MEVE013

MEVE-013
Environmental
Indira Gandhi National Open University Biotechnology
BLOCK 1
Overview of Environmental Biotechnology 9
BLOCK 2
Biodegradation of Natural and Xenobiotic Compounds 99
BLOCK 3
Bioremediation 195
BLOCK 4
Ecofriendly Bioprocesses 279
PROGRAMME DESIGN COMMITTEE
Dr. Himanshu Pathak, Director, Prof. P.A. Azeez, Director, Prof Indu Shekhar Thakur,
ICAR-National Rice Research Salim Ali Centre for School of Environmental
Institute Cuttack, Odisha Ornithology and Natural Sciences, JNU, New Delhi
History, Coimbatore
Prof Uma Melkania, Dean, Prof. Nidhi Rai, University Prof. Jitendra Pandey, Centre of
College of Basic Sciences and College of Science, M.L. Advanced Study in Botany,
Humanities, GBPUAT, Sukhadia University, Udaipur BHU
Pantnagar
Prof. R. Baskar, Department of Prof. Jaswant Sokhi, School of Prof Neera Kapoor, School of
Environmental Science & Science, IGNOU Science, IGNOU
Engineering, Guru
Jambheshwar University of
Science & Technology, Hisar,
Haryana
Prof. P.K. Biswas, STRIDE, Prof. S.K.Yadav, School of Prof. Nandini Sinha Kapur,
IGNOU Agriculture, IGNOU School of Interdisciplinary and
Trans-disciplinary Studies,
IGNOU
Prof. Shachi Shah, Director Prof. B. Rupini, School of Dr. Surendra Singh Suthar,
School of Interdisciplinary and Interdisciplinary and Trans- School of Environment &
Trans-disciplinary Studies, disciplinary Studies, IGNOU Natural Resources, Doon
IGNOU University, Dehradun
Prof. Vijay Kumar Baraik, SOS, Dr.Tanushree Bhattacharya, Dr. Pulak Das, School of
IGNOU Department of Civil and Human Ecology, Ambedkar
Environmental Engineering, University, Delhi
Birla Institute of Technology,
Mesra, Ranchi
Prof. Shubhangi Vaidya, School Dr. Sadananda Sahoo, School of Dr.Sushmitha Baskar, School of
of Interdisciplinary and Trans- Interdisciplinary and Trans- Interdisciplinary and Trans-
disciplinary Studies, IGNOU disciplinary Studies, IGNOU disciplinary Studies, IGNOU
Dr. V. Venkat Ramanan, School Dr. Deeksha Dave, School of Dr. Y.S.C. Khuman, School of
of Interdisciplinary and Trans- Interdisciplinary and Trans- Interdisciplinary and Trans-
disciplinary Studies, IGNOU disciplinary Studies, IGNOU disciplinary Studies, IGNOU
PROGRAMME COORDINATORS
Prof. Shachi Shah, School of Dr. V. Venkat Ramanan, School Dr. Deeksha Dave, School of
Interdisciplinary and of Interdisciplinary and Interdisciplinary and
Transdisciplinary Studies, Transdisciplinary Studies, Transdisciplinary Studies,
IGNOU IGNOU IGNOU
COURSECOORDINATOR
Prof. Shachi Shah, Environmental Studies School of Interdisciplinary and Transdisciplinary Studies,
IGNOU
BLOCK PREPARATION TEAM
Prof. Shachi Shah, Environmental Studies, School of Interdisciplinary and Transdisciplinary Studies,
IGNOU
COURSE CONTRIBUTORS
Unit Unit writer
BLOCK 1- OVERVIEW OF ENVIRONMENTAL BIOTECHNOLOGY
Unit 1: Introduction to Environmental Dr. Jaspal Chauhan, Head, Department
Biotechnology of Himalayan Aquatic Biodiversity
H.N.B. Garhwal University, Srinagar, Garhwal,
Uttarakhand, India
Unit 2: Environmental Biotechnology in Dr. Maitreyie Narayan,
waste water treatment Kumaun University, Nainital
Uttarakhand
Unit 3: Environmental Biotechnology for Dr. Jaspal Chauhan, Head, Department
solid waste management of Himalayan Aquatic Biodiversity
H.N.B. Garhwal University, Srinagar, Garhwal,
Uttarakhand, India
Unit 4: Biotechnological Processes Dr Ashok Nadda
Department of Biotechnology and Bioinformatics,
Jaypee University of Information Technology,
Waknaghat, Solan - 173 234
Himachal Pradesh
Dr Deepak Pant Professor, Chemical Sciences
Central University of Haryana
Mahendragarh (Haryahna) 123029
BLOCK 2 –BIODEGRADATION OF NATURAL AND XENOBIOTIC COMPOUNDS
Unit 5: Degradation of Natural Compound Dr. Pratibha, JSS Academy of Technical
Education, Noida, Uttar Pradesh
Unit 6: In Silage Production from Waste Dr. Pratibha, JSS Academy of Technical
Education, Noida, Uttar Pradesh
Unit 7: Microbes in Greenhouse Gases Dr. Pratibha, JSS, Academy of Technical
Mitigation Education, Noida, Uttar Pradesh
Unit 8: Degradation of Xenobiotic Dr. Pooja Ghosh,Centre for Rural Development
Compounds and Technology (CRDT) Indian Institute of
Technology, Hauzkhas, New Delhi 110016
BLOCK 3- ENERGY RESOURCES
Unit 9: Principles of Bioremediation Prof. Bhawana Pathak, Dean, Department of
Environment and Sustainable Development,
Central University of Gandhinagar, Gandhinagar,
India- 382030, Gujrat
Unit 10: Bioremediation for Soil Environment Dr. Monika Verma, Indian Institute of
Technology, Hauzkhas, New Delhi 110016
Unit 11: Bioremediation for Air Environment Dr. Piyush Malviya, Dr. Khalida and Dr. Rozi
Sharma, Department of Environmental Science,
University of Jammu, Jammu
Unit 12: Phytoremediation Dr. Vikas Singhal, Assistant Reginal Director,
IGNOU, New Delhi 110068
BLOCK 4 – NATURAL RESOURCE CONSERVATION AND MANAGEMENT
Unit 13 Biofuel (Adopted From MEV-023) Dr. Shiv Prasad, Principal Scientiest, Center for
Environmnetal Science and Climate Resileient
Agriculture, Indian Agricuture Reasearch
Institute, New Delhi
Dr Sandeep Kumar. Center for Environmnetal

