with other lignocellulosic
feedstocks as switchgrass (18.4 MJ/kg) and big bluestem (18.6 MJ/kg) [4].
Due to its chemical and power characteristics, sweet sorghum bagasse can
be used for the paper and animal feed production, in soil applications as
a fertilizer, for the power co-generation, and as a raw material for fuel
cellulosic ethanol [17].
12.3 CELLULOSIC ETHANOL PRODUCTION
Sweet sorghum bagasse requires some pretreatment steps, a long processing

time, and a large investment to make the sugar present in its lignocellulosic
fraction available for fermentation [7]. The main cellulosic ethanol produc-
tion steps involve: biomass pretreatment, lignocellulosic material saccharifi-
cation, and released sugars fermentation.
12.3.1 BIOMASS PRETREATMENT
Pretreatment is the most critical bioprocess step in the cost-efficient conversion

of biomass to ethanol and other bio-based products [18], since it is responsible
for preparing the cellulose for the enzymatic and biological action, therefore
influencing the productivity and economy of the fermentative process. This
step requires a low biomass constituents’ degradation and a low inhibitors
formation for being considered adequate [7]. The pretreatment aims to reduce
cellulose recalcitrance by removing lignin and releasing hemicellulose, prefer-
ably, with an energy requirement as low as possible [19]. Production costs,
including ethanol recovery costs, are directly affected by the pretreatment [20].
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These costs normally increase with a decrease in the solids load of the treated
material (dilute solution form) [19].
The main pretreatments used with lignocellulosic materials include
physical, chemical, physical-chemical, and biological methods. Many of
these treatments can be combined to improve cellulose accessibility and

sugar yield in the enzymatic hydrolysis step. The main technologies for the
sweet sorghum bagasse treatment were summarized, according to previous
studies (Figure 12.4).
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FIGURE 12.4 Main methodologies for the sweet sorghum bagasse treatment.
12.3.1.1 PHYSICAL
Physical pretreatment of sweet sorghum bagasse involved mechanical

methods of preparing biomass, responsible for increasing the surface area of
the material, by reducing the particle size and the cellulose crystallinity [18].
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The main physical methods investigated were: washing, drying, grinding,

and sieving [7].
Sweet sorghum bagasse is usually chopped, milled in hammer or rotary
mills, washed, and dried at 50–60°C to reduce the moisture content to about
10 to 15% [21]. The grinding reduces the particle size by mechanical shear,
which can be classified according to their diameter by the sieving process.
The particle size is a parameter related to the material’s saccharification
efficiency, as it is inversely proportional to its surface area and, therefore,
represents the contact area available to the action of the hydrolysis agents. In
several studies, the size range of the sweet sorghum bagasse particles after
grinding and sieving was 0.2 to 1.2 mm. Cao et al. [22] used a three-roller
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mill to separate broth and sweet sorghum bagasse, which was air-dried,
ground, and sieved at 0.42 mm. Chen et al. [23] obtained the sweet sorghum
bagasse by crushing in a roller press, drying at 20°C to maintain the final
moisture content at 20%, grinding, and sieving in three fractions: 9.5–18,
4–6, and 1–2 mm. Darkwah et al. [24] used a mechanical extractor to sepa-
rate the juice from the bagasse, which was air-dried and ground with a 1 mm
mesh in a knife mill. Lavudi et al. [25] submitted the sorghum bagasse to
oven drying to reduce the moisture content to 9–10%, being subsequently
chopped, ground, and sieved at 0.6 mm.
12.3.1.2 CHEMICAL
Chemical pretreatment is based on the application of solvents and chemical

compounds capable of breaking down lignocellulolytic structures with high
efficiency. During this process, the production of the toxic compounds, with
carbohydrate loss, can happen, being it the main limitation of the method.
Furthermore, it is a high cost and environmental risk technology [12].
The main chemical pretreatment methodologies used with sweet sorghum
bagasse were: acid and base treatment, organic solvent treatment, and ionic
liquid treatment [7].
12.3.1.2.1 Acid and Base Treatment
Acid treatment is based on the application of organic (formic, acetic, and

propionic) and inorganic acids (sulfuric, nitric, hydrochloric, and phosphoric)
to remove lignin and, mainly, hydrolyze vegetable fibers, with the removal
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of hemicellulose. The acids can be employed in the concentrated (greater

than 30% v/v) or diluted (up to 10% v/v) forms. The concentrated acid-based
process typically occurs at 100°C, for 2 to 10 h, and at atmospheric pressure.
It is considered slow, toxic, and corrosive, requiring corrosion-resistant
equipment, which increases the capital, operational, and maintenance costs.

Conversion rates for cellulose and hemicellulose can reach values above 90%
[18, 26, 27]. Diluted inorganic acid is used at 100–240°C and a pressure greater
than 10 atm for a few seconds or minutes [27]. The process is considered
fast, with no need to recycle the acid. However, there is the formation of
biomass degradation products that can compromise the fermentation process
[12]. The application of different acids and pretreatment conditions were
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responsible for different yields of released sugar. The treatment with 0.5%
(v/v) H2SO4 at 180°C for 5 min allowed a release of 66% glucan, with total
xylan removal. Compared to untreated bagasse, the available glucan amount
increased by 51% with treatment based on diluted acid [28]. In another
investigation, an improvement in the enzymatic hydrolysis step was verified
with the application of 1.75% (v/v) H2SO4 at 121°C for 40 min, which
favored the release of 14.22 g/L xylose and 2.42 g/L glucose. A small amount
of inhibitory compounds was also produced: 1.34 g/L acetic acid, 0.90 g/L
phenol, 0.12 g/L hydroxymethylfurfural (HMF), and 0.98 g/L furfural [29].
In the treatment based on H3PO4 (85% v/v) at 40–85°C, it was not observed
the HMF and furfural formation. After acid pretreatment at 50°C for 43
min, followed by enzymatic hydrolysis, the released glucose yield was 85%
[30]. The diluted H2SO4 treatment was investigated at 150°C for 1 h, at high
pressure in four different concentrations: 0.5; 1.0, 1.5 and 2.0% (v/v). A
greater recovery of glucan and xylan, around 88% and 91%, respectively,
was observed in the pre-treatment with 2% (v/v) H2SO4 [31].
Basic treatment is based on the application of hydroxides (NaOH, KOH,
NH4OH and Ca (OH)2), Na2CO3 and similar alkaline compounds, usually at
140–200°C, for a residence time of minutes to hours [19], which are respon-
sible for the saponification reactions of the ester bonds between lignin and
other carbohydrates [18]. These reactions reduce the polymerization degree
and the lignocellulosic material crystallinity, due to the lignin destruction [27]
and the cellulose and hemicellulose swelling, with consequent degradation
of their crystals [12]. Basic treatment causes lesser sugar loss than acid treat-
ment, and it is usually carried out with diluted alkalis [27]. Different alkalis
have been used to treat sweet sorghum bagasse. Treatment with NaOH at
121°C, for 1 hour at 15 psi, led to an approximate 37% lignin removal, with
88% cellulose recovery. In this method, the furfural and HMF formation
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was not verified [32]. The treatment based on alkaline distillation using 10%
(w/w) NaOH was carried out at 100°C, for 30 min at 0.04 MPa. A recovery
of approximately 94.5% glucan and 86% xylan, and a removal of about 72%
lignin were obtained [33]. The alkaline hydrogen-peroxide treatment was
investigated in two conditions: at 35°C, 2% (w/v) H2O2, for 24 h, in a dark

room (mild condition); and at 121°C (autoclave), 2% (w/v) NaOH, for 1 h +
5% (w/v) H2O2, at 20°C, for 24 h, in a dark room (severe condition). In both
conditions, more than 90% of cellulose was recovered. However, the severe
treatment was more efficient in obtaining fermentable sugars, with higher
concentrations in the hydrolysis step, reaching a value 2-fold higher than
the mild treatment [34]. The NaOH application under the same conditions
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established by Yu et al. [33], but without biomass washing, increased the
fermentable sugar conversion from 44% to 65% [35]. The residual alkalis
use is an economical and environmental method alternative for the basic
treatment of sweet sorghum bagasse. The black liquor, resulting from the
basic pretreatment of the empty fruit cluster, was used at 150°C for 30 min.
This residue contained about 76% NaOH and pH 13, which contributed to
approximately 62% bagasse delignification [20]. The green liquor, generated
in kraft pulp mills, consisting of Na2CO3 and NaS2 (simulated composition),
was used at 160°C for 110 min, with 18% total titratable alkaline charge and
40% sulfidity. A sugar yield of 82.6% was achieved after this treatment [36].
The comminated treatment is an efficient alternative for the treatment
of the lignocellulosic material, especially between acid and base, since
together, these methodologies can remove lignin and hemicellulose, making
cellulose available with greater purity. The combination of 1.4% (v/v) H2SO4
at 120°C for 47 min, and 0.25 mol/L NaOH at 120C for 5 min, resulted in
92% hemicellulose removal 90% lignin removal, with only 2.3% cellulose
lost, whose composition in the bagasse after treatment was approximately
79% [37]. The acid treatment with 1.5% (v/v) H2SO4 in autoclave for 33
min followed by the basic treatment with 4% (v/v) H2O2 for 45 h, pH 11.5,
allowed almost 77% hemicellulose and lignin removal, making available
about 79% cellulose for the reducing sugars generation [38].
12.3.1.2.2 Solvent-Based Treatment
The treatment using organic solvents is known as organosolv or solvolysis.

This method is capable of removing lignin and hemicellulose via solubiliza-
tion, characterized by the cleavage of α-O-aryl, β-O-aryl bonds, and esters
of 4-O-methylglucuronic acid [18]. The most used solvents are ethanol,
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methanol, acetone, organic acids (formic and acetic), and ethylene glycol [19].
Although expensive, most organic solvents can be reused after recovery by
evaporation and other extractive technologies, thereby reducing the adverse
effects of their presence in the fermentation step [27]. When treated with 50%
ethanol and 1% H2SO4, at 140°C for 30 min, sweet sorghum stem showed a
sugar yield of 77%, 2-fold greater than untreated bagasse [39]. Acetone treat-
ment at 180°C for 60 min, improved the enzymatic hydrolysis step by 94.2%
in a process intended for the joint production of acetone, butanol, and ethanol
(ABE process), allowing the removal of 143 g lignin and the release of 250 g
hemicellulose for each 1 Kg bagasse treated [40]. Concerning the biorefinery
concept, treatment with an aqueous solution of 60% (v/v) ethanol and 20%
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(v/v) isopropanol at 140°C for 30 min, with 1% sulfuric acid as catalyst,
contributed to a hydrolysis yield of 90.3%, producing 24.9 g/L glucose [41].
12.3.1.2.3 Ionic Liquids (ILs) Treatment
Ionic liquids (ILs) are salts formed by a large volume organic cation and an
inorganic anion of different size, usually smaller than the cation, which are
liquids at room temperature [19]. ILs are considered special solvent types,
being classified as green solvents, as they do not release toxic or flammable
substances during the cellulose treatment. These compounds are capable of
dissolving lignin and other carbohydrates by forming hydrogen bonds with
the hydroxyls of the sugars present in the lignocellulosic material [42]. The
sweet sorghum bagasse treated with 1-Butyl-3-methylimiazolium chloride
([BMIM] Cl) at 110°C for 120 min, showed a reduction in the hemicellulose
composition from 26.3% in untreated bagasse to 16.7% after treatment
[43]. ILs such as 1-allyl-3-methylimdazolium acetate ([AMIM] OAc),
1-ethyl-3-methylimidazolium formate ([EMIM] Fmt) and 1-ethyl-3-methy-
limidazolium acetate ([EMIM] OAc) showed high capacity dissolving the
lignocellulosic fraction of sweet sorghum, without causing damage to the
cellulase enzymatic action. A sugar yield 7.5-fold higher was verified with
the treatment using [EMIM] Fmt, compared to untreated bagasse, which also
favored a bacterial nanocellulose productivity of 0.71 g/(L.d) [44].
12.3.1.3 PHYSICAL-CHEMICAL
Physical-chemical treatment is a special type of combined treatment,

resulting from the union between mechanical and chemical methods of
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treating lignocellulosic material. The physical-chemical methods most used

with sweet sorghum bagasse have been: SE, LHW, AFEX, and microwave-
or ultrasound-assisted technologies [7].
12.3.1.3.1 Steam Explosion (SE) Treatment
Steam explosion (SE) is a hydrothermal pretreatment capable of hydrolyzing

the acetyl group present in hemicellulose, and promoting structural changes
in cellulose by the hydrogen bonds rupture, increasing its digestibility [27].
The biomass is rapidly heated by saturated steam at high temperatures and
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pressures [45], often in the range of 160–270°C and 20–50 bar, respectively,
for a few seconds or minutes [12], without the addition of a chemical reagent.
Therefore, it is a lower environmental impact method, responsible for the
complete sugar recovery when compared to other pretreatments [27]. SE can
be associated with water and SO2 impregnations to increase the lignocellu-
losic fiber fracture efficiency. The application of the SE treatment increased
in 2.5-fold the maximum cellulose conversion and the glucose release from
sweet sorghum bagasse treated in a reactor with 0.25 t/h high pressure, at
160°C, for 5 min [43]. Sweet sorghum bagasse samples impregnated with
SO2 gas were treated with steam at 190C for 5 min. Approximately 54%
glucan, 10% xylan, and 26% lignin were obtained after treatment [46]. The
sweet sorghum bagasse submitted to steam at 200°C for 5 min released
leachate, which had its liquid and solid fractions separated by a cyclone. A
composition of 52% cellulose, 9% hemicellulose, and 25% lignin was veri-
fied for the released solid fraction [47]. Impregnation with water was also
investigated in the SE treatment of dry sweet sorghum bagasse. After being
subjected to steam at 215°C for 2 min, about 37.3% of total sugars could be
recovered from the biomass treated [48].
12.3.1.3.2 Liquid Hot Water (LHW) Treatment
Liquid hot water (LHW) or hot-compressed water acts as a solvent and

reaction medium capable of breaking down cellulose, by combining retention
time, temperature, and pressure [26]. Usually, the method is carried out at
200–230°C, and at high pressure, so that the water can penetrate the biomass
and, thus, favor the cellulose hydrolysis, in addition to the removal of the
hemicellulose [27]. Sweet sorghum bagasse, when hydrolyzed with water
at 60°C for 75 min, showed a sugar yield of almost 68% [21]. When it
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was treated at 200°C for 6.5 min, hemicellulose solubilization of 85% was
achieved, being greater than the 74% yield obtained with the treatment at
190°C for 20 min [49].
12.3.1.3.3 Ammonia Fiber Expansion (AFEX) Treatment
Ammonia fiber expansion (AFEX) is a pretreatment based on the applica-

tion of liquid anhydrous or gaseous ammonia at elevated temperatures
(90–100°C) and high pressures (1–5.2 MPa) [27]. With the pressure release,
ammonia is quickly evaporated, which promotes the cellulose crystallinity
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reduction, hemicellulose depolymerization, and deacetylation of acetyl
groups [45]. In AFEX treatment, the fermentation inhibitors formation and
a liquid current are not verified being, therefore, considered a dry-to-dry
process [18]. Ammonia can be reused in this method, reducing process costs,
and avoiding environmental problems with its disposal [27]. The optimi-
zation of AFEX treatment conditions was defined as 140°C, 30 min, 2:1
for ammonia/biomass ratio, and a mixture content of 120%. Under these
conditions, the glucan and xylan conversion reached 90% for sweet sorghum
bagasse. Free sugars destruction was observed during the AFEX treatment,
which was solved with the previous biomass washing [50].
12.3.1.3.4 Microwave- or Ultrasound-Assisted Treatment
Microwave and ultrasound irradiation methods are considered energetical

efficient, simple to operate, and capable of heating the lignocellulosic material
quickly [27]. These treatments can accelerate the chemicals released and the
biological and physical processes action, through collisions of polar and ionic
molecules promoted by heat [45]. Although microwave treatment improves the
cellulose accessibility to hydrolytic enzymes, its investment cost (equipment
cost) is higher than other technologies [27]. Microwave treatment assisted by
ammonia heated to 130°C for 1 h promoted the release of 42 g glucose for
each 100 g dry sweet sorghum biomass [23]. The association of microwaves
and ultrasound treatments with other methods contributes to the increase in
cellulose digestibility. The optimization of the microwave-assisted alkali
pretreatment conducted at 1,000 W as 0.1 g lime, 10 ml water content, and
exposure time of 4 min resulted in a sugar yield of 52.6%. In the lime absence
and maintaining the water content and exposure time, this yield increased
to 65.1% [51]. For the microwave treatment of sweet sorghum bagasse at
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180 W for 20 min, equivalent to a power of 43.2 kJ/g of dry biomass, in the
sulfuric acid solution (50 g/Kg of bagasse) presence at 82°C, a yield of 820 g
sugar for each 1 Kg treated bagasse was obtained after the process [52]. The
microwave-assisted acid treatment at 180 W and microwave-assisted alkali at
300 W employing two different microorganisms in a co-fermentation process

at different cell ratios were investigated. Better ethanol yields were achieved
through microwave-assisted acid treatment, regardless of the cell ratio used.
Microwave-assisted acid pretreatment is more efficient, requiring less energy
[53]. A sugar yield of 57% was achieved by treating sweet sorghum bagasse
with diluted aqueous ammonia and assisted by 90 W ultrasound [54].
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12.3.1.4 BIOLOGICAL
Biological treatments are based on the action of enzymes and microorganisms

such as bacteria and fungi, capable of carrying out the fermentation in the
solid-state and producing several lignocellulolytic enzymes, responsible for
degrading the sweet sorghum biomass [7]. Microbial consortia, use of fungal
species, and enzymatic technologies are examples of biological treatments
[45]. The biological method is effective in lignin degradation, having as
advantages the use of mild conditions, low energy consumption, and low
environmental impact. However, it is limited by long-term cell cycles and
consumption of cellulose and hemicellulose for the growth of some fungi,
high degradation conditions for bacteria, and low activity of lignocellulolytic
enzymes, which limits its industrial application [27]. Coriolus versicolor
fungus is widely used in the sweet sorghum bagasse treatment due to its
selective lignin degradation capacity. In an investigation using this fungus
with CuSO4-syringic acid supplements, the synergistic effect of CuSO4
and serum acid was evaluated. An approximate degradation of 36% lignin
was achieved, which resulted in a sugar production 2.2-fold greater in the
enzymatic hydrolysis step [55]. In another study using the C. versicolor,
supplements of serum acid and gallic acid were added to the biological
treatment. The synergistic action of these acids resulted in the degradation of
about 31% lignin, contributing again to a sugar production 2.2-fold greater in
the enzymatic hydrolysis step [56]. C. versicolor was also used in a solid-state
fermentation in a Mesh Tray Bioreactor. In this investigation, an increase
in the production of the lignocellulolytic enzymes laccase and xylanase
was observed, due to the proper conduction of the fermentation in the tray
mesh, and in the ideal airflow in the bioreactor. The lignin degradation was
approximately 46%, with an almost 8% cellulose loss. Enzymatic hydrolysis
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produced about 2.5-fold more fermentable sugars than the control assays
using untreated sweet sorghum bagasse in a meshless bioreactor and without
a humid airflow [57].
12.3.2 SACCHARIFICATION AND FERMENTATION
After initial treatment, sweet sorghum bagasse is submitted to enzymatic and

microbial action to convert cellulose and hemicellulose into ethanol. Treated
biomass saccharification can be carried out simultaneously with the fermen-
tation process in the same bioreactor (SSF), or in the different bioreactors
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(SHF). When the SSF process steps take place at relatively large intervals,
the process is classified as DSSF. Treated biomass can also be saccharified
and fermented together with sweet sorghum juice in a process classified as
SSCF (Figure 12.5).
FIGURE 12.5 Saccharifications and fermentation approaches for the cellulosic ethanol
production from sweet sorghum bagasse. [Abbreviations: SHF: separate hydrolysis and fermen-
tation; SSF: simultaneous saccharification and fermentation; DSSF: delayed simultaneous
saccharification and fermentation; SScF: simultaneous saccharification and co-fermentation].
12.3.2.1 SIMULTANEOUS SACCHARIFICATION AND FERMENTATION (SSF)
Simultaneous saccharification and fermentation (SSF) process contribute to

the reduction of operating and capital costs, avoiding the cellular inhibition
caused by the sugar-released excess after lignocellulosic material enzymatic
hydrolysis. Although advantageous to ethanol yield and low biomass loss,
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the SSF process is limited by the recovery difficulty and reuse of the micro-
organisms and enzymes, also requiring distinct favorable conditions for
each step such as optimal temperature and pH [58]. Treated sweet sorghum
bagasse can be submitted to the SSF process, using different enzymatic
loads and microorganisms, for the cellulosic ethanol production. The sweet
sorghum bagasse treated with diluted H2SO4 was fermented in two steps.
Firstly, Neurospora crassa DSM 1129 fungus was used to produce lignocel-
lulolytic enzymes. Posteriorly, the SSF process was performed using these
enzymes, with cellulase and β-glucosidase supplementation at 6 FPU/g.
About 27.6 g/L ethanol was produced, with a yield of 84.7% [59]. The
SSF process optimization was performed to the efficient cellulosic ethanol
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production. At 37°C, 25 FPU/g enzymatic load, and 1.4 g/L Saccharomyces
cerevisiae ATCC 24858, about 38 g/L ethanol, with 89.4% yield and 1.28
g/L/h productivity, were produced from biomass treated with dilute H2SO4
[28]. Approximately 85 g/L ethanol was achieved in 21 h fermentation by
S. cerevisiae JP1 at 37°C, after 6 h enzymatic hydrolysis at 50°C, with 32.8
FPU/g enzymatic load, and combined acid-base pretreatment. An ethanol
yield of 63% and 4 g/L/h productivity were obtained [37]. At severe alkaline
treatment using NaOH and H2O2, about 19.3 g/L ethanol was obtained by
S. cerevisiae CICC1308 at 36°C, after the pre-saccharification at 60 FPU/g
cellulase and 80 IU/g β-glucosidase, at 50°C for 12 h. Ethanol yield reached
more than 88% in 48 h of SSF [34]. The combined acid-base treatment
followed by hydrolysis at 20 IU/g, at 60°C for 58 h, and fermentation at
35°C by Pichia kudriavzevii HOP-1 resulted in 26.8 g/L ethanol in 48 h. P.
kudriavzevii HOP-1 was considered advantageous for the SSF process due
to its thermotolerant capacity, which confers economic advantages to the
commercial cellulosic ethanol production [25]. The black liquor application
for 30 min followed by enzymatic hydrolysis with 30 FPU/g enzymatic load
and fermentation at 32°C by commercial S. cerevisiae resulted in 45.06 g/L
ethanol in 72 h [20]. The alkaline treatment based on Na2CO3, resulting from
the reaction between NaOH from the bagasse basic treatment and the CO2
released from the sweet sorghum juice fermentation, was associated with
saccharification and fermentation at 32°C for 72 h. A theoretical ethanol
yield of almost 82% glucan by S. cerevisiae was obtained [60]. The DSSF
process was also investigated after hydrothermal treatment associated with
liquefaction-saccharification at 50°C, with 10 FPU/g enzymatic load, for 24
h, and addition of extra enzymes. About 48.3 g/L ethanol by baker’s yeast at
30°C was reached. The ethanol yield and productivity under these conditions
were equal to 71.2% and 2.2 g/L/h, respectively [61].
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SSF process can be performed by a fed-batch approach, as verified in a

study using sweet sorghum bagasse treated with acetic acid. This process was
carried out at 37°C for 96 h, resulting in 53.1 g/L ethanol by S. cerevisiae
ATCC 24858, which was 2-fold higher than the batch SSF production,
equal to 25.7 g/L. Fed-batch SSF improved the final ethanol production and
contributed to reduce the cell inhibition caused by the high fermentation
medium viscosity containing a high solid load content [24].
SSF process can also be combined with biological pretreatment by solid-
state fermentation of sweet sorghum bagasse, in which filamentous fungi are
used to degrade the lignocellulosic fraction. In a study using Mucor indicus
CCUG 22424, the solid-state fermentation product was submitted to the SSF
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process, with 15 FPU/g cellulase and 30 IU/g β-glucosidase, and fermen-
tation at 37°C for 48 h. Compared to the SSF process without biological
pretreatment, the solid-state fermentation allowed a 4.3-fold higher ethanol
yield, equal to 85.6% [62].
12.3.2.2 SEPARATE HYDROLYSIS AND FERMENTATION (SHF)
The separate hydrolysis and fermentation (SHF) process is widely used in the
ethanol production from sweet sorghum bagasse, although its yield achieved
is slightly lower than the SSF process yield [58]. The separation of these
steps allows each process to be conducted under optimal temperature and
pH conditions, thus contributing to an efficient conversion of cellulose and a
high ethanol production [8]. SHF process also allows the cellular biomass and
hydrolysate recovery, being widely used with filamentous fungi. In a study
using M. hiemalis CCUG 16148, the sweet sorghum bagasse treated by the
ultrasound-assisted NaOH was submitted to the SHF process at 32°C for 24 h,
and 81% yield and 0.70 g/L/h ethanol productivity have been achieved [63].
In another investigation, the sweet sorghum bagasse treated by microwave-
assisted diluted ammonia was submitted to 60 FPU/g cellulase and 64 CBU/g
hemicellulase, at 55°C for 24 h. After 48 h fermentation by S. cerevisiae
(D5A) at 30°C, about 21 g ethanol/100 g treated biomass were produced [23].
Cellulosic ethanol was also obtained by Dekkera bruxellensis GDB 248, yeast
capable of assimilating cellobiose and glucose. After the alkaline treatment
using H2O2 and enzymatic hydrolysis at 50°C, with 20 FPU/g commercial
cellulase, for 48 h, and ethanol yield of 0.44 g/g was achieved in 7 h fermenta-
tion at 32°C [64]. The acid treatment based on H2SO4 was optimized and used
for the sweet sorghum bagasse hydrolysis, whose hemicellulosic fraction
generated was submitted to fermentation at 30°C by Scheffersomyces stipitis
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ATCC 58376. About 22 g/L ethanol, 0.40 g/g yield, and 0.34 g/L/h produc-
tivity were obtained after 55 h [29]. The cellulosic ethanol production was
also investigated without pre-treatment. For an enzymatic hydrolysis with
8.32 FPU/g load and a fermentation at 30°C by Trichosporon fermentans CBS
439.83, approximately 20.7 g/L ethanol were obtained. The external nitrogen
supplementation increased ethanol production to 23.5 g/L, with 0.118 g/g
yield and 0.196 g/L/h productivity in 120 h [65].
Enzymatic hydrolysis step is usually performed in batch approaches.
However, some studies suggest that the fed-batch approaches can improve
the glucose release during the saccharification process. The sweet sorghum
bagasse treated with hot liquid water at 180°C, for 20 min at 4 MPa, was
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submitted to saccharification in fed-batch, with supplemented Tween80.
The solid loads were fed in 24 h and 48 h, together with 20 FPU/g and 30
FPU/g cellulase at 50°C, to reach a final load of 20% and 30%, respectively.
Approximately 89 g/L glucose was released after 120 h, and, then, submitted
to fermentation by S. cerevisiae Y2034 at 30°C for 72 h. About 43.4 g/L
ethanol was produced [66].
SHF processes are generally compared to SSF processes, considering the
same biomass pretreatment, to identify and differentiate their advantages
and limitations. The SSF and SHF processes were used after sweet sorghum
bagasse steam-treated to produce cellulosic ethanol by S. cerevisiae (Tembec
1). In the SSF process, the hydrolysis step was conducted at 50°C for 12 h,
and the fermentation step at 37°C for 120 h. About 23.3 g/L ethanol was
produced, with a yield of almost 64%. In the SHF process, the hydrolysis step
was conducted at 50°C and the fermentation step at 30°C. Approximately
21.2 g/L ethanol was obtained, with a yield of almost 58% [46]. The ethanol
produced by P. kudriavzevii HOP-1 was investigated in the SSF and SHF
processes, after the acid-base pretreatment of sweet sorghum bagasse. Both
methods resulted in just over 26 g/L ethanol, however, the SSF productivity
(0.56 g/L/h) was 40% higher than the SHF productivity (0.40 g/L/h), due to
the time required for the maximum ethanol production, which was 48 h for
SSF and 66 h for SHF [25]. S. cerevisiae TISTR 5606 was used to convert
sweet sorghum bagasse treated by H2O2 and NaOH into cellulosic ethanol.
The saccharification and fermentation of the hydrolysate were carried out by
SSF, SHF, and DSSF. In the SSF process, the substrate, inoculum, and cellu-
lase were mixed in the same bioreactor, at 37°C, for 72 h, which resulted in
22.3 g/L ethanol. In the DSSF process, the hydrolysis step was carried out
at 50°C for 6 h, followed by fermentation at 37°C for 66 h. About 28.3 g/L
ethanol was produced by DSSF. The DSSF productivity was 26% higher
than the SSF and SHF productivities of 0.31 g/L/h [8].
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Combined fermentation using different microorganisms was also verified

in the cellulosic ethanol production from sweet sorghum bagasse. The micro-
bial consortium is applied to ensure that 5- and 6-carbon sugars are converted
to ethanol. The treated hemicellulosic fraction of sweet sorghum by diluted
H2SO4, self-hydrolyzed by SE, and detoxified by the over-liming process,

was converted into 38.7 g/L ethanol, with 82.5% yield, by the synergic
action of P. stipitis NCIM 3497 and Debaryomyces hansenii sp. at 30°C.
About 92 g/L sugars, mainly xylose and glucose, were assimilated by both
microorganisms [67]. Zymomonas mobilis ATCC 31821 and commercial S.
cerevisiae were used in the sweet sorghum bagasse assimilation, after it was
treated by microwave. The combined fermentation was performed at 32°C
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for 24 h, which resulted in 94% conversion efficiency and 480 g ethanol/Kg
treated biomass. Concerning only lignocellulosic fraction, about 33 m³/ha
ethanol can be produced from sweet sorghum [52]. Z. mobilis ATCC 31821
and commercial S. cerevisiae were also used for the sorghum hydrolysate
fermentation, treated by microwave-assisted acid/base. For 10 g/L Z. mobilis
and 5 g/L S. cerevisiae, a yield of 0.50 g/g ethanol was obtained after 24 h
fermentation at 32°C [53].
12.3.2.3 SIMULTANEOUS SACCHARIFICATION AND CO-FERMENTATION