Science and Climate Resileient Agriculture
Unit 14 Bioplastics Prof. Shachi Shah, Environmental Studies School
of Interdisciplinary and Transdisciplinary Studies,
IGNOU, New Delhi 110068
Unit 15 Biofertilizers Dr. Rashmi Yadav and Dr. Harinder
Vishwakarma, ICAR, National Bureau of Plant
Genetic Resources, New Delhi
Unit 16 Mining and Bioleaching Dr. Garima Kaushik, Central University of
Rajasthan, Rajasthan
Unit 17 Biomarkers Prof. Praveen Garg, Department of Botany,
Swami Shraddhanand College, Alipur, Delhi.
Dr. Narbir Singh ,Department of Chemistry
Swami Shraddhanand College, Alipur, Delhi
CONTENT EDITOR
Prof. Shachi Shah, Environmental Studies, School of Interdisciplinary and Trans-disciplinary Studies,
IGNOU, New Delhi.
FORMAT EDITOR AND LANGUAGE EDITOR
Prof. Shachi Shah, Environmental Studies, School of Interdisciplinary and Transdisciplinary Studies,
IGNOU, New Delhi
PRODUCTION TEAM
Mr. Rajiv Girdhar Mr. Hemant Parida
Assistant Registrar, MPDD, IGNOU, New Delhi Section Officer, MPDD, IGNOU, New Delhi
February, 2023
© Indira Gandhi National Open University, 2023
ISBN:
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CONTENTS
Page No.
BLOCK 1- OVERVIEW OF ENVIRONMENTAL BIOTECHNOLOGY 9
Unit 1 Introduction to Environmental Biotechnology 11
Unit 2 Environmental Biotechnology in Wastewater Treatment 24
Unit 3 Environmental Biotechnology for Solid Waste Management 61
Unit 4 Biotechnological Processes 79
BLOCK 2 BIODEGRADATION OF NATURAL AND XENOBIOTIC 99
COMPOUNDS
Unit 5 Degradation of Natural Compound 101
Unit 6 In Silage Production from Waste 132
Unit 7 Microbes in Greenhouse Gases Mitigation 156
Unit 8 Degradation of Xenobiotic Compounds 178
BLOCK 3 BIOREMEDIATION 195
Unit 9 Principles of Bioremediation 197
Unit 10 Bioremediation for Soil Environment 211
Unit 11 Bioremediation for Air Environment 233
Unit 12 Phytoremediation 259
BLOCK 4 ECOFRIENDLY BIOPROCESSES 279
Unit 13 Biofuel 281
Unit 14 Bioplastics 296
Unit 15 Biofertilizers 310
Unit 16 Mining and Bioleaching 319
Unit 17 Biomarkers 335
COURSE INTRODUCTION
Dear learner,
Welcome to the course MEVE-013 Environmental Biotechnology!
Environmental pollution and waste generation has become a major global

concern mainly due to rapid industrialization, urbanization, modern
agricultural development, energy generation and due to overexploitation of
Natural resources. This has resulted in disturbing the ecological balance on
which the quality of environment depends.Environmental biotechnology is
the use of science and technical knowledge to remediate the environment
polluted environment. Environmental biotechnology is an approach where
biotechnological principals/methods are applied to addresses environmental
problems, such as the removal of pollution, renewable energy generation or
biomass production, by exploiting biological processes. The significance of
environmental biotechnology grew in in the 1980s, as a result of the
establishment of industry guidelines, regulation of conformity, and the
introduction of laws for environmental protection.At present, environmental
biotechnology has immense potential in environmental cleanup process,
biomonitoring, and bioprocess technologies and also in environmental
sustinability.
The course gives an overview of environmental Biotechnology, give a brief
accunt of biodefgradation of natural and xenobiotic compound, and explain
principles and applications of bioremediation and covers ecofriendly
bioprocesses.
We hope that after studying this course, you will acquire an understanding of
environmental biotechnology, its application in environmental clean up and
obtaining the value added products.
Wishing you success in this endeavour!
BLOCK 1
OVERVIEW OF ENVIRONMENTAL
BIOTECHNOLOGY
BLOCK 1 OVERVIEW OF ENVIRONMENTAL
BIOTECHNOLOGY
Environmental biotechnology has very wide scope in environmental safety.

Environmental biotechnology is an integrate approach that includes many
disciplines like biochemistry, microbiology, environmental engineering,
molecular biology, and ecology. Environmental biotechnology offers the
remarkable new tools for pollution prevention that have not been widely
available before now. These new tools not only prevent pollution but can also
significantly cut energy demand, natural resource consumption, and
production costs while creating high-quality intermediates or consumer
products.
Unit 1 “Introduction of Environmental Biotechnology” gives a brief

introduction of Environmental biotechnology, its application and scope
specially in pollution prevention and detection, monitoring, waste
management, clean up process, energy etc. and explain various tools and
techniques
Unit 2 “Environmental Biotechnology in waste water treatment” covers the

role of environmental biotechnology in waste water treatment, its principles
and practices, treatment of waste water by aerobic and anaerobic process e.g.
activated sludge, trickling filters and other new advance technology.
Unit 3 “Environmental Biotechnology for solid waste management”
elaborates upon thetypes of solid waste, sources, treatment, landfill,
composting, vermicomposting, aerobic digestion, anaerobic digestion,
advance technology, biomonitoring etc.
Unit 4 “Biotechnological Processes” gives a brief introduction
ofbiodegradation of macromolecules, biodegradation of xenobiotics,
biotechnological innovations for recovery of food, feed and energy from
natural waste and biosolids, bioreactors designing, process parameters
optimization, cell immobilization, application of nanotechnology for
bioremediation and recovery of product.
We hope that after studying this block, you will acquire an understanding of
the concept, scope and application of environmental Biotechnology
Introduction to
UNIT 1 INTRODUCTION TO Environmental
Biotechnology
ENVIRONMENTAL
BIOTECHNOLOGY
Structure
1.1 Introduction
1.2 Objectives
1.3 What is Environmental Biotechnology?
1.4 Scope of Environmental Biotechnology
1.5 Application of Environmental Biotechnology
1.6 Environmental Biotechnology for Environmental Clean-up
1.6.1 Bioremediation
1.6.2 Waste Treatment
1.7 Environmental Biotechnology and Alternative Solutions
1.7.1 Biomass Energy Production
1.7.2 Biopesticides
1.7.3 Biofertilizers
1.7.4 Bio-enzymes
1.8 Pollution Control
1.9 Waste Water Treatment
1.9.1 Activated Sludge Process
1.9.2 Tricking Filters
1.9.3 Membrane Bioreactors
1.9.4 Anaerobic Treatment
1.9.5 Oxidation Pond
1.10 Biodiversity Conservation
1.11 Biomonitoring
1.11.1 Biomarker
1.11.2 Bioindicator
1.12 Let Us Sum Up
1.13 Key Words
1.14 Suggested Further Reading/References
1.15 Answers to Check Your Progress
1.1 INTRODUCTION
Increasing population, limited resources, depleting biodiversity, increasing
pollution and climate change are some of the important problems that the
earth is facing today. The pollution of air, water, and soil has increased as a
result of human advancements in industry, transportation, agriculture,
11
Overview of residential space, and other areas. Different tools, techniques and approaches
Environmental
Biotechnology are used to tackle these major problems. Environmental biotechnology had
proved to be an important tool to solve or restrict such problems and
maintained sustainable development as well. It has significantly contributed
in the preservation, conservation improvement and protection of the
environment.
1.2 OBJECTIVES
After studying this unit, you should be able to:
• Define environmental Biotechnology and its scope;

• Describe application of environmental Biotechnology in environment
clean up and pollution control etc.;
• Explain Environmental biotechnology role in providing alternate
soltuions for sustainable Development.
1.3 WHAT IS ENVIRONMENTAL

BIOTECHNOLOGY?
Environmental Biotechnology is an approach of biotechnology for the
abatement of different issues of the environment (air, water and soil) for the
service of mankind. It is actually use of microbiology for environmental
cleaning, restoration and protection. It is used to detect, analyze, remediate
and monitor the contaminants released in the environment due to different
anthropogenic activities. There are many contaminants that are released from
the different sources in the environment. Few of major contaminants of
environment are shown with the help of the figure (1).
Figure 1.1: Different contaminants in the environment.

12
Introduction to
Objectives of Environmental Biotechnology Environmental
Biotechnology
The major objectives are:
• To adopt biotechnology for controlling contaminants at the source level

before they enter the environment.
• To remediate contaminants when they enter environment by any means.
• To develop eco-friendly approaches for the testing, analysis and
detection of contaminants in the environment.
Overall, the major aim of environmental biotechnology is to imply

biotechnology for the protection of environment, maintaining ecological
integrity and to promote sustainable development.
1.4 SCOPE OF ENVIRONMENTAL

BIOTECHNOLOGY
With the pressure of increasing population, the necessity to fulfill the demand
of every individual in terms of food, shelter, clothes have also increased. For
fulfilling these demands development in the industries sectors is continuously
increasing. This created a lot of changes in the environment and
simultaneously polluted each part of it i.e. air, water and soil. During such
time there is an urgent need for an eco-friendly technique that could protect
the environment from the contaminants released from the different sources.
In such conditions, environmental biotechnology has to play an important
role to solve this problem efficiently and keep the future safe. Environmental
biotechnology is an integrate approach that includes many disciplines like
biochemistry, microbiology, environmental engineering, molecular biology,
and ecology. Environmental biotechnology has very wide scope in
environmental safety. Some major scopes of Environmental biotechnologies
are shown in figure 1.2.
Fig1.2 : Major Scope of environment biotechnology. 13

Overview of
Environmental 1.5 APPLICATION OF ENVIRONMENTAL
Biotechnology BIOTECHNOLOGY
Microbiology plays an important role in Environmental biotechnology.
Different forms of microorganisms are widely used for tackling
environmental problems like waste management, wastewater treatment,
xenobiotic degradation, etc. A list of microbes employed for abatement of
environmental contaminants is listed in table 1.
Different sets of genetic manipulations are conducted to develop strains that

are capable of degrading or removing a particular contaminant from the
environment. This is achieved by a particular approach of biotechnology
known as genetic recombination technique.
Table 1.1 List of microbes employed for abatement of environmental

contaminants
S.No Environmental Micro-organism References

problem employed
1. Trinitrophenol Bacillus cereus PU Singh et al.2011
2. Organophosphates, Pseudomonas putida Gong et al.
pyrethroids, and KTUe 2018
carbamates
3. Mercury Rhodopseudomonas Deng and
palustris Jia(2011).
4. Arsenic Bacillus subtilis 168 Huang et
al.2015
5. Plastic bags Bacillus sp. BCBT21 Dang et al.
2018
6. Radionuclides Microbacterium Ortet et al. 2017
oleivorans Strain A9
7. Oil spills Brachybacterium sp Angelim et al.
Pseudomonas sp. 2013
8. Industrial wastes Brevibacterium Ziganshina et
epidermidis EZ-K02 al. 2017
9. Degradation of PAHs Pseudomonas putida
Naphthalene and strains Chauhan et al.
Phenanthrene 2008
10. Heavy metals Microbacterium spp Corretto et
al.2015
14
Introduction to
1.6 ENVIRONMENTAL BIOTECHNOLOGY FOR Environmental
ENVIRONMENTAL CLEAN-UP. Biotechnology
Bioremediation is the process that employ biological organism for the
removal of different type of pollutants from the environment (water, soil and
air). The environment is full of different type of pollutants which are threats
for both the living and non-living organisms. These pollutants should be
cleaned up from the environment regularly so that their concentration may
not pile up, creating a serious impact on environment.Different organisms are
used for cleaning up these pollutants from the environment. Mostly
microorganisms and plants are employed for the remediation of the
contaminated sites. To improve the capacity of these organisms, favorable
conditions are also provided such as fertilizers, oxygen and other nutrients.
Bioremediation is efficient against contaminants like heavy metals,
xenobiotics, oil spills, polyaromatic hydrocarbon, plastics, etc. The
bioremediation process can clean the hazardous substances up into non-toxic
compounds. Bioremediation can be done at In-situ or Ex-situ mode and are
commonly of three types; plant assisted (Phytoremediation), Myco-
remediation (fungi assisted) and Bacterio-remediation (bacteria assisted).
Bioremediation is a thriving technique for eradicating environmental
contaminants like heavy metals, crude oil, pesticides, hydrocarbon, and much
more. Biotechnology has improved this process by designing specific
microorganism for particular type of waste or contaminant, through genetic
engineering. Dr Anand Chakrabarty, one of the scientists of Indian origin was
successful in creating a genetic engineered oil eating bacteria which can
degrade oil into individual hydrocarbons. These bacteria include
Pseudomonas aureginos’, where a gene for oil degradation has been
introduced into the Pseudomonas.
1.6.2 Waste Treatment