(SSCF)
All sweet sorghum components (grain, juice, and bagasse) can be used to
increase ethanol production from its lignocellulosic fraction, since the low
concentration of fermentative products is the main limitation to the industrial
production of the cellulosic ethanol [49]. The bagasse saccharification and
fermentation together with the juice fermentation can improve ethanol
yield from different feedstocks, making the bioprocess economically
advantageous. A previously treated and dehydrated sweet sorghum juice and
bagasse was responsible for 53 g/L ethanol production in 168 h. The nutrients
present in the sweet sorghum juice were beneficial to the fermentation
process, improving in 92% the final ethanol, in lesser time of 72 h [49]. The
fermentative integration was also performed for the sweet sorghum bagasse
obtained from the solid-state process. This biomass was treated by NaOH
and submitted to SScF by Z. mobilis TSH-ZM-01 at 32°C. Under optimized
conditions, about 69.5% yield was achieved, which can reduce the capital
costs and energy consumption, making the ethanol bioprocess commercially
viable on a large scale [33]. The bagasse, treated by SE-assisted dilute
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phosphoric acid, and submitted to liquefaction, followed by SScF at 37°C

using an engineered ethanologenic E. coli strain, was converted into 27.5
g/L ethanol in 96 h. Concerning to juice and bagasse parts, about 10,600 L/
ha ethanol has been achieved from sweet sorghum [68]. The SScF integrated
process was investigated after H2SO4 treatment. Under optimized conditions

of 2% H2SO4, 20 FPU/g cellulase, and fermentation at 37°C using S.
cerevisiae M-HT 3013, about 120.4 g/L ethanol was produced in 216 h. This
production corresponded to a yield of 67.75 g ethanol/Kg treated biomass
[31]. The SE treatment followed by the fermentation of the free fermentable
and the lignocellulosic sugars resulted in more than 90% ethanol yield, with
low inhibitor formation such as 5-HMF, furfural, and levulinic acid, by a
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wild-strain S. cerevisiae. The detoxification step was not necessary, which
reduces the production costs [69]. The co-fermentation of juice and bagasse
from sweet sorghum was also investigated by the cellular consortium
fermentation. Firstly, the lignocellulosic fraction was treated by H2SO4
(98% v/v) at 120°C for 1 h. Next, the treated biomass and the juice were
fermented at 30°C for 96 h, using S. cerevisiae ATCC 7754 and Z. mobilis
ATCC 29191. For each 1 Kg treated sweet sorghum (variety SS-301), about
160 mL ethanol was produced [14].
12.4 BIOTECHNOLOGY EVOLUTION
Cellulosic ethanol production from residual sweet sorghum has been exten-
sively studied in recent years, as a promising alternative to sugarcane ethanol.
In 2009–2020, several investigations were carried out, using different
treatment technologies and saccharification and fermentation approaches.
Approximately 32% of scientific articles investigated physical, chemical,
physical-chemical, and biological treatments to make cellulose digestibility
more efficient and to available the sweet sorghum bagasse sugars for fermen-
tation. Of the 68% scientific articles addressing the fermentation stage, about
54.3% corresponded to the SHF process, 30.4% to the SSF process, and
15.2% to the SScF process (Figure 12.6). Although lesser efficient than the
SSF process, the SHF process has been the most used method in recent scien-
tific research for converting sweet sorghum bagasse into ethanol. Possibly,
the conducting of saccharification and fermentation steps under respective
optimal conditions can explain this predominance. In turn, the promising
SScF process was the least reported method for sweet sorghum processing
since it is still a recent technology, starting its investigations in 2017.
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FIGURE 12.6 Main saccharifications and fermentation approaches reported by scientific
articles in 2009–2020.
The three fermentation approaches reported were preceded by the

treatment of sweet sorghum biomass. For the SSF process, the acid
pretreatment was the most used technology, followed by the combined
treatment, in 2009–2016 [24, 28, 37, 59, 70, 71]. The main used acid was
the diluted H2SO4, and its combination with NaOH was considered one of
the most efficient pretreatments for the cellulose release. In 2017–2020,
the alkaline treatment gained prominence, accounting for 60% of reported
articles employing the SSF process [8, 20, 60]. Again, NaOH was the most
used alkali for processing the lignocellulosic fraction of sweet sorghum
(Figure 12.7).
In 2009–2012, the following treatments were investigated in 60% of
articles using the SHF process: alkaline, SE, and combined. In addition
to NaOH, Ca(OH)2 [32] and H2O2 [22] were used in basic treatments as
alkaline compounds. The SE treatment was conducted independently [46,
72] or combined with H2SO4 [67], and it did not require an enzymatic
hydrolysis step, since it was able to release glucose efficiently. The
microwave-assisted NaOH [63] and microwave-assisted diluted NH3
treatments [23] were considered important combined treatments without
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the saccharification step. In 2013–2016, the alkaline and combined treat-

ments predominated, being used NaOH and H2O2 as alkalis, with, and
without NaOH washing [35, 73]. The association between NaOH, H2O2,
and H2SO4 [74], and the SE-associated diluted H2SO4 [52] were the main
combined treatments used in the period. In 2017–2020, the acid, the alka-
line, and the acid-base treatments were also wildly investigated [8, 25, 29]
(Figure 12.8).
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FIGURE 12.7 Main methods for the sweet sorghum treatment using the SSF approach.
FIGURE 12.8 Main methods for the sweet sorghum treatment using the SHF approach.
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SScF has been recently investigated, begging in 2013. In 2013–2016,

the alkaline treatment using NaOH [33], the hot liquid water [49], and the
biological-assisted NaOH treatments [75], were the most methods investi-
gated. However, in 2017–2020, 50% of scientific articles employed the SE
independently [48, 69] or assisted by diluted acid [68] to release glucose to

the fermentation step (Figure 12.9).
FIGURE 12.9
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Main methods for the sweet sorghum treatment using the SScF approach.
In all fermentative processes, the fermentations have been mainly

performed by yeasts such as S. cerevisiae, which was the most used micro-
organism in the scientific investigations developed between 2009 and 2020.
Approximately 65% of articles reported this yeast, in cellular consortium
or isolated one, for the cellulosic ethanol production from sweet sorghum.
Z. mobilis was the second most used microorganism, according to 14% of
articles reported. It is a gram-negative bacterium that has a high bioethanol
production capacity, reaching a productivity 2.5-fold higher than that
obtained by S. cerevisiae. It is also able to tolerate up to 120 g/L ethanol
presents in the fermentation broth [53] (Figure 12.10).
Concerning the sweet sorghum bagasse treatment, without the fermenta-
tion step, there was an increase in the use of combined treatments, and a
decrease in the acid and the SE treatments over the years (Figure 12.11).
While the acid and the SE treatments are reported in the main articles, going
from 25% (2009–2012) to 20% (2013–2016), and from 25% (2009–2012)
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to 9% (2017–2020), respectively, the combined treatment grew from 25%

(2009–2012) to 55% (2017–2020). The association of different treatments
can assist the cellulose digestibility since they integrate the advantages
observed in each separate technology in a single process. Therefore, an
increase in the saccharification step efficiency can be reached. The opera-

tional combination can also reduce some technological problems such as
environmental pollution, energy consumption, and reaction time, associated
with a single treatment methodology [27], justifying, thus, its large applica-
tion in recent years.
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FIGURE 12.10 Main producer microorganism of cellulosic ethanol from sweet sorghum
bagasse.
12.5 CONCLUSIONS
The bioprocess for the sweet sorghum bagasse ethanol production has
changed in recent years, especially due to the development of more effi-
cient, cheaper, and eco-friendlier treatment technologies and fermentation
approaches. The combination of different treatments will be increasingly
used, since it can take advantage of the separated methodologies benefits
to improve the ethanol conversion and yield. Furthermore, the simultaneous
saccharifications and fermentation processes using cellular consortia and the
free sugar co-fermentation can contribute to an optimized and economically
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feasible large-scale implementation. Therefore, the full sweet sorghum

bagasse potential should be used with biotechnology evolution, consolidating
this biomass as an important and economical biorefinery crop.
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FIGURE 12.11 Evolution of the main methods for the sweet sorghum treatment.
KEYWORDS
• biotechnology evolution
• cellulosic ethanol
• fermentation approaches
• pretreatment methodologies
• saccharification and fermentation
• sweet sorghum bagasse
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CHAPTER 13
Bioethanol Production Using

Agave americana L. Leaves Wastes
from Coahuila
CÉSAR D. PINALES-MÁRQUEZ, SHIVA, ROHIT SAXENA,
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ROSA M. RODRÍGUEZ-JASSO, and HÉCTOR RUIZ LEZA
Biorefinery Group, Food Research Department, Faculty of Chemistry
Sciences, Autonomous University of Coahuila, Saltillo–25280, Coahuila,
Mexico, Phone: (+52)-1-844-416-12-38,
E-mail: rrodriguezjasso@uadec.edu.mx (R. M. Rodríguez-Jasso),
hector_ruiz_leza@uadec.edu.mx (H. A. Ruiz)
ABSTRACT
Currently, there is a tendency to mitigate through advances in science and

technology the environmental problems caused by the abuse of burning
fossil fuels, and this resulted in accelerated climate change that has nega-
tively impacted the quality of human life and countless species. The resi-
dues from the Agave industry in Mexico represent an excellent opportunity
to adapt large amounts of LCB into the biorefinery sustainable processing
model to produce various bioproducts, including bioethanol, a fuel that
can improve the substantivity of combustible compared to those produced
by hydrocarbons. In this study, residues from Agave americana L leaves
were used as biomass for the production of bioethanol under the following
processing line: drying, milling, hydrothermal pretreatment, enzymatic
hydrolysis, and fermentation. Also, a rich pretreated solid of cellulose
(48.21%) was obtained after hydrothermal pretreatment (190C/50 min).
In this study, pre-saccharification, and fermentation strategy was applied
in the bioethanol production, 33.78 g/L of sugars were produced at 16 h
of hydrolysis and 13.58 g/L of ethanol. Therefore, the development of this
process allowed the use of a promising raw material in the production
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of biofuels and high added-value compounds in terms of biorefinery in

Mexico.
13.1 INTRODUCTION
In recent decades, civilization has had an accelerated growth, resulting in

different environmental problems, one of them being a large number of
emissions into the atmosphere, causing high levels of air pollution, resulting
in one of the most critical issues in the public health worldwide [1]. The
World Health Organization released a statement in which nine out of 10
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people are exposed to environments with high levels of pollutants in the air
and it is resulting in the death of 4.2 million people worldwide per year due
to stroke, heart disease, lung cancer, and chronic respiratory diseases [2].
Air pollution is derived from a complex mixture of particle matters
(PM), vapors, and gases that can be emitted in different ways, both natural
and synthetic. PM is generally classified into particles of 10 (PM10) and 2.5
(PM2.5) micrometers and is formed by the conglomerate of suspended solids,
humidity, and atmospheric conditions, PM10 particles tend to accumulate in
the nasal concavities and the respiratory tract superior. At the same time, the
finer particles (< PM2.5) can also be absorbed in the lower respiratory tract,
causing diseases such as lung cancer, ischemic heart disease, respiratory tract
infections, allergies, and type 2 diabetes due to saturation of suspended solid
particles in the environment [1, 3]. On the other hand, gases released into the
atmosphere have produced anormal amounts of CO2, CO, NO(x), CH4, which
in addition to decreasing air quality and deteriorating human health, is the
cause of climate change because these gases cause the greenhouse effect,
which is the main reason of the increase in the temperature of the earth. The
increase in air contamination is directly related to the excessive use of fossil
fuels and industrial growth [1, 4].
Based on this problem, efforts have been made to try to mitigate or control
climate change. One of these efforts is The Paris Agreement of the United
Nations, whose main objective is to regulate polluting emissions that cause the
greenhouse effect and to keep the increase in world temperature below 2°C,
limiting this increase to 1.5°C and this in a time frame that allows ecosystems
to adapt to climate change and enable sustainable economic progress [5].
The biorefinery concept fits as an emerging solution to this problem
because it seeks the substitution of hydrocarbon-produced energy compounds
for biofuels produced from renewable sources such as biomass, some of
the proposed fuels are bioethanol, biogas, biohydrogen, biodiesel, bio-oil,
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Bioethanol Production Using Agave americana L. Leaves 373
vegetable oils, biosynthetic gas, bio-char among others, increasing the

substantivity of energy sources, which are also produced through increas-
ingly sustainable and efficient strategies [6].
The operation of a biorefinery is similar to a conventional refinery which
the raw material is oil, since different products, including energy compounds,
are produced from a petroleum complex mixture through various stages,
however, given the non-renewable nature of this material and the environ-
mental problems that result from the excessive use of these products, make
the concept of biorefinery an attractive alternative, since using different types
of biomass, which can come from various sources and have meager costs due
to its high availability, and result in a wide variety of compounds with novel
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applications in addition to bioenergetics [7].
In general, biomass is defined as all material from a living organism,
and its use in biorefineries has generated a classification as generations,
depending on the biomass used. Currently, there are four generations of
biomass, the first generation (1G) comprises those of biorefineries that use
food crops as fuels, due to their high content in sugars, starch, and natural
oils, resulting in the first-generation biofuels, this generation represents
a significant concern because it uses edible resources creating direct
competition between food and fuel production [8]. The second-generation
biorefinery (2G) includes biorefineries whose raw material is LCB, that
is, those that are composed of hemicellulose, cellulose, and lignin. These
biorefineries present significant advantages since the raw material is found
in large quantities and is usually the residue of agricultural, forestry, and
food industries, so it does not compete with the crops generated for the food
sector, and due to the main components of the matrix it is possible to produce
a wide variety of byproducts and biofuels [9]. The third-generation (3G)
is characterized by the use of aquatic biomass as raw material, specifically
microalgae and macroalgae, this alternative is very attractive since it does
not generate competition with arable land, it also has a high carbohydrate
content and it does not have the strong union of terrestrial biomasses, so
there is a great opportunity to produce a wide variety of different products
and biofuels [10]. The fourth-generation (4G) consists of microorganisms
and genetically modified plants to have high amounts of carbon for the
production of fuels and various biochemicals [8].
Finally, the use of biomass has become a sustainable alternative to the
future for the supply of renewable products. It generates an alternative to
non-renewable energy sources, promoting the growth of a biologically based
economy, of a sustainable nature, also affected circular bioeconomy [8].
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13.2 LIGNOCELLULOSIC MATERIALS: A NOVEL PROPOSAL FOR

RAW MATERIAL IN BIOREFINERIES
13.2.1 OVERVIEW OF LIGNOCELLULOSIC BIOMASS (LCB) FOR

BIOREFINERIES
In general, lignocellulosic materials are mainly composed of cellulose

(30–50%), hemicellulose (20–40%), and lignin (20–30%) [11]. Cellulose is
made up of a polymer of linked β-1,4 glucose units, Hemicellulose by the
polymer of pentoses, hexoses, and uronic acids, where its main component
is xylan, and lignin is constituted by polymers cross-linked phenolic which
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function as the main structuring agent of plants [12, 13]. This matrix of
components is an opportunity for the creation of different compounds from
a single raw material, through different chemical, physical, biological, and
enzymatic processes [12]. Finally, annual worldwide production of LCB of
200 × 109 tons per year is estimated, making abundant feedstocks available
for the production of biofuels and biochemicals [8, 14].
Some examples of possible products from the second generation
biorefineries are formic acid, ethylene glycol, acetic acid, lactic acid,
glycerol, glycolic acid from cellulose, but the primary designated use for this
polymer is for the production of bioethanol under the model of a biorefinery.
In the case of lignin, the transformation of this polymer can lead to quinones,
phenol benzene, syringaldehyde, pyruvate, and different lipids. And finally,
hemicellulose can be transformed into furfural, hydroxymethylfurfural (HMF),
levulinic acid, pentane, among others [15]. However, numerous investigations
have been directed towards the use of xylan from hemicellulose, because the
compounds derived from this polymer have various food applications such
as xylitol and xylose (Low-calorie sweetener substitutes) [13, 16], but with a
particular interest in the production of oligomers such as xylooligosaccharides
(XOs), which have multiple uses in food and pharmaceutical technology due
to their prebiotic, antioxidant, and cytoprotective activity [17], especially with
those of lower molecular weight or with a lower degree of polymerization
(DP = 2–4, oligomers made up of 2 to 4 xylose units) [13, 16] and those with
higher molecular weight, such as biopolymer with (DP > 5–10) which have
a wide variety of applications such as its use in the development of food
covers [18]. Finally, the design of operational chains that promote an integral
use of biomass, through the combination of the production of biofuels and
high added value co-products, because all these biochemicals promote the
economic projections of the second generation biorefineries. For this reason,
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it is essential to highlight the importance of the great variety of products that

can be produced from LCB and its economic and social contribution [19, 20].
One of the common obstacles when it comes to LCB is its resistance
to fractionation, this factor is known as recalcitrance, and it also worsens
stages such as the saccharification of the constituent polymers of lignocellu-

losic materials such as glucan and xylan. Nevertheless, there are processing
models that improve the availability of the material using mechanisms such
as autohydrolysis in treatments such as LHW and SE, which use only water
as a catalytic medium to promote the separation of the lignocellulosic matrix
and improve the later stages [21].
Nowadays, biorefineries around the world mainly produce biodiesel and
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bioethanol using first-generation raw materials, due to its high content of
sugars, starches, or natural oils [22]. As reported by the Renewable Fuels
Association, 15,776 million bioethanol gallons were produced in 2019
using primarily grains like corn and sorghum [23]. However, although
first-generation biofuels diversify the raw materials with which biofuels are
produced, they are surrounded by environmental and social contradictions
such as environmental degradation due to the change in land use derived
from the expansion of agriculture, affecting indirectly or directly biodiversity
and the amount of CO2 produced, and also the creation of direct competition
with agriculture for food generation [22].
In addition, the environmental problem tends to worsen, because as the
population grows, there will be a higher demand for resources, specifically
energy and food, by 2030 it is expected that there will be 8.5 billion people
in the world, at a growth rate 83 million people each year, which projects an
increase of 70 million ha to meet global food demand by 2050 [24]. All these
factors drive towards a transition that first generates a renewable alterna-
tive for the production of fuels and does not compromise the use of food
resources and the environment, taking into account the raw material and the
sustainability of the process. Finally, this promotes the concept of second-
generation biorefineries, whose main objective is to generate bioeconomic
circular routes (Figure 13.1) where non-edible lignocellulosic waste is used
with increasingly optimal and sustainable processes [22, 24].
13.2.2 SECOND GENERATION BIOREFINERY DEVELOPMENT

OPPORTUNITY IN MEXICO
Mexico is one of the most promising developing countries, due to its

geographical position, this country has a great variety of favorable climates
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for agricultural production, and also is a sunny country, which also has various
seas, rivers, and lakes. For these reasons, Mexico has enormous potential to
develop more sustainable alternative energies such as solar, wind, hydraulic,
and thermal energy, but also with large amounts of biomass available due to
its vast agricultural activity [25].
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FIGURE 13.1 Overview of second-generation biorefinery and its applications.
According to the National Institute of Statistics and Geography (INEGI),

in 2017, there was a total of 18.900,000 ha for agricultural activity in Mexico,
of this available land 83% was destined for annual crops and the rest for
perennial plants. The five main crops in the country during that year were:
sugar cane (56,354,945 ton), white grain corn (23,142,203 ton), yellow grain
corn (8,071,840 ton), wheat grain (3,214,047 ton), red tomato (3,008,036
ton) [26]. As a result, the availability of lignocellulosic residues in Mexico
is high, given the level and the great variety of agricultural products in the
country.
According to the Mexican National Energy Secretariat (SENER) and
the National Energy Balance databases, the national energy consumption
is 9,236,858 PJ, of which the energy consumption for fuels corresponds
to 5,283,705 PJ or 57.2%. Only 6.01% of use for fuels was produced by
renewable sources in 2018, where firewood (249,084 PJ or 4.71%) and cane
bagasse (55,716 PJ or 1.05%) are the primary sources of energy genera-
tion from biomass. On the other hand, petroleum products corresponding
to 57.07% (3,015,637 PJ) plus the contribution derived from coal of 3.54%
(186,931 PJ) [26]. This demonstrates that the current generation of fuels in
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Mexico is still highly inclined towards the generation of energy through non-
renewable sources and where the burning of fossil fuels has a great presence
in the country [25].
Today, despite political limitations and the legal framework for the
use of biomass for the production of biofuels is still not defined, there
has been a growing interest in the development of renewable energy in
the country. In 2016 the Mexican Center for Innovation in Bioenergy
(CEMIE-Bio) was created to carry out research and development of
technologies for the sustainable production of fuels from biomass, said
the group works in 5 specialized groups for each type of biofuel: solid
biofuels, bioalcohol, biodiesel, biogas, and bioturbosin. The results of
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these research groups are expected to increase the use of biomass in
Mexico and contribute to the generation of at least 35% of clean energy
by 2024 [27].
13.2.3 AGAVE WASTE AS RAW MATERIAL UNDER BIOREFINERY
The use of agave has a strong presence in Mexico since it is used in various
artisanal and industrial activities, this generates large amounts of solid waste
with enormous potential for its use [28]. Various of agave species contribute
to the economy of Mexico via the production of contrasting products such
as in northern Mexico, where agave is used for the production of textile
fibers, while, in the southern region tradition Mexico developing tequila,
mezcal, and pulque [28]. For example, Agave tequila weber is distinguished
for the production of tequila in central Mexico through cooking and crushing
this plant to create juices with high sugar content, which are fermented and
distilled to create tequila; however, this process leaves large amounts of solid
residue that can be used for the creation of biofuels and high added-value
compounds [29, 30].
Through the biorefinery model this solid bagasse can be used to
produce the biofuels and biproducts. Agave bagasse contains the cellu-
lose, hemicellulose, and lignin in (31–43%), (11–22%), and (11–20%)
w/w respectively. Biofuel refineries using the agave bagasse with the
maximum concentration of amorphous cellulose and hemicellulose, but
the recalcitrance property of lignin make it difficult to extract which act
as a barrier [31].
In addition to the presence of the Agave tequilana weber, the climate of
Mexico allows different species of Agave to grow such as Agave americana,
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Agave angustifolia, Agave fourcroydes and Agave sisalana, and these also
form in the economic activity of the country [32]. Agave species reflects
the low rate of transpiration, have crassulacean acid metabolism capability
which upgrade water using efficiency in semiarid region [33].
13.2.3.1 AGAVE AMERICANA L.
The Agave americana has broad leaves, and its color is green with slightly
gray with a whitish color, and also, is commonly used as an ornamental
plant in gardening or for the extraction of mead also known as “Aguamiel.”
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These plants have very resistant fibers, used to make handcraft textile items.
Agave americana has an average size of 1.0 m to 1.4 m, with leaves 80 to
120 cm long and 15 to 20 cm wide, and its leaves are fleshy and smooth to
the touch [32].
The origin of this plant is Southern Africa and it is reported that the
size of this plant could reach a height of up to 2 meters. In Mexico, the
Agave americana L. is used in various activities, which makes a plant with
great importance for the economy and culture of the country. In Mexico,
generates the sustenance of 38,000 families, which also maintains a product
range in the country of 412,900 tons to 998,400 tons and wherein 2008 there
was a maximum in the production of this input with 1,125,100 tons, which
shows that it is a company that promises and has remained within Mexican
culture [34].
The Industrial Scientific Research Council (CSIR) studied about the
processing of Agave americana L. and highlighted the enormous potential
for fiber production and paper manufacturing, and also conducted important
market research on the global demand for inulin as a by-product which
could be derived from respected processes [34]. All this to carry out the
development of the technology to establish an industry based on the Agave
americana L. in Southern Africa, although studies have also been carried
out in our country to use in a more optimal way this plant and that can also
adapt to the process of manufacturing textile fibers since according to the
CSIR, the contribution of fiber in this plant is 1.5% resulting in the rest of
the plant having no use and also with these conditions prevents the exclusive
processing from this raw material to be unreachable. For the production of
mead Agave americana, L. is cut and generates a hole at the center of the
plant, and allow to collect the liquid of the plant also from the rain later it
could be supplied to tequila industries for the purification and sterilization
of the mead [34].
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13.3 PRELIMINARY STAGES FOR THE CONVERSION PROCESS OF

AGAVE WASTES TO BIOMASS
13.3.1 DRYING AND GRINDING OF BIOMASS AS A PRELIMINARY

STAGE
The first stage contemplated for the biomass transformation process consists
of a series of mechanical treatments, which can be drying and crushing. This
process aims to improve the disposition of the material to the later stages of
the process. Generally, the dry and crushed materials have higher surface
areas and lower crystallinity, which can promote, for example, hydrolysis
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or mass transfer phenomena within the biomass. Mechanical pretreatment
as the only pretreatment is not very useful for LCB es because it cannot
fractionate hemicellulose, cellulose, and lignin, so a series of subsequent
steps are necessary which can separate the biomass. However, mechanical
pretreatment is required to achieve essential characteristics in the process,
such as the appropriate particle size for later stages of the process, making
this an indispensable step for biorefineries [8].
The mechanical pretreatment of Agave Americana L consisted of a primary
stage where the leaves were cut into thin strips to facilitate drying. Subse-
quently, sunlight was used to dry the biomass partially, and then the strips were
cut in 1 cm2 pieces to be dried in a laboratory oven at 75°C until reaching an
internal humidity of 8% (w/w). Finally, a blade mill (Thomas Wiley, Swedes-
boro, NJ, USA) was used to achieve a particle size ≈ (1–0.3 mm) [36].
13.3.2 CHEMICAL CHARACTERIZATION OF BIOMASS
Only with the biomass, which had a particle size of (0.5–0.3 mm) the
characterization of the raw material was carried out. The analysis considered
were: a humidity determination at 121°C, ash determination, protein content
(N × 5.67) by Kjeldahl method, and solvent and aqueous extractives (through
a Soxhlet type extraction with acetone and water respectively). Also, it was
necessary to make a physicochemical characterization to determine the
cellulose, hemicellulose, and lignin content, which was made according to the
standard analytical procedures of the National Renewable Energy Laboratory
(NREL/TP-510-42618), through the quantification of monomers and acid by
high-performance liquid chromatography (HPLC) (Section 13.3.2.1). Finally,
Kalson lignin was quantified by the gravimetry method (Table 13.1) [36, 37].
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TABLE 13.1 Characterization of Agave americana L. Leaves (% on Total Dry Weight) [38, 36]
Component Agave americana Agave tequilana Agave tequilana
L. Leaves [38] Bagasse [36]
Cellulose 29.89 ± 3.80 34.81 20.85
Hemicellulose 14.61 ± 0.10 17.98 17.31

Xylan 13.65 ± 0.02 16.49 –
Arabinan 1.31 ± 0.54 1.5 –
Klason lignin 13.65 ± 0.13 9.89 17.31
Ashes 10.59 ± 0.51 – 7.67
Protein 6.018 ± 0.0 8.35 –
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Solvent extractives 2.55 ± 0.10 – 1.53
Aqueous extractives 43.55 ± 1.21 12.48 8.36
Although the composition of Agave americana L and Agave Tequilana is

similar, there is a clear difference in the amount of soluble aqueous extractives.
However, some reports indicate that the soluble extracts in Agave Americana
L are approximately 55.5% (w/w) [39]. Due to the natural morphology of the
leaf, The Agave Tequilana leaf is quite thin. In contrast, the Agave Americana
L leaf is much thicker, resulting in much more soluble material inside.
13.3.2.1 ANALYTICAL METHOD (HPLC)
For the physicochemical characterization and the reading of monomers in

the various stages of the process, they were quantified under the same condi-
tions, using an Agilent 1260 Infinity II HPLC with a refractive index for
glucose, xylose, arabinose, acetic acid, ethanol, and degradation compounds.
All these compounds were detected and quantified using calibration curves
made with pure agents. Besides, a MetaCarb 87 H column (300 × 7.8 mm,
Agilent) was used for the analyzes; the column temperature was 60°C and
a mobile phase with a concentration of 0.005 mol/L of sulfuric acid, with a
flow rate of 0.7 mL/min and an injection volume of 20 µL [36].
13.3.3 GENERAL ASPECTS OF AVAILABLE PRETREATMENTS FOR

BIOMASS CONVERSION
Because drying and grinding treatments are not capable of breaking the
complex union that exists between the components of lignocellulosic
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material, it is necessary to add a stage to the process that has this task
as the main function. This stage is known as pretreatment. This opera-
tion is crucial since it directly influences the subsequent steps because it
also changes the adaptability of the raw material, such as for enzymatic
hydrolysis, fermentation, inhibitor production, filtration of flows, and the

treatment of the waste to be produced during the process. For this reason,
it is estimated that the choice of pretreatment constitutes 40% of the total
cost of the process [40].
In addition to mechanical treatments, there are physical treatments
that can modify the structure of the biomass. Such is the case of
microwaves, ultrasound, high-energy electron radiation, pretreatment,
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and high-temperature pyrolysis. There are also chemical pretreatments
such as acid and alkaline treatments, oxidative treatments, ionic liquid,
Organosolv, among others. Another branch of pretreatments is made up
of biological processes carried out by microorganisms to structurally
change biomass and, finally, physicochemical treatments, in which there
are processes such as steam or CO2 explosion and finally hydrothermal
pretreatments [41].
13.4 EFFECTS OF HYDROTHERMAL PROCESSING ON THE BIOMASS
13.4.1 FUNDAMENTALS OF HYDROTHERMAL PRETREATMENT
Hydrothermal processing or also known as liquid-hot water pretreatment

is a process widely used in lignocellulosic materials. This stage is applied
to a wide variety of operational conditions and strategies. Usually, the
operating parameters are 150–230°C in isothermal regime (maintaining
the set-point for 10–50 min) and non-isothermal, at pressures ranging from
4.9 to 20 bars [21, 42].
In this range of temperatures, the hydrogen bond of the water begins
to weaken, which results in the autoionization of the water in hydronium
ion (H3O+) which acts as a catalyst due to its acid potential, and the
hydroxide ions are formed equally (OH–) whose potential is naturally
basic. Thanks to the hydrogenation of the acetyl groups contained in the
hemicellulose present in the biomass, it is how the ions formed by water
act, causing that at the end of the reaction between 40 or 60% of the total
mass on dry base have been lost, diluting the aqueous compounds and
hemicellulose [43].
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13.4.2 AN OVERVIEW OF THE USES OF HYDROTHERMAL

PRETREATMENT (LIQUID HOT WATER (LHW) AND STEAM
EXPLOSION (SE) PROCESS)
Nowadays, the hydrothermal process is a favorable process among the

researchers which could hydrolysis the polysaccharides into monomers and
oligomers. During the hydrothermal pretreatment, hydronium ions act as
a catalyst and are able to eradicate the rigidity of the biomass as well as
prevents the corrosion in the equipment which results in a reduction of the
capital cost of the end product. Pretreatment by water supports the reduction
in the capital cost of chemicals likewise the cost of expensive non-corrosive
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metals. The Autohydrolysis technique reflects the economic feasibility
because the process does not require the acid, neutralizing agents, expensive
corrosive metals for the support of degradation of biomass, and prevention
from corrosion respectively [21, 42, 44].
The solid-liquid ratio of biomass and water composes the medium
complex and hike the processing scale which requires a greater amount of
the power consumption to heat the slurry mixture. Recent studies use the
heat exchanger to recover these amounts of energy which could enhance
the overall cost of the process because of the use of heat exchanger [45,
46]. Autohydrolysis technique adequate to improve the quality of solid
also removal of the metal component from the lignocellulosic feedstock.
Mainly removal of metal from the lignocellulosic raw material plays
a significant role during the production of bio-oil [47]. Studies state the
preference of the autohydrolysis technique reduces the chances of develop-
ment of inhibitors that would not disrupt the downstream processing and
complement its accessibility for efficient saccharification using hydrolyzes
enzymes [45, 48].
13.4.2.1 SEVERITY FACTOR AS PARAMETER
The effectiveness of the hydrothermal treatment can be related to the severity

factor (R0). This parameter describes the relation between temperature and
reaction time; this relationship is described with the following Eqns. (1)
and (2) [49].
t  T − 100 
Log ( R0 ) = ∫ exp   (1)
0
 w 
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where; T is the process temperature (°C); t is the reaction time (min); 100
is the reference temperature in degree Celsius (°C); and w is an empirical
parameter related to the activation energy, assuming kinetics of first-order
(w=14.74) [50]. However, this equation in isothermal regime considers the
contributors to heating, steady-state, and cooling during the process, resulting

in the following Eqns. (2) and (3).
[ Heating ( R0 )] + [ Isothermal ( R0 )] + [Cooling ( R0 )]
Log ( R0 ) = (2)
 t1  T ( t ) − 100  t2  T (t ) − 100  t f  T (t ) − 100  

LogRo = ∫0   + ∫t1 exp   + ∫t2    dt (3)
  w   w   w  
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When the hydrothermal pretreatment is in an isothermal regime, it is
necessary to make a distinction in the stages of the temperature profile. In
the case of the second equation, the first term refers to the heating stage,
so the value of t1 is the time in which the temperature setpoint is reached.
The isothermal period begins, which is described by the second term of the
equation and is evaluated from t1 to t2. With this temperature t2, the cooling
stage begins and ends with the final time of the process tf [36].
13.4.3 HYDROTHERMAL PRETREATMENT OF AGAVE AMERICANA

L. LEAVES
13.4.3.1 DESCRIPTION OF THE LIQUID HOT WATER (LHW)

PRETREATMENT REACTOR AND ITS OPERATION
The autohydrolysis of the Agave americana L biomass was carried out in a

stainless-steel batch reactor with a stirring propeller and a maximum capacity
of 750 mL (design by biorefinery group: http://www.biorefinerygroup.com).
The pre-treatment was carried out with the biomass whose particle size was
≈ (1–0.3 mm), the particle size distribution in was 0% > 2 mm, 16% > 1 mm,
54% > 0.5 mm and 28% 0.3 mm and finally 2% fines (% w/w). The liquid/
solid ratio was 10:1 (% w/v), with a maximum working volume of 300 mL,
with a maximum temperature of 190°C and a minimum of 150°C, a maximum
isotherm duration of 50 min and a minimum of 10 min, and a stirring of 120
rpm. The reactor had a PID controller to regulate the temperature during
the isotherm with the intensity of the heating jacket and a stainless-steel
thermocouple and the rotations with a sensor in the propeller shaft located
outside the reactor [51].
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13.4.3.2 EXPERIMENTAL DESIGN FOR THE HYDROTHERMAL