With the development of industries are huge amount of solid and liquid waste
is discharge into the environment. These wastes are either untreated or
partially treated and hence, when released in the environment, cause threats
to living organism. Therefore, it is very important to treat such type of waste
before discharging into the environment. Environmental biotechnology plays
an important role in reduction of waste by treating it with different set of
microorganisms in the treatment plant. The treatment of solid and liquid
waste generated from various industries with the help of specialized
microorganism designed by biotechnology helps to reduce the load of
pollutant in the environment.
15
Overview of
Environmental 1.7 ENVIRONMENTAL BIOTECHNOLOGY AND
Biotechnology ALTERNATIVE SOLUTIONS
1.7.1 Biomass Energy Production
Fossil fuels are one of the major contributors of pollutants in the
environment. Different air pollutants like CO, HC, PAH, Soots, etc. are
released due to use of fossil fuels. Environmental biotechnology approach has
helped in the production of alternative fuels through different biomass. The
biomass may be of any living matter or its residue that has potential to form
energy. One of the important alternatives used against fossil fuels is ethanol
that is being utilized in blended mode with other fuels. Biomass employed by
biotechnology is also a good source of biogas which is quite compatible for
the environment. The biogas which majorly contains CO2 and methane are
produced from the organic matter, kitchen waste, Courtyard Waste,
sugarcane waste and many other carbon sources. This biogas is again a good
source of energy and can be used in different sectors, thereby reduces
pressure on fossil fuels.
1.7.2 Biopesticides
For consistent high yields, the traditional agricultural system applies
numerous chemicals, such as insecticides and fertilizers, on a huge scale.
Nevertheless, alternatives to this system are needed due to worries about
environmental preservation and issues related to human health. A great
substitute for conventional chemical pesticides is biopesticides. Biopesticides
are classes of compounds that are derived from natural substances including
plants, animals, microbes, or specific minerals that can be used to directly or
indirectly suppress, inhibit, damage or kill a pest. Thus, biotechnology is a
fast-expanding area of biology that has a variety of applications in the
sustainable agriculture, starting with the creation of chemical-free
biopesticides.
Microorganisms are used to create pesticides that are extremely effective,

species-specific, and environmentally friendly. These microorganisms are
the important part of integrated pest management techniques. Few of the
microorganism used as biopesticides and considered safe are Bacillus
thuringiensis, Bacillussphaericus, Pseudomonas aeruginosa, Nostoc
piscinale, Chlamydopodium fusiforme, Chlorella vulgaris, etc.
1.7.3 Biofertilizers
Biofertilizers are biological products that employs microorganism to improve
the soil fertility and enhance plant growth naturally. The activities like
nitrogen fixation, phosphorus solubilization, synthesis of plant growth
hormones, etc. is triggered with application of such biofertilizers. The
microorganism used for biofertilizers includes Symbiotic Nitrogen-Fixing
Cyanobacteria and Free-Living Nitrogen-Fixing Bacteria. The commonly
16
Introduction to
utilized biofertilizers are Rhizobium, Azolla pinnata, Clostridium, Environmental
Azotobacterand Azospirillum. Other biofertilizers are plant growth-promoting Biotechnology
Rhizobacteria, phosphate solubilizers such as Alcaligenes, Serratia,
Rhizobacteria, Erwinia, etc. Biotechnology helps to culture these organisms
in a large amount so that they can be used as biopesticides and further by
means of genetic engineering their efficiency is also enhanced.
1.7.4 Bio-Enzymes
Bio-enzymes are an effective biological instrument that supports
environmental cleanliness in a number of ways. The bio-enzyme are used in
agriculture and soil stabilization, for treating the water bodies, to purify the
air, monitoring the pollutants and for managing different type of waste. Bio-
enzymes can completely alter a contaminant's toxicological structure or
completely turn a harmful chemical into a harmless inorganic end product.
The bio-enzymes are specific for a specific type of contaminants and has
potential to transform and detoxify it efficiently. Enzymes like
Hydrolases, Esterases, nitrilases, lipase, cutinase, etc are few enzymes that
are found effective against xenobiotics such as herbicides, pesticides,
organophosphorus compounds, nitrile compounds, and other polymers.
Numerous enzymes from diverse microorganisms have been reported to be
essential in applications for wastewater treatment. Enzymes can precisely
target resistant pollutants and remove them by precipitating and transforming
them into other products. They can also alter the features of a given waste to
make it more amenable to treatment or help turn trash into goods with
additional value. The likelihood of enzyme leaching into solution is reduced
while the mechanical and thermal stability of the enzymes is increased
through immobilization.
1.8 POLLUTION CONTROL

Biotechnology is an essential tool to deal with the problem of environmental
pollution as it has potential to provide new approaches for analyzing,
managing, preserving, and restoring the environment. It can transform
pollutants into valuable substances and also generate renewable energy
sources from biomass. Further, environmental biotechnology can develop
environmentally safe manufacturing thereby reducing pollutants at source
level. Environment is polluted by different substances continuously that are
released due to various anthropogenic activities. Most of these activities
cannot be banned like Food industries, Pharma Industry etc. since these are
important for fulfilling the basic needs of the population. These industries
release different forms of air water and soil pollutants which are needed to be
taken care so that they may not accumulate in the nature. For sustainable
future these contaminants should be continuously eliminated from the
different component of environment.Natural bioenzymes can be utilized to
detoxify toxic compounds being discharged into the environment with the aid
of environmental biotechnology. Environmental biotechnology enables
17
Overview of biological solutions to common problems with water and waste treatment,
Environmental
Biotechnology such as sludge management, the breakdown of resistant substances, and the
production of biogas. Additionally, it aids customers in setting up more
environmentally friendly wastewater treatment methods.
1.9 WASTE WATER TREATMENT

A large amount of wastewater is being generated continuously from different
industries and domestic sector.This wastewater is needed to treated before
discharging in the environment otherwise it will pollute the receiving body.In
industries many of treatment processes are based on biotechnological
approaches. The process includes the use of activated sludge, Oxidation
Ponds, Tricking Filters, Membrane bioreactors, Oxidation ponds and
Anaerobic Treatment. Each method is employed as part of biological
treatment to treat organic waste of the wastewater into carbon dioxide, water,
and other end products. It protects the environment and the populace from the
harmful effects of wastewater discharge. Additionally, biotechnology is an
effective tool in utilizing the sludge into biogas and compost. The secondary
wastewater treatment includes different process where biotechnology play an
important role. Major of these is discussed below:
1.9.1 Activated Sludge Process

In the activated sludge process, wastewater is biologically digested by a
microorganism including bacteria, algae, fungi and protozoa. The wastewater
is passed in the aeration chamber where microorganisms are seeded. One
feature of this process is the flow of liquid waste called activated sludge,
which settles in the bottom of a clarifier tank with the solid wastes. After that,
the activated sludge is put back into the aeration chamber, where it is
repeated with the subsequent batch of sewage.
1.9.2 Tricking Filters

Trickling filter process is an aerobic wastewater treatment with a fixed-bed
bioreactor. A biological filter is prepared with the stones, bricks, any rough
media on which a slimy layer of microorganisms are seeded. This filter is
porous so that air can easily pass through it.Waste waterflushes downward
through rotary distributor that evenly distribute the wastewater over the filter
bed and microorganism degrade organic waste from it. With this approach,
both suspended solids and the biological oxygen demand are decreased.

It is aerobic suspended growth process where microorganism remains
suspended in the reactor with the wastewater. This mixture is continuously
stirred and high compressed air is sparged under pressure from below to keep
the aggregates in suspension.
18
Introduction to
1.9.4 Anaerobic Treatment Environmental
Biotechnology
The degradation of organic waste with the help of microorganisms without
using oxygen is known as anaerobic treatment. Mostly anaerobic degradation
of wastes release carbon dioxide and methane which are used as biogas.
1.9.5 Oxidation Pond

Large and shallow ponds used for stabilization or degradation of organic
matter from wastewater by the action of sunshine, bacteria, and algae are
called oxidation ponds. These are also known as lagoons or stabilization
ponds. During photosynthesis, algae in the pond uses sunlight to create
oxygen. Aerobic bacteria in the oxidation pond use this oxygen to break
down the organic trash from the wastewater. The ponds provide place where
the most of the waste is treated, broken-down materials settled, producing
well-treated effluent.
Check Your Progress 1

Note: a) Use the aspace given below for your answers.
b) Check your answer with the one given at the end of this unit.
1. What is the role of environmental Biotechnology in Environmnetal Clean
up?
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2. Discribe the scope of Environmental biotechnology.
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1.10 BIODIVERSITY CONSERVATION

Biodiversity is an important aspect for sustainability of an ecosystem and
overall nature. Unfortunately, due to extensive exploitation imposed by ever
increasing population many of the plant species have been extinct from the
nature. Other species which are at the verge of extinction (endangered),
requires to be conserved. In this regard biotechnological advancement like
tissue culture, Germplasm Bank or Gene Bank has shown very impressive
results. These Banks are places for conservation of seeds or vegetative tissue
19
Overview of of endangered species in storehouses maintained at low humidity and
Environmental
Biotechnology temperature, to help maintain the genetic diversity.
Different plants which were very important for an area i.e. they played role of
keystone species, were removed due to the different environmental stresses.
This resulted in the ecological imbalance in that area and degradation of
diversity. Biotechnological approaches like tissue culture have played a
significant role in restoration of such keystone species thereby reviving the
ecology of degraded region. Different biotechnological strategies like
micropropagation, bio-fertilizer, biopesticides, bio enzymes, etc. were helpful
in restoration of biodiversity in the degraded land.
1.11 BIOMONITORING
To keep environment safe and sustainable it is very important to identify the
contaminants disturbing its integrity. Not only identification but they are
regular monitoring is quite important to restrict their value up to permissible
level. Environmental biotechnology provides many biological approaches
that can detect and monitor contaminant concentration in the environment.
Such instruments are named as biosensors or biological sensors. Biosensors
are analytical device that has potential to detect changes in the biological
processes and convert them into an electrical signal which is measured
finally. Biological process may include materials like enzyme,
microorganisms, cells, tissues, etc. Biomonitoring is basically conducted by
the help of biomarkers and bioindicators.
1.11.1 BIOMARKER
The term "biomarker" refers to biochemical, cellular, physiological, or
behavioural changes in tissue, body fluids, or the entire body of an organism
that serve as indicators of exposure to chemical pollutants and may or may
not also point to the presence of a harmful consequence. A few of the often-
employed biomarkers, including pigments, cytochrome P4501A enzyme
induction, acetylcholinesterase inhibition, DNA integrity 16, and metallothiones.
1.11.2 Bioindicator
A bioindicator is an individual, species or community whose function,
population, or status can provide information about the state of the
environment's quality. A slight change in the ecosystem’s variable will show
a correlation with the indicator species. These bioindicator may be any living
organisms such as lichens, moss, plants, planktons, animals, and microbes,
Comparable to an instrument measurement; bioindicators use living
organisms to measure pollutants. Bioindicators give information on the state
of an ecosystem or organism's health as well as the environment's quality.
The examples of bioindicators are earthworm, Cyclops,Vogesella Indigofera,
Hylocomium splendens, etc.
20
Introduction to
Check Your Progress 2 Environmental
Note: a) Use the aspace given below for your answers. Biotechnology
1. Explain the importance of biofertilizer in the environmental safety.
……………………………………………………………………………
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2. How biotechnology is playing role in waste water treatment?
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1.12 LETS US SUM UP

With the development of the society, State, Country and World, new
challenges will arise for the environment. In future these challenges would be
very significant for human beings and tools like biotechnology will be very
helpful in in dealing them. Environmental biotechnology is a rapidly growing
and significant field of study that has implications for both pollution
prevention and clean-up in different environmental components. It was
introduced to resolve many issues of environmental degradation and
protection. It played successful role to complete the entire assigned task that
were important for the environmental safety. The approach of genetically
modified organisms for the environmental safety and balance was a key for
sustainable development. This approach has proved efficient and eco-friendly
for the human beings. With the ever-increasing population, rise in
industrialization and expanding urbanization environmental biotechnology
will have to play a key role to safeguard the environment for the welfare of
human beings in future also.
Biotechnological techniques like metagenomics (functional), proteomics,

transcriptomics and metabolomic will have to play an important role in
environmental protection and at the same time parallel development. Major
sectors which needed to be work upon by biotechnology in future are
• Alternative energy resources