PRETREATMENT OF BIOMASS
A central composite was used to identify the conditions in which the highest
cellulose content was found in the pre-treated solid phase. To determine the
number of conditions was calculated using the following equation [36, 51].
N = 2k + 2 * k + 1 (4)
where; k is equal to the number of variables to manipulate during the experi-
ment (k = 2, time, and temperature). Resulting in a total of 9 conditions with
3 extra repetitions of the central value (170°C–30 min) [36, 51].
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13.4.3.3 CHANGES IN BIOMASS DUE TO HYDROTHERMAL
PRETREATMENT
Finally, after the operation, 2 streams were generated, the first a solid phase
with a high concentration of cellulose and lignin with which the ethanol will
be produced, and a liquid one where the presence of XOs and monomers
will be checked during the treatment with the HPLC (Section 13.3.2.1) [51].
During the pretreatment stage, the heating time, isothermal, and cooling
time was captured each 10C (Figure 13.2). With this information, it was
possible to make a temperature profile and calculate the area under the curve.
The severity factor was calculated with the software Polymath v6.0., between
100°C of heating and 100°C of the cooling stage, for each pretreatment to
obtain the severity factor (Log(R0)) (Figure 13.3).
From biomass pretreatment and the calculation of the severity factor, it
was possible to measure the contribution of the operation in the transforma-
tion of the biomass and to relate this change to the increase in the severity
factor, as the pretreatment conditions were more intense. This factor is an
interesting relationship between the parameters of the pretreatment of LHW,
serving this same as a guideline to relate the behavior of biomass in different
reactors or the scaling-up of the processes (Table 13.2).
With the results of the previous table, we can see how hemicellulose
dilutes as the severity of the treatment increases. Furthermore, it is possible
to observe an increase in the percentage of cellulose and lignin in the solid
phase, starting from 36.67% to 50.66% (w/w) in the case of cellulose, and for
lignin from 18.38% to a 33.06% (w/w).
Besides, it is possible to see the reduction of factors such as pH, checking
the acidification of the medium by acetylation of the medium. XOs are an
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exciting factor since it is possible to increase their production in treatments

where the severity factor is not as high, such as 170°C for 10 min, 190°C for
10 min, approximately with severity factors of ≈ 3.8. Finally, monomers and
XOs in the liquid phase tend to degrade under severe treatment conditions,
resulting in some acids, such as acetic acid and degradation compounds such
as (HMF) or furfural [52].
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FIGURE 13.2 Temperature profiles of hydrothermal pretreatments.
It was determined that the best treatment for ethanol production, after
data analysis, is the condition with higher cellulose production and lower
hemicellulose content. For this reason, the state of 190°C and 50 min was
directly chosen for enzymatic hydrolysis, due to avoid inhibition of the
enzyme by traces of compounds derived from hemicellulose.
This behavior can be compared to that obtained by Aguirre-Fierro et
al. [53] where they used agave bagasse for ethanol production, which was
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pretreated by high-pressure CO2-H2O. In this study, it was found that at

temperatures from 150–190°C (10–50 min), they produced 110.5 g/L of
reducing sugars.
FIGURE 13.3 Glucose production from enzymatic hydrolysis condition. Author Copy
13.5 ENZYME SACCHARIFICATION OF AGAVE AMERICANA L. WASTE
13.5.1 USE OF ENZYMES IN SECOND-GENERATION BIOREFINERIES
Enzymes used as biological catalysts derives from their versatility, accepting

a great variety of complex molecules, including synthetic structures and
found in nature. Enzymes stand out for their selectivity, and factor of great
application within the chemical industry. Consequently, enzymes have gained
relevance within different sectors and processes, are usually characterized as
a sustainable technology that promotes a transition towards processes with
higher environmental principles. Finally, science has directed the use of
enzymes as a substitute for chemical processes, with low efficiency or where
there is a high generation of waste [54].
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TABLE 13.2 Operational Conditions for Hydrothermal Pretreatment and Chemical Composition of Pretreated Solid and Liquid Phase
Temperature (°C) 150 170 190
Time (min) 10 30 50 10 30 50 10 30 50
Log(R0) 3.76 3.93 4.00 3.85 4.03 4.22 3.96 4.10 4.17
pH 5.64 5.01 4.88 4.70 4.56 4.46 4.40 4.32 4.28
Heating Rate 2.31 2.10 2.38 2.57 2.37 2.29 2.15 2.63 2.21
Solid Phase Composition (% on Total Dry Weight)
Cellulose 36.67±0.92 38.085±1.90 40.026±1.77 37.692±0.99 43.935±1.03 48.215±0.84 37.755±0.95 44.933±0.90 50.660±0.82
Lignin 18.38±0.42 22.57±1.72 25.590±0.55 27.472±0.38 29.270±0.38 31.568±0.52 28.642±0.52 32.069±0.23 33.066±0.09
Hemicellulose 11.22±0.00 9.3953±0.33 8.745±0.472 7.514±0.472 3.088±0.177 2.587±0.14 2.273±0.13 2.273±0.13 00.000±0.00
Liquid Phase Composition (g/L)
Glucose 4.438±0.05 4.161±0.01 4.132±0.14 3.912±0.23 2.472±0.36 1.940±0.27 1.698±0.01 1.403±0.00 1.117±0.01
Bioethanol Production Using Agave americana L. Leaves
Xylose 4.602±0.00 4.754±0.01 5.196±0.14 6.648±0.00 6.543±0.01 4.105±0.02 6.147±0.01 4.366±0.01 3.234±0.11
Arabinose 1.166±0.04 1.140±0.00 1.193±0.21 1.387±0.00 1.082±0.00 0.000±0.00 0.000±0.00 0.000±0.00 0.000±0.00
XOs 2.581 2.787 3.221 4.349 3.938 1.313 4.17 2.409 1.475
Acetic acid 0.117 0.129 0.144 0.124 0.126 0.128 0.157 0.185 0.215
Levulinic acid 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
Formic acid 0.000 0.000 0.000 0.000 0.000 0.025 0.253 0.034 0.035
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Hydroxy- 0.000 0.000 0.000 0.000 0.000 0.001 0.023 0.045 0.054
methylfurfural
387
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In second-generation biorefineries, the depolymerization of the polymer

constituents of biomass to monomers or sugars represents a technological
barrier. The way enzymes can be produced from various sources, such
as fungal taxa, bacteria, and archaea. In the case of lignin, there are non-
hydrolytic ligninase enzymes (laccases, lignin-peroxidases, and manganese

peroxidases (MnPs)) that act through the generation of highly reactive free
radicals that break the bonds between carbon-carbon and ether units in the
structure of the lignin. In the case of hemicellulose, due to its heterogeneous
nature, there are a great variety of hydrolytic and non-hydrolytic enzymes
with different specificities that work cooperatively, which attack the main
chains of hemicellulose polymers, working as debranching enzymes,
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such as α-arabinofuranosidase, ferulic acid esterase, acetyl xylan esterase,
and α-glucuronidase. Finally, the most common enzymes for cellulose
degradation are endo-glucanases, exo-glucanases, cellobiohydrolases, and
β-glucosidases; these enzymes can be found as a set for the hydrolysis of
cellulose to glucose [55].
13.5.2 ENZYMATIC HYDROLYSIS IN AGAVE AMERICANA L.
With the biomass pretreated at 190°C and 50 min, enzymatic hydrolysis

was carried out for the saccharification of glucan. The enzyme consisted
of 125 mL shake flasks at 50°C under 150 rpm agitation for 72 h with
enzyme concentration of 5 and 15 FPU/g of glucan of Cellic CTec2 enzyme
cocktail, with a 50 mM citrate buffer to maintain the reaction at a pH of
4.8. sodium azide was added at a concentration of 0.2% (w/v) to prevent
microbial growth [56]. The operating volume was 50 mL. The sampling was
performed at 0, 12, 24, 48, and 72 h with a sample size of 2 mL. The amount
of glucose produced was determined using the methodology described in
Section 13.3.2.1. The experiments were carried out in triplicate, with the use
of blanks to compare that there is no conversion of glucan to glucose without
the enzyme [36, 57].
13.5.2.1 CELLIC CTec2 COMMERCIAL ENZYME COCKTAIL
The enzymatic cocktail used for the conversion of Agave americana L to biomass
was the cellulase Cellic® CTec2 by Novozymes (North America, USA). This
cocktail is composed of Exo-1,4-β-D-glucanase, Endo-1,4-β-D-glucanase, and
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1,4-β-D-glucosidases. This mixture of enzymes manages to break the cellu-

lose chains through different mechanisms (Figure 13.4). Finally, the enzyme
activity was determined using 0.5 mL of a diluted enzyme solution, 50 mg of a
Whatman No. 1 filter paper (1 cm × 6 cm) and 1 mL of 50 mM citrate buffer at
pH 4.8. Finally, reducing sugars were quantified with Miller’s method (DNS),
resulting in 123 FPU/mL [52, 56, 58].
13.5.2.2 YIELD CALCULATION FOR ENZYMATIC HYDROLYSIS
Through the following Eqn. (5), the yield of enzymatic hydrolysis is
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calculated [56].
Enzymatic saccharification yield (%) =

[Glucose] + 1.053[Cellobiose] *100
(5)
f × [ Biomass ] × 1.111
where; [Glucose] is glucose concentration (g/L); 1.053 is the conversion

factor of cellobiose to glucose; [Cellobiose] is the concentration of cello-
biose (g/L); f is the cellulose fraction in dry biomass (g/g); [Biomass] is
dry biomass concentration at the beginning of the enzymatic saccharification
(g/L); and 1.111 is the conversion factor of cellulose to Glucose.
13.5.2.3 ENZYMATIC HYDROLYSIS OF PRETREATED SOLID BIOMASS
The quantification of the transformation of the pretreated biomass of

Agave America L was measured and analyzed using three main param-
eters: Production of glucose from glucan (g/L), the yield of the reac-
tion (%), and the initial reaction rates of each treatment. Each of these
measures was analyzed individually, to choose which of the conditions
would be the best candidate for the production of bioethanol. Within the
analysis, it was a priority to maximize the amount of glucose produced
(Figure 13.4), as well as a reasonable reaction yield. Still, the factor of the
initial reaction rate was an indispensable factor in choosing the condition
since the ethanol produced during the reaction would greatly influence
fermentation.
At this point, the condition of 10% of solid loading and 15 FPU/g glucan
is the treatment where there was a higher conversion of glucan to glucose.
However, it was also necessary to check if there was a significant difference
appropriate for the choice of this condition (Table 13.3).
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FIGURE 13.4 Graphical representation of the enzyme cocktail.
TABLE 13.3 Single-Factor Analysis of Variance for Yield and Initial Reaction Rate
Effect Degrees of Sum of Mean Sum F-Value Significant
Freedom Square of Square (p<0.005)
ANOVA-Enzymatic Saccharification Yield
Treatment 5 2257.729 451.546 206.153 F5,6 = 4.39
Error 6 13.142 2.19 – –
Total 11 2270.871 – – –
ANOVA-Initial Reaction Rates
Treatment 5 7.53 1.506 408.436 F5,6 = 4.39
Error 6 0.022 0.004 – –
Total 11 7.55 – – –
When this statistical difference was verified, a comparison of means was

made for the reaction speed and yield (Table 13.4).
By analyzing the parameters, the idea of using the treatment of 10%
pretreated solids loading and 15 FPU/g of glucan was reinforced, since it
has the second-best reaction yield of all the conditions, the highest amount
of glucose produced, and finally, was the condition with the highest glucose
conversion rate. Finally, the results of the blanks showed that it was not
possible to convert glucan to glucose without enzymes since no trace of
glucose was detected in these blank tests.
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TABLE 13.4 Enzymatic Conversion Yield, Initial Rates and Mean Comparison of the Hydrolysis
Hydrolysis Enzymatic Comparison of Initial Rate of Comparison
Conditions Saccharification Means (95%) Reaction of Means
Yield (%) (g L–1 h–1) (95%)
1%–5 FPU/g 96.24±2.83 a 0.327±0.011 e

5%–5 FPU/g 65.97±0.33 c 0.7405±0.015 e
10%–5 FPU/g 60.88±0.38 d 1.783±0.145 d
1%–10 FPU/g 94.98±0.65 a 0.302±0.008 c
5%–10 FPU/g 73.67±1.67 b 1.259±0.017 b
10%–10 FPU/g 70.46±1.27 b 2.487±0.0103 a
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These results can be compared with the information reported by Láinez et al.
[59], who from Agave Salmiana leaves residues produced bioethanol using the
model of a second-generation biorefinery. Through an acid-alkaline pretreat-
ment, where the hemicellulose was removed, and enzymatic hydrolysis with a
solid load was 5.62% and 10 FPU/g of glucan, it resulted in 49.04 g/L of glucose
released and also a behavior similar to leading in the enzymatic hydrolysis of
Agave Americana L. The enzyme used during this experiment was Celluclast
1.5L (Novozymes), an enzymatic complex of cellulases and β-glucosidase.
13.6 BIOETHANOL FERMENTATION OF THE RESIDUES
13.6.1 BIOETHANOL FERMENTATION
Fermentation is a biological process in which microorganisms, mainly yeasts,

and bacteria, convert fermentable monomeric sugars into acids, gases, and
ethanol. Saccharomyces cerevisiae yeast (baker’s yeast) is the most widely
used microorganism in these processes for the production of alcohol, due
to its high productivity and performance with different raw materials. The
stoichiometric equations characterize the fermentation of hexose (C6H12O6)
and pentose (C5H10O5) to ethanol with yields close to (51.1%) since CO2 is
an integral part of the reaction [35].
13.6.2 EXPLORATORY FERMENTATION OF AGAVE AMERICANA

L. RESIDUES
According to the results obtained from the two previous stages, the isothermal
hydrothermal pretreatment of 190°C for 50 min, and the enzymatic hydrolysis
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of 10% solid-loading and 15 FPU/g of glucan, an exploratory fermentation

was carried out as to demonstrate the conversion of glucose produced
during hydrolysis to ethanol. The pre-saccharification and fermentation
strategy (PSSF) experimentation was carried out in 125 mL flasks with
a 25 mL working volume. Each flask contained 10% of pretreated solids

loading, the enzyme concentration was maintained at 15 FPU/g glucan,
5% (v/v) citrate buffer to maintain the pH of the reaction at 4.8. This flask
was placed in an incubator with a shaking of 150 rpm at 50°C for 16 h so
that the pre-hydrolysis step was carried out. Subsequently, the temperature
of the incubator would be lowered to 32°C; at this temperature, 1.5 mL of
suspended yeast Saccharomyces cerevisiae PE-2 was added. From this point,
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samples were taken every 12, 24, 36, and 48 hours to monitor fermentation.
Each of the tests was carried out in triplicate [52]. The conversion of glucose
to ethanol was measured as described by (Section 13.3.2.1) using HPLC.
13.6.2.1 SACCHAROMYCES CEREVISIAE PE-2 INOCULUM
The yeast strain PE-2 was grown during this experiment in a 500 mL flask,
with a total working volume of 125 mL. The medium contained 10 g/L
yeast extract, 10 g/L peptone, 10 g/L agar, 10 g/L dextrose, and 0.5 g/L of
(NH4)2HPO4 and MgSO4·7H2O. The glucose was dissolved in an aqueous
solution and sterilized separately. The flask with the medium and the yeast
was incubated at 30°C, 150 rpm for 12 h. The yeast suspension was made
from the pre-inoculum centrifugation at 5,000 rpm at 4°C for 10 min. Finally,
it was suspended in a 0.4% NaCl solution [35].
13.6.2.2 FERMENTATION CONVERSION YIELD
According to the following equation, the ethanol production yield is

calculated [57].
 EtOH f 
Ethanol yield ( % )
= × 100 (6)
0.51× f × [ Biomass ] × 1.111
where; [EtOH] is the final concentration of ethanol (g/L); 0.51 is the theoretical
conversion of glucose to ethanol based on stoichiometric biochemistry
of yeast; [Biomass] is dry biomass concentration at the beginning of the
enzymatic saccharification (g/L); f is the cellulose fraction in dry biomass
(g/g); and 1.111 is the conversion factor of cellulose to glucose.
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13.6.2.3 PRODUCTION OF BIOETHANOL FROM PRETREATED AND

HYDROLYZED BIOMASS OF AGAVE AMERICANA L.
From the operation route outlined during this article, it was possible to
produce bioethanol from the biomass produced by the waste of Agave

Americana L leaf, within the concept of a second-generation biorefinery.
The maximum glucose concentration reached in the pre-hydrolysis stage
was ≈33.78 g/L at 16 h. Later the yeast was added, and a maximum level of
ethanol was reached ≈13.53 g/L after 12 h (Figure 13.5), which is equivalent
to an ethanol yield of 43.15±0.48%. A decrease in ethanol concentration
can also be observed after 12 hours; this may be due to the consumption of
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ethanol by microorganisms as a carbon source.
FIGURE 13.5 Conversion of glucose to ethanol from hydrolyzed biomass.
13.7 CONCLUSIONS AND PERSPECTIVES
The search for different sources of energy leads to the development of

emerging technologies that solve the environmental problems that the use
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of fossil fuels implies. From this study, it was observed that the remnants
of different processes, such as the use of Agave americana L, are an option
for the production of biofuels such as bioethanol, revaluing the residues of
this economic activity. Also, the use of waste from desert biomass implies
generating lignocellulosic materials with less use of water and cares in their
production. For this and other reasons, it is necessary to continue in the
search for new technological alternatives that reduce the cost and increase the
production volumes of fuels such as bioethanol as well as it is also necessary
to generate options that integrate sustainable processes such as the use of
enzymes and hydroelectric pretreatment. Finally, the use of the residues of
Agave americana L, reinforces the concept of adaptability of biorefineries
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and gives rise to a new area of application of resources that avoid competition
with food crops, an essential factor in developing countries such as Mexico.
ACKNOWLEDGMENTS
This project was funded by the Secretary of Public Education of Mexico-

Mexican Science and Technology Council (SEP-CONACYT) with the Basic
Science Project-2015-01 (Ref. 254808). We gratefully acknowledge support for
this research by the Energy Sustainability Fund 2014-05 (CONACYT-SENER),
Mexican Center for Innovation in Bioenergy (Cemie-Bio), Cluster of Bioalcohols
(Ref. 249564). César D. Pinales-Márquez, Shiva, and Rohit Saxena thank the
National Council for Science and Technology (CONACYT, Mexico) for theirs
Master and PhD Fellowship support, respectively, (grant number: 1001882).
KEYWORDS
• biofuels
• biomass
• enzymatic hydrolysis
• fermentation
• hydrothermal processing
• lignocellulosic material
• pretreatment
• second-generation biorefinery
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CHAPTER 14
Sustainable Ethanol Production from

Lignocellulosic Biomass: A Water
Footprint Analysis over Pretreatment
Technologies
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OZNUR YILDIRIM, DOGUKAN TUNAY, BESTAMI OZKAYA, and
AHMET DEMIR
Department of Environmental Engineering, Yildiz Technical University,
Turkey, Davutpasa Campus, Istanbul–34220, Turkey
ABSTRACT
Lignocellulosic biomass (LCB) has a complex body consisting of hetero-

polymers such as lignin, cellulose, and hemicellulose with high tolerancse
to biodegradation, which is abundant and cheap in nature, and significantly
reduces CO2 emissions in the atmosphere when used in ethanol production.
With the increase of global warming (GW), the concept of lignocellulosic
biorefinery has become a critical focus. There are many pretreatment
methods which have been developed to disrupt this structure even they
are durable to biodegradation. The most important parameters affecting
the sustainability and economy of the biorefinery industry are the amount
of water and chemical consumption. The pretreatment process is the most
critical step in the production of valuable products from LCB in terms of
water and chemical consumption. This chapter aims to contribute to reducing
costs during the production of high value-added products by examining all
available pretreatment technologies in terms of water, chemical, and energy
consumption.
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Graphical abstract
14.1 INTRODUCTION
In recent years, ethanol production from LCB has gained great momentum.
LCB mostly consists of agricultural wastes and forestry residues [1]. Energy
production from LCB is more significant in the economic, social, and environ-
mental aspects for all over the world. Agricultural wastes are substantial focus
area for renewable energy production because of containing high amounts
of carbohydrates. Special pretreatment applications should be applied to
make LCB open to biological activities by breaking the rigid structure of the
lignocellulosic matrix [2, 3].
Cellulose, a kind of polymer, found abundantly in nature, formed as a
result of glucose monomers combined by glucosidic bonds [4]. Cellulose
polymers of different lengths and structures bond to each other with weak
hydrogen bonds to form amorphous or crystalline cellulose fibrils [5].
As the number of cellulose chains formed increases, the strength of the
cellulose increases, and an indigestible crystal structure is formed, which
makes it arduous to break down. Similarly, as the Glucose number of cellu-
lose increases, it is assumed that the DP increases which is proportional
to the glucose number [6]. The most significant limiting factor affecting
the efficiency of hydrolysis is the cellulose crystallinity. The amorphous
cellulose chains can decompose faster than those with a crystalline struc-
ture [7]. Hemicellulose, which is formed by the combination of different
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Sustainable Ethanol Production from Lignocellulosic Biomass 403
compounds such as pentoses, hexoses, and uronic acids, attaches to cellu-

lose fibers and binds cellulose to lignin and gives the biomass hardness
[8]. This situation also prevents enzymes from accessing cellulose during
enzymatic hydrolysis. The use of some chemicals during the pretreatment
of hemicelluloses can cause the formation of inhibitors such as furfurals and

hydroxymethylfurfural, which may adversely affect microorganisms during
fermentation [9–11]. Lignin, a heteropolysaccharide consisting of three
separate phenyl propane units and having an irregular structure, enables
plants to be resistant, impermeable, and resistant to microbial attacks [12].
There is no order between the units in the lignin structure formed by phenyl
propane units and ether bonds and carbon-carbon bonds [13].
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With the use of lignocellulosic ethanol in the transportation sector, the net
CO2 emissions released into the atmosphere are decreasing [14]. However,
it is no longer sufficient to produce the maximum amount of ethanol by
increasing lignocellulosic ethanol production efficiency with different
pretreatment processes. Due to the rapid reduction of water resources, water
usage during the process and its reduction have become one of the most
important parameters. It is known that most water usage in the lignocellulose-
based ethanol production occurs during the pretreatment stage [15]. Water
usage should be reduced in the pretreatment stage to make this process more
sustainable. It is anticipated that if the water resources in the world continue
to be consumed at the current rate, it will be able to manage for about 30
more years. Water demand is expected to increase by 40% by 2050, and 25%
of people will not have adequate access to freshwater [16].
For the production of ethanol, water is essential, especially in the
grinding, liquefaction, and fermentation processes. In recent years, water
consumption per gallon of ethanol has decreased significantly. According to
the plants that were operated in 2002, older plants consuming much more
water. For instance, water usage of older plants were more than the 15-Gal
water/Gal etOH. However, it was declined from 1 gallon to 11 gallons and
on average 4.7 gallons for production of 1 gallon of ethanol from the 2002
data (Figure 14.1) [17].
There are two types of fermentation processes that is used in the ethanol
industry, which are continuous and batch. 27% of ethanol was produced with
the continuous fermentation process, and the remaining was produced with
the batch process according to the 2002 survey of ethanol plants. When the
ethanol plants were analyzed due to their process selection, it can be said
that the continuous fermentation system was more preferred in large plants.
While 4 of these large plants were operated continuous, 17 out of 21 plants
were operated with batch system [17].
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FIGURE 14.1 Gallons of water usage per gallon of ethanol produced for the 21 ethanol
plants in 2002 [17].
Although lignocellulosic ethanol production is important in terms of

slowing the effects of GW by reducing CO2 emissions, if a large amount of
fresh water is used during the process, a sustainable fuel production cannot
be obtained when the whole process is examined. Therefore, it is increasingly
important to conduct water footprint studies to compare the water consump-
tion data of different pretreatment processes. This section details the water
footprint data of different pretreatment types in detail and introduces more
sustainable methods.
14.2 WATER FOOTPRINT
Rise in water consumption due to population increase and industrialization

have a significant effect on the environment and ecological life. Even though
almost 70% of the Earth's surface is covered by the water, only 2.5% of this
water is fresh and can be directly be used [18]. It is an undeniable fact that
these limited water resources will be run out one day in the future unless
precautions are not taken. For this reason, water consumption should be
reduced and the water cycle should be maintained by recycling the used
water. Water footprint is a measurement of water required for the production
of any tools, products, goods, or services that takes a place for daily life.
There are three main components of water footprint which are called blue,
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green, and gray water footprints. Green water footprint can be explained as
the water that is deposited, transformed, evaporated by plants, or consump-
tion of water from precipitation that is held by the roots of the soil. In short,
everything is incorporated with plant, agriculture, and forestry taking charge
of water cycle called the green water footprint. On the other hand, surface
and groundwater sources that are used for domestic, industrial, and irriga-
tion water for agricultural purposes are related to the blue water footprint.
Bluewater consumption estimations need detailed analyzes and study since
there could be many reasons for the water usage directly or indirectly. For
instance, there is a study regarding the water footprint for coffee and tea
consumption in the Netherlands. If the virtual and real water consumptions
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were compared, there will be a huge difference. For example, while the real
water consumptions were 0.125 L and 0.250 L water for the coffee and tea,
respectively. On the other hand, virtual water consumptions rise to approxi-
mately 140 L and 34 L for coffee and tea [19]. Greywater footprint is the
other component that is considered the freshwater usage for the assimilation
of pollutants according to the water quality standards. It is considered as a
point source pollution discharge through a pipeline, runoff, or impervious
surfaces.
14.2.1 BLUEWATER FOOTPRINT FOR BIOREFINERY PURPOSES FOR

LIGNOCELLULOSIC BIOMASS (LCB)
Industrialization, energy need, and increasing living standards had significantly

affected to studies related with the biorefinery in last decades [20–22]. However,
water scarcity is another vital issue that should be considered because of the
limited freshwater sources. It is estimated from a national research council
(NRC) that water consumption for the ethanol production from corn kernels
and cellulosic feedstocks are 4- and 9.5-Gal water/Gal etOH [23]. Based on
these predictions, it can be said that approximately 256 billion gallons of
fresh water are needed in a year for the required 36 billion gallons of ethanol
in 2022 [24]. For the production of biofuels from woody biomass, there are
different approaches. The first approach supports the production of the crops
for direct fuel production such as (biogas, bioethanol, biohydrogen, biochar,
etc.) [25, 26]. In the first approach, the problem is the usage of feedstocks that
is already used as a food for the bioethanol production. The use of agricultural
land for fuel production can also be of concern for the future. Therefore, the
requirement of fuel production should be carefully analyzed compared to other
demands. In the second discernment, the biofuel production can be carried out
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depending on the usage of a lignocellulosic waste or biomass that is not to be

used as a food or food products. Nevertheless, these feedstocks could need
more water to break down lignin, and biofuel production yields can be dropped
down [6, 27].
Process water which is used in the pretreatment step is evaluated as blue

water. Besides, LCB holds some amount of water in their fabrics which is
interpreted as green water. Green water can decrease the water usage for
the preparation operations; however, it is also part of a water cycle and
it should be considered in terms of process water. Figure 14.2 shows the
components of the water footprint during biofuel production from LCB.
Chiu et al. [8] investigated that water consumption according to bioethanol
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production stage and irrigation need for different locations of the United
States. Bioethanol production consumes 0.5–28% of the total water which
includes process water and irrigation water. It shows that growing crops for
bioethanol production has a much higher water footprint due to the lignocel-
lulosic waste or uncontrolled growing herbs or plants usage.
FIGURE 14.2 The components of water footprint during biofuel production from lignocel-
lulosic biomass.
Irrigation should be considered as green water is considered as a kind of

green water source in the water that plants hold in themselves, even if the
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water used directly from freshwater sources (process water) is gained, it is

included in the current water consumption within the water cycle. The water
used in the pretreatment process is used as process water for fuel production
and is therefore considered blue water. Therefore, in this section, studies on
the amount of water consumed during the pretreatment, in other words, the
determination of the blue water footprint for pretreatment will be examined
in current studies.
14.3 PRETREATMENT METHODS
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The carbohydrate polymers in lignocellulose are tightly bonded to lignin
with hydrogen bonds and covalent bonds, creating a complex and crystalline
structure that prevents the carbohydrate in the raw material from being used
by microorganisms and enzymes [28]. The object of the pretreatment is to
decrease the crystallinity of lignocellulose, break their resistance to enzymatic
depolymerization, and disrupt the heterogeneous structure of lignocellulosic
materials. For obtaining valuable products from lignocellulosic materials,
their complex structure must be disrupted and broken down into smaller
carbohydrate components. Pretreatment types are basically classified as
physical, chemical, physicochemical, and biological.
For a conventional pretreatment process to be efficient; It would be
expected to increase the accessible cellulose surface area, break the lignin
barrier and cellulose crystallinity, prevent the formation of toxic by-products
that will cause inhibition in the fermentation stages, to reduce the loss of
reducing sugar resulting from the process and to be economically efficient
[29]. However, new issues to be considered in addition to these items have
become a current issue. Now, in addition to the requirements such as high
sugar conversion rate of the pretreatment process, being economical, and
providing low inhibitor production, lower water usage has become one of the
most important parameters. Due to the depletion of water resources and GW,
efforts to develop new methods that reduce water consumption during the
process and wastewater production after the process to make environmen-
tally friendly waste-to-fuel production processes such as biofuel production
from lignocellulosic wastes more sustainable.
The literature includes different approaches for pretreatment. Several
methods of pretreatment were tested by applying them to different lignocel-
lulosic wastes. In this chapter, studies that mostly conduct water footprint
studies and indicate how much water is consumed per waste are included.
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14.4 WATER CONSUMPTION IN PHYSICAL PRETREATMENT
In physical pretreatment, the crystallinity of lignocellulosic material is

broken, so the surface area accessible by microorganisms is increased by
reducing the size of wastes by cutting, grinding, and milling [30]. Methods
such as mechanical extrusion, grinding, and microwave are in the physical
pretreatment category. In addition, there are methods such as ultrasound and
pulsed electric field, which have recently been applied.
In milling and chipping methods, which are the most preferred types of
physical treatment, size reduction is performed with the help of grinders and
cutting tools without any water usage. However, it may be necessary to use
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water in some methods such as microwave. One of the physical processes
that are used for biofuel production from lignocellulosic feedstocks is the
cooling necessity. Wu and Sawyer [31] found that 65% of the total process
of water consumption for renewable diesel blendstock (RDB) is caused by
cooling. Water necessity for the pretreatment and enzymatic hydrolysis were
found 0.16 L water/L RDB and 0.18 L water/L RDB, respectively.
14.5 WATER CONSUMPTION IN CHEMICAL PRE-TREATMENT
Chemical pretreatment includes acid, alkaline, ozone, and ionic liquid

methods. At the end of the chemical pretreatment process, the hemicellulose,
cellulose, and lignin matrix split from each other and becomes explicit to the
attacks of enzymes [32]. Large cellulose and hemicellulose chains are divided
into shorter pieces due to chemical pretreatment after lignin hemicellulose
and cellulose are segregated from each other and enzymes can act. However,
the use of chemicals in pretreatment processes is an important disadvantage
in terms of the cost-effectiveness. Furthermore, usage of chemicals also
increases water consumption during application. The most preferred methods
are acid and alkaline pretreatment. Acid and alkaline applications are
examined under two subtitles as concentrated and diluted [33]. Dilute acid
and alkaline applications are more preferred because the use of concentrated
acid causes more chemical consumption. However, a considerable amount
of clean water is spent even to purify only the pretreated wastes from acid
or base residue [34]. After pretreatment with acid or base, some inhibitory
by-products can be formed due to harsh pretreatment conditions. If these
inhibitors remain on the pre-treated fermentation raw material, it may damage
the fermentation microorganisms. In addition, as a result of pretreatment with
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acid and base, the washing is a significant step to bring the pH to a neutral
level. Approximately 10 L of water is consumed during the 1 L of ethanol
production process [35].
In a study [36], a greenhouse gas (GHG) and water use inventory were
created in case of use of cholinium lysate ([Ch] [Lys]) chemical known as

ionic liquids (ILs) in the lignocellulosic biofuel production process. They
compared two different methods, the traditional water washing (WW) method
and the integrated high gravity (iHG) method, which does not require water
use. As a result of the study, they observed that pretreatment of corn stover
using [Ch] [Lys] resulted in inadmissible high GHG in the WW process,
while the iGH process could reduce GHG by 45% as per gasoline. When
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compared in terms of water consumption, WW and iHG processes have been
observed to have almost similar water consumption (about 0.30–0.40 L/MJ).
The reason the water usage values are similar for the WW process and the
iHG process is that all the water used in the WW stage is eventually recovered
and recycled through highly efficient evaporative dehydration process.
According to the National Renewable Energy Laboratory Technical
Report, Aden et al. [37] investigated the ethanol process design and econom-
ical approaches for corn stover biomass. Water balances were derived from
the ASPEN simulations. It was found that 0.252 kg ethanol can be produced
from 1 kg of corn stover with the 0.224 kg of water consumption for the
pre-hydrolysis (0.113 kg of water/kg ethanol produced) and saccharification
(0.111 kg of water/kg ethanol produced). According to ASPEN simulations,
it was proven the pretreatment applied during ethanol generation from the
corn stove affects the water usage by 2.62%.
14.6 WATER CONSUMPTION IN PHYSICOCHEMICAL