• Waste degradation
• Clean up sites 21
Overview of • Clean water and air
Environmental
Biotechnology
1.13 KEYWORDS
Bioremediation : The use of microrganisms to consume or help remove toxic
chemical wastes and metals from contaminated soil and water, is cleaning up
the environment using enzymes or living organisms such as naturally-
occurring and genetically modified microrganisms such as bacteria and fungi.
Waste : Materials that are not prime products (that is, products produced for
the market) for which the generator has no further use in terms of his/her own
purposes of production, transformation or consumption, and of which he/she
wants to dispose. Wastes may be generated during the extraction of raw
materials, the processing of raw materials into intermediate and final
products, the consumption of final products, and other human activities.
1.14 SUGGESTED FURTHER READING/

REFERENCES
• Singh, B., Kaur, J., and Singh, K. (2011). 2, 4, 6-Trinitrophenol
degradation by Bacillus cereus isolated from a firing range. Biotechnol.
Lett. 33, 2411–2415.
• Gong, T., Xu, X., Dang, Y., Kong, A., Wu, Y., Liang, P., et al. (2018).
An engineered Pseudomonas putida can simultaneously degrade
organophosphates, pyrethroids and carbamates. Sci. Total. Environ. 628,
1258–1265.
• Deng, X., and Jia, P. (2011). Construction and characterization of a
photosynthetic bacterium genetically engineered for Hg2+ uptake.
Bioresour. Technol. 102, 3083–3088.
• Huang, K., Chen, C., Shen, Q., Rosen, B. P., and Zhao, F. J. (2015).
Genetically engineering Bacillus subtilis with a heat-resistant arsenite
methyltransferase for bioremediation of arsenic-contaminated organic
waste. Appl. Environ. Microbiol. 81, 6718–6724.
• Dang, T. C. H., Nguyen, D. T., Thai, H., Nguyen, T. C., Tran, T. T. H.,
Le, V. H., et al. (2018). Plastic degradation by thermophilic Bacillus sp.
BCBT21 isolated from composting agricultural residual in Vietnam.
Adv. Nat. Sci.: Nanosci. Nanotech. 9:015014.
• Ortet, P., Gallois, N., Piette, L., Long, J., Berthomieu, C., Armengaud, J.,
et al. (2017). Crossm draft genome sequence of bacterium isolated from
chernobyl radionuclide-contaminated soil. Genome Announc. 5, 18–19.
• Angelim, A. L., Costa, S. P., Farias, B. C. S., Aquino, L. F., and Melo,
V. M. M. (2013). An innovative bioremediation strategy using a bacterial
consortium entrapped in chitosan beads. J. Environ. Manage. 127, 10–17.
• Ziganshina, E. E., Mohammed, W. S., Doijad, S. P., Shagimardanova, E.
22
Introduction to
I., Gogoleva, N. E., and Ziganshin, A. M. (2018). Draft genome Environmental
sequence of Brevibacterium epidermidis EZ-K02 isolated from Biotechnology
nitrocellulose-contaminated wastewater environments. Data Br. 17, 119–
123.
• Chauhan, A., Fazlurrahman., Oakeshott, J. G., and Jain, R. K. (2008).
Bacterial metabolism of polycyclic aromatic hydrocarbons: strategies for
bioremediation. Ind. J. Microbiol. 48, 95–113.
• Corretto, E., Antonielli, L., Sessitsch, A., Kidd, P., Weyens, N., and
Brader, G. (2015). Draft genome sequences of 10 Microbacterium spp.,
with emphasis on heavy metal-contaminated environments. Genome
Announc. 3:e00432-15.
• https://www.yourarticlelibrary.com/biotechnology/use-of-biotechnology-
for-cleaning-up-our-environment/29370
1.15 ANSWERS TO CHECK YOUR PROGRESS

1. Refer Section Number 1.6

2. Refer section Number 1.3,1.4
1. Refer Section Number 1.7.3
2. Refer section Number 1.9
23
Overview of
Environmental UNIT 2 ENVIRONMENTAL Bi
Biotechnology
BIOTECHNOLOGY IN WASTE
WATER TREATMENT
Structure
2.1 Introduction
2.2 Objectives
2.3 Principles of biotechnology for wastewater treatment
2.4 Practices of biotechnology for wastewater treatment
2.5 Use of Biotechnology in Wastewater Treatment
2.5.1 Activated Sludge
2.5.1.1 Sequential Batch Reactors (SBRs)
2.5.1.2 Oxidation Ditches
2.5.1.3 Deep Shafts
2.5.2 Trickling Filters
2.5.4 Anaerobic Wastewater Treatment
2.5.4.1 Anaerobic Lagoons
2.5.4.2 Anaerobic Sludge Blanket Reactors
2.5.4 .3 Anaerobic Filter Reactors
2.6 Some Recent Development of Advanced Biotechnology for
Wastewater Treatment
2.7 Let us Sum Up
2.8 Sugested Further Reading /References
2.9 Key Words
2.1 INTRODUCTION
Environmental Biotechnology is the multidisciplinary integration of sciences
and engineering in order to utilize the huge biochemical potential of
microorganisms, plants and parts thereof for the restoration and preservation
of the environment and for the sustainable use of resources. Biotechnology is
a use of living microbes to transform undesirable and harmful substances into
non-toxic compounds.
“Cleaning of environment through nature’s scavengers”
Environmental biotechnology employs a diverse set of methodological

approaches to explore and exploit the natural bio diversity of microorganisms
and their enormous metabolic capacities.
24
Environmental
The field includes the application of microorganisms for: Biotechnology in Waste
Water Treatment
• Improvement of environmental quality.
• Discovery of microorganisms with metabolic potentials that can be
employed for industrial applications.
• Use of molecular methods for assessing the natural distribution of
microbes in the environment and the ecological function they perform.
2.2 OBJECTIVES
• Define biotechnological processes used for wastewater treatment;

• Also, trace the features and scope of these processes for the production
of biogas and hydrogen as new energy resources;
• Knowledge of advanced technologies applied in the treatment plants that
improved to increase their performance and reduce energy consumption;
• Able to know about recent advanced technologies of wastewater
treatment.
2.3 PRINCIPLES OF BIOTECHNOLOGY FOR

WASTEWATER TREATMENT
Concern about the environmental impact of genetically engineered
microorganisms has greatly constrained the possibility of deliberately
releasing recombinant microbes for environmental remediation. Using such
recombinant microorganisms may be possible within contained bioreactors
but, their broader environmental applications will depend upon new
understanding of ecological functions and risk assessments related to
population of introduced organisms.
Technological advancements in the environmental biotechnology sectors are

concerned with the industrial processing of materials by microorganisms to
provide desirable products or serve other useful purposes. It forms part of
biotechnology, which emphasizes the application of biological systems to the
manufacturing and service industries or the use of biological processes within
the framework of technical operations and industrial productions.
2.4 PRACTICES OF BIOTECHNOLOGY FOR