PRE-TREATMENT
The processes where physical and chemical pretreatment processes are used
together are defined as a physicochemical pretreatment method [2]. In the
methods applied in this type of treatment, treatment is done in harsh condi-
tions such as high temperature and pressure. LHW, wet oxidation, ammonia
fiber explosion (AFEX), and CO2 explosion are the most used techniques.
As the name suggests, methods such as LHW and wet oxidation are methods
that cause too much water consumption. In terms of process efficiency, they
have less sugar conversion efficiency compared to acid and base applications
under high temperature and pressure.
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Dong et al. [38], produced ethanol using sulfite pretreated Monterey pine
and completed the water and energy footprint analysis of the whole process.
As a new pretreatment in the study, the process of breaking down oven-
dried (OD) wood chips with sulfuric acid and sodium bisulfite was applied
in a rotary electric heated digester. They proved that the newly pretreatment
process they performed when they examined the water footprint of the
process provides 3.65 tons/ton of dry biomass water consumption and this
value provides 25–51% less water consumption than pretreatment processes
such as steam explosion (SE). They also claimed in their report that the
highest water consumption of the SE, diluted acid, and organosolv pretreat-
ment types where organosolv > dilute acid > steam explosion, respectively.
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Organosolv pretreatment leads not only to the highest water consumption,
but also to energy consumption. Pretreatment with dilute acid consumes less
energy than steam putting process. It is also stated that the acid pretreatment
process mostly results in higher sugar conversion efficiency than SE [39]. In
this case, if both the efficiencies of energy, water consumption, and pretreat-
ment are compared, the most preferred pretreatment form, dilute acid, tends
to be more advantageous.
In another study [40], while investigating the effect of reactor filling and
solid-liquid ratio on ethanol yield during the production of ethanol from corn
stover, water, and steam consumption amounts were investigated. At the end
of the process, it was observed that the steam consumption was significantly
reduced and no acidic waste containing acid was produced. in pretreatment
conditions where the highest sugar content and minimum water consumption
occur; 190°C; 3.00% sulfuric acid; 3 min, water, and steam consumption,
respectively, 97.3 g water and 44 g steam per g dry waste were used.
Pan et al. [41], after dilute acid pretreatment for corn stover based ethanol
production, water footprint analysis of various conditioning processes were
carried out. These methods were; overliming, ammonia addition, two-stage
treatment, and membrane separations. In overliming with conditioning
application, 4.94 kg/kg dry biomass direct process water was used in total
and approximately 35% of this was used during pretreatment. In conditioning
with ammonia and two-stage conditioning application, a total of 4.58 kg and
4.25 kg of water were used respectively, and the most water was used in the
pH adjustment stage. In the air conditioning application with membrane, the
total water usage (2.54 kg) has the lowest value compared to other processes.
The results showed that the amount of steam and water used in the dilute acid
pretreatment stage ranged from 14% to 74% for the four methods applied.
The 4.94 L of water per kg-DB was used in conventional overliming. With
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membrane separations of approximately 2.54 L per kg-DB, water use

was found to be the lowest application, and almost half the use of water
compared to overliming. Also, a considerable amount of water was used for
the hydrolyzate neutralization. Membrane applications as a conditioning
method could be offered as a solution to accomplish water-efficient both

dilute acid removal and pH adjustment. In addition to the blue WF, which
is defined as direct/indirect water use in the study, gray WF was determined
by considering process discharge in all scenarios. Among the four different
treatment processes, both blue and gray WF values were the lowest applica-
tion membrane separations.
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14.7 WATER CONSUMPTION IN BIOLOGICAL PRE-TREATMENT
Biological pretreatment, also known as microbial pretreatment, is considered

as the most environmentally friendly method since it is performed without
using chemicals. Microorganisms and rotting fungi are used in biological
pretreatment. Microorganisms often secrete extracellular enzymes to
disrupt lignin and hemicellulose. Enzymes from different species such as
Aspergillus and Streptomyces are used for biodegradation of lignocellulosic
materials [42]. The degradation of lignin by fungi occurs by the effect of
lignin-destructive enzymes, such as peroxidases and laccase. Rotting fungi
are examined in two groups as white and brown. While brown rot fungi can
only degrade cellulose, white-rot fungi can break down both cellulose and
lignin [43]. Biological pretreatment is suitable for lignocellulosic material
pretreatment due to its low energy usage and the absence of toxic by-product
production as in chemical pretreatment. However, biological pretreatment
has also some drawbacks such as consuming some of the raw material and
long process time.
In a study [44], a new procedure was developed to increase hydrolysis
efficiency and reduce the water footprint. In the newly developed method,
both solvent and water are used to separate the confectionery and lignocel-
lulose components. By adding trace amounts of inorganic acid, lignin is
dissolved and hemicellulose is hydrolyzed under the appropriate reaction
temperature and pressure. In the conventional method, it is known to wash
with plenty of water to remove the solid phase solvent after pretreatment. In
the newly developed method, after hydrolysis with acid and solvent, drying
is carried out with a relatively low-temperature air suction using the relative
volatility difference between solvent and water to remove solvents in the
solid phase.
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Thus, while achieving high efficiency in enzymatic hydrolysis, water

consumption and wastewater generation are reduced. As a result of the
pretreatment proposed by the present invention, the lignin removal rate can
be increased to 90% and the enzymatic hydrolysis efficiency of the cellulose
solid residue to 80–90%. In addition, the amount of water used is saved about
10 to 20 times the weight of the cellulose solid residue, which significantly
reduces the water used in production, resulting in an important decrease in
the water footprint. As in the mentioned study, the original efficiency of the
process is preserved and even increased, while water consumption can be
reduced by new methods (Table 14.1).
As a result, there are many pretreatment methods used for lignocellulose-
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based ethanol production. The sugar conversion efficiency of the pretreatment
method used varies according to the type, content, location, and harvesting
method. In order to figure out which pretreatment process would result
in maximum efficiency, different parameters such as the amount of water
consumption and output in the production of ethanol need to be examined.
Table 14.1 shows the pretreatment type applied, the LCB used, the amount of
water needed for ethanol production, and the ethanol production efficiency
of some studies.
14.8 CONCLUSION
Traditional ethanol production, which accelerated because of depletion

of fossil fuels and the damage it caused to the environment, became
uncontrollable, and was replaced by lignocellulosic ethanol production as a
result of the anticipation of the depletion of food sources due to the increase
in the world population. In order to make the process more sustainable, it is
of great importance to conduct studies to examine and reduce the amount of
water consumed during the process. Using LCB as a raw material, the amount
of water consumed in biofuel production is larger (10 L water/L ethanol)
compared to the use of corn as a raw material [59]. When thermochemically
biofuel production and crude oil production processes are evaluated, it is
seen that water consumption is 2 L/L and 2.5 L/L, respectively [60]. From
a different perspective, the incineration and landfilling of agricultural
wastes are not accepted in the circular economy. Ethanol production
from lignocellulosic wastes not only provides sustainable management of
agricultural wastes but also reduces the amount of net CO2 emission released
into the atmosphere compared to gasoline. As a result, lignocellulosic
ethanol production draws attention as an environmentally friendly method in
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TABLE 14.1 Water Demands for the Process of Ethanol Production from Different Lignocellulosic Biomass
Pretreatment Methods Biomass Water Demand (m3 Water/t Ethanol Yield References
Lignocellulosic Biomass) (t/t Dry Matter)
Physical Pretreatment Liquid hot water N/A 8.55 0.174 [45, 46]
Liquid hot water Palm-oil residues 2.62–7.62 0.09–0.136 [47, 48]
Steam explosion Corn stover 1.5 N/A [49]
Chemical Pre-Treatment Diluted acid (0.5–2%) N/A 4.76 0.216 [45, 46]
SO2 stem explosion N/A 4.80 – [45, 46]
Acid Olive tree biomass 2.13–2.42 0.097–0.11 [47, 50]
Lime N/A 11.39 0.202 [45, 46]
Ozonolysis Oil palm 0.5 N/A [51, 52]
Ionic liquid Palm biomass 15 N/A [51, 53]
Mild NaOH Raw cogon grass 46 0.134–0.174 [47, 54]
Physicochemical Ammonia fiber explosion N/A 2 0.220 [45, 46]
Sustainable Ethanol Production from Lignocellulosic Biomass
Pretreatment
Ammonia fiber explosion Corn stover 1 N/A [51, 55]
Soaking in aqueous ammonia N/A 5.53 0.148 [45, 46]
(SAA)
Biochemical conversion (diluted Corn stover 8.55 0.252 [56]
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acid 1.1% + enzymatic hydrolysis)
Biological Pretreatment Irpex lacteus CD2 Corn stalks 2.5 N/A [57]
Ganoderma boninense Water hyacinth 1.43 N/A [58]
413
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all aspects. By developing new methods that will reduce the high freshwater
consumption, which is one of the handicaps of this process, the process will
be made more preferred. More novel technology development and techno-
economic analysis of these methods are required for lignocellulosic ethanol
production.
KEYWORDS
• biorefinery
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• ethanol
• ionic liquids
• lignocellulosic biomass
• pretreatment
• water footprint
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CHAPTER 15
Biodegradation and Biocatalysis Aspects

of Direct Bioethanol Production by Fungi
in a Single Step Named Consolidated
Bioprocessing
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LUIS FERNANDO AMADOR CASTRO and DANAY CARRILLO NIEVES
Tecnologico de Monterrey, Escuela de Ingenieria y Ciencias,
Av. General Ramon Corona No. 2514, Zapopan–45201, Jal., Mexico
E-mail: danay.carrillo@tec.mx (Danay Carrillo Nieves)
ABSTRACT
The increasing industrialization and population growth are coupled with

rising energy demand. However, using fossil fuels to satisfy this demand is
not a feasible strategy as climate change has intensified due to anthropogenic-
related emissions. The previous triggered the search for cleaner energies, and
among the options, ethanol was presented as a potential alternative fuel; but
its success was clouded as its production was considered a threat for food
security. To assess this problem, the use of lignocellulosic residues and non-
edible crops has been suggested, but its production process is more expensive.
Consolidated bioprocessing (CBP) recently surged as a solution as it reduces
the number of steps involved in ethanol production, thus reducing the cost of
the final product. However, the search for suitable microorganisms that can
be employed in this type of process continues, following that they need to be
able to degrade the recalcitrant structure of the cell walls and ferment sugars.
Fungi are promising microorganisms, as they can produce lignocellulosic
enzymes and can be engineered for efficient fermentation. This chapter aims
to give the reader some aspects regarding the use of lignocellulosic materials
for ethanol production.
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15.1 INTRODUCTION
Currently, climate change poses a risk to modern societies. It has been

estimated that a global temperature increase by 1.5°C from pre-industrial
levels will result in negative effects on several aspects, including human

health, economic growth, food security, biodiversity, and natural ecosystems
[1]. Anthropogenic-related emissions are the greatest contributor to climate
change, being carbon dioxide (CO2) emissions the most abundant [2]. Given
that around 90% of CO2 emissions proceed from fossil fuel usage and indus-
trial sources [3], the search for alternative energy sources stands as a viable
strategy for mitigating the increasing effects of climate change [4, 5]. The
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use of biomass has been proposed as one of the alternatives to fossil fuels
being lignocellulose of particular importance as it is known to be the most
abundant biomass on Earth [6].
Lignocellulose is an organic material that constitutes the plant cell walls.
It can be readily obtained from feedstocks and forestry as well as from their
wastes [7]. Biofuels that can be obtained from this material include ethanol,
butanol, and biodiesel; among the previous ethanol has the highest relevance
[8]. Ethanol can be readily mixed with gasoline reducing hydrocarbon and
carbon monoxide emissions [9]. Ethanol-gasoline blends can also contribute
to decreasing the effects of climate change given that feedstocks used for
ethanol production sequester CO2 for its growth [10]. Additionally, the use
of lignocellulosic wastes and non-edible crops for ethanol production can
favor rural economic development and at the same time does not comprise
food security [11]. However, the implementation of this technology faces
significant challenges.
Serving as a defense mechanism against pathogens, lignocellulose is
highly recalcitrant, and its hydrolysis requires the employment of chemical
or enzymatic procedures. Furthermore, to improve the ethanol yield
biomass should be pretreated either by physical or chemical means [12].
The requirement of a multiple-step process for ethanol production from
LCB makes the process not economically competitive when compared
to currently existing fuels [11]. Aiming towards cost reduction, multiple
strategies have been proposed, including the elimination of the pretreat-
ment procedures, the increase in cellulose hydrolysis yield by optimizing
the enzymes involved in the process and improving the ethanol yields [13].
However, one of the most promising strategies is to perform the conver-
sion of lignocellulose into ethanol by implementing a single-step process
known as CBP [14].
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Biodegradation and Biocatalysis Aspects 421
CBP possesses multiple advantages when compared to separate hydro-

lysis and fermentation (SHF) processes. But this technology is still under
development and finding or engineering an appropriate microorganism
for the process has resulted to be not an easy task. Therefore, we aim to
provide the reader with some insights regarding the structure of the plant cell
walls and the enzymes that are involved in their synthesis and degradation.
Further, we explain the concepts, advantages, and challenges around CBP;
and finally, we review some fungi that have been used or have the potential
to be used in this type of process as they are known to be the most efficient
lignocellulosic enzymes producers.
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15.2 LIGNOCELLULOSIC MATERIALS, THE STRUCTURE, AND
RECALCITRANCE OF CELL WALL
The cell wall is a critical component of plant cells. Apart from encasing cell’s
protoplasm, it participates in biological processes such as differentiation,
intracellular communication, water, and nutrient transport and as a defense
mechanism against pathogens [15]. Cell wall composition varies greatly
among different plant species and within cells from the same plant depending
on the physiological role they exert. Furthermore, there are some plant cells
that contain two cell walls, primary, and secondary, which composition is
also different [15]. Plant cell walls have a complex structure consisting of a
mixture of carbohydrates, proteins, and aromatic compounds. Carbohydrates
that form the plant cell walls include cellulose, hemicellulose, and pectin and
can account for as much as 90% of the content of the primary cell wall [16].
Aromatic compounds like lignin which are present in the walls of many plant
cells provide plants with further structural support. Proteins present on the
cell walls can serve as structural molecules or signaling receptors which are
essential for plant development. This section will briefly describe the main
components of the plant cell walls.
15.2.1 LIGNIN
Lignin is a complex aromatic polymer mainly present in secondary thickened

plant cell walls. It is covalently bound to hemicellulose and its function is to
provide the cell wall with strength and rigidity, allowing plants to grow upward
and serve as a vascular system for transport of water and solutes. This highly
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cross-linked polymer is commonly synthesized from the oxidative coupling

of p-hydroxycinnamyl alcohol monomers, often referred to as monolignols
[17]. The three most common monolignols are p-coumaryl alcohol, coniferyl
alcohol, and sinapyl alcohol, molecules from which the hydroxyphenyl (H),
guaiacyl (G), and syringyl (S) lignin subunits, respectively derive [18].
Recent evidence indicates that apart from hydroxycinnamyl alcohols, lignin
can be synthesized to a less extent from other phenolic monomers, further
increasing the complexity of this polymer [19]. Still, all currently known
lignin building blocks derived from the general phenylpropanoid pathway
in which phenylalanine serves as the substrate for this pathway in plants and
tyrosine as an additional substrate in grasses. The amount of lignin as well as
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its composition vary significantly among and within plant species cell types
and even cell walls; being influenced by diverse environmental factors [19].
Generally, lignin polymers from dicotyledonous angiosperms are composed
mostly of G and S subunits having only traces of H subunits. Gymnosperm
lignins lack S subunits and are composed as much as 90% from G subunits.
Lignins from grasses contain G and S subunits at similar levels and a lower
amount of H [20]. In lignin, these subunits are typically connected by
carbon-oxygen ether linkages and carbon-carbon linkages being β-O-4 is
the most common linkage. Other molecular bonds include α-O-4, β-5, 5-5,
4-O-5, β-1, and β-β linkages [20]. The interest in the study of lignin arises as
this polymer, which accounts for up to 30% of LCB, can greatly hinder the
process of the bioconversion of LCB into ethanol. Therefore, reducing its
content or changing its composition can represent significant improvements
regarding the use of lignocellulosic materials in multiple industries.
15.2.2 CELLULOSE
Cellulose is the most abundant polymer of the plant cell being present in both
primary and secondary walls. It provides structural support allowing plants
to withstand physical stress and act as a barrier against other organisms.
Cellulose is not exclusive to plants as certain bacteria, fungi, and animals
can synthesize it [21]. This polymer consists of chains of repeating D-gluco-
pyranose molecules which are linked by β-1,4-glycosidic bonds. Chains of
cellulose comprise a non-reducing end, which is a glucose molecule with
its original C4-OH group and a reducing end where C1-OH is present; the
rest of the polymer contains anhydroglucose molecules containing three
hydroxyl groups. Each anhydroglucose molecule is rotated 180° in the plane,
being two adjacent molecules, the cellulose monomer called cellobiose [22].
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In nature, cellulose can take more complex supramolecular structures which

include crystalline and amorphous areas. Crystalline cellulose, which is
denser than amorphous cellulose, can account for approximately 50–90% of
the total cellulose and not readily accessible to water [23]. Structures include
elementary fibrils, microfibrils, and microfibrillar bands [22]. Cellulose

microfibrils constitute the main component of plant cell walls. Microfibrils
are inelastic and surround cells providing resistance to osmotic pressures.
These cable-like structures are regularly composed of about 36 chains
containing from 500 to 14,000 β-1,4-linked glucose molecules [24]. Molec-
ular weight and DP, which is the number of glucose residues in the chain,
vary depending on its location within the cell. Primary cell wall cellulose
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chains have a lower molecular weight and less DP when compared to those
of the secondary cell wall [21]. Being the main polymer present in LCB,
cellulose regularly accounts for 30–50% of its dry weight. Further, glucose
molecules proceeding from cellulose degradation can undergo fermentation,
thus being an important substrate for biofuel production.
15.2.3 HEMICELLULOSE
Hemicellulose is a diverse group of polysaccharides that accounts for about

one-third of the total dry weight of LCB. Its structure and amount, as in the
case of previously mentioned polymers vary depending on the plant species
and within the same plant depending on the tissue type, developmental stage,
and its physical location (primary or secondary cell wall) [25]. The term
hemicellulose is not as clearly established as it is the case of cellulose or
lignin and some polysaccharides like galactans, arabinans are sometimes
included within this group. However, here we consider hemicellulose as
a group of polysaccharides which include xyloglucan, xylans, mannans,
glucomannans, and mixed linkage glucans [26]. Xyloglucan is present on all
land plant species, being the most common hemicellulose in dicots primary
cell wall. Xyloglucan comprises a β-1,4-linked glucan backbone partially
substituted with side chains at the O-6 positions [25]. Xylans are a group of
polysaccharides that share the feature of having a backbone of β-1,4-linked
xylose residues. In grasses, xylans, following cellulose, represent the second
most abundant polysaccharides of the primary cell wall with content around
20% [26]. Mannans and glucomannans are also β-1,4-linked polysaccha-
rides. Its backbone can contain solely mannose, as in the case of mannans
and galactomannans, or consist of a combination of mannose and glucose
in a non-repeating pattern like it can be observed for glucomannans and
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galactoglucomannans. They are the main hemicellulose in charophytes and

galactoglucomannans are the main components of the secondary walls of
gymnosperms [26]. In higher plants, mixed linkage glucans are limited to the
cell walls of grasses. Hemicelluloses often interact with the other cell wall
components, thus adding more complexity to the plant cell walls.
15.2.4 PECTIN
Pectin is considered the most structurally complex family of plant polysac-

charides [27]. It can be found in the cell walls of plants and certain algae,
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constituting about 35% of primary walls in non-graminaceous monocots
and dicots, 2–10% of grass primary walls, and as much as 5% of woody
tissue walls. As previous cell wall constituents, pectin is involved in vital
plant functions including growth processes, morphology, development,
and defense against environment and pathogens. In the cell walls, it also
provides strength and flexibility [28]. Pectin polysaccharides include
homogalacturonan, rhamnogalacturonan I (RGI), rhamnogalacturonan
II (RGII), and xylogalacturonan (XGA). The total pectin content and the
relative proportion of the polysaccharides that constitute it varies depending
on environmental factors, from plant to plant and within same the plant.
Homogalacturonan is the most common polysaccharide, it can account for
up to 65% of pectin; RGI constitutes from 20% to 35%. XGA and RGII
are present in minor amounts, each accounting for less than 10% of pectin
[27]. All previous polysaccharides contain galacturonic acid linked at the
O-1 and the O-4 position. Homogalacturonan is a linear polymer consisting
only of α-1,4-linked galacturonic acid molecules. The common length of the
chain is of about 100 galacturonic acid molecules, however, shorter chains
can be found. RGII structure is far more complex, it consists of a backbone
of homogalacturonan that contains branches formed from different sugars.
XGA is composed of a homogalacturonan chain substituted at O-3 with a
β-linked xylose. Finally, RGI consists of a backbone of the disaccharide
repeats of rhamnose and galacturonic acid, that can be substituted with side
chains containing L-galactose and L-arabinose residues [27].
15.2.5 EXTRACTIVES AND ASHES
Apart from the cited polysaccharides and lignin, plant cell walls contain other
minor components that may be taken into consideration when designing a
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process where LCB is the raw material. Plants contain a variety of minerals,
sometimes referred to as ashes, which are important for their development.
The presence of high contents of ashes can have negative effects on enzymatic
hydrolysis and fermentation processes [7]. Plants also contain pigments such as
flavonoids and anthocyanins that, when extracted, can affect the bioconversion
of lignocellulose into certain products, therefore pretreatment processes should
be considered depending on the final use of the biomass. A variety of proteins
can also be found on the plant cell wall, these proteins exert mainly structural
functions, although some are involved in morphogenesis having signaling
functions. Most cell wall proteins are glycosylated and contain hydroxyproline
except for glycine-rich proteins [29]. The presence of proteins adds to further
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increase the complexity of the plant cell walls, which degradation will involve
the participation of multiple enzymes as we well address in further section.
15.3 LIGNOCELLULOLYTIC ENZYMES PRODUCED BY FUNGI ACT

AS DEGRADERS AND BIOCATALYST OF THE MAIN POLYMERS OF
THE CELL WALL
The recalcitrance of the cell wall is one of the first challenges for the
bioprocessing of lignocellulosic materials for their use in biofuel production.
As aforementioned, plant cell wall is composed mainly of lignin, cellulose,
hemicellulose, and pectin; therefore, its complete enzymatic degradation
entails the participation of several lignocellulosic enzymes. However, nature
can give us the solution, as lignocellulosic microorganisms like fungi possess
different enzymes that can make feasible the degradation of this structure.
Lignin recalcitrance to degradation is due to its complex structure and linkage
heterogeneity [30]. Lignin-degrading enzymes can be classified mainly into
two groups namely heme peroxidases and laccases [31, 32]. Ligninolytic heme
peroxidases comprise lignin, manganese, and versatile and dye decolorizing
peroxidases (DyP) [31]. Laccases assist lignin degradation and are part of
the multicopper oxidase superfamily [30]. Additional enzymes that produce
hydrogen peroxide (H2O2), which is required for peroxidase-based lignin
degradation, include glyoxal oxidase, pyranose-2 oxidase, and aryl alcohol
oxidase [33]. Cellulose and hemicellulose contain most of the sugars of the
plant cell walls, but to make these sugars available for fermentation, the
complex structures of these polymers need to be degraded. The hydrolysis
of these cell walls’ components requires the synergistic action of multiple
cellulases and hemicellulases. Pectin degradation is performed by a set of
pectinases, enzymes that have found applications in different industries.
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Besides directing their attention to the enzymatic cell wall degradation,

some scientists have also focused on engineering feedstocks that can be
easily degraded by previously cited lignocellulosic enzymes [34]. Pursuing
this strategy requires extensive knowledge of the processes involved in the
formation of the cell wall; an area that, despite extensive investigations

remains with many questions. This section will briefly describe some of
the enzymes that are involved in plant cell wall degradation and give some
insights into cell wall formation and fermentation processes.
15.3.1 LIGNIN DEGRADING PEROXIDASES
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Peroxidases are widely distributed among plants, animals, and microorgan-
isms. These enzymes catalyze the oxidation of different substrates, using
H2O2 or other peroxides as electron acceptors [35]. Most of the peroxidases
are heme enzymes that contain an iron protoporphyrin IX prosthetic group,
although there are many nonheme peroxidases [36]. Heme peroxidases can
be mainly classified into two superfamilies animal and non-animal (plant)
peroxidases [37, 38]. Non-animal peroxidases are further subdivided into
three classes. Class I are intracellular peroxidases that have been found in
bacteria, fungi, plants, and some protists, examples include cytochrome c,
catalase, and ascorbate peroxidases. Class II refers to extracellular fungal
peroxidases in which lignin, manganese, and versatile peroxidases (VPs) are
included. Class III are secreted peroxidases only found in plants [39]. Animal
peroxidases have also been subclassified as in the case of plant peroxidases,
however, its subclassification is more complex [37]. Some heme peroxidases
such as DyPs do not fit within the previously mentioned superfamily clas-
sification and constitute its family [40]. Different peroxidase phylogenetic
classifications have also been proposed [41, 42].
15.3.1.1 LIGNIN PEROXIDASE (LiP)
Lignin peroxidase (LiP) (EC 1.11.1.14) can catalyze lignin depolymeriza-

tion using H2O2 as an electron acceptor. These enzymes have been found on
different white-rot and brown-rot fungi as well as in some types of bacteria
[43]. Various LiPs isoenzymes have been encountered in some fungi, and
up to 16 different forms of LiPs have been identified in Trametes versicolor
[44]. LiPs are monomeric hemoproteins that have a molecular mass of about
40 kDa, an isoelectric point around 3.3 to 4.7, and an optimal acidic pH
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of approximately 3 [45–47]. LiPs are capable of oxidizing different non-

phenolic lignin compounds for example arylglycerol β-aryl ethers [31]. The
oxidation of β-O-4 linked compounds requires the formation of a radical
cation through one-electron oxidation, the process is followed by cleavage of
the side chain, demethylation, intramolecular addition, and some molecular

rearrangements [47]. Additional to the oxidation of non-phenolic molecules,
LiPs can also oxidase different phenolic compounds such as ring- and
N-substituted anilines [48]. The catalytic cycle of LiPs is similar to that of
other peroxidases such as the horseradish peroxidase [49]. The cycle can be
divided into three steps. First, the native ferric enzyme [Fe (III)] undergoes
two-electron oxidation to form an intermediate iron porphyrin radical cation
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[Fe (IV)] using H2O2 as the electron acceptor. In the second step, the interme-
diate, known as compound I, is reduced by a substrate which translocases one
electron to it to form another intermediate referred to as compound II. The
final step compound II receives another electron from the reduced substrate
and consequently, the enzyme returns to its original oxidation state [50].
15.3.1.2 MANGANESE PEROXIDASE (MnP)
Manganese peroxidase (MnPs) (EC 1.11.1.13) is another heme extracellular

fungal peroxidase capable of degrading lignin as well as other phenolic
substrates by oxidizing Mn2+ to Mn3+ in a peroxide-dependent manner [51].
MnP is one of the major lignin-degrading enzymes produced by certain
basidiomycetes, particularly white-rot fungi [35, 52]. Same as with of LiPs,
different isoforms of MnP exists among fungi and up to 11 different forms
of MnPs have been described in Ceriporiopsis subvermispora [53]. MnPs
are monomeric hemoproteins with masses between 32 and 62.5 kDa with
an optimum pH of 4–7 and a temperature of 40–60°C [43]. The catalytic
cycle of MnP is like that of LiPs. First, the native ferric enzyme reacts
with H2O2 to yield an intermediate Fe4+-oxo-porphyrin radical complex,
that receives the name of complex I. Further Mn2+ oxidation into Mn3+
allows the one-electron reduction of compound I to form compound II. In
the last step, Mn2+ oxidation is used for the final reduction of compound
II to return the enzyme into its native oxidation state [54]. Chelated Mn3+
ions derived from previous reactions act as diffusible redox mediators that
allow the oxidation of lignin phenolic structures [51]. For the oxidation of
non-phenolic substrates by Mn3+ reactive radicals like superoxide (O2–) or
peroxyl radicals must be formed in the presence of a second mediator such
as oxalate and malonate [51, 55].
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15.3.1.3 VERSATILE PEROXIDASE (VP)
Versatile peroxidases (VPs) (EC 1.11.1.16) are ligninolytic heme peroxi-

dases that are not only specific to Mn2+ as in the case of MnPs, but also can
oxidase phenolic and non-phenolic substrates that are commonly employed

by LiPs without the presence of manganese [56]. VPs have mainly been
reported to be produced natively by fungi species of the genera Bjerkandera
and Pleurotus [56, 57]. Similarly, to lignin and MnPs, multiple isoforms of
VPs exists [58]. The molecular structure of VP possesses characteristics of
both LiPs and MnP, however, it is more like LiP than MnP [59]. VPs have a
mass around 37 kDa, an isoelectric point of ~3.5, and an acidic optimum pH
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of about 4.5 [60]. This type of peroxidases can oxidize substrates in a broad
range of potentials, including low and high redox potentials compounds. The
catalytic cycle of VP is like that of previously described lignin and manga-
nese heme peroxidases. It involves the first two-electron oxidation followed
by two reduction reactions in which intermediate compounds are formed
to return the enzyme to its native oxidation state. The main difference is
that these enzymes have the versatility of oxidizing either Mn2+ or other
substrates to carry out their reduction reactions [59].
15.3.1.4 DYE DECOLORIZING PEROXIDASE (DyP)
Dye decolorizing peroxidases (DyPs) (EC 1.11.1.19) are heme-containing

peroxidases that comprise a peroxidase family that differ from other plant
peroxidases due to their primary and tertiary structures as well as their
specific reaction characteristics [40]. DyPs are produced mainly by some
species of bacteria, although they have been reported to be present in
fungi and some archaea [61, 62]. DyPs have a mass between 40–60 kDa,
an isoelectric point of around 3.8, and an acidic optimum pH around 2–3
depending on the employed substrate [62, 63]. DyP has been named after
certain enzymes that belong to this family were identified as capable of dye
degradation. Apart from dyes DyPs have been shown to oxidize a variety of
substrates. DyP from fungi Auricularia auricula-judae has been observed to
degrade the nonphenolic β-O-4 lignin model compound and oxidize veratryl
alcohol at low pH [64]. However, the physiological role of these enzymes
is not completely elucidated, but it has been associated with the oxidation
of nonphenolic substrates like lignin and toxic aromatic products or as a
defense mechanism against oxidative stress [62, 63].
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15.3.2 LACCASES
Laccases (EC 1.10.3.2, benzenediol: oxygen oxidoreductase) are multi-

copper enzymes produced by some species of fungi, plants, bacteria, and
insects [65]. Laccases are involved in numerous biological processes; in

plants, they contribute to the radical-based mechanisms for the formation
of the lignin polymer. In fungi, there have been related to immunity, patho-
genesis, morphogenesis, and lignin degradation [65, 66]. These enzymes
are of biotechnological importance due to their capacity of oxidizing a wide
variety of substrates by only using molecular oxygen (O2) and producing
water as a by-product [67]. In contrast with ligninolytic peroxidases which
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are only extracellular, laccases can be localized extra or intracellularly. The
localization of these enzymes may be related to the physiological function
they exert, and this localization also determines the variety of substrates
that can be used by them [66]. Generally, fungal laccases have masses
around 60–70 kDa, an isoelectric point about 4, and an acidic optimum pH
between 2.5–4 [66].
The catalytic cycle of laccases reduces one molecule of O2 to two
molecules of water with the concomitant oxidation of four molecules of a
substrate to produce four radicals. This redox process is aided by a cluster
of four copper atoms that serve as the catalytic core of the enzyme [67].
Laccases can catalyze direct oxidation of phenolic compounds and certain
amines [68]. Nevertheless, oxidization of high redox potential compounds
such as non-phenolic model compounds and β-1 lignin dimers requires the
presence of a mediator. Some commonly used mediators are 3-hydroxyan-
thranilic acid (HAA), N-hydroxybenzotriazole, and 2,20-azino-bis-(3-ethyl-
benzothiazoline-6-sulphonic acid) (ABTS) [67]. However, the use of these
mediators increases the overall cost of the process.
15.3.3 DEGRADATION OF CELLULOSE AND HEMICELLULOSE
As described earlier, cellulose and hemicellulose are major constituents

of the plant cell walls. Like in the case of lignin, the degradation of these
compounds is challenging due to their complex structure, linkage strength,
and high molecular weight [69]. However, microorganisms have developed
a set of enzymes called cellulases and hemicellulases that can degrade these
compounds into simpler carbohydrate monomers. Their degradation capacity
has made these enzymes of biotechnological importance in various sectors
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including textile, wine, and brewery, food, pulp, and paper, agriculture, and
biofuel industries [70, 71].
15.3.3.1 CELLULASES
Cellulases are a family of three groups of enzymes, endoglucanases (EC

3.2.1.4), exoglucanases (EC 3.2.1.91), and β-glucosidases (EC 3.2.1.21)
[72]. Cellulases are expressed natively by different microorganisms
including fungi and bacteria. However, most commercially available cellu-
lases are produced from aerobic cellulolytic fungi due to the high yields that
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some engineered strains have in producing these enzymes [73]. In bacteria,
cellulases are present as extracellular aggregated structures attached to the
cells called cellulosomes [74]. Fungal cellulases have a relatively simple
molecular structure containing a catalytic domain and a cellulose-binding
domain. Extensive reviews describing the structure and characteristics of
these enzymes are available on refs [75, 76]. The catalytic domain is linked
to the cellulose-binding domain by a linker peptide. The anchoring of the
cellulose-binding domain to cellulose permits the enzyme to optimally
perform its catalytic function [75]. Cellulases degrade cellulose using
different synergistic approaches. Endoglucanases, also called non-processive
cellulases, can cleave the internal O-glycosidic bonds producing polysac-
charide chains of variable lengths. Exoglucanases or processive cellulases
act by coupling to the cellulose chain ends and hydrolyzing cellulose into
cellobiose. β-glucosidases can then cleave cellobiose into glucose that can
be used by the microorganism [76].
15.3.3.2 HEMICELLULASES
Hemicelluloses are a diverse group of enzymes capable of degrading

hemicellulose. Due to the variety of monomers that conform to hemicellulose,
its complete degradation requires the synergistic interaction from different
enzymes [77]. Hemicellulases can be classified according to their catalytic
modules in either glycoside hydrolases that can hydrolyze glycosidic bonds
or carbohydrate esterases (CEs), which are enzymes capable of hydrolyzing
ester linkages of acetate or ferulic acid side groups [76]. Among hemicellulose
degrading enzymes we can find xylanases (EC 3.2.1.8), β-β-mannanases (EC
3.2.1.78), α-L-arabinofuranosidases (EC 3.2.1.55) and α-L-arabinanases (EC
3.2.1.99), α-glucuronidases (EC 3.2.1.139), β-xylosidases (EC 3.2.1.37) and
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acetyl xylan esterases (EC 3.1.1.72) [78, 79]. Many of these hemicellulolytic
enzymes have been found on various types of microorganisms, including
fungi and bacteria [78]. Describing the catalytic mechanisms and structure
of the previously mentioned enzymes is beyond the scope of this chapter.
15.3.4 PECTINASES
Pectin, as formerly mentioned, is a complex polysaccharide of the cell walls

that contributes to their strength and flexibility. It contains acidic sugars that
are involved in some cell wall characteristics including adhesion, extensi-
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bility, porosity, and electrostatic potential [80]. Because of its contribution to
porosity and charge, pectin has been a target for studies aiming at cell wall
degradation [81]. Pectinases are a group of enzymes capable of degrading
pectin that has found application in the fruit, paper, and pulp, textile, and
biofuel industries [81, 82]. Pectinases are natively produced by a variety
of organisms including fungi, bacteria, insects, protozoa, and plants [83].
However, for the industrial production of these enzymes, fungi, and bacteria
are the most commonly employed organisms [84]. Different classifications
for pectinases have been proposed that are based on their preferred substrate,
their mode of action, and cleavage characteristics [85]. Microorganisms can
produce different forms of pectinases that have been encountered to work
in a wide range of pH and temperature values [82]. Optimum pH values
may vary between 4–10 and optimal temperature from 30 to 70°C [82].
Such variability can be related to the natural environmental factors to which
the microorganism is exposed [83]. Because of the previous recombinant
pectinases are produced from different sources so that they can be applied in
processes that involve harsh temperature and pH conditions [84].
15.3.5 PLANT CELL WALL BIOSYNTHESIS
Even though lignocellulose is considered the most abundant biomass on