Environmental biotechnology offers the remarkable new tools for pollution
prevention that have not been widely available before now. These new tools
not only prevent pollution but can also significantly cut energy demand,
natural resource consumption, and production costs while creating high-
quality intermediates or consumer products. Accelerated uptake of new 25
Overview of industrial biotechnology processes could lead to further pollution prevention,
Environmental Bi
Biotechnology waste reduction, and energy cost savings in related services such as waste
disposal or energy production.
According to an OECD report, biotechnology for the treatment of waste

materials is stated as “fermentation using bioreactors, bio-processing, bio-
bleaching, bio-pulping, bioleaching, bioremediation, infiltration and
phytoremediation.” The microbiological processes use living organisms such
as bacteria to degrade waste and convert it into usable forms.
• Bioreactors
Bioreactors are vessels that have been designed and produced to provide
an effective environment for enzymes or whole cells to transform
biochemicals into products. In some cases, inactivation of cells or
sterilization is carried out in the bioreactor such as in water treatment.
Many different bioreactors and bioreactor applications are described,
including those for cell growth, enzyme production, biocatalysts,
biosensors, food production, milk processing, extrusion, tissue
engineering, algae production, protein synthesis, and anaerobic digestion.
• Bio-processing
A bioprocess is a specific process that uses complete living cells or their
components (e.g., bacteria, enzymes, chloroplasts) to obtain desired
products. Transport of energy and mass is fundamental to many
biological and environmental processes. Areas, from food processing
(including brewing beer) to thermal design of buildings to biomedical
devices, manufacture of monoclonal antibodies to pollution control and
global warming, require knowledge of how energy and mass can be
transported through materials (momentum, heat transfer, etc.)
• Bio-bleaching
The use of bacteria or enzymes or biological agents in the removal of
color is termed as biobleaching.
• Bio-pulping
Biopulping is the treatment of wood chips with lignin-degrading
microorganisms to alter the lignin in the cell walls of wood, making the
wood chips softer. This treatment not only improves paper strength and
remove wood extractives but also reduces the energy consumption in the
process of pulping.
• Bioleaching
Bioleaching (or biomining) is a process in mining and
biohydrometallurgy (natural processes of interactions between microbes
and minerals) that extracts valuable metals from a low-grade ore with the
help of microorganisms such as bacteria or archaea.
26
Environmental
• Bioremediation Biotechnology in Waste
Water Treatment
Bioremediation is a branch of biotechnology that employs the use of
living organisms, like microbes and bacteria, in the removal of
contaminants, pollutants, and toxins from soil, water, and other
environments.
• Biofiltration
Biofiltration is a pollution control technique using a bioreactor
containing living material to capture and biologically degrade pollutants.
• Phytoremediation
Phytoremediation basically refers to the use of plants and associated soil
microbes to reduce the concentrations or toxic effects of contaminants in
the environment. Phytoremediation is widely accepted as a cost-effective
environmental restoration technology.
Notes: a) Use the space given below for your answer.
1. Explain principles and practices of biotechnology for wastewater
treatment.
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2.5 USE OF BIOTECHNOLOGY IN

There are several wastewater treatment methods based on biotechnology that
are used worldwide. It includes activated sludge, oxidation ponds, tricking
filters, bio-filters and anaerobic treatment. All the methods are part of the
biological wastewater treatment and use microbes to treat organic waste in
sewage.
2.5.1 Activated Sludge

The term activated sludge refers to suspended aerobic sludge consisting of
flocs of active bacteria, which consume and remove aerobically
biodegradable organic substances from screened or pre-settled wastewater.
Activated sludge systems can treat black water, brown water, greywater,
faecal sludge and industrial wastewater as long as the pollutants to be treated
are biodegradable.
27
Overview of
Environmental Bi
Biotechnology
Fig. 2.1Schematic of the aeration tank and secondary settling tank (clarifier) of an
activated sludge system.
Activated sludge reactors are aerobic suspended-growth type processes.

Different configurations of the activated sludge process can be employed to
ensure that the wastewater is mixed and aerated in an aeration tank,schematic
of the aeration tank and secondary settling tank is shown in Fig. 1.
Aeration and mixing can be provided by pumping air or oxygen into the tank
or by using surface aerators. The microorganisms oxidize the organic carbon
in the wastewater to produce new cells, carbon dioxide and water. Although
aerobic bacteria are the most common organisms, facultative bacteria along
with higher organisms can be present. The exact composition of bacteria
depends on the reactor design, environment, and wastewater characteristics.
The flocs (agglomerations of sludge particles), which form in the aerated

tank, can be removed in the secondary clarifier by gravity settling. Some of
this sludge is recycled from the clarifier back to the reactor. The effluent can
be discharged into a river or treated in a tertiary treatment facility if necessary
for further use.In the view of reuse of the effluent in agriculture, it is not
beneficial to remove all nutrients while standards for pathogen removal are
barely met.As the system is also of high complexity and strongly
mechanised, it is mainly adapted for centralised systems where energy,
mechanical and technical spare equipment and skilled staff are available.
Design Considerations
Activated sludge processes are one part of a complex wastewater treatment

system (U.S. EPA 2002). They are usually used following primary treatment
( including screening that removes settleable solids) , include one or more
main aerated treatment chambers, aeration devices, a device for appropriate
mixing to keep the sludge in suspension, a secondary clarifier to separate the
biomass from the treated effluent and collect settled biomass, generally a
non-linear, highly complex circulation regime (e.g. recirculation loops, by-
passing etc.) and are sometimes followed by a final polishing step. The
biological processes that occur are effective at removing soluble, colloidal
28
Environmental
and particulate materials. The reactor can be designed for biological Biotechnology in Waste
nitrification and denitrification, as well as for biological phosphorus removal. Water Treatment
The design must be based on an accurate estimation of the wastewater