Earth [86], some of the processes that are related to its biosynthesis and
the construction of the plant cell walls remain to be elucidated [87, 88].
Cell wall synthesis is a complex process that involves numerous enzymes
and processes such as intracellular trafficking of cell wall precursors and
proteins, the extracellular assembly of cell wall polymers, the remodeling of
these polymers, and the recycling of some cellular sugars [87]. The assembly
of the nucleotide sugars which are required for cellulose and hemicellulose
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synthesis is catalyzed by several glycosyltransferases, enzymes capable of

forming glycosidic bonds by transferring sugar moieties between the donor
and accepting molecules [87]. On the other hand, the building blocks for
lignin biosynthesis proceed from the general phenylpropanoid pathway, being
phenylalanine and sometimes tyrosine the substrates for this pathway [19].
Furthermore, laccases, and peroxidases, enzymes that are involved in the
lignin degradation process, are also involved in lignin biosynthesis by
catalyzing the oxidation of phenolic monolignols into phenolic monomers,
a process essential for the initiation of in vivo polymerization of lignin [30].
The study regarding cell wall synthesis is of great interest due as one
strategy to overcome its recalcitrance may be to develop feedstock varieties
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with more labile cell walls that apart from maintaining is agricultural yields
can be useful for biofuel production [89]. Some of the enzymes that partici-
pate in the degradation of the main components of the plant cell wall have
also been identified to be involved in its biosynthesis. This is the case of the
membrane-anchored endoglucanase Korrigan which appears to be related to
cellulose biosynthesis in the primary and secondary cell walls of Arabidopsis
thaliana. Mutant genotypes have exhibited defects in cell wall formation,
including reduced cellulose content and increased pectin synthesis deriving
in abnormal plant morphology [90, 91]. Comprehensive reviews can provide
the reader with a complete picture of the current understanding of the biosyn-
thesis of the main components of the cell wall [19, 25, 80, 92].
15.3.6 INSIGHTS INTO ALCOHOLIC FERMENTATION
Once that lignocellulosic materials have been pretreated and lignocellulo-

lytic enzymes have degraded the main components of the cell wall, sugars
are fermented to produce ethanol [93]. Sugars derived from cellulose and
hemicellulose degradation include hexoses such as glucose, mannose,
fructose, and galactose, as well as pentoses, namely xylose and arabinose
[94]. Some microorganisms like yeast, fungi, and bacteria possess the
mechanisms for fermenting these sugars into ethanol; being yeasts the most
studied and industrially-employed organisms [95]. Saccharomyces cerevi-
siae can natively ferment hexoses; fermentation of glucose, mannose, and
fructose occurs via the Embden-Meyerhof pathway of glycolysis, and galac-
tose fermentation via the Leloir pathway [93]. The Entner-Doudoroff (ED)
pathway is an additional pathway that allows glucose fermentation bacteria
such as Zymomonas [96]. However, the use of these species of bacteria may
not be suitable for industrial bioethanol production as it can only ferment
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glucose, fructose, and sucrose [96]. Wildtype S. cerevisiae is not capable of

fermenting neither xylose nor arabinose. But some species of Candida have
been identified to ferment these substrates into ethanol [93]. Rhamnose,
6-deoxy sugar, can also be available for fermentation as it is a constituent
of the rhamnogalacturonan part of pectin and hemicellulose. However, as its

alcoholic fermentation pathway is not natively expressed in most organisms,
metabolic engineering will be required to express a route for its fermentation
in microorganisms that are currently used for industrial fermentation [93].
The use of genetic engineering can greatly increase the yield of bioethanol
from LCB by allowing the engineered organism to ferment most of the avail-
able sugars. Furthermore, the integration of lignocellulosic enzymes will
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make able to develop a consolidated bioprocess to simultaneously perform
the saccharification and fermentation processes.
15.4 CONSOLIDATED BIOPROCESSING (CBP)
Ethanol production from lignocellulosic materials is a multiple-step process

that includes biomass collection and storage, pretreatment, saccharification,
fermentation, and ethanol recovery [13]. The need for multiple steps is trans-
lated into a higher cost of production when compared to traditional ethanol
production from starch- or sugar-based crops [97]. Conventionally bioethanol
production process involves that saccharification and fermentation processes
are performed separately, which results in a time consuming and costly
process [98]. Apart from the previous disadvantage, SHF processes have the
drawback that cellulases activity during the hydrolysis process is inhibited
by their product, limiting the efficiency of the hydrolysis process [98].
The necessity to reduce costs and optimize the process has led to the
development of simultaneous saccharification and fermentation (SSF)
processes [13]. In SSF processes biomass hydrolysis and fermentation are
performed within the same bioreactor; this allows to reduce costs and obtain
higher ethanol yields as sugars are fermented as they appear in the solution
[99]. However, the SSF process is focused on fermenting only hexoses
leaving potentially fermentable sugars unused. To overcome this problem,
simultaneous saccharification and co-fermentation (SSCF) processes have
been developed in which fermentation of hexoses and pentoses occur in a
single step [100]. Nevertheless, the SSCF process still requires the addition
of lignocellulolytic enzymes into the vessel, which represents a disadvantage
as the cost of the biomass, its pretreatment, and the enzymes used for
hydrolysis account for about two-thirds of the ethanol production cost,
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being the cost of the enzyme the most significant [101]. All the previous
has led to the development of CBP, where enzyme production along with
saccharification and fermentation are produced simultaneously within the
same vessel [101]. For CBP to be feasible, it is required that either multiple
or a single microorganism be able to produce both lignocellulosic and

fermentative enzymes [102].
Despite being a promising strategy to optimize the process for ethanol
production, CBP remains in its early development stages. One known
limiting factor is that the optimal temperature for saccharification (45–50°C)
differs from that required for efficient fermentation which is around 28–35°C
[94, 96]. Another limitation is that ethanol has the capacity of inhibiting the
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growth of its producing microorganisms once a certain concentration has
been reached; thus, discontinuing the fermentation process which may result
in low ethanol yield [99]. These factors have led to the quest of finding micro-
organisms that are optimal for this type of bioprocess. However, microbes
capable of overcoming previous difficulties and that possess the enzymatic
machinery to perform both saccharification and fermentation efficiently have
not yet been encountered in nature [101, 102]. Therefore, different strategies
have been proposed to develop these ideal microorganisms. Mainly there is a
focus on two strategies, the native strategy, and the recombinant strategy. The
first one aims to genetically engineer a microorganism natively capable of
producing lignocellulosic enzymes to make it ethanologenic. The second is
targeted to engineer an ethanol-producing microorganism to make it capable
of degrading lignocellulose [97, 102].
Following the native or recombinant strategies has some challenges. By
engineering microorganisms according to the native strategy, scientists need
to use the currently available genetic engineering tools to produce a strain
capable of yielding high ethanol concentrations and that is robust enough for
its industrial application [102]. Most promising candidate microorganisms
for following this strategy are lignocellulosic fungi and bacteria, as microor-
ganisms like bacterium Clostridium thermocellum and a variety of white-rot
fungi have been already used in ethanol production via CBP [103, 104]. On
the other hand, the production of organisms by the recombinant strategy
faces the challenge that the selected organisms need to produce enough
quantities of a wide variety of enzymes to degrade the main components of
the plant cell walls [97]. The recombinant strategy has focused mainly on
modifying yeasts and bacteria, with most of the advances being performed
in S. cerevisiae [102, 105].
Besides previously mentioned challenges, other aspects limit the full
potential of CBP. Genetically engineered microorganisms may present
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problems regarding their genetic stability, and productivity [106]. Ethanol

yields are lower, and fermentation is longer (taking between 3 to 12 days)
when using organisms like bacteria and fungi [107]. Further, lignocellulose
degradation rates of some modified non-lignocellulolytic microorganisms
remain notably lower than those exhibited by lignocellulolytic fungi [106].

Additionally, the simultaneous expression of multiple cellulose-degrading
genes at the appropriate levels is difficult and is still under investigation
[106]. Lastly, it is important to consider that biomass pretreatments can
release toxic compounds that may reduce the process performance including,
furan derivatives, pentose, and hexoses, weak acids, and lignin-derived
phenolic compounds [95]. Therefore, the ideal microorganisms would need
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to be engineered to overcome these difficulties. In the following section, we
will present some fungi that are good candidates or that already have been
employed in the CBP of ethanol production from LCB.
15.5 ROT FUNGI FOR DIRECT ETHANOL PRODUCTION FROM

LIGNOCELLULOSE MATERIALS
At the hand of the abundant presence of biomass and the pressing need for
different fuel sources, the finding of fungi with the potential to produce
lignocellulolytic enzymes is a viable solution, regarding the problem. The
fungi are grouped in relation to the type and characteristics of produced
rot, in spite of the classification system is not accurately aimed. Table 15.1
depicts the following groups of fungi-rot.
TABLE 15.1 Examples of Fungi Belonging to Different Groups

Fungi-Rot Examples
White rot Agrocybe aegerita, Auricularia auricula-judae, Bjerkandera adusta, Coprinellus
fungi radians, Ceriporiopsis subvermispora, Heterobasidium annosum, Irpex lacteus,
Mycena haematopus, Phanerochaete chrysosporium, Pleurotus eryngii, Phlebia
radiata, Pleurotus ostreatus, Pycnoporus cinnabarinus, Trametes versicolor,
Stropharia rugosoannulata, and Xylaria hypoxylon [108–112]
Soft rot Alternaria alternata, Chaetomium globosum, Ceratocystis, Kretzschmaria
fungi deusta, Paecilomyces spp, Thielavia terrestres, and Ustulina deusta
[108, 113, 114]
Brown rot Coniophora puteana, Laetiporus sulphureus, Gloeophyllum trabeum, Piptoporus
fungi betulinus, Phaeolus schweinitzii Postia placenta, and Serpula lacriman [95, 114]
Stain fungi Ophiostoma, Ceratocystis spp., Aureobasidium pullulans, Phialophora spp.,
and Trichoderma spp [113]
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15.5.1 WHITE ROT FUNGI
White-rot fungi produce a multi enzymatic system allowing for break down
the three principal polymers of the natural lignocellulosic substrates: lignin,
hemicellulose, and cellulose in progressive form. This complex system may

work separately or cooperatively. In particular, the ligninolytic enzymes
provided by white-rot fungi have high specificity to the substrate and they are
LiP, VPs, MnPs, and copper-containing phenol oxidases (laccase) [108–112].
15.5.2 SOFT ROT FUNGI
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Ascomycetes and Deuteromycetes belong to soft-rot fungi and they are the
noblest fungi in terms of the level of decomposition they cause. Even, they
emerge before the rotting process carried out by white and brown fungi. Despite
the low degradation, soft-rot fungi degrading wood in severe environmental
conditions. These kinds of fungi require fix nitrogen to synthesize cellulase
enzymes, in charge of depolymerizing the cellulose of the secondary cell
walls, making huge cavities [108, 113, 114].
15.5.3 BROWN ROT FUNGI
Numerous fungi cause white rot compared to brown rot. However, they
play an important role in the degradation of deadwood, especially, in
coniferous ecosystems. The brown rot fungi firstly attack the lignin through
demethoxylation, occurring a lignin partial modification, without produce
lignin-degrading enzymes. Posteriorly, the cellulose tackle is carried out by
the transformation of methanol to peroxide with a methanol oxidase over-
expression via Fenton chemistry. Thanks to the accelerated fractionation of
cellulose, the wood loses strength rapidly [95, 114].
15.5.4 STAIN FUNGI
Stain fungi are a small group of Ascomycetes and Deuteromycetes, colo-

nizing more favorably ray parenchyma cells and resin canals with a mild
degradation. The degradation impacts on the water-soluble components and
extractives components as fatty acids, diterpenoids resin acids, and triglyc-
erides of the cell wall [113].
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KEYWORDS
• hydrogen peroxide
• lignin peroxidase
• manganese peroxidase
• rhamnogalacturonan I
• separate hydrolysis and fermentation
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CHAPTER 16
Bioelectrosynthesis of Ethanol via

Bioelectrochemical Systems: Future
Perspectives
SILVIA YUDITH MARTÍNEZ AMADOR,1
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LEOPOLDO JAVIER RÍOS GONZÁLEZ,2
MIGUEL ANGEL MEDINA MORALES,2
MÓNICA MARGARITA RODRÍGUEZ GARZA,2
ILEANA MAYELA MARÍA MORENO DÁVILA,2
THELMA KARINA MORALES MARTÍNEZ,3 and
JOSÉ ANTONIO RODRÍGUEZ DE LA GARZA2
1
Botany Department, Agronomic Division, Antonio Narro Autonomous
Agrarian University, Mexico
2
Department of Biotechnology, School of Chemistry, Autonomous
University of Coahuila, Mexico, Phone: +52 (844) 415-5752–Ext 120,
E-mail: antonio.rodriguez@uadec.edu.mx (J. A. R. D. L. Garza)
3
Bioprocess and Microbial Biochemistry Group, School of Chemistry,
ABSTRACT
Microbial electrosynthesis has gained much attention in the last decade in the
scientific community due to that this novel process allows the production of
commodity chemicals sustainably. MES (Microbial electrosynthesis cell), as
commonly referred to as this technology, is a bioelectrochemical system that
utilizes the reducing power generated from the anodic oxidation to produce
high value-added products in the cathode. When coupled with a renewable
source of electricity and organic or inorganic substances to produce biofuels,
such as alcohols (ethanol, butanol, etc.), significant advantages stand out,
such as the avoidance of fossil fuels to provide the energy that drives the
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process and food crops that serve as feedstock. The present chapter will
give an insight into the novel perspectives of bioethanol production through
bioelectrosynthesis as well as the more recent and relevant developments in
MES technology for this purpose.
16.1 INTRODUCTION
2020 has been a year that has not only have cause several health issues all
around the world but also has shown the fragility of the global economy
[1] and its dependence on fossil fuel. This dependence has been more of
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a reminder of the need to search, and develop sustainable fuel production
processes to fulfill our energy needs and, additionally, options that have
higher market stability than oil prices [2–5]. In this regard, biofuels were
considered an alternative to gradually replace fossil fuel over the next 30
years; however, many concerns have arisen mainly due to the feedstocks
used to produce biofuels, generating food security issues all around the
globe. In the past decade, it has been clear that future energy demand will be
met by a combination of renewable energy (solar, hydro, thermal, biofuels,
and eolic) and fossil fuels [6–8].
As stated initially, this year (2020) has presented several challenges;
however, also it has opened new opportunities. The Contraction in global
oil consumption has opened the windows for the renewable energy market;
additionally, the energy market is being subjected to pressure to lower its gas
emissions to fulfill the objectives of reverting or at least tackling the climate
change issue [2–5].
The use of renewable energy, combined with bioenergy, will allow us
to be more independent in energy matters and fight climate change. Among
bioenergy, bio-alcohols as high value-added products from agro-industrial
waste as an organic carbon source are well established in the energy market.
Among bioalcohols, bioethanol is by far, the most produced biofuel in the
globe, being Brazil and the United States the dominant players in bioethanol
production, representing almost 80% of the global output. However, as
mentioned earlier, conflicts over food supply and land use have made its
production and utilization a controversial issue. To overcome this issue, in
the last decade, second-generation bioethanol production technology has
focused on the use of non-edible (human consumption) biomass sources,
using a wide range of feedstocks, such as LCB, energy crops, cellulosic
residues, and, particularly, wastes [9–11]. The quality of bioethanol produced
is majorly dependent on the production routes. Bioethanol production, in
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Bioelectrosynthesis of Ethanol via Bioelectrochemical Systems 447
general, consists of several sequential procedures, namely pre-treatment,

hydrolysis, fermentation, and distillation. Each stage can have more than
one possible route, and each alternative route can render different results in
ethanol quality, as well as overall production cost [12].
Bioelectrosynthesis is a novel hybrid biobased process that involves

electrochemical approaches to utilize bioelectrochemical systems to
convert dissolved CO2 into value-added organic compounds. In this regard,
bioethanol can be produced by microbial reduction of acetate as the main
intermediate of anaerobic digestion (AD) with hydrogen as an electron donor
[13–15]. Bioethanol production via microbial bioeletroshyntesis (MES)
from CO2-waste streams, could alleviate the concern of food security-related
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issues mentioned before. Although ethanol can be produced directly from
CO2, its production from acetate is thermodynamically and energetically
more favorable, providing that the undissociated acid exists in a slightly
acidic medium [13–15]. Still, ethanol production through MES is far from
becoming a feasible process, yet as titers and efficiencies are relatively
low. The following book chapter will be focused on describing briefly on
describing the current development on BES and its multiple application,
followed by exploring the use of BES for ethanol production by means of
MES. It will be discussed how can BES compete, in terms of energy usage,
with conventional bioethanol production technologies by exploiting the
energy content of some of the chemicals present in the waste biomass and
CO2 waste streams.
16.2 BIOELECTROCHEMICAL SYSTEMS OVERVIEW
In the early XX century, Potter [16] reported the production of electricity

associated with microbial catabolism, for the first time experimentally
demonstrating the capability of microorganisms to transport electrons extra-
cellularly when degrading organic compounds [17–20]. The first microbial
fuel cell (MFC) was built by Cohen [21]. However, the importance of the
work was not recognized until the energy crisis got worse globally in the
late 1990s [4, 5], re-stimulating the concerns in biofuel cells; since then, the
research activities with bioelectrochemical systems as topics began to blossom
resulting in an exponential increment in the number of publications, as shown
in Figure 16.1 [22]. The concept of microbial electrolysis cell (MEC) was
originated in 2005. Two different research groups independently noticed the
production of hydrogen gas in an electrolysis-type process modified from
MFC nearly at the same time [23, 24]. This process has previously been
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named as “electrochemically assisted microbial reactor (BEAMR),” then

“biocatalyzed electrolysis.” Finally, it was defined as “electrohydrogenesis”
or “microbial electrolysis” to distinguish the hydrogen evolution in an MEC
from dark fermentation [25–30]. Shortly afterwards, the function of MEC
for CO2 bioconversion to methane was discovered as well, this is, “elec-
tromethanogenesis” that we now know [15, 31–37]. Based on these initial
efforts, a great deal of MEC configurations with different purposes have been
designed and proposed very recently, such as microbial electrodialysis cell
(MEDC), microbial reverse electrodialysis electrolysis cell (MREC), micro-
bial desalination cell (MDC), etc., substantially extending the scope of MEC
applications. From then onwards, a new era belonging to MEC is approaching
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(Figure 16.2) [29, 30].
FIGURE 16.1 Number of published journal articles on MES containing the phrases
“microbial fuel cell;” “microbial electrolysis cell;” “microbial electrosynthesis;” or “microbial
desalination cell.”
Source: Adapted from: 2013–2019 PubMed Central.
Source: Based on figure presented by Wang and Ren [22].
16.3 APPLICATIONS OF BIOELECTROCHEMICAL SYSTEMS
The different types of BES can distinguish from one another by the main func-
tion and the output of the BES. For example, a microbial fuel cell (MFC) is the
most characteristic type of BES, whose main function is the generation of direct
electricity. When an external power supply is added to an MFC reactor to reduce
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the potential of the cathode, the system becomes a microbial electrolysis cell
(MEC), where hydrogen gas and other products are generated [22]. Addition-
ally, the potential input required in a MEC can be provided by a MFC so that
hydrogen can be produced directly from biomass and waste sources [38]. If the
primary objective of the BES is the cathode to reduce oxidized contaminants,

such as uranium, perchlorate or chlorinated solvents, it can be called a microbial
remediation cell. If the main objective of the system is to synthesize value-added
chemicals through cathodic reduction by microorganisms, the system can be
named microbial electrosynthesis (MES). Another application for BES is water
desalination, in this type of BES an additional chamber between the anode and
cathode is required and this type of BES has been named as microbial desalina-
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tion cell or MDS [22]. Nutrient removal or recovery can also be carried out by
a BES, in the case of nitrogen, it can be removed through bioelectrochemical
denitrification or recovered vía ammonium migration driven by electricity
generation (Figure 16.3) [20, 39, 40].
FIGURE 16.2 Overview of various types of bioelectrochemical systems and main products.
16.4 MICROBIAL FUEL CELLS (MFCS)
A MFC is a bioreactor that focuses on the production of electricity from

biodegradable materials through microbial catalytic reactions [29, 42–44].
Electricity is produced by the oxidation of organic carbon in the anode under
anaerobic conditions, generating electrons, protons, and products such as
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volatile fatty acids, biomass, and carbon dioxide (CO2) [45]. This type of
BES has the potential of a dual application, treating different types of waste
streams, such as wastewater, and during this process, an electric current will
be produced. In this approach, it generates an alternative energy source from
wastes [46, 47].
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FIGURE 16.3 Schematic overview of various types of bioelectrochemical systems [41].
16.5 MICROBIAL ELECTROLYSIS CELLS (MECS)
A MEC takes advantage of the property of bacteria to transform the chemical

energy stored of compounds, and with the addition of an external power
applied onto the electrical circuit of MEC, this will drive electrons from
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anode to the cathode. In the presence of a catalyst, electrons, and protons

are chemically reduced at the cathode to form hydrogen gas. Contrary to the
MFC, the cathode of MEC operates under anaerobic conditions that facilitate
hydrogen production. However, the anoxic environment in MEC, along
with high concentrations of hydrogen production, can also promote methane

production once CO2 and methanogens are available [15, 34, 48–53]. A few
of the methods to mitigate methane production includes the aeration of the
cathode chamber between batches, lowering of the pH, operation at short
retention times and giving a heat shock to the inoculum, or adding chemicals
that inhibit the growth of methanogens [37, 53–56]. Higher electric currents
are typically observed in MEC when compared to MFC, which is due to
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the additional applied voltage that helps overcome the cathode limitations.
The energy required for MEC operation can also be provided by another
separate MFC as a power source [28, 57–60]. In a MEC with bioanode and
biocathode, expensive metals like platinum are not required as catalyst, and
preferably, the enrichment of microbes on the carbon cathode decreases the
start-up time and produces comparable current densities to those of bioanode
[61–67]. Furthermore, the hydrogen synthesized in MEC can also drive the
biochemical production of other high value-added chemicals [22, 63, 68, 67].
16.6 MICROBIAL ELECTROSYNTHESIS (MES)
MES, also known as bioelectrosynthesis, is a new-fangled perspective of

BES which utilizes the reducing power generated from the anodic oxidation
to produce value-added products at the cathode. Cathodic biocatalysts (with
attached cathodic biofilms) reduce the available terminal electron acceptor to
produce value-added products [8, 29, 69]. The bioelectrosynthesis process can
be highly specific, depending on the biocatalyst catalyzing the redox reaction
and the terminal electron acceptor involved in the process, along with the
electrochemically active redox mediators or suitable reducing equivalents.
Biocathodes are the key components of microbial electrosynthesis, where
the electrode oxidizing microorganisms are involved in the formation of
reduced value-added products such as acetate, ethanol, butyrate [8, 65, 69].
The production process of high added-value products in MES it through an
electrochemical cell by electricity-driven CO2 reduction as well as reduction/
oxidation of other organic feedstocks using microbes as biocatalyst [31, 65,
67, 69–73]. The feasibility of using MES as a technology into multi-carbon
organic compounds using electrical current at the cathode has been reported
widely. Additionally, MES technology also has the potential to harvest electrical
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energy in covalent chemical bonds of organic products. Mes technology has

been extensively studied recently, highlighting the different aspects of that
make it unique, such as microbiology, technology, and economics, and other
practical consideration [29, 32, 34, 63, 67, 74–77].
16.7 ETHANOL PRODUCTION IN BES
Ethanol via the Wood-Ljungdahl pathway can be produced either directly

from CO2 reduction or by acetate reduction or from both. CO2 reduction via
WL pathway is highly energetically efficient, in where more than 95% of
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carbon and electron flow is channeled to the production of extracellular end
products rather than to the microbial growth and biomass production [15, 33,
49]. The thermodynamics of ethanol production by CO2 or acetate reduction
indicates that lower pH and excess H2 can shift the production to ethanol.
The selectivity of the products of microbial CO2 reduction has been shown to
be limited by micro-elements of trace metal and vitamin components due to
their effect on the activity of NADPH/NADP and Acetyl-CoA enzymes [15,
32, 78, 79]. By limiting micronutrient and trace elements (namely pantothenic
acid and cobalt), higher concentrations of ethanol can be produced rather
than acetate, suggesting that larger multicarbon compounds can be produced
by means of applying strategies like those use in the gas fermentation process
in where gaseous feedstocks such as CO, CO2, syngas, CH4 or biogas, into
platform chemicals, fuels, polymers, etc. Thermodynamically, the reduction
of acetate to ethanol is more favorable than the reduction of bicarbonate
to ethanol and this has been reported when acetate accumulated and goes
through further reduction (Figure 16.4). However, ethanol concentrations
achieved are quite low [14, 34, 49, 56, 73, 80, 81].
One aspect that still needs further study is the mechanism of bacterial
interaction with solid electrodes for CO2 reduction. In this regard, the
overpotential of the electrode will create conditions that much likely require
a lower potential in the cathode. The necessary overpotential so that CO2
reduction can take place in the cathode will require much lower cathode
potential than the hydrogen evolution reaction (HER) [82]. Additionally,
in the case of using a mixed culture, most likely, the HER will take place
first, followed by CO2 reduction mediated by H2 [6, 34, 67, 69, 72]. On the
other hand, when direct electron transfer takes place, it could be possible to
achieve higher production rates of the targeted compound. Cytochrome-c
for extracellular electron transfer on homoacetogenic bacteria has shown the
potential of different of using multiple mechanisms of electron transfer, and
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this will depend of the biocatalyst chosen (Figure 16.5). In most of the litera-
ture, the bioelectrochemical reduction of CO2 has been reported to take place
at lower cathode potentials than the theoretical HER potential (0.61 V when
reference electrode is Ag/AgCl). MES experiments showed a considerable
production of acetate only when the potentials were more negative than 0.8
V (using as reference electrode Ag/AgCl) and whenever the potentials were
above or equal to 0.8 V, acetate, and butyrate were degraded instead of CO2
reduction. In this regard, acetate production occurs only below 0.9 V, in
this case H2 should mediate the CO2 reduction reaction. Bioelectrocatalsys
combined with the HER can take place when higher current densities (up to
10 ampers per m2) are obtained at a potential of 0.9 to 1 V and when abiotic
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cathode is being used. However, when the biocathode is a mixed culture, it
would not be clear what kind of mechanisms for electron transfer will be
taking place [32, 33, 49, 52, 62, 76, 78, 81, 83–90].
FIGURE 16.4 Schematic of the Wood-Ljungdahl pathway for CO2 reduction for ethanol
production.
16.8 CONCLUSIONS
MES can offer a wide range of opportunities that go from, energy production
from wastes, to the production of high added value products from inorganic
sources. MES technology offers new approaches that other heavily
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FIGURE 16.5 Extracellular electron transfer mechanism of Geobacter sulfurreducens [41].
biomass-relaying technologies that require large extension of land to obtain

its feedstocks, on the other hand, MES land-use needs are low. Additionally,
MES technologies, coupled with existing photovoltaic technologies can
offer an almost non-polluting alternative to obtain a wide range of chemicals
and energy. Operating MES system with existing photovoltaic technologies
can offer higher efficiencies per surface area than conventional technologies.
Additionally, fresh water and nutrients requirement can be lowered
substantially in the bioproduction of chemicals compared to agricultural
production. These features make MES technology very attractive, not just
from an economic point of view, but also for being an almost a non-polluting
technology as mentioned earlier. In these regard, MES can operate entirely
without of the high input of hazardous and non-renewable raw materials
in the unsustainable manner (high temperature/pressure, acid/alkaline
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solutions, etc.). Inexpensive and abundant carbon sources including CO2 and
waste streams can be reutilized for bioproduction with the input of electrical
energy only (renewable energy). In addition, MES offers unique possibilities
to promote cell growth and the controlling of redox state in bioprocesses by
steering the metabolic pathways electrically. Therefore, this is a promising

technology that provides prospects of renewable energy-driven waste to
fuel/chemical to establish a circular economy. Still in development, this
technology will require further understanding that will allow to compete in
the near future with the existing commercial technologies.
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KEYWORDS
• hydrogen evolution reaction

• microbial desalination cell
• microbial electrodialysis cell
• microbial electrolysis cell
• microbial electrosynthesis
• microbial fuel cell
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CHAPTER 17
Challenges and Perspectives on

Application of Biofuels in the Transport
Sector
F. G. BARBOSA,1 S. SÁNCHEZ-MUÑOZ,1 E. MIER-ALBA,1
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M. J. CASTRO-ALONSO,1 R. T. HILARES,2 P. R. F. MARCELINO,1
C. A. PRADO,1 M. M. CAMPOS,1 A. S. CARDOSO,1 J. C. SANTOS,3
and S. S. DA SILVA1
1
Bioprocesses and Sustainable Products Laboratory,
University of São Paulo (EEL-USP), Lorena–12.602.810, SP, Brazil,
E-mails: salvador.sanchez@usp.br;
sanchezmunoz.ssm@gmail.com (S. Sánchez-Muñoz)
2
Material Laboratory, Catholic University of Santa Maria (UCSM),
Yanahuara, AR–04013, Peru
Bioprocesses, Biopolymers, Simulation, and Modeling Laboratory,
3

ABSTRACT
Environmental and economic impacts caused by the use of fossil fuels have
been demanding the development of renewable energy technologies based on
low carbon fuels. In the last times, large-scale investments in the production of
biofuels have increased in many countries intending to achieve the Sustainable
Development Scenario. However, policies, economic competitors, regional
feedstocks availability, technological challenges of biomass conversion, and
bottlenecks for current and future biofuels productions, represent key issues
to be considered for their incorporation into the transport sector.
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17.1 INTRODUCTION
In the transport sector, there is the dominance of the use of fossil fuels that are
derived from oil sources. With global economic development, the demand
for fuels is growing proportionately. The increasing use of these fuels has
been pointed to as responsible for the occurrence of climate changes due to
an increased concentration of CO2, the main greenhouse gas (GHG), in the
atmosphere. Reports inform us that the transport sector consumes 30% of
global energy and is responsible for 21% of the annual GHG emissions [1].
As a consequence of environmental concerns, the search for sustainable
fuels (biofuels) production with low CO2 emission and minimal climate
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changes has grown. In the world, the countries with the highest production
of biofuels are the USA, Brazil, and China, and it is expected that these
countries will double or triple their consumption of biofuels by 2030 [2, 3].
Worldwide, the biofuels most commonly used in motor vehicles,
marine vessels, and aviation are bioethanol, biodiesel, and biogas [4, 5].
Bioethanol and biodiesel are mainly produced by USA and Brazil, while
for biogas, the largest production is observed in Europe. Even though,
policies and technologies need to be improved to allow and encourage the
application of these biofuels in transportation. For this, government projects
and programs are developed to intensify the production and use of these
sustainable fuels. Also, financial subsidies are provided to manufacturers
aiming for the production of sustainable supplies from available biomass to
support bioeconomy development [6]. Due to these financial subsidies and
government programs, biofuels classified as the first-generation dominate
the sector of renewable fuels applied to transportation. Their production is at
an advanced stage, from the processing of the biomasses involved until the
industrial infrastructure. However, aspects related to land availability and
the competition of feedstocks with the food sector, make second and third-
generation technologies gaining attention worldwide [7, 8].
In this context, the perspectives and challenges faced for the production
and application of biofuels in the transport sector will be discussed in this
chapter.
17.2 BIOFUELS AS ALTERNATIVE TRANSPORT FUEL:

WORLDWIDE CURRENT STATUS AND THE USE OF BIOETHANOL
Biofuels can replace conventional fuels derived from oil, such as gasoline
and diesel in the transportation sector [187]. The obtaining processes of
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Challenges and Perspectives on Application of Biofuels 465
those renewable forms of fuel and their use must be environmentally friendly
[9–11]. Available biofuels are bioethanol, biodiesel, biogas (biomethane),
and biohydrogen [12, 13].
Biofuels are classified as first-generation (1G), second-generation (2G),
and third-generation (3G) [14–19, 188]. This classification relates to the type
of feedstocks used in their production, for example, 1G biofuels have as
feedstock energy crops like rice, wheat, corn, sugarcane, and also vegetable
oils, among others [20]. On the other hand, 2G biofuels are obtained from
wood residues, inedible vegetable oil, non-food crops, and crop byproducts
such as wheat straw, corn straw, sugarcane bagasse [21]. In the 3G biofuels,
the biomass of organisms is used as raw material, such as microalgae and
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seaweed [22, 23]. Currently, industrial global production is led by 1G biofuels
from traditional biomass, and it has been estimated that this technology will
still be responsible for the development of biofuels for years to come. For
example, bioethanol production is expected to use 14% and 24% of global
corn and sugarcane production by 2028 [24].
In 2018, about 14% of the energy in the world came from renewable
sources, and the other 86% corresponds to non-renewable energy, including
coal, crude oil, natural gas [25]. From whole renewable energy, approximately
3.7% was directed to the transport sector [26]. Thus, biofuels contribute only
with a small part of the energy supply in transportation, but their use has
been increasing in parallel with the growth of the vehicle fleet [4, 27].
In 2019, the world production of biofuels was approximately 154.5 billion
liters, and it is expected to increase by 25% during the 2019–2024 period,
reaching about 190 billion liters of biofuels [28]. USA and Brazil were the
largest producers, generating around 66.8 and 37 billion liters, respectively
[25, 29]. Among the biofuels available in top producer countries for trans-
portation, here will be highlighted biogas, biodiesel, and bioethanol [4].
Biogas corresponds to a mixture of combustible gases that can be produced
by AD of manure, farm wastes, wastewater, and other residues [30–32]. It
is used for the domestic generation of energy/heat, but also can be used as
a motor fuel in traditional vehicle designs [33, 34]. Europe, Asia, America,
and Oceania are the main biogas producers, as shown in Figure 17.1 [35].
This biofuel is mostly used in energy conversion into electricity, but its use
in vehicles for transportation still has limitations [32, 36]. Although, when
biogas is upgraded to biomethane, its use as a transport fuel in vehicle engines
for natural gas has been successful. Due to the low emission of toxic gases,
the use of biomethane in vehicles is considered one of the best renewable fuel
options in transportation [36, 37].
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Nowadays, biomethane is a common transportation fuel in gas filling

stations in Europe, especially Germany and Sweden, either as 100% methane
(CBG100) or blended with natural gas CBGXX (where XX is the percentage
by volume of biomethane present in the mixture, ex. CBG10 and CBG50)
[38, 39]. In Sweden, it is estimated that more than 4,000 light vehicles and
fleets of public transport busses are moved by biomethane [32, 40]. On the
other hand, biodiesel and bioethanol are the biofuels most used in transport
vehicles in other countries. Biodiesel is a fuel formed by mono-alkyl esters of
long-chain fatty acids, produced from vegetable oils [41]. It can be used pure
(B100) or in diesel fuel mixtures, following the requirements of standards
allowed by the policies of each country (e.g., ASTM D6751) [5, 32, 42].
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USA and Brazil are one of the main producing countries of biodiesel
in the world. In 2019, these countries produced around 6.5 and 5.9 billion
liters of biodiesel, behind Indonesia, responsible for the production of 7.9
billion liters [43] (Figure 17.1). Currently, the biodiesel industry in the USA
is the fastest-growing in the world, and it is expected to obtain favorable
investments and taxes in the coming years to encourage its expansion [33].
This biofuel is also widely used in the transport sector by the European
Union (EU), which in 2015 used approximately 12 billion liters [37, 44].
Among biofuels, bioethanol is the most promising to be used in the
worldwide transport sector in the short and medium-term (approximately
66% of the bioethanol produced in the world is used for transportation) [45,
46]. Bioethanol of first-generation is produced by a fermentative process of
feedstocks such as sugarcane, present in tropical and subtropical areas such
as in Brazil, India, and Colombia, or from corn, wheat, and tubers as occurs
in the USA, EU, and China [33, 47–50]. The bioethanol obtained from
sugarcane has approximately 70% lower GHG emissions than fuels formed
by conventional hydrocarbons, considering the life cycle balance [46].
In 2019, the USA produced the highest quantity of bioethanol in the
world, generating 59.73 billion of liters, followed by Brazil (32.48 billion
liters), EU (5.45 billion liters), China (3.41 billion liters), Canada (1.89
billion liters) (Figure 17.1) [51]. Although the USA is the largest producer
of bioethanol in the world, it still uses gasoline as its main transport fuel.
In 2019, petroleum fuels such as gasoline and diesel accounted for about
91% of the total energy use of the transportation sector in the USA [52]. In
this country, it is common to find a mixture of gasoline with 10% and 15%
bioethanol (E10–E15), the exact amount added varying by region [49, 53].
In this context, the current way of using biofuels is as additives for
gasoline and diesel, commonly without requirements of modification in the
vehicle engines. In its composition, bioethanol contains oxygen and using
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it as an additive to gasoline turns possible to reduce gas emissions by 25%

compared to the use of pure gasoline, being beneficial for the environment
[54, 55]. Recent research of Liu et al. [56], used life cycle assessment
(LCA) tools to investigate three new LCB refinery systems using bioethanol
mixtures E10 and E85, compared with fossil gasoline. Fuels combined with
bioethanol performed better compared to gasoline, in terms of depletion of
fossil fuels, with E10 and E85 were 6% and 64% to 70% lower, respectively.
To increase the use of bioethanol as an automotive biofuel, industries have
been working on the development of vehicles with a Flex system. In Brazil,
since 2003, automobiles with Flex engines have been sold, which have a
dual supply system for fuel, bioethanol, and/or gasoline. Those vehicles are
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capable of using 100% bioethanol (E100), or gasoline, or any mixing rate
between them [29, 57]. Vehicles with this technology have become widely
used in Brazil, where about 95% of new cars sold have a Flex system [46, 58].
Although biofuels have been produced in many countries, the rate of
their adoption is still low. According to information from the International
Sustainable Development Strategy Group, in order to reduce the emission of
GHGs and fight against climate change, it is necessary to have sustainable
growth in the production of biofuel of 10% per year until 2030. Thus, political
support and innovation are needed to favor the use of available feedstocks,
to reduce industrial production costs, and to increase the advanced consump-
tion of biofuels [2, 189]. The current environmental impact caused by the
use of fossil fuels and the production of biofuels for the near future will be
discussed below.
17.3 PERSPECTIVES IN BIOFUELS PRODUCTION: CURRENT

ENVIRONMENTAL IMPACT, PROJECTIONS, AND SUSTAINABILITY
The strong economic development is the direct reason for the increasing use
of fossil fuels, which causes the increase in global warming (GW) of the
planet and the harmful effects of natural destruction, such as the accelerated
melting of glaciers, floods, and long-term rains [34, 59, 60]. In addition, the
concentration of CO2 in the Earth’s atmosphere has grown 1.48 times from
280 ppm in 1,750 to 415 ppm in 2019, representing an increase of 0.9% per
year [34, 61].
To decarbonize the energy sector, several strategies are under development.
One of the established strategies is the utilization of renewable energy, which
production is expected to increase by 24% in the next decade [62]. However,
the dynamics of production of renewable energy and CO2 emissions in the
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FIGURE 17.1 Global distribution of the main biofuels.
Source: Biodiesel and biogas production data taken from the Statista database (production of biogas worldwide in 2017, by region); (leading
biodiesel producers worldwide in 2019, by country (in billion liters); [186].
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world may vary according to economic, political, or social global events [63].
For example, the CO2 emissions slowdown in 2019 was of 0.5%, but it would
need to be analyzed in the context of the strong increase in carbon emissions
(2.1%) due to the energy production boost in 2018 led especially by Russia,
India, and USA [64]. Another issue that can be considered when analyzing
future trends would be the pandemic COVID-19 crisis, which occurred in
2020. This health crisis brought momentary environmental benefits, such
as the drastic decrease of global carbon emissions (expected to be 8%), but
also challenges as a decrease in global investment and energy demand (6%).
These factors together with economic issues, such as land distribution and
higher food prices, can also influence the production of biofuels [65, 66]. It
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has been estimated that in the first period of 2020, global use of renewable
energy and electricity increased about 1.5% and 2%, respectively, compared
to the same period in 2019. However, for the first time in decades, it is
expected to decrease during this year [67].
It is important to highlight that modern renewable energy share has
increased only around 10% of the final global consumption of energy for
the last 10 years [3]. Thus, to achieve our global goals for the use of cleaner
energies and environmental improvement, the efforts must still focus, more
than ever, to improve the production and use of renewable energies as biofuels.
17.3.1 CURRENT BIOFUELS PRODUCTION AND FUTURE TRENDS
In the world, the biggest contributors to global energy growth in 2019 were
China, followed by Indonesia and India. China led the expansion of growth
and energy consumption, accounted for about 75% of total growth, and
achieved a greater increase in demand for each energy source [64]. Recent
reports show that with the growth of renewable energies in the world, fossil
fuel consumption fell for the first time to its lowest level. At the same time,
an increase of more than 60% in the global production of biofuels has been
observed since 2010, with a forecast to reach a consumption of 298 million
tons of oil equivalent (Mtoe) by 2030 [2, 3, 64]. Countries with the highest
production of biofuels are expected to double or triple their consumption of
biofuels in the next 10 years compared to their current production [2].
Biofuels such as bioethanol, biodiesel, and others (biomethane and
biohydrogen) are the most promising renewable energies to be produced on
a large scale and for being applied in the transport sector. For this reason,
countries have been increasing their investments to improve the production
of these biofuels to address the economically, environmentally, and socially
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worldwide perspectives [30, 68]. Advantageously, conditions and feedstocks

for their production can be adapted according to local characteristics like
environmental parameters and available biomass [3, 30, 69].
In the first place, bioethanol with a production of 120,000 million liters
is estimated to increase more than 1% during the next decade [68]. As seen
before, USA and Brazil account for about 90% of bioethanol production
and approximately 85% of the global production of liquid biofuels [33, 69].
Both countries have policies that have encouraged the production and use
of bioethanol for decades. Many of these initiatives motivated other devel-
oping countries of America like Mexico, Colombia, and Argentina, and also
Asia, to introduce smaller scale bioethanol programs that are so beneficial
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and necessary to achieve the international goals of sustainability [49]. As a
consequence, Argentina (forecasted total production growth of more than
46% by 2030) and other Asian countries (forecasted total production growth
between 19% and 50% by 2030) become large bioethanol producers. Nowa-
days, developed countries produce 58% of bioethanol in the world, and they
could increase their production by 6% until 2028 [68]. Other important top
producers are China (actual production of 10 billion liters), India (actual
production of 2 billion liters), and the EU (actual production of almost 7
billion liters), as is shown in Table 17.1.
Another liquid biofuel with positive impacts on the environment is
biodiesel. Nowadays, developed countries are responsible for 57% of the
world’s production of biodiesel, and it is expected by 2028 their produc-
tion should increase by 9% [68, 69]. Table 17.1 shows actualized data of
production and use of biodiesel around the world, having as greater producer
USA, followed by the EU. In addition, it was calculated that global biodiesel
production and consumption would increase by around 2% until 2030 [68, 69].
However, some of the developed countries do not produce and/or commer-
cialize biofuels, thus they do not appear in future production statistics.
Additionally, reports show that biogas is another potential biofuel, for
example, Dos Santos et al. [189], assessed energy potential and reduced
emissions by applying biogas energy formed from the biodigestion of
different organic wastes. It was found that potential power was between 4.5
and 6.9 GW, which would reduce CO2 emissions at a rate of 4.93% per year.
It is worth to note there are some specifications for each biogas applica-
tion or use. For example, for heat and electricity production usage biogas
requires the removal of water vapor and H2S. Its use in transportation, on
the other hand, requires an upgrading process that consists of impurities
removal (mainly CO2), conserving high energy content to reach each coun-
try’s quality standards [39, 70–72]. Fuels produced from biogas, such as
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TABLE 17.1
Bioethanol and Biodiesel Projections: Production, Use, Price, and Emission Saving
Bioethanol Biodiesel
Pa (ML) Pa (ML) EPa (ML) EPG (%) AGa (%) EGF (%) Pa (ML) Pa (ML) EPa EPG (%) AGa (%) RG (%)
(ML)
2018 2019 2028 2018–2028 2019–2020 2018–2028 2018 2019 2028 2018–2028 2019–2020 2018–2019
b
United States 60,911 59,718 62,062 1.8 1.85 4.7 7,700 6,500 8,693 12.90 –1.85 –4.8b
Brazil 32,085 30,432 37,155 15.8 1.52 16.1c 5,400 5,358 5,916 9.56 1.10 8.5c
d
China 10,000 10,185 14,773 47.7 0.85 142.6 1,140 1,153 1,279 12.19 1.13 4.8d
e
European Union 6,860 6,822 7,406 7.9 –1.88 –1.62 13,522 13,605 12,871 –4.81 –0.73 3.0e
India 2,637 2,637 3,132 18.7 –3.41 –0.31f 184 199 238 29.35 2.15 2.4f
g
Canada 2,030 2,026 2,047 0.8 –1.05 –5.67 550 558 611 11.09 1.10 5.2g
Thailand 1,923 2,000 2,810 46.1 –14.03 60.61h 1,490 1,517 2,320 55.70 4.84 12.8h
i
Argentina 1,235 1,206 1,802 45.9 –0.36 46.05 2,450 2,505 2,768 12.98 1.40 12.82i
Challenges and Perspectives on Application of Biofuels
Global Total 127,584 126,302 143,111 12.1 –0.51 – 40,251 43,131 43,931 9.14 0.10 –
Global Consumption 125,864 127,250 143,190 13.7 – – 39,927 43,556 44,227 10.77 – –
World1 Emission – – 2.3j – – – – – 1.7j – – –
Saving (%)
Word Price (US Dollar 39.41 40.93 52.63 – – – 89.40 87.31 94.68 – – –
per Hectoliter)a
Overview of the annual and expected production, consumption, and price projections of bioethanol and biodiesel between the period 2018–2028,
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for larger producers and global (P: production, EP: expected production, EPG: expected production growth, AG: annual growth, EGF: expected
growth in use as fuel, RG: on-road use growth).
Note: 1. Provides a Well-to-Wheel (WTW) emission savings using the baseline of the 2018 OECD-FAO Agricultural Outlook for the period
2015–2017 and by 2030 for major biofuel consuming countries.
Sources: Refs. [69, 75–83].
471
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liquefied biogas (LBG) and compressed natural gas (Bio-CNG) have been
commercially produced via biogas liquefaction and compression, respec-
tively [73]. Currently, there is a global production of around 3.5 Mtoe of
biomethane (obtained from biogas upgrading). Actually, biomethane repre-
sents about 0.1% of natural gas demand [74].

Regardless of biogas has been produced, consumed, and applied in
several fields, its use in transportation is not well-established. However, IEA
[74] forecasted that power and heat usage of biogas is going to increase from
70 to 85% between 2018–2040.
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17.3.2 SUSTAINABILITY AND ENVIRONMENTAL BENEFITS OF
BIOFUELS
Even with government efforts for the constant increase of biofuels produc-
tion and promising future trends, there still are several challenges to be faced
to achieve the Sustainable Development Scenario (a major transformation
of the global energy systems, which is derived or aligned from other energy
scenarios). According to IRENA and IEA, the increase of approximately 3%
in the use of biofuels and renewables energies per year is a goal that would
allow environmental and energy sustainability in the next decades [2, 3]. For
the Sustainable Development Scenario, renewable energy share in the final
energy supply would increase by 17% by 2030 and 25% by 2050. There is
also a Transforming Energy Scenario that would bring sustainability in a
shorter time-lapse and reduction of total carbon emissions. To achieve this
goal, it would need an increase of six-fold and almost twice the actual trends
to achieve 28% of the renewable energy share in the final energy supply by
2030 and 66% by 2050 [3].
In the context of the Sustainable Development Scenario and taking
as an example the largest producers of biofuels and their forecast annual
production growth during the next decade, all these countries need at least
duplicate their predicted growth to achieve sustainable goals. For example,
in the case of USA, its actual production is of 35 Mtoe of biofuels represents
the 5% of its transportation energy sources [53], and have to increase to 95
Mtoe to get in the Sustainable Development Scenario, which means, this
country has to get its forecasted annual production of 1.9% to 7% of annual
production growth every year during the next decade. Other countries as
China and India would need to increase their actual production 8-fold to
fulfill the expected sustainable consumption of biofuels by 2030. According
to statistics, the EU would be the top producer with the hardest challenge, it
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would have to increase 18-fold the annual growth of production to achieve a

sustainable scenario (Table 17.2) [2].
TABLE 17.2 Expected Biofuel Production Growth to Achieve Sustainable Development

Scenario by 2030
Biofuel Production SDS Biofuel Forecast Annual SDS Annual
2019 (Mtoe) Consumption Production Production
(Mtoe) Growth (%) Growth (%)
United States 35 95 1.9 7
Brazil 22 37 1.7 5
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China 3 26 15.3 19
European 16 51 0.5 9
India 1 8 11.8 22
Source: International Energy Agency (IEA) [2]; Transport biofuels, IEA, Paris.
Another important goal involved in sustainability is the mitigation of

GHG emissions. In this context, the transportation sector is responsible
for a large portion of the global use of fossil fuels, which contributes to a
quarter of global CO2 emissions producing 10 Gton/year of CO2. Moreover,
the number of cars on the road (4.6 ton CO2/year/car) and the kilometers
flown by airplanes (918 Mton in 2018 just for commercial operations) will
be close to doubling by 2040 [84–87]. Hence, this sector will need to reduce
50% of its CO2 emissions by the year 2050 to acquire the Planned Energy
Scenario (perspective based on countries’ current energy plans under the
Paris Agreement) or 75% for the Transformation Scenario (perspective
based on the renewable energy to keep GW below 1.5–2.0°C this century).
In both scenarios, the production and use of renewable sources represent
around 50% of the mandatory improvement [3, 190].
Forecasts from IEA [74] anticipate that clean energy technology invest-
ment would grow to 40% of total investment, due to the great decrease in
fossil fuel investment. This scenario would mean an important increment
since it has been stagnant at 33% in recent years. In this context, a transi-
tion to more electrified forms of transport and heat, together with greater
production of renewable energy as biofuels, would reduce 60% of the CO2
emissions related to the energy needed by 2050 [3].
Based on the actual panorama for getting the sustainable goals, there are
some other limitations on the biofuels supply chain, that involves political,
economic, and social activities, besides, to biomass logistic and technological
operations.
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17.4 CHALLENGES IN THE APPLICATION OF BIOFUELS IN

TRANSPORT SECTOR
17.4.1 POLICIES FOR BIOFUELS AROUND THE WORLD

Due to environmental, social, or economic issues, many nations started to

create political strategies to reduce dependence on petroleum and increase
the share of biofuels in the transport sector [88]. Regulations and guidelines
are created to encourage producers and manufacturers to develop this
form of sustainable energy. These documents set goals on the quantities of
biofuels that must be produced, the levels of GHG emissions, in addition to
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the restriction of biomass cultivation in areas intended for food planting [89].
Policies involving the production and use of biofuels have been under
development for many years in different countries. For example, Brazil
in 1975, developed the national fuel alcohol program (ProÁlcool), which
aimed the production of bioethanol from sugarcane, in response to the
oil crisis [90], while the National Biodiesel Production and Use Program
(PNPB) was launched in 2004 [191]. The development of these programs led
to the creation of the RenovaBio program, which integrated the production
of the most used biofuels (bioethanol, biodiesel, biogas), aiming at the
decarbonization of the Brazilian energy matrix and creating methodologies
that promote the stability of biofuels prices [91, 92].
China was another country that developed important programs for
biofuels production and application. In 1986, the 863 plan was created with
the National High Technology Research, which aimed to develop bioethanol
production from sweet potatoes and industrialize the biomass liquid fuel [8,
93, 94, 188]. Later in 2005, the Bioethanol Use Plan and the Renewable
Energy Law were created, which focused on the importance of biofuel,
promoting resources, and tax subsidies for its production [95, 96]. As part
of the bioethanol implementation plan, China has adopted different national
standards (GB 18350 and GB 18351) related to the production and use of
biofuels. A GB 18351 standard allows a mixture of 10% (E10) of ethanol with
gasoline [97]. In 2018, this mixture was already available in six provinces,
such as Liaoning, Heilongjiang, Henan, Jilin, Guangxi, and Anhui [98].
In 2006, the Ministry of Finance of China created financial support policies
for biofuels that encourage the development of biodiesel. These policies
provided subsidies for biodiesel plants that used non-food raw materials
[99–101]. The government strictly prohibited the use of used oil as cooking
oil, but illegal use persists due to the high profit [98]. The purchase price of
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biodiesel based on residual oil is lower when compared to biodiesel based

on cooking oil. In 2010, the development program started using B5 with help
from China National Offshore Oil Corporation (CNOOC). However, there
is no mandatory blending requirement for biodiesel in China [99, 102, 103].
USA government has already elaborated several political projects

involving the production of different biofuels. The Clean Air Act was the
first and the most influential environmental law to regulate air quality, estab-
lished in 1963. From this policy, others such as the US Energy Independence
and Security Law of 2007 and the Biomass Program in 2008 were created
targeting to reduce gasoline consumption to 70% by 2030. As consequence,
biofuels emerged as a potential alternative for transportation [92]. Currently,
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there is the standard renewable fuel program (RFS), which was established
by the energy policy act (EPA) in 2005, to increase the volume of renewable
fuel that is mixed with transport fuels. In 2007, this program was revised to
meet the requirements of the energy independence and security act (EISA),
that established specific targets for increase the production of cellulosic
biofuels, biomass diesel, and advanced biofuel in increasing volumes until
2022 [55, 65, 192].
In European Union (EU) countries, policies to encourage biofuels
application have evolved over the years. In 2008, was passed in the EU a
legislation that allowed the use of biofuels in the transport sector. One year
later, in 2009, as part of its “Climate Change Package,” the EU adopted
the Renewable Energy Directive (DRE 2009/28/EC). In this directive, each
Member State must achieve a minimum usage quota of 20% of energy from
renewable sources, reduce GHG emissions by 20% and increase energy
efficiency by 20% [104, 193]. In 2018, an agreement on the new Renewable
Energy Directive (REDII) was determined by the Council of the EU, which
will be in force between 2021 and 2030. This new directive includes the use
of advanced biofuels, and estimates consumption of total renewable energy
in the European energy matrix in 2030 should be 32% and in the transport
sector 14% [105, 106].
As seen above with the top biofuel producer countries, global govern-
ments have had initiatives to enhance the development of the biofuels
market. Among the most important initiatives created are:
1. Global Bioenergy Partnership (GBEP): Intergovernmental initia-
tive, with more than 70 members, including members of civil society,
government, and the private sector. It aims to support “the deploy-
ment of biomass and biofuels, particularly in developing countries
where the use of biomass is predominant” [107].
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2. Biofuture Platform (BFP): An initiative that supports global actions

to combat climate change, fostering solutions in low carbon transport
and the bioeconomy [108].
3. Innovation Mission-Challenging Innovation in Sustainable Bio-
fuels (SBIC): A global initiative with the objective of promoting the

drastic acceleration of innovation in clean energy, which includes
23 countries. Through this initiative, participating countries pledged
to double their investments in research and development of clean
energy by the government over five years (2015–2020).
4. Low-Carbon Technology Partnerships Initiative (LCTPi) – Below
50: The main objective is to accelerate the development and diffusion
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of low carbon technologies. New fuel production technologies are
starting to be deployed at new production facilities around the world.
All these projects aim to intensify the use of clean energy in the form of
biofuels; however, other limitations involve the economic barriers between
fossil and renewable fuels.
17.4.2 ECONOMIC CONSIDERATIONS
Even with fluctuations in the price of oil, biofuels still cost more than conven-
tional fossil fuels. The productive process of biofuels is the main barrier to
making these technologies economically viable. For this purpose, it is required
public support to encourage feedstock supply, cultivation of biomass, and
reduce the capital cost of biofuel processing [109, 110]. Moreover, the cost of
biofuel production depends mainly of process involved, industrial logistics,
obtaining biomass, storage, and distribution of the final product [111–114].
In the case of 1G and 2G biofuels the most important economic
consideration is related to the dependence on biomass [10]. The share in
cost of raw materials is 40% to 70% of the total production costs. Like
most of biofuels available, 1G biofuels depend on obtaining biomass,
involved in the food industries, which account for more than two-thirds
of total production costs [10]. Meanwhile, costs of 2G biofuels are also
dependent on additional steps (pretreatments) for the biomass involved
in the production processes [115]. For example, in the production of 2G
bioethanol, the highest costs are observed in the biomass conversion
stage [116, 193]. This step involves the use of enzymes for the hydrolysis
process, and these are responsible for about 20% to 40% of the total cost
of one liter of second-generation bioethanol [194].
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When comparing the total cost of liquid biofuels (bioethanol and

biodiesel) with fossil fuels cost, as is shown in Figure 17.2, it is observed
that biofuels demonstrated higher cost. However, USA, and Brazil have been
the top producer countries with the lower costs during the last years, because
their production is at an advanced stage (from the processing of the biomass

involved until the industrial infrastructure) [7, 8].
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FIGURE 17.2 Comparative cost analysis of main biofuels vs fossil fuels.
Source: [114, 117–121, 195].
In this context, all processes related to biomass have an important impact

on the cost of the biofuel supply chain that can vary according to regional
availability, especially for those regions with the need of importation [2, 190].
17.4.3 BIOMASS FEEDSTOCK AVAILABILITY FOR BIOFUELS

PRODUCTION
Many of the political projects developed to date encourage the production and
increase availability of biofuels in society. Research carried out by special-
ized agencies, such as the International Energy Agency, shows that in 2030,
biofuels will contribute approximately 4% to 10% of the total energy involved
in the planet’s road transport. Thus, it will be necessary to use between 3.8%
and 4.5% of the arable land available worldwide for the cultivation of crops
directed to the production of biofuels, against only 1% of the total used [122].
According to the Global Renewable Energy Roadmap [30], the use
of biomass in several sectors in the world grows approximately 3.7% per
year between 2010 and 2030. It is estimated that in 2030 a total of 108 EJ
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(Energy Joules) of biomass will be harvested, of which 31 EJ (28% of the

total biomass) will be used to obtain liquid biofuels. From the total amount
of available biomass, is expected a global production of approximately 650
billion liters of liquid biofuels per year until 2030 [30].
The use of biomass to produce first-generation biofuels has certain

limitations due to its concomitant use as feed, causing great impacts on food
prices [10]. In addition, the increase in demand of these feedstocks generates
deforestation of natural reserves [123, 124]. These limitations have opened
opportunity for the development of other generation biofuel technologies. It
is estimated that by 2050, approximately 2150 Mtoe of agro-industrial waste,
955 Mtoe of forest materials, 2388 Mtoe of grains, 1194 Mtoe of waste, and
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478 Mtoe of algae will be available worldwide, favoring the production of
2G and 3G biofuels [125, 196].
To get the expected demand for biofuel production in 2028, it will be
necessary to increase the availability of feedstocks involved in the production
process (Table 17.3). For example, in USA to obtain 59.72 ML of bioethanol
in 2019, about 374.02 kton of maize were used and to achieved the forecasted
amount for 2028, 388.70 kton will be required. Thus, countries that produce
and use biofuels must take action to ensure biomass availability, such as the
S2Biom Study, a project carried out in the EU that aims to provide sustainable
supplies (non-food biomass) to support the development of the bioeconomy [6].
Lignocellulosic biomass (LCB) stands out among the several available
feedstocks for the production of biofuels. This is an abundant organic resource
in many countries around the world, with the produce of an estimated 181.5
Gton annually [126–129]. Currently, about 8.2 Gton are used, of which
approximately 7 Gton of biomass are obtained from agricultural land, grass,
and forest, and 1.2 Gton are derived from agricultural waste [128]. During
2018/2019, Brazilian mills harvested around 620 Mton of sugarcane, gener-
ating about 180 Mton of wet bagasse and 43 Mton of cane straw [6, 197].
LCB represents a suitable low cost and eco-friendly alternative for the
production of biofuels and byproducts, however some aspects make difficult
the conversion of this material at large-scale that corresponds to its recalci-
trance, heterogeneity, and technical problems in its bioprocessing [130].
17.4.4 TECHNOLOGICAL CHALLENGES FOR LCB CONVERSION TO

BIOFUELS: BIOGAS AND BIODIESEL
Bioprocessing of feedstocks is an early step of the biofuel production process

and an important stage for the biorefinery value chain. The improvement of
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TABLE 17.3 Feedstock Used for Bioethanol and Biodiesel Production: Production, Use, and Projections
Bioethanol Biodiesel
Biomass Production Expected Production Biomass Production Expected Production
Used as (Kton) Use as Estimate Used as (Vegetable Use as Required
Feedstock 2016–2019 Biofuel for Biofuel Feedstock Oil-Kton) Biofuel for Biofuel
Feedstock Production 2016–2019 Feedstock Production
2030 (%) (2028) 2030 (%) (2028)
United States Maize 374,019 10 388.699 Soybean oil 13,432 25 11.915
Brazil SugarCane 33,891 85 41,378 Soybean oil 9,312 40 10.282
China Maize 260,005 8 377.128 Waste oils 28,254 – –
European Union Maize 63,635 8 69,082 Rapeseed oil 14,474 40 13.693
Challenges and Perspectives on Application of Biofuels
a
Thailand Sugarcane 28,600 68 40,183 Palm oil 3,410 50 5.215
Argentina Maize 49,157 9 73,450 Soybean oil 9,289 70 10.264
Global Consumption – 121.9 mln.L – – – 37.6 mln.L – –
as Biofuel Feedstock
Overview of the biomass used as feedstocks and production required of biomass for production of bioethanol and biodiesel between the period
2018–2028.
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Source: Refs. [69, 131]; OECD‑FAO Agricultural Outlook 2019–2028.
479
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this key stage would mean a future large-scale application of biofuels in the
transport sector since it impacts in the global cost of the process. Furthermore,
other benefits would be market competition with fossil fuels, enhancement
of technology for energy transition, and reduction of negative environmental
impacts. Even thought, biorefineries still face technical/process challenges

for biomass (LCB) conversion and utilization. The technical challenges
involved in the use of these biomass as feedstocks to produce biofuels will
be discussed in this section.
LCB can be used for biogas and biodiesel production through different
processes. In the case of biogas, anaerobic digestion (AD) is the core process
where occur all syntrophic mechanisms for biomass conversion. However,
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the high carbon and nitrogen ratio (C:N) of LCB negatively affects methane
production, and the mono-digestion of this substrate is not an adequate
strategy [126, 127, 129]. There are several methods to improve the use of
lignocellulosic feedstock as substrate in the AD process, such as anaerobic
co-digestion, bioaugmentation, and nutrient supplementation established
to optimize the C:N to 20–30:1 [132]. For example, Kainthola et al. [133]
evaluated the methane production of the anaerobic co-digestion of rice straw
and food waste. It was shown that methane yield increased around 94%
with respect to control in the optimized process (C:N 30, pH 7.32, food/
microorganism ratio 1.87).
In biodiesel production, LCB can be hydrolyzed and used for lipids
synthesis by oleaginous yeast, such as Meyerozyma guilliermondii and
Pichia kudriavzevii or filamentous fungus-like Mortierella isabellina for
subsequent obtaining of biodiesel [19, 134, 135]. Those microorganisms
could accumulate up to 60–80% lipids of their cell dry weight and can grow
exponentially while utilizing cheap substrates, becoming suitable candi-
dates for biotechnological purposes [136, 137]. The use of cheap carbon
sources like crop byproducts, significantly diminishes the cost of yeast oil
production [109].
Due to the complexity of the lignocellulosic matrix, the crystallinity of the
cellulose and the inhibition of metabolic operations by the lignin by-products,
other strategies have been established to improve the use of LCB in the
production of biofuels. Thus, pretreatments aim to modify the structural and
compositional barriers of this biomass, in order to improve digestibility by
enzymes to provide fermentable sugars for microorganisms [132, 138–140].
LCB pretreatment methods are widely known and can be classified in
physical, chemical, physicochemical, and biological processes, and can be
used alone or in combination.
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The most common physical pretreatments are mechanical. One advantage

of their use is the increase of the biochemical potential for biofuel production.
For example, when pretreatments, such as roll milling, extrusion, pelletiza-
tion, and hammer milling were applied to wheat straw, it was observed an
increment in the maximum daily methane production and enzymatic hydro-

lysis yield related to the amount of released glucan content [141]. In another
research, higher methane production was obtained from rice straw in AD as
the result of particle size reduction, which improves the cellulose degrada-
tion rate [142, 143]. In this context, the most common mechanical method
to increase the surface area and reduce the crystallinity of LCB is milling.
The particle size is an important parameter that could lead to supplementary
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costs in the overall process of bioenergy production from lignocellulose. For
example, Moiceanu et al. [144] used Miscanthus biomass in a 10 mm orifice
sieve, which means energy consumption of 50–67 kJ/kg, that led to the
increase of processing costs [144]. In addition, the energy consumption and
susceptibility of milling machines in the presence of large materials such as
stones and metals mixed with LCB, are some inconveniences [145]. Milling
is also a currently used pretreatment on biodiesel production, but its purpose
is centered on the release of lipids from marine algae species. A study with
Chlorella vulgaris shows that from 208 mg/L of biomass could be obtained
a lipid release of 10% when using bed milling [146].
On the other hand, chemical pretreatments like acid, alkaline, oxidative,
and organo-solvent processes have also been extensively studied for
pretreatment of LCB. Acid pretreatment causes the rupture of chemical bonds
present in the biomass matrix, mainly removing the hemicellulose fraction
[132]. One example of this pretreatment was shown by Taherdanak et al.
[147]. In this study was observed the decrease of the crystallinity index of the
wheat biomass accompanied by the solubilization of surface layers’ structure.
Moreover, Amnuaycheewa et al. [148] showed that biogas production and
saccharification from rice straw can be enhanced by pretreatment with organic
acids like oxalic and citric. Also, Huang et al. [149] used this pretreatment
to obtain sulfuric acid-treated rice straw hydrolysate (SARSH) for microbial
(Trichosporon fermentans) oil synthesis with potential for the production of
biodiesel. During fermentation using SARSH without detoxifying, 1.7 g/L
of lipid was obtained, a low yield compared to that obtained by fermentation
with detoxified hydrolyzate, generating a 40.1% lipid content. Despite of the
acid process is an efficient method for LCB, the main disadvantages are the
high cost of acids, drastic operation conditions (pressure and temperature),
and the formation of potential biological inhibitors [127, 150].
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Looking to face the limitations of the most used pretreatments, researchers

have found that chemical pretreatments assisted by alternative technologies
are a promising way to unlock technological bioprocessing challenges.
Unlike acid pretreatment, alkaline pretreatment utilizes an alkali solution
capable of promoting the solubilization of lignin present in biomass [127, 129].

As mentioned above, this pretreatment can be combined with the hydrogen
peroxide pretreatment [151, 152], ultrasound [153], and hydrodynamic cavi-
tation (HC) [154–157]. HC technology is a new and promising option for
the pretreatment of LCB. Cavitation is the phenomenon of the formation of
vaporous microbubbles or cavities growing and collapsing in a flowing liquid
[156–158]. These collapsing cavities generate localized hot spots with very
Author Copy
high temperatures and pressures in addition to intense shear [159]. Conse-
quently, highly reactive oxygen species (ROS) named hydroxyl radicals (OH)
are formed which produce oxidative degradation of LCB, turning more suit-
able for subsequent enzyme action and for microbial metabolization, which
could result in higher yields of biofuels [158, 160–163].
17.4.5 CHALLENGES FOR BIOETHANOL TECHNOLOGIES:

1st, 2nd, AND 3rd GENERATION
With the implementation of first, second, and third-generation bioethanol

production, also appeared some biotechnical and economic challenges to
make these technologies viable, aiming to enhance their use in the transport
sector. In this topic, some issues that companies have been facing on a large-
scale bioethanol production will be discussed (Figure 17.3).
17.4.5.1 FIRST-GENERATION BIOETHANOL
Despite the first-generation bioethanol is a bioprocess well implemented in

some countries such as USA, Brazil, and India, the industrial production
of bioethanol by this technology has some challenges to be overcome. The
main problems in 1G bioethanol production are the following:
1. Climatic Problems for Crops: With changes in climate dynamics in
recent years, energy crops have been showing deficits due to drought.
In addition to harming energy crops, this factor also interferes with
the food and biofuel production chain related to them [164]. For
example, in 2019, the severe drought that hit central-south Brazil for
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more than four months lit the warning signal for the 2020 sugarcane
harvest. Sugarcane has an annual cycle and, after its harvest, depends
on a regular water regime for the full development of the plant until
the next harvest [165]. As a consequence, this fact affected feedstock
availability and led to a reduction of bioethanol production and an

increase in price level.
FIGURE 17.3 Bottlenecks for bioethanol production. Author Copy

2. Environmental Impacts Caused by Agricultural and Industrial
Bioethanol Production: Bioethanol production can also cause soil,
water, and air pollution, a fact unknown to most of the population. The
main environmental impacts encountered by agricultural activities are
soil compaction by tractors and cultivation implements, contamina-
tion of water and soils by intensive use of fertilizers and pesticides,
uncontrolled application of vinasse (as fertilizer), filter cake residues,
and air pollution resulting from burning straw and other wastes.
Furthermore, during industrial processes, electricity is generated from
the burning of bagasse, excess straw, and other residues. Electricity
generation also produce air and soil contamination by accumulation
of GHGs and fly ash. Besides, the inadequate disposal of feedstocks
residues and equipment washing water, condenser, and multi-jet and
domestic wastewater also cause water pollution [166].
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3. Devaluation of the Price of Oil: Besides, in some countries, low

government investment in the sector and the increase in taxes could
make the survival of some bioethanol plants even more difficult. If
government interventions in the fuel price table are not effective, the
competitiveness between gasoline and bioethanol may decrease, which

would cause losses to biofuel plants [167]. Besides, in some countries,
low government investment in the sector and the increase in taxes could
make the survival of some bioethanol plants even more difficult.
17.4.5.2 SECOND-GENERATION BIOETHANOL
Author Copy
The problems reported related to 2G bioethanol production are based on
global industrial experiences. Industries such as Raízen, Granbio, Poet, Beta
Renewables, Abengoa (Synata Bio), and DuPont have found difficulties in
the production phase regarding the pretreatment processes, microorganisms
used, even in the mechanics and equipment costs of the unit operations
involved in the process [168].
Substantially, the production of 2G bioethanol is based on the use of
lignocellulosic and starchy biomass, which main challenges are pretreat-
ment and hydrolysis processes that are magnified on an industrial scale.
According to specialists working in 2G bioethanol plants, none of the
pretreatments and hydrolytic processes available today are more advanta-
geous than the other, and each varies in the production process, depending
on the type of raw material and several other factors [168]. For these stages,
enzymatic processes are preferred since they do not form microbial inhibitor
compounds, such as furans and phenolics. However, the enzymatic cocktails
used can make the process more expensive, due to their prices and the high
enzymatic load required. Thus, it is essential to improve the efficiency of
enzymes for reducing the price of this biofuel [168]. As stated by Schubert
[169], cellulase cocktails cost around 15–20 cents per gallon bioethanol as
compared to only 2–4 cents per gallon bioethanol for amylases used in the
bioethanol production process by starch (1G bioethanol). The adoption of
each type of enzyme in the pretreatment and enzymatic hydrolysis stages is
totally dependent on the structure of feedstocks.
It is well documented that the high crystallinity of cellulose, high levels
of lignin, hemicellulose, acetyl group, and degree of polymerization (DP) are
undesirable characteristics of biomass that directly interfere in the perfor-
mance of different enzyme cocktails [19, 170–175]. In an attempt to solve
such problems, companies such as Novozymes and Genencor International
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have been studying and improving cellulose cocktails obtained by Tricho-

derma reesei and Cellulomonas fimi [173].
Recently, Santos et al. [176], modified the enzyme β-glycosidase, of
the family 1 of the glycoside hydrolases (GH1) expressed by Trichoderma
harzianum. With the site-directed mutation, the active site of the engineered
enzyme became smaller and similar in size to that of β-glycosidases GH1.
The results of the analyzes indicated that the modified enzyme showed a
catalytic efficiency 300% higher than that of wild protein and became more
tolerant of glucose, providing a significant increase in the release of sugar
from all sources of plant biomass tested and also increase of thermal stability
during fermentation. This engineered β-glycosidase can be used to supple-
Author Copy
ment the enzymatic cocktails commercialized today for the degradation of
biomass and the production of second-generation biofuels. Besides, this
study shows that this enzyme can be produced by a homologous expression,
which makes possible its production on a large scale and with low costs.
In the case of the fermentation stage, the main challenge is the selection
of an appropriate microorganism to achieve the expected yield of bioethanol
production on an industrial scale. There are microorganisms more efficient
in the metabolism of hexoses from cellulose, than in pentoses from hemicel-
lulose hydrolysis [177]. In this context, there are several studies to select
wild yeast and produce engineered microorganisms also capable of ferment
pentoses.
Furthermore, other important difficulties are operational and mechanical
processes. GranBio reported the corrosion of the equipment in one of its
plants that used the steam explosion (SE) pretreatment in which the LCB
was subjected to very high temperature and pressure conditions. When the
system was suddenly decompressed, the biomass hit the walls of the equip-
ment causing routine damage and possibly interrupting the production. The
solution to this problem was the use of the liquid hot water (LHW) process
associated with the mechanical treatment of the fibers [177].
Raízen plant shows other problems related to the high level of dirt in the
biomass, which required a pre-cleaning step. It was also necessary to redesign
the process for the separation of sugars and lignin, introducing more filters
and centrifuges. In this 2G bioethanol plant, the following problems were also
presented: (I) the corrosion of pretreatment equipment, due to the silica present
in the sugarcane straw, (II) the excessive use of water when working with wheat
straw as feedstock, (III) the high water absorption by the sugarcane bagasse,
making it a high viscosity material, clogging of pipes and valves [177].
Also, according to Lynd [178], the success of the industrial implementa-
tion of 2G bioethanol depends on investments in research and development.
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One of the errors noticed in current biorefineries is the excessive investment

in plant infrastructure, instead of concerning with supporting technological
advances capable of reducing production costs.
17.4.5.3 THIRD-GENERATION BIOETHANOL
As analyzed previously in 1G and 2G technologies, the third-generation

also presents bottlenecks. The main steps to 3G bioethanol production are
microalgae cultivation, cell collection, pretreatment of biomass, fermenta-
tion, and recovery of bioethanol by distillation [179]. Algal biomass could be
Author Copy
pretreated by mechanical or enzymatic methods to break cell walls, making
carbohydrates available. After this step, yeasts are added to convert ferment-
able sugars into bioethanol [180–182].
Some issues in the production of 3G bioethanol are reported as follows.
The selection of the microalgae strain is one of the most important
parameters. The carbohydrate composition of the cells must be considered
since the glycosidic portion acts as a substrate for the fermentation process.
As reported by Ueno [183], Chlorella vulgaris species is commonly used due
to its capacity of carbohydrate accumulation higher than 65%. According to
Kose and Oncel [184], the main parameters should be considerers for micro-
algae selection including (I) tolerance to oxygen gas levels, (II) resistance to
photo-inhibition, (III) high rates of cell growth, (IV) low need for nutrients
to develop, (V) high photosynthetic activity, (VI) easy adaptation to harsh
environmental conditions (for open systems), (VII) high survival rates, and
(VIII) genetic stability.
Temperature of microalgae cultivation is also an important factor that
influences the production of 3G bioethanol. As reported by Costa and De
Morais [180], it was observed that when Chlorella sp. was cultivated at
30°C, showed higher yields (448.0 μmol.g–1) of dry mass, in comparison
with cultures grown under 20°C (196.0 μmol.g–1). It is emphasized that the
drastic reduction in yield was also observed when the culture was performed
at 35°C and 45°C.
The microalgae cultivation mode also influences 3G bioethanol production.
Despite open lagoons are popular for being simple projects, contamination,
temperature control, sun exposure, and other factors, cannot be controlled,
highlighting the need for a careful selection of the bioreactor type [184].
Supply and physical and chemical characteristics of water are of funda-
mental importance for the seaweed cultivation during bioethanol 3G produc-
tion. A large volume of water is required to produce algae. Thus, this resource
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can be optimized using wastewater, brackish, and with high levels of organic
nutrients [184].
17.5 CONCLUSION AND FUTURE PERSPECTIVES
Biofuels are the main alternative to reduce environmental impact and cope
with the energy crisis, and their global production has significantly increased
in the last decade. In agreement with global sustainable goals, it is expected
to achieve a biofuels production growth by about 10% by 2030, using 28%
of available biomass as feedstock. Thus, it is extremely important to continue
Author Copy
investing and encouraging the development of technologies to make biofuels
an achievable option with a better cost-benefit for those countries without
oil reserves and refineries. Policies and programs, like RenovaBio in Brazil,
have been established to improve biofuel production and application in the
transportation sector. Consequently, biofuels developing countries could
support their future policies based on top producers’ experiences. Despite the
large number of biofuels manufactured in some countries, the actual industrial
production does not reach sustainable goals and still faces environmental,
economic, and technological challenges. At this moment, several strategies
for biofuel technologies need to be applied by the integration of different
biorefinery modes, such as 1G-1G integration (e.g., sugarcane-corn), 1.5G
(e.g., 1G-2G), and other possibilities based on 2G and 3G. In this context,
technological advances will be the core to boost biofuels to achieve a secure
transition from fossil fuels to an energy sustainable panorama.
KEYWORDS
• biofuels
• biomass conversion
• European union
• global warming
• greenhouse gas
• life cycle assessment
• technological challenges
• transport sector
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190. OECD/FAO (2020), OECD-FAO Agricultural Outlook 2020–2029, FAO, Rome/OECD
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CA8861EN.pdf.
191. Laursen, K. (2015). Revealed comparative advantage and the alternatives as measures of
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An Overview. Retrieved from: https://crsreports.congress.gov/product/pdf/R/R43325
193. Subramaniam, Y., Masron, T. A., & Azman, N. H. N. (2020). Biofuels, environmental
sustainability, and food security: A review of 51 countries. Energy Research & Social
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from: https://www.conab.gov.br (accessed on 8 December 2021).
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Author Copy
Index
β Agricultural wastes, 106, 402

Agro-industrial
β-glucans, 83
residues, 65, 67, 275
β-glucosidase, 5, 239, 249, 355, 356, 388,
waste, 7, 8, 29, 446, 478
391, 430
Alanine, 64, 103, 149
2 Alcohol (EtOH), 3, 6, 11, 16, 23, 24, 29, 30,
Author Copy
32, 33, 46, 47, 55, 57, 58, 60, 63, 69, 71,
2-keto-3-deoxy-6-phosphogluconic acid 85, 104, 113, 123, 142, 145–147, 155,
(KDPG), 28 162–165, 172, 181, 195, 196, 198–200,
210–212, 219, 272, 274, 275, 279, 286,
3 325, 339, 391, 422, 425, 428, 474
3-hydroxyanthranilic acid (HAA), 429 acetyltransferases, 63
dehydrogenase (ADH), 6, 16, 24, 32, 46,
5 47, 55, 57, 58, 60, 69, 71, 85, 104, 113,
5-hydroxymethylfurfural (HMF), 59, 104 123, 155
ADH II (ADH2), 55, 57, 59, 68, 123, 145
A enzymes, 6, 86
Alcoholic
Abiotic agents, 75 beverage, 2, 14, 21–23, 143
Acetaldehyde, 6, 24, 31, 32, 47, 55, 57, 63, fermentation, 16, 24, 26, 30, 65, 197, 240,
75, 85, 104, 155 254, 432, 433
aldehyde dehydrogenase (ALDH), 155 Aldehyde, 6, 57, 58, 61, 63–65, 73, 76–78,
dehydrogenase, 47 117, 118, 181
Acetate kinase, 47, 121
dehydrogenase, 32, 58, 63, 69, 118, 155
Acetic acid, 45, 61–64, 70, 104, 105, 113,
gene, 32
115–117, 170, 181, 348, 356, 374, 380,
groups, 63
385
Algal biomass, 486
Acetyl-CoA reduction, 155
Alphaproteobacteria, 27
Acid
base treatment, 347 American civil war, 162
tolerance, 27 Amino acid, 7, 29, 30, 32, 57, 59, 67, 70,
tolerant mutants, 115 73, 76, 99, 103, 105, 118, 145, 147, 155,
Acidity, 1, 3, 33, 321 345
Acidophilic mesophiles, 10 Ammonia, 8, 180, 284, 339, 352, 353, 356,
Adaptation laboratory evolution, 105 409, 410, 413
Adhesives, 14 fiber expansion (AFEX), 180, 339, 340,
Aerobic 351, 352, 409
conditions, 6, 31, 35, 70, 76 recycling percolation (ARP), 180
gram-negative bacteria, 74 Ammonium
Agave reductase, 8
americana, 371, 378, 380 salts, 6, 8
hydrothermal pretreatment, 383 Amorphous macromolecule, 107
waste, 377, 379 Amyloglucosidase, 181, 278
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502 Index
Anaerobic Biodiesel, 25, 161, 162, 165, 174, 316, 344,

bacteria, 47, 75, 183 372, 375, 377, 420, 464–466, 468–471,
conditions, 1, 2, 6, 31, 57, 63, 70, 75, 76, 474, 475, 477–481
278, 449, 451 Biodigestion, 470
Bioeconomy, 237, 373, 464, 476, 478
digestion (AD), 58, 59, 62, 155, 199,

201–203, 210, 213, 447, 465, 480, 481 Bioelectrochemical systems
growth, 11 applications, 448
microorganisms, 48 overview, 447
Anhydroglucose molecule, 422 Bioenergetics, 373
Antioxidant Bioenergy, 25, 122, 273, 277, 289, 294, 316,
enzymes, 61 331, 344, 377, 394, 446, 475, 481
proteins, 106 Bioethanol, 14, 22, 29, 53, 97, 108, 109,
Apoptosis, 76 113, 116, 120, 161, 233, 256, 315, 320,
Author Copy
Aquatic biomass, 373 329, 330, 339, 371, 406, 419, 446, 447,
Aqueous two-phase systems (ATPS), 222 464, 466, 471, 474, 479, 483
Arabinose, 73, 107, 115, 178, 236, 286, 380, challenges
424, 432, 433 first-generation bioethanol, 482
Aromatic second-generation bioethanol, 484
amino acids (AAA), 30, 149 third-generation bioethanol, 486
molecules, 65 commercialization, 161, 162
Artificial fermentation, 181, 391
antioxidant defense system, 106 production, 22, 109, 113, 116, 120, 161,
modification, 107 256, 329, 371, 406, 419, 446, 447, 483
Aspartic acids, 8 enzymatic hydrolysis, 238
Autohydrolysis, 375, 382, 383 fermentation, 240
Autophagy, 100 genetic engineering strategies, 108
Azeotrope, 196, 197, 199, 202, 203, 206 microorganisms, 25
Azeotropic pretreatment, 237
composition, 196–199, 203, 206, 207, process, 26, 236
211, 213 substrates, 25
distillation, 195, 196, 199–201, 210, 216, yield, 26
yield, 26, 33, 183, 243, 325, 327–329
282
Biofilm reactor, 36
Biofuel, 21–23, 26, 44, 48, 49, 56, 98–100,
B
102, 104, 105, 119, 123, 162, 165, 166,
Bacterial 170, 172, 173, 175, 234, 246, 272, 273, 283,
gene expression, 74 295, 324, 343, 377, 405–409, 412, 420,
pathway, 29 423, 425, 430–432, 446, 447, 463–467,
Batch, 14, 282, 451 469–471, 474–478, 481, 482, 484, 487
system, 14, 250, 403 challenges, 474
Beta vulgaris, 165, 166 production, 98, 102, 105, 108, 122, 123,
Binary permeability, 207 170, 176, 255, 316, 325, 341, 467, 469,
Biocatalysis, 99 472, 474, 487
Biocatalyst, 21, 143, 425, 451, 453 solvent tolerance, 102
Bio-char, 373 sustainability and environmental benefits,
Biochemical, 373, 374 472
degradation pathways, 24 Biofuture platform (BFP), 476
Biodegradability, 180 Biogas, 117, 282, 344, 372, 377, 405, 452,
Biodetoxification, 118 464, 465, 468, 470, 472, 474, 478, 480, 481
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Index 503
Biohydrogen, 25, 172, 372, 405, 465, 469 C

Bioinformatic analysis, 83
Caffeic acid 3-O-methyltransferase
Biological macromolecules, 102
(COMT), 107
Biomass, 170, 175, 235, 242, 249, 255, 256,
Caldanaerobacter, 45
275, 292, 295, 296, 343, 373, 389, 392,

Carbohydrate, 9, 12, 21, 23, 29, 46, 74, 78,
401, 413, 464, 475, 479
83, 84, 111, 112, 119, 174, 181, 184, 248,
chemical characterization, 379
274, 283, 284, 286, 347, 348, 350, 373,
conversion, 175, 184, 241, 316, 317, 380, 402, 407, 421, 429, 430, 486
463, 476, 480, 487 active module, 83
drying and grinding, 379 esterases (CEs), 46, 430
feedstock, 15, 122, 184, 273, 477 stress, 12
hydrothermal Carbon
pretreatment, 384
Author Copy
dioxide (CO2), 1, 165, 273, 420, 450
processing, 381 metabolic flux, 102, 103
Biomethane, 465, 466, 469, 472 rich raw materials, 274
Biomolecules, 54, 98–100, 174 corn, 276
Bionic acid, 28 sugarcane, 276
Bioprocesses, 98–100, 271, 297, 455 source, 7, 22–25, 29, 32–34, 54–57, 59,
Bioreactor, 14, 142, 216, 221, 234, 235, 65, 67, 68, 70, 73, 74, 85, 274, 393,
237, 238, 241–248, 250–255, 257, 340, 446, 455, 480
353, 354, 357, 433, 449, 486 Carbonate compounds, 11
design, 235, 253, 257 Carboxylic acids, 32
enzymatic hydrolysis process, 244 Catabolism, 29, 30, 32, 59, 74, 447
operation mode and geometric configu- Catalytic zinc-binding motif, 60, 71
ration, 244 Cell
process conditions, 247 collection, 486
fermentation process, 241, 250 cycle, 67, 353
geometric configuration and instru- densities, 3
mentation, 250 division, 11, 105, 145
process conditions, 253 equilibrium, 61
Biorefineries, 16, 36, 98, 100, 107, 123, filtration, 220
195, 196, 199, 213–215, 255, 272–277, functions, 142
279–283, 287–297, 316, 340, 344, 350, growth, 13, 14, 57, 77, 106, 110, 118,
364, 371–377, 379, 383, 386, 388, 391, 216, 218, 219, 255, 455, 486
393, 394, 401, 405, 414, 478, 480, 486, 487 harvesting, 14
Biosynthesis, 59, 67, 76, 79, 103, 107, 108, mass, 12, 143, 145, 150
111, 115, 147–149, 179, 431, 432 membrane, 28, 86, 118, 142
Biosynthetic motility, 77–79
gas, 373 population, 13
genes, 76 recycling, 3, 14, 281
Biotechnological applications, 28, 75 repair, 67
Biotechnology, 28, 99, 287, 340, 359, 364 separation, 14
evolution, 359, 364 wall, 77–79, 107, 118, 119, 146–149, 174,
Biotin, 27 181, 236, 419–426, 429, 431, 432, 434,
Bluewater footprint, 405 436, 486
Bottlenecks, 100, 102, 107, 277, 297, 463, integrity (CWI), 147
486 Cellobiose, 5, 45, 49, 83, 85, 119, 122, 178,
Brown rot fungi, 180, 411, 436 183, 235, 239, 344, 356, 389, 422, 430
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504 Index
Cellodextrins, 83, 239 Chromatin, 55, 72

Cellular Chromosome, 31, 75, 119, 120, 151
adaptation, 75 Cider sickness, 27
biology, 53 Cinnamate 4-hydroxylase (C4H), 107
damage, 64, 104, 105 Circular plasmids, 31
dynamics, 75 Citric acid cycle, 6

functions, 153 Climate change package, 475
growth, 103 Closed
lifespan, 64 circuit system, 14
maintenance, 64 circulating fermentation system (CCCF),
mechanisms, 111 224
membranes, 102 Clostridium, 23, 43–46, 53, 54, 80–84, 87,
metabolism, 54, 73, 103, 104, 111, 254 97–99, 105, 108, 109, 119, 120, 141, 144,
Author Copy
proteins, 110 156, 178, 183, 434
recycling batch fermentation, 14 thermocellum, 43–46, 53, 54, 80–84, 87,
stress, 9 97–99, 108, 109, 119, 120, 141, 144,
thiol-redox pathways, 61 156, 178, 434
Cellulases, 46, 48, 100, 183, 238, 239, 244, genetic regulation, 83
248, 391, 425, 429, 430, 433 physiology, 46
Cellulolytic Clustered regularly interspaced short palin-
bacteria, 45 dromic repeats (CRISPR), 102, 153
enzymes, 238, 248, 344 Co-factor balancing, 98
activities, 248 Combinatorial method, 152
Cellulose Computer science, 79
hemicellulose Consolidated
degradation, 429 bioconversion process, 182, 183
hydrolysis, 183, 246, 250, 283, 351, 420 bioprocessing (CBP), 26, 43, 48, 50, 80,
Cellulosic 142, 245, 286, 297, 419, 433, 437
agricultural residues, 25 Continuous
biofuels, 475 membrane fermenter separator (CMFS), 224
ethanol, 170, 286, 287, 339–341, 345, stirred tank reactors (CSTR), 252
354–359, 362–364 Conventional
biomass pretreatment, 345 distillation, 197, 199, 201, 203, 209, 210,
production, 170, 339, 341, 345, 354, 213, 216
355, 357, 358, 362 dry-grind process, 277
Cellulosomal enzymes, 80 separation, 195, 197, 199, 201
Cellulosome, 43, 45, 48, 80, 81, 83, 85, 119, solvent, 204–206
430 Copolymers, 211
genetic regulation, 80 Corn gluten
Central metabolism, 5, 29, 46 feed (CGF), 168
Chaperones, 102, 105 meal (CGM), 168
Chemical Corrosion, 203, 205, 250, 286, 348, 382, 485
hydrolysis, 3 Cost-effective bioproducts, 98
stress, 64 Crabtree
Chemotaxis, 76 negative competitors, 35
China National Offshore Oil Corporation positive
(CNOOC), 475 species, 35
Chitosan (CS), 211, 225 yeast, 35
Chlorella vulgaris, 174, 175, 481, 486 Cross-protection, 9
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Index 505
Crystalline cellulose, 49, 85, 402 Electrochemical effects, 2

Crystallization, 3, 205, 251, 282 Electrofusion, 116, 118, 152
Cyclohexane, 200 Electron
Cytoplasm, 55, 103 flux, 48, 121
Cytoplasmic membranes, 64 transfer mechanism, 46, 454
Cytoprotective activity, 374 Embden-Meyerhof-Parnas (EMP), 21, 24,

Cytoskeleton proteins, 105 29, 30, 46, 64, 75, 85, 143
Cytosolic acidification, 61 Endocytosis, 32
Endoglucanases, 5, 239, 248, 430
D Energy
Decarboxylation, 30, 75 cogeneration system, 282
Deep eutectic solvents (DESs), 222 independence and security act (EISA), 475
Degree of polymerization (DP), 180, 284, policy act (EPA), 475
Author Copy
374, 484 Enolase, 69
Dehydration, 115, 200, 201, 206, 210–214, Entner-Doudoroff (ED), 21, 74, 115, 179, 432
288, 289, 409 Environment parameters, 8
Dehydrogenase, 30, 58, 69, 102, 105, 108, Environmental
115, 143 factors, 1, 35, 75, 143, 422, 424, 431
Delayed simultaneous saccharification and pollution, 107, 141, 142, 161, 185, 234,
fermentation (DSSF), 340, 354 315, 363
Deodorizers, 14 stress response (ESR), 9
Detoxification, 57, 59, 61, 63–65, 70, 77, Enzymatic
105, 115, 123, 359 antioxidant defense system, 106
Dextrins, 278 binding, 247
D-glucopyranose molecules, 422 catalysis, 247
Dietary fibers, 345 hydrolases, 246
Dihydroxyacetone hydrolysis, 3, 5, 13, 14, 180, 181, 183,
kinase, 69 184, 233–236, 238, 240, 244, 246–248,
phosphate (DHAP), 32 255, 257, 283–286, 291, 296, 340, 346,
Diploidization, 151 348, 350, 353–357, 360, 371, 381, 385,
Direct microbial conversion (DMC), 48, 183 386, 388, 389, 391, 394, 403, 408, 412,
Disaccharides, 7, 86 413, 425, 481, 484
Distillation, 10, 45, 167, 174, 195–200, pretreated solid biomass, 389
203, 204, 206–208, 210, 212–216, 222, yield calculation, 389
277–279, 282, 288, 289, 349, 447, 486 Enzyme
Distillers dried grains (DDGs), 279, 297 cocktail, 287, 288, 388, 390, 484
Down-regulating genes, 98 expression, 65
Downregulation, 107, 149 saccharification, 386
Downstream process, 99, 216, 382 Equilibrium, 98, 197, 203, 206, 208–210,
Dry 212, 217, 246, 247
distillation grain, 289 Ergot alkaloids, 13
weight basis, 167, 174 Error-prone polymerase chain reaction, 153
Dye decolorizing peroxidase (DyP), 425, 428 Escherichia coli, 23, 25, 78, 99, 101, 102,
177, 178
E Ethanogenic bacterium, 33
Economic substrate, 122 Ethanol
Economical fluctuations, 98 adaption (EA), 144
Ehrlich pathway, 29, 30 affinity, 208
Electric flux, 47 aqueous solution, 203, 209
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506 Index
biorefinery, 277, 279, 288 improving methods, 146

commercialization nutritional strategy, 149
bottlenecks, 184 small RNA (sRNA) engineering, 154
dehydration, 196, 200–203, 206, 210, tolerant strains, 111, 144, 145, 148, 150,
211, 213, 216 153–155
hydrophilic membranes, 210 Ethanolic fermentation, 23

fermentation, 13, 49, 75, 217, 219, 221, Ethanologenic
222, 255, 278 bacteria, 29, 73, 74, 80, 86
fermentative bioprocess, 23 genetic regulation, 73
history, 162 Clostridium thermocellum, 80
industrial importance, 14 Zymomonas mobilis, 74
production, 1, 4, 21, 34, 43, 46, 66, 67, yeast, 53, 55
161, 166, 175, 177, 178, 275, 292, 401, gene regulation, 55
Author Copy
412, 413, 433 Ethanol-tolerant mutant strains, 110, 118
biochemistry, 3 Eukaryotes, 6, 153
Clostridium thermocellum, 119 Eukaryotic cells, 54
first generation bioethanol, 165 European Union (EU), 3, 164, 466, 471,
fourth generation bioethanol, 175 475, 479, 487
genetic engineering, 100 Exogenous
intermediate enzymes, 57 efflux pumps, 103
key genes, 68 enzymes, 49
organic feedstocks, 165 Exoglucanases, 5, 239, 248, 430
Saccharomyces cerevisiae, 56, 110 Exopolysaccharides (EPS), 145, 156
second generation ethanol, 170 Exothermic reaction, 10
strain improvement, 46 Exploratory fermentation, 391
third generation ethanol, 170 Exponential phase, 13, 325
Expression
transcription factor genes, 57
analysis, 70
Zymomonas mobilis, 112
dynamics, 75, 86
recover, 45, 184, 195–197, 199, 201, 208,
profile, 30, 77
209, 213, 216, 345, 433
Extractive distillation (ED), 195, 202, 204,
azeotropic distillation (AD), 199
205, 225
conventional distillation and separation
Extrinsic factors, 8
technology, 197
dehydration, 201
F
extractive distillation (ED), 202
nonconventional technologies, 202 Fatty acid, 1, 2, 7, 9, 11, 12, 64, 119, 144,
organophilic membranes, 208 146, 153, 436, 450, 466
selective membranes, 208 metabolism, 67, 147
separation membrane technology, 207 Feedstock, 15, 44, 45, 48, 49, 98, 99,
pervaporation (PV), 207 122, 162, 167, 170, 172, 173, 175, 176,
stress, 32, 35, 64, 73, 78, 111, 142, 143, 184, 185, 233, 235, 273, 282, 283, 286,
145, 146, 149, 154 289–291, 295, 296, 344, 382, 432, 446,
tolerance, 23, 26, 32, 35, 47, 59, 65, 78, 465, 476, 477, 479, 480, 483, 485, 487
80, 83, 85, 86, 111, 114–116, 118, Fermentation
141–147, 149–156 approaches, 339, 340, 354, 359, 360, 363,
AdhE mutation, 155 364
evolutionary engineering, 150 conversion yield, 392
genes involved, 147 microorganism used, 177
genetic approaches, 151 pathway, 34, 433
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Index 507
Fermentative Gene
growth, 7 activation, 62, 106
yeasts, 10 coding, 68
Ferredoxin oxidoreductase, 43, 47 encoding, 32, 46–48, 59, 61, 63, 83, 103,
Filamentous 105, 106, 108, 111, 113, 121

fungi, 8, 356 protein kinases, 63
growth, 13 expression, 53, 55, 61, 70, 73–76, 78, 80,
First-generation, 44, 56, 108, 165, 170, 271, 98, 111, 116, 147, 155
273, 276, 282, 287, 289, 297, 316, 320, modules, 154
326, 331, 373, 375, 464–466, 478, 482 regulation, 54–57, 61, 64, 67, 68, 72, 80,
bioethanol, 108 86, 87, 154
starch biomass, 167 targeting, 100
sugar biomass, 165 Generally recognized as safe (GRAS), 115,
Author Copy
biofuels, 108, 170, 373, 375, 478 240
ethanol, 44, 56, 165, 287 Genetic
Flavoprotein, 76 approaches
Flex-fuel engines, 15, 199 genome editing and shuffling, 152
Fluorescent in situ hybridization (FISH), 252 global transcription machinery
Fluxomics, 65 (gTME), 153
Food industry, 2, 276 over-expression, 153
Forkhead transcription factor, 72 random mutagenesis, 151
Formic acid, 75, 374 engineering, 3, 23, 32, 49, 55, 99, 100,
Fossil fuels, 98, 122, 123, 141, 142, 161, 102, 104, 105, 107, 108, 110, 111, 122,
170, 216, 233, 234, 272, 315, 331, 371, 144, 151, 167, 279, 433, 434
372, 377, 394, 412, 419, 420, 445, 446, expression, 87
463, 464, 467, 473, 476, 477, 480, 487 mechanisms, 59
Fractionation, 172, 174, 375, 436 mutation, 9
Freeze-thaw treatment, 151 perturbations, 152
Fructans, 73 regulation, 53–56, 59, 67, 73–75, 77, 78,
Fructose, 7, 27, 34, 69, 325, 343, 432, 433 80, 81, 83, 85, 87
Fumaric acid, 65 stability, 181, 435, 486
Functional Genome, 3, 6, 31, 54, 59, 74, 75, 86, 106,
group, 144, 206 113, 115, 117, 118, 145, 150, 152–155
protein association networks, 69 analysis, 83
Fungal editing, 106, 152, 154
biomass, 13 Genomics, 65, 85, 146
cells, 13 Geothermal areas, 86
plasma membranes, 10 Global
Fungi, 5, 10, 11, 13, 99, 108, 141, 180, 284, bioenergy partnership (GBEP), 475
340, 353, 411, 421, 422, 425–432, 434–436 metabolic networks, 53
Furan aldehyde stress, 65 sorghum production, 319
sustainable goals, 487
G transcription machinery, 111, 124, 153
Galactans, 423 engineering (gTME), 111, 124
Galactomannans, 423 warming (GW), 142, 234, 401, 467, 487
Gasoline, 2, 8, 15, 162–164, 196, 199, 201, Glucan, 348, 349, 351, 352, 355, 375,
206, 234, 272, 316, 325, 409, 412, 420, 388–392, 423, 481
464, 466, 467, 474, 475, 484 Glucokinase, 58
Non Commercial Use

508 Index
Glucomannans, 423 Hemicellulases, 425, 429, 430

Glucose Hemicellulolytic enzymes, 48, 431
depletion, 58 Hemicellulose, 12, 45, 48, 54, 80, 106, 107,
tolerance, 34, 143 115, 119, 167, 170, 174, 178, 180, 181, 235,
Glutamate, 69, 73
236, 238, 283, 284, 287, 288, 291, 345,

dehydrogenase, 69 348–354, 373, 374, 377, 379–381, 384,
Glutamic acid, 147 385, 387, 388, 391, 401–403, 408, 411,
Glutaraldehyde, 219 421, 423–425, 429–433, 436, 481, 484, 485
Glutaredoxin, 61 Heterogeneous azeotropic distillation
Glutathione, 58, 59, 61, 67 (HTAD), 199
pathway, 59 Heterologous expression, 75, 101, 120, 121,
Glyceraldehyde-3-phosphate (GAP), 28, 149
69, 102 Hexane-isooctane-cyclohexane, 201
Author Copy
Glycerol
Hexokinase, 58, 69, 143
3-phosphate (G3P), 32
Hexoses, 5, 7, 23, 24, 86, 107, 165, 167,
assimilation, 24
176, 181, 183, 184, 236, 240, 250, 253,
pyruvic fermentation, 24
254, 286, 340, 374, 403, 432, 433, 435,
Glycine, 64, 147, 425
Glycogen, 9, 11 485
Glycolaldehyde, 63 High
Glycolysis, 5, 24, 29, 30, 46, 57, 64, 67, 68, osmolarity glycerol, 32
73, 75, 85, 110, 122, 432 performance liquid chromatography
Glycolytic genes transcriptional activator, 72 (HPLC), 379
Glycosidases, 485 Homeostasis, 6, 11, 61, 67, 69, 143, 146, 153
Glycoside hydrolases, 430, 485 Homoethanol pathway, 75
Glycosylation, 55 Homogenization, 247
G-protein, 149 Homogenous azeotropic distillation (HAD),
Gram-negative 199
anaerobic bacterium, 74 Homologous
bacteria, 27, 112, 143 expression, 485
Gram-positive recombination (HR), 67
anaerobic bacterium, 119 Hopane, 28
bacterium, 143 Hoponaid (hpn), 151
thermophilic microbe, 45 Hybrid
Greenhouse gas (GHG), 162, 234, 331, 409, distillation-pervaporation (PV) columns,
464, 487 213
Growth separation, 213
conditions, 12, 16, 26, 83, 154 Hydrocarbon, 201, 372, 420
inhibitor, 10, 75 Hydrochloric acid (HCl), 284, 297
Hydrodynamic cavitation (HC), 242–244,
H 284, 482
Heat reactors (HCR), 243
shock, 10, 35, 149 Hydrogen
factor protein, 62, 72 evolution reaction (HER), 452, 455
proteins (HSP), 10, 35, 36, 75, 102, peroxide (H2O2), 238, 244, 425, 437, 482
149 Hydrogenase deletion, 47
stress, 102 Hydrolytic
transcription factor, 32, 35 enzyme, 44, 45, 48, 119, 352, 388
stress, 9, 72, 75, 105, 106 linkage, 44
Non Commercial Use

Index 509
Hydrophilic Internal membrane, 223

membranes, 210, 211 Interseason period, 274, 276
portion, 110 Intracellular
Hydrophobic interactions, 10 accumulation, 65
detoxification, 59
Hydrothermal
liquefaction reactors, 241 metabolites, 118
pretreatment, 351, 371, 381–385, 391 redox potential, 105
fundamentals, 381 Ionic
processing, 381, 394 liquids (ILs), 204, 205, 222, 284, 350, 409,
Hydroxycinnamoyl transferase (HCT), 107 414
Hydroxymethylfurfural, 238, 348, 374, 403 stress, 61
Hyperbranched polymer (HyPol), 196, 206, Iron-sulfur clusters (ISC), 153
207, 225 Isoamyl acetate, 12
Author Copy
Isobutanol, 30, 45, 47, 102, 104, 105
I Isoleucine, 29, 64, 103, 147
Isomerase pathway, 112
Immobilization, 219, 221
In situ J
ethanol recovery
hybrid fermentation technology, 216 Jalview software, 60, 71
product recovery (ISPR), 216
Incubation, 27, 99, 151, 180, 325
K
period, 180 Keto acids, 29
Industrial Ketones, 6, 61, 63, 219
fermentation, 2, 12, 36, 105, 142, 433 Klebsiella pneumoniae, 103
scientific research council (CSIR), 378 Kluyveromyces marxianus, 3, 4, 54, 65–73,
Industrialization, 404, 419 87, 112, 256
Inhibitor, 3, 12, 14, 57, 59, 61, 63, 64, 73, genetic regulation, 67
75, 77, 78, 104, 115, 118, 123, 141, 181, metabolomic analysis, 73
238, 240, 283, 284, 295, 345, 352, 382, molecular strategies, 67
403, 408, 481
compounds, 110, 484 L
tolerance, 104, 108, 181 Laccases, 388, 425, 429, 432
genetic engineering, 104 Lactate dehydrogenase (LDH), 43, 46, 47,
Inhibitory compound, 57, 59, 65, 73, 246, 50, 121
295, 348 Lactic acid, 10, 43, 374
stress, 77 Lag phase, 13, 104
Inner membrane proteins, 103 Lallemand biofuels, 56
Inorganic membranes, 209–212 Laminaribiose, 81, 83
Integrated Large-scale fermenters, 16
corn, 288 Leucine, 29, 103
high gravity (iHG), 409 Life cycle assessment (LCA), 467, 487
Intelligent microbial heat-regulating engine Lignin, 107, 174, 235, 236, 242, 387, 403,
(IMHeRE), 106 421, 425, 426
Intensifiers, 70 degrading peroxidases, 426
Interfacial tension, 218 dye decolorizing peroxidase (DyP), 428
Intermediary lignin peroxidase (LiP), 426
enzymes, 68 manganese peroxidase (MnP), 427
molecules, 55, 64 versatile peroxidase (VP), 428
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510 Index
peroxidase (LiP), 426, 437 M

polymer, 170, 422, 429
Macronutrients, 6
Lignocellulolytic
Magnesium-activated aldehyde dehydroge-
enzymes, 353, 355, 425, 432, 433, 435
nase, 58
microorganisms, 246, 435

Malate
Lignocellulose, 43, 75, 115, 118, 119, 172,
dehydrogenase (Mdh), 30, 58, 69, 120
235, 240, 254, 255, 283, 295, 407, 411,
shunt pathway, 122
420, 425, 431, 434, 435, 481 Maleic acids, 180
hydrolysates, 184 Maltose-permease enzyme, 7
materials, 435 Maltotriose, 7
Lignocellulosic Manganese peroxidase (MnP), 388, 427, 437
biomass (LCB), 3, 16, 43, 54, 100, 101, 113, Mannans, 423
119, 170, 171, 233, 235, 242, 244, 274,
Author Copy
Matrix, 178, 211, 283, 373–375, 402, 408,
339, 374, 401, 405, 406, 413, 414, 478 474, 475, 480, 481
overview, 374 Mechanical resistance, 107
ethanol production, 53, 70, 240, 243, 403, Membrane
404, 412, 414 assisted vapor stripping (MAVS), 216, 225
feedstocks, 142, 295, 345, 408 biogenesis, 77–79
hydrolysates, 12, 53, 64, 65, 115 composition, 119, 145, 147
materials, 3, 5, 7, 8, 13, 44–46, 49, 104, organization, 147
108, 119, 170, 175, 176, 178, 180, 235, permeability, 111, 145, 146
236, 238, 246, 248, 274, 283, 284, 286, permeabilization, 145
291, 295, 340, 345, 346, 348–352, 354, perturbation, 115
374, 375, 381, 394, 407, 408, 411, 419, selectivity, 207, 208, 210–212, 225
421–425, 432, 433 transporters, 103, 112
cellulose, 422 Metabolic
extractives and ashes, 424 activities, 9, 102, 110, 142
hemicellulose, 423 analysis, 64, 80
lignin, 421 branching, 6
pectin, 424 engineered microorganisms, 110, 123
pretreatments, 107 engineering, 29, 36, 46, 75, 80, 85,
residues, 25, 36, 63, 283, 376, 419 97–101, 113, 115, 116, 118–120, 123,
substrates, 16, 83, 253, 436 124, 172, 433
Lipid, 46, 57, 73, 144, 147, 151, 174, 374, fluxes, 68
480, 481 function, 70, 102
peroxidation, 106 heat, 106
Liquefaction, 181, 242, 243, 277, 278, 355, pathway, 6, 13, 24, 28–30, 32, 33, 54, 55,
359, 403, 472 57, 64, 67, 74, 77, 86, 98, 119, 122,
Liquefied biogas (LBG), 472 123, 143, 167, 177, 455
Liquid reactions, 80, 122, 219
hot water (LHW), 180, 351, 382, 383, regulation analysis, 68
413, 485 Metabolism, 5, 7, 11, 13, 21, 28–31, 35, 57,
liquid extraction (LLX), 197, 217–222, 225 59, 63, 67, 68, 73, 76, 78, 85, 99, 104,
phase, 11, 199, 203, 385 110, 115, 119, 123, 143, 378, 485
hydrolysis, 5 Metabolome, 64, 73
Liquor tax imposition, 162 Metabolomic, 64, 65, 85, 99, 122, 144, 146
Logarithmic cells, 13 analysis, 64, 73, 146
Long-term productivity, 14 data, 79
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Index 511
Metallic residues, 280 oxygen, 11, 429

Methionine, 29 sieves, 195, 196, 201, 202, 216, 279
Mexican center for innovation in bioenergy size, 201
(CEMIE-Bio), 377, 394 techniques, 67
Microalgae, 174, 177, 184, 273, 373, 465, 486 tools, 100, 106, 142
Microalgal weight, 64, 423
biomass, 174, 181 Monoethylene glycol, 282
cells, 174, 181 Monomers, 44, 239, 379, 380, 382, 384,
Microarray, 110 385, 388, 402, 422, 429, 430, 432
analysis, 78, 111 Monosaccharides, 100, 165, 174
Microbes, 45, 98, 141, 142, 147, 176, 295, Morphological abnormalities, 105
434, 451 MspI gene, 106
Microbial Mucor indicus, 25, 356
Author Copy
bioeletroshyntesis (MES), 447 Multidrug resistance transporters, 63
cells, 53, 144 Multienzymatic systems, 80
consortium, 122, 176, 237, 358 Multiple
desalination cell (MDC), 448, 449, 455 genetic tools, 36
electrodialysis cell (MEDC), 448, 455 metabolic pathways, 55, 64
electrolysis cell (MEC), 447–450, 455 sequence alignment analysis, 59, 60, 70, 71
electrosynthesis (MES), 445, 448, 449, Multi-round atmospheric and room tempera-
451, 455 ture plasma (mARTP), 115
fermentation, 141, 142, 162, 174, 253 MUSCLE algorithm, 60, 71
fuel cell (MFC), 447–449, 455 Mutagenesis, 4, 9, 49, 75, 104, 105,
growth, 23, 26, 35, 104, 295, 388, 452 115–119, 144, 146, 151–153
heat shock stress, 35 Mutants, 101, 115, 118, 148, 149
reverse electrodialysis electrolysis cell Mutation, 111, 115, 118, 151, 152, 155, 485
(MREC), 448 Mutational pathways, 150
strain, 7, 105, 141
tolerance, 47, 104 N
transcriptional profiles, 102, 153
Microbiological control agent, 10 Nanoparticle, 212
Micronutrients, 6, 27 National
Microwave, 241, 283, 284, 339, 340, 352, Biodiesel Production and Use Program
353, 358, 360, 408 (PNPB), 474
irradiation reactor (MWIR), 241, 242 Research Council (NRC), 405
reactors (MWR), 241 Native xylokinase, 68
Milling, 241, 277, 283, 294, 371, 408, 481 N-butanol tolerance, 103, 155
Mitochondria, 6, 57, 104 Near-universal stress factor, 142
Mitochondrial Negative regulation, 54, 55
enzymes, 68 Next-generation sequencing (NGS), 75
isozyme, 57 Nicotinamide
transcription factor, 72 adenine dinucleotide, 76
Molasses, 164, 167, 198, 273, 274, salvage pathway, 105
279–281, 289, 317 Nitrogen
Molecular metabolites (NCR), 30
biology, 31, 99 source, 1, 325
mechanism, 61, 64, 147, 150 Nitrogenous compounds, 8
methods, 3 Nonconventional solvents, 202
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512 Index
Non-enzymatic antioxidant defense system, P

106
Palmitelaidic acid, 64
Nonhydroxy myristic acid, 28
Paris agreement, 372, 473
Non-optimal fermentation, 12
Particle matters (PM), 372
Nonrenewable resources, 44
Paulownia tomentosa, 108

Novel
genetic strategies, 123 Pectin, 84, 167, 174, 421, 424, 425,
genome sequence, 29 431–433
strategies, 123 Pectinases, 425, 431
Nuclear RNA molecules, 31 Pencibacillus, 45
Nucleic acids, 10, 63 Penicillin, 13
Nucleotide, 70, 74, 116, 117 Pentacyclic triterpenoids, 28
mutations, 151 Pentose, 5, 23, 24, 86, 107, 112, 165, 167,
Author Copy
sequence site, 74 176, 181, 183, 184, 236, 250, 254, 286,
Nucleus, 55 340, 374, 403, 432, 433, 485
Nutrient starvation, 72 phosphate pathway (PPP), 67, 104
Periplasmic linkers, 103
O Permeability, 24, 62, 110, 111, 144, 146,
207, 208, 219, 225
Octadecanoic acids, 64
Peroxiredoxin, 69
Oligosaccharides, 181, 278
Pervaporation (PV), 196, 207, 208,
Omics, 85, 87, 99, 146, 151, 154, 156
212–215, 223
technique, 73, 86, 87, 156
Pervaporative fermentation, 197, 222
Optimal growth temperature, 10, 105
Pesticides, 14, 317, 328, 483
Organic
pH, 4, 8, 10, 11, 13, 24, 27, 31, 33, 34, 45,
acids, 10, 57, 61, 65, 67, 100, 120, 180,
61, 63, 74, 86, 110, 111, 115, 118, 121,
218, 222, 281, 350, 481
143, 146, 147, 153, 181, 182, 209, 218,
compound, 5, 447, 451
interface, 218 225, 234, 239, 240, 243, 244, 246–249,
membranes, 210 251, 253, 255, 277, 278, 322, 325, 326,
nitrogen, 8 329, 349, 355, 356, 384, 387–389, 392,
solvents, 102, 218, 219, 339, 340, 349, 350 409–411, 426–429, 431, 451, 452, 480
Organization of Arab petroleum exporting Pharmaceutical, 99
countries (OPEC), 162 products, 14
Organophilic membranes, 208, 222 Phenolic
Osmoprotectants, 147 alcohols, 77
Osmostress response, 62 aldehyde, 75, 77, 78, 118
Osmotic adaptation, 78
pressure, 32, 35, 144, 423 stress, 78
stress, 23, 24, 34, 72, 240 compounds, 1, 2, 104, 105, 427, 429, 435
Osmotolerance, 100 inhibitors, 63
Overexpression, 30, 57, 67, 68, 76, 85, 86, Phenols, 57, 61, 65, 75
100, 102–105, 108, 111–114, 116, 118, Phenotype, 8, 64, 111, 142–144, 146, 151,
119, 149, 436 153–155
Oxidation, 5, 6, 57, 63, 68, 70, 180, 409, Phenylalanine, 29, 73, 236, 422, 432
426–429, 432, 445, 449, 451 Phlegma streams, 198, 213, 214
Oxidative stress, 35, 58, 61, 63, 72, 75, 104, Phosphatidic acid (PA), 155
105, 144, 149, 236, 428 Phosphatidylcholine, 73, 146, 155
Oxidoreductase, 6, 104, 105, 112, 121, 429 Phosphatidylethanolamine, 58, 65, 73
Oxygen surface tension, 11 Phosphoenolpyruvate carboxykinase, 69, 120
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Index 513
Phosphofructokinase (Pfk), 30, 69 Proline, 58, 62, 65, 73

Phosphogluconate dehydrogenase (Pgd), utilization trans-activator, 58
30, 69 Propionaldehyde, 63
Phosphoglycerate mutase, 69 Protective oxidative disaccharide, 9
Phosphoric acids, 10 Protein
Phosphorylation, 28, 118 engineering, 98

Phosphotransacetylase (PTA), 43, 46, 47, kinase A (PKA), 149
50, 120, 121 misfolding, 102
Photosynthetic activity, 486 Proteobacteria, 27
Physicochemical properties, 3, 218, 222 Proteomics, 65, 85, 99, 122, 146
Physiological Protoplast, 116, 118, 152
growth, 6 fusion, 152
maturity, 324, 327 Pseudomonas putida, 103
Author Copy
Pigments, 1, 2, 99, 425 Pyruvate
Plant cell wall biosynthesis, 431 carboxylase, 100, 121
Plasma membrane, 11, 61, 73, 111, 144, decarboxylase (PDC), 6, 68, 69, 115, 121
146, 219 ferredoxin oxidoreductase, 46, 47, 85
Plasmids, 31, 75, 103 kinase, 69, 105, 117, 120
Pleiotropy, 153 Pyruvic acid, 24, 28
Polar
flagella, 27 Q
solvents, 217 Quorum-regulating system, 106
Poly(1-trimethylsily-1-propyne) (PTMSP),
209 R
Poly(ethylene glycol), 206
Polydimethylsiloxane (PDMS), 209, 225 Raw material, 2, 12, 14, 16, 22, 32, 53, 59,
Polyglycerol, 206, 207 77, 108, 141, 161, 168, 184, 234, 240,
Polymer, 29, 99, 107, 206, 209, 235, 236, 273–277, 287, 289, 292–294, 315–317,
238, 277, 284, 374, 375, 388, 402, 407, 321, 323, 325, 339, 345, 371, 373–375,
421–425, 431, 436, 452 377–379, 381, 382, 391, 407, 408, 411,
Polymeric membranes, 209, 210, 213 412, 425, 454, 465, 474, 476, 484
Polypeptide, 55, 76 Reactive oxygen species (ROS), 57, 113,
Polysaccharide, 9, 32, 81, 83, 178, 236, 248, 144, 482
382, 423, 424, 430, 431 Recalcitrance, 107, 344, 345, 375, 377, 421,
Portable digital refractometer, 329 425, 432, 478
Positive regulation, 54 Redox balance, 6
Posttranslational regulation, 68 Reflux ratios, 196, 197
Potassium aluminosilicates, 196, 201 Renewable
Potential protein-encoding genes, 31 diesel blendstock (RDB), 408
Prefoldin (PFD), 108, 124 energy, 22, 26, 29, 46, 316, 373, 377, 402,
Pre-saccharification, 26, 340, 355, 371, 392 446, 455, 463, 465, 467, 469, 472, 473,
pre-saccharification and fermentation 475
strategy (PSSF), 392 sources, 44
Pressure swing adsorption (PSA), 201 fuel
Pretreatment methodologies, 347, 364 association, 3, 375
Primary yeast taxa, 56 program (RFS), 475
Principal ethanol-producing yeasts, 2 Resistant yeast, 63
Production systems, 324, 331 Respiratory growth, 7
Prokaryotes, 28, 153 Restriction-modification system, 106
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514 Index
Rhamnogalacturonan I (RGI), 424, 437 Sequencing batch reactor (SBR), 253

Ribose-5-phosphate isomerase (RKI1), 68 Severity factor, 382, 384, 385
Ribosome, 13, 76, 145 Shearing forces, 100
Rice metallothionein, 149 Short-chain
aldehyde reductase enzymes, 63
Roger, 326
Rot fungi, 180, 284, 411, 426, 427, 434–436 aliphatic acids, 104
Signaling pathways, 67, 68, 146
S Simultaneous saccharification and,
co-fermentation (SSCF), 13, 182, 183,
Saccharides, 7, 278
245, 297, 340, 354, 358, 433
Saccharification, 9, 13, 26, 59, 105, 108,
fermentation (SSF), 13, 26, 105, 182,
174, 182, 183, 244–248, 250, 277, 278,
183, 245, 278, 340, 354, 433
284, 295, 297, 340, 345, 347, 354, 355,
Small RNA (sRNA), 32, 103, 154
Author Copy
357–359, 361, 363, 364, 371, 375, 382,
Soaking aqueous ammonia (SAA), 180
386, 388, 389, 392, 409, 433, 434, 481
Socio-economic troubles, 22
and fermentation, 9, 13, 26, 105, 182,
Soft rot fungi, 180, 436
183, 245, 246, 250, 278, 297, 339, 340,
Solid
354, 355, 357–359, 364, 392, 433, 434
biomass, 389
Saccharomyces, 2–4, 7, 21, 24, 29–31, liquid interfaces, 5
33–36, 44, 54, 56, 59–62, 64, 87, 98, 99, Solvent
101, 103, 108–111, 119, 141, 143, 144, stress, 103
167, 177–179, 181, 240, 252, 253, 255, tolerance, 102
278, 281, 286, 317, 325, 328, 355, 391, Sorghum, 66, 166, 167, 249, 254, 256, 273,
392, 432 276, 315–327, 331, 339–364, 375
cerevisiae, 2–4, 7, 21, 29–31, 33–36, 44, bioenergy production, 344
54, 56, 59–62, 64, 87, 98, 99, 101, 103, sweet sorghum bagasse, 343, 345
108–111, 119, 141, 143, 167, 177–179, world production, 341
181, 240, 252, 253, 255, 278, 281, 286, Spt-Ada-Gcn-5 acetyltransferase, 154
317, 325, 328, 355, 391, 392, 432 Stagnant fermentations, 12
adaptation and tolerance, 61 Stain fungi, 436
genetic regulation, 59 Starch integration, 294
metabolomics analysis, 64 Stationary phase, 13, 70, 325, 329
stress tolerance, 64 Steam explosion (SE), 180, 237, 284, 351,
uvarum, 7 382, 410, 413, 485
Saturation, 252, 372 Sterols, 7, 9, 11, 12, 144
Secondary Stirred tank
fungal metabolic compounds, 13 bioreactors (STBR), 243
metabolism, 13 reactor, 252
Second-generation Stoichiometric conversion, 241, 254
bioethanol, 16, 22, 108, 170, 250, 283, Strain improvement, 43, 46, 50, 151, 152
284, 286, 288, 297, 321, 373, 376, 391, Stress
393, 394, 446, 476, 484 conditions, 10, 34, 58, 59, 67–69, 100,
biorefineries, 386, 388 110, 111, 149
development, 375 factors, 55, 57, 142, 150, 154
Selective milling technology (SMT), 287 response program, 9
Separate hydrolysis and, tolerance, 64, 78, 146, 153, 343
co-fermentation (SHCF), 26 Substrate utilization, 6
fermentation (SHF), 13, 26, 182, 245, Succinate dehydrogenase, 69, 103
278, 297, 354, 356, 421, 437 Succinic acid, 45
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Index 515
Sucrose, 7, 22, 27, 34, 56, 73, 108, 165, Thermal

273, 274, 276, 279, 294, 316, 322, 325, combustion, 2
343, 344, 433 energy, 206, 207, 213, 215, 282, 376
Sugar shock, 10, 201
acids, 107 Thermoanaerobacter, 45, 86, 122, 178, 183

beet pulp (SBP), 167 Thermobacterium mobile, 27
Sugarcane bagasse, 107, 243, 273–275, 283, Thermodynamic limitations, 196
284, 286, 289–291, 294, 465, 485 Thermophiles, 45, 47, 54, 86, 106
Sulfur compounds, 76 Thermophilic
Sulfuric acid-treated rice straw hydrolysate anaerobe, 45, 46, 145
(SARSH), 481 anaerobic
Superoxide dismutase, 61 bacteria, 46, 49, 86, 122
Supreme cells, 152 cellulolytic bacteria, 49
Author Copy
Surface tension, 8, 11 bacteria, 86, 145
Surplus, 7, 296 cellulolytic
Suspended solid particles, 372 bacteria, 49
Sustainable microorganisms, 45
alternative fuels, 2 Thermo-protection, 10
biofuels, 100 Thermo-robustness, 106
genetic engineering, 100 Thermostability, 105, 106, 123
economic progress, 372 genetic engineering, 105
energy, 474 Thermotolerance, 65, 105, 106
power source, 98 Thermotolerant, 10, 35, 73, 105, 106, 111,
renewable natural sources, 25 254, 355
scenario, 473 Thiamine metabolism regulatory protein, 58
Sweet sorghum, 254, 315–327, 331, Thiol-specific peroxidases, 61
339–341, 343–364 Thioredoxin, 61, 68
advantages, 320 Threshold maltose, 7
bagasse, 249, 339, 340, 345–359, Titanium dioxide (TiO2), 212
362–364 Tolerance, 1–3, 7, 12, 23, 24, 26, 27, 31–35,
biology, 318 47, 59, 61, 63–65, 74, 78, 80, 83, 85, 86,
basic races, 318 102–106, 108, 110, 111, 113–118, 123,
intermediate, 319 141–147, 149–156, 176, 181, 216, 240,
conversion, 325 316, 321, 340, 343, 486
production, 321, 324 mechanism, 80, 146, 155
energy use, 324 Toxic
varieties, 315, 317, 320, 326, 331, 343 agents, 77
Synthetic biology, 29, 59, 98, 100, 106, 123, compounds, 57, 58, 65, 74, 76, 103, 117,
124, 156 118, 347, 435
Syringaldehyde, 77, 374 metabolites, 13
molecules, 67
T Traditional distillation schemes, 196
TALEN assisted multiplex editing (TAME), Transaldolase (Tal), 30, 68
152 Transamination reaction, 29
Target mRNA interactions, 32 Transcription
Technological challenges, 272, 294, 463, activator-like effector nuclease, 152
478, 487 factor (TFS), 57–59, 61, 62, 70, 72, 111,
Temperature tolerance, 35 113, 116, 149, 153, 154
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516 Index
Transcriptional W
activation, 149
Water
activator protein, 62, 72
consumption
factors, 64
biological pre-treatment, 411
regulation, 76–79
chemical pre-treatment, 408

regulator, 62, 72, 80
physical pretreatment, 408
regulatory physicochemical pre-treatment, 409
network, 62, 72, 80 footprint, 401, 404–407, 410–412, 414
protein, 72 resources, 403, 404, 407
Transcriptomics, 65, 68, 85, 99, 122, 146 selectivity, 210, 211
Transmembrane lipids, 144 washing (WW), 409
Transmission electron microscopy (TEM), 151 Weak acid resistance protein, 58, 62
Transport sector, 22, 463–466, 469, 474, White rot fungi, 436
Author Copy
475, 480, 482, 487 Wild yeasts, 2
Trehalose, 9, 11, 32, 36, 65, 105, 111, 143,
147, 149, 151 X
6-phosphate
phosphatase (TPP), 32, 36 Xylan, 49, 84, 236, 348, 349, 351, 352, 374,
synthase (TPS), 32 375, 388, 423, 431
degradation, 111 Xylitol dehydrogenase (XDH), 68, 112
Tricarboxylic acid cycle, 100 Xylogalacturonan (XGA), 424
Tryptophan, 73, 111 Xyloglucan, 423
Xylooligosaccharides (XOs), 374
U Xylose, 3, 24, 34, 46, 49, 68, 73, 105, 107,
110, 112–115, 119, 122, 165, 167, 178,
Ubiquitin, 105 181, 236, 240–242, 246, 250, 286, 344,
Ultrasound-assisted treatment, 352 348, 358, 374, 380, 423, 424, 432, 433
United adaptation, 68
Nations (UN), 319, 341, 372 isomerase (XI), 112, 122
States (US), 3, 15, 22, 23, 164, 165, 209, reductase (XR), 68, 112
273, 275, 287, 342, 406, 446, 471, 473, heterologous (XYL1), 68
479 Xylulokinase, 112, 122
Uronic acids, 107, 374, 403
Y
V Yeast
Vacuolar acidification, 145, 146 cells, 2, 10, 11, 14, 16, 24, 63, 111, 143,
Vacuole membranes, 104 147, 150, 197, 219–224, 282
Valine, 29, 64, 65, 103, 114, 147 physiology, 8, 12, 16, 57
Vanillin, 77, 117, 181
Vapor-phase adsorption, 201 Z
Variety ROGER, 315, 326, 328, 329, 331 Zea mays, 166, 276
Vegetable biomass, 106 Zeolite, 201, 209, 210, 212, 216, 219
molecular tools, 106 adsorption units, 201
Velocity, 247 Zero population growth, 13
Versatile peroxidase (VP), 426, 428 Zinc
Viscosity, 206, 217, 218, 244, 247, 356, 485 finger
Volatility, 2, 196, 197, 202, 203, 206, 207, 411 nuclease (ZFN), 152
Volumetric productivity, 12 protein, 72
Vortexes, 246 starvation, 58
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Index 517
Zing finger transcription factor, 62 bioethanol yield production, 33

Zymomonas, 21, 24, 26–31, 33–36, 44, 48, features, 28
53, 54, 74–77, 79, 87, 97, 99, 102, 108, fermentation, 34
109, 112, 115, 121, 141, 144, 156, 177, fermentative pathways, 29
178, 181, 254, 358, 432 genetic feature and regulation, 31, 75,
mobilis, 21, 26–31, 33, 34, 44, 48, 53, 54, 77, 78
74–79, 87, 97, 99, 102, 108, 109, 112, overview, 26
115, 121, 141, 144, 156, 177, 178, 181, physiological advantages, 30
254, 358
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Biochemistry and Biotechnological Advances

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Library and Archives Canada Cataloguing in Publication

CIP data on file with US Library of C

ISBN: 978-1-77463-849-1 (hbk)

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Ayerim Y. Hernández Almanza, PhD

Nagamani Balagurusamy, PhD

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(SNI-CONACYT, Level I distinction) and also a regular member of Mexican

Héctor Ruiz Leza, PhD

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he participates in the Editorial Advisory Board of Industrial Crops and Prod-

Campillo” of the Mexican Society of Biotechnology and Bioengineering in

Cristóbal N. Aguilar, PhD

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2. Physiology of Ethanol Production by Zymomonas mobilis.........................21

3. Physiology of Ethanol Production by Clostridium thermocellum..............43

4. Genetic Regulation of Principal Microorganisms

5. Metabolic Engineering of Yeast, Zymomonas mobilis, and Clostridium

7. Challenges in Developing Sustainable Fermentable Substrate

8. Emerging Strategies for Ethanol Purification...........................................195

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9. Design and Engineering Parameters of Bioreactors for

Héctor Hugo Molina Correa, Cristian Emanuel Gámez-Alvarado,

11. Conversion of Sweet Sorghum Juice to Bioethanol..................................315

12. Biotechnology Development of Bioethanol from Sweet

13. Bioethanol Production Using Agave americana L. Leaves

14. Sustainable Ethanol Production from Lignocellulosic Biomass:

15. Biodegradation and Biocatalysis Aspects of Direct Bioethanol

16. Bioelectrosynthesis of Ethanol via Bioelectrochemical Systems:

17. Challenges and Perspectives on Application of Biofuels in the

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Silvia Yudith Martínez Amador

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Luis Fernando Amador Castro

Héctor Hugo Molina Correa

Cristian Emanuel Gámez-Alvarado

José Antonio Rodríguez de la Garza

Mónica Margarita Rodríguez Garza

Miriam Soledad Valenzuela Gloria

Leopoldo Javier Ríos González

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School of Biological Science, Autonomous University of Coahuila, Torreón–27000, Coahuila, Mexico,

Inty Omar Hernández-De Lira

Thelma Karina Morales Martínez

Perla Araceli Meléndez-Hernández

Miguel Angel Medina Morales

Danay Carrillo Nieves

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David Francisco Lafuente Rincón

Cindy Nataly Del Rio-Arellano