composition and volume. Treatment efficiency can be severely compromised
if the plant is under- or over-dimensioned. Depending on the temperature, the
solids retention time (SRT) in the reactor ranges from 3 to 5 days for BOD
removal, to 3 to 18 days for nitrification.
The excess sludge requires treatment to reduce its water and organic content
and to obtain a stabilized product suitable for end-use or final disposal. It is
important to consider this step in the planning phase of the treatment plant.
Fig. 2.2 Complete overall process flow scheme of a conventional large-scale activated
sludge system. Source: ENDRESS+HAUSER (2002)
Fig. 2.3 Example of a complete activated sludge treatment system (London). Source:
CITY OF LONDON (n.y.) 29
Overview of Large amounts of injected oxygen allow maintaining aerobic conditions and
Environmental Bi
Biotechnology optimally mixing the active biomass with the wastewater to be treated. To
maintain a relatively high amount of active microorganisms useful in
removing organic substances from the wastewater, the sludge is separated
from the effluent by settling in a secondary clarifier or by membrane
filtration and kept in the process by recirculation to the aeration tank. Several
modifications of this basic process have been developed, including different
aeration devices, different means of sludge collection and recycling to the
aeration tank or primary clarifier, and process enhancement trough the
addition of an inert media area on which biofilm can grow (combined fixed-
film/suspended-growth process).
Although aerobic bacteria are the most dominant microorganisms in the

process, other aerobic, anaerobic and/or nitrifying bacteria along with higher
organisms can be present. Thus, besides the removal of organic matter,
nutrients (organic ammonia, phosphorus) can also be removed biologically
by nitrification/denitrification and biological uptake of phosphorus. The exact
composition of microorganisms depends on the reactor design, the
environment and the wastewater characteristics. To achieve optimal
conditions for both, organic and nutrients removal, a sequences of changing
aerobic and anaerobic chambers are used.
Detailed Treatment Process
After screening sand and similar heavy particles are removed next in a grit
chamber where they settle to the ground. This chamber only wants to remove
coarse grit and the wastewater spends only a relatively short period (some
minutes) in it. Smaller solids are removed in a settling or sedimentation tank.
In this unit, the wastewater spends more time (about one hour) to allow for a
good separation. The sludge from this mechanical primary treatment
(including screening and settling in the grit chamber and the sedimentation
tank) is called primary sludge and, as all excess sludge, requires an advanced
further treatment chain.
After this primary treatment, the main unit containing the activated sludge
follows. The pre-treated wastewater is mixed with the concentrated
underflow activated sludge from the secondary clarifier in an aerated tank.
Aeration is provided either by mechanical surface agitators or by submerged
diffusers of compressed air. Aeration provides oxygen to the activated sludge
and at the same time thoroughly mixes the sludge and the wastewater. During
aeration and mixing, the bacteria form small clusters or flocs. Under these
conditions, the bacteria in the activated sludge degrade the organic substances
in the wastewater. They use the organic substance for energy, growth and
reproduction. The end products are carbon dioxide (CO2), water (H2O) and
new cells.
After a few hours in the aeration chamber, the mixture then enters the
secondary settling tank (clarifier), where the flocculated microorganisms
30
Environmental
settle and are removed from the effluent stream. The settled microorganisms Biotechnology in Waste
(the activated sludge) are then recycled to the head end of the aeration tank to Water Treatment
be mixed again with wastewater and continue to grow and form new sludge
and to degrade organics. To maintain an optimal amount of sludge in the
system, the rate of recirculation of settled sludge varies from 20 to 100%.
Excess sludge produced each day (waste activated sludge) must be processed
in a further treatment chain together with the sludge from the primary
treatment facilities. A conventional excess sludge treatment chain consists in
anaerobic digestion, thickening, incineration and the safe disposal, e.g. in a
landfill. A more sustainable way would be to compost the sludge (either
before or instead of digestion) in order to reuse the nutrients in agriculture.
Hydraulic retention times in the whole systems range from some hours up to
several days for the liquid phase. Proceeding of excess sludge can take
somehow longer depending on the type of thickening and anaerobic digestion
applied. The effluent from a properly designed and operated activated-sludge
plant is of high quality, usually having BOD and TSS concentrations equal to
or less than 10 mg/L. The removal of both, biological oxygen demand (BOD)
and suspended solids (TSS) generally lies within 80 to 100% depending on
the influent concentrations, the system set-up and temperature.
Nutrients such as nitrogen and phosphorus are also removed in activated
sludge process but require a set-up of different aerated and non-aerated
chambers in hybrid activated sludge systems. Biological removal of nitrogen
is first achieved by the transformation of organic nitrogen into ammonia,
followed by the aerobic conversion of ammonia (NH4+) to nitrite (NO2) and
then nitrate (NO3-) and the anaerobic transformation of nitrate to gaseous
nitrogen (N2), which is then released to the atmosphere. The transformation
of ammonia to nitrate via an intermediate step of nitrite is called nitrification.
The transformation of nitrate to gaseous nitrogen is referred as denitrification.
Thus, a combination of both, aerobic and anaerobic (anoxic) processes are
required to achieve complete elimination of nitrogen from the wastewater. In
many activated sludge treatment systems, an anaerobic tank is either
integrated after the aerated basin and before clarification (post-
denitrification); or just before the aeration tank (pre-denitrification). In the
case of pre-denitrification, nitrification takes place in the aerated tank after
the aerobic pre-tank. Denitrification only occurs when the effluent from the
aerated tank, containing nitrite, is re-circulated like the sludge.
The removal of phosphorus in activated sludge systems can be done
chemically or biologically. Biological elimination of phosphorus in
conventional wastewater treatment system occurs through the uptake of
phosphorus by some bacterial cells. However, only little phosphorus can be
removed this way, as the phosphorus mass fraction in volatile sludge is only
about 2.5%. This results in an effluent concentration of about 2 to 7 mg /L for
municipal sewage with a COD concentration of 500 mg/L. However, it will
in general be required to lower the effluent phosphorus concentration to a
value ≤ 1 mg /L.
31
Overview of Another biological process is the enhanced biological phosphorus removal.
Environmental Bi
Biotechnology Enhanced biological phosphorus removal is based on the cultivation of some
special phosphorus accumulating bacteria, which, compared to 2.5% in
conventional activated sludge, can lead to up to 38% of accumulation in the
sludge.
When both nitrogen and phosphorus are to be removed, the combination

becomes even more complex. Enhanced biological phosphorus removal
requires generally an anaerobic stage (for PAO cultivation), an anoxic stage
(for denitrification) and an aerobic stage (for nitrification and phosphorus
accumulation) in series.
Nowadays, activated sludge systems, where valuable nutrients (phosphorus

and nitrogen) and organic matter are incinerated instead of re-circulated to
the food production in agriculture are not perceived as sustainable any more.
The introduction of nitrogen removal into an activated sludge plant increases
the reactor volume significantly and leads to higher energy consumption of
approximately 60 to 80% for aeration. The elimination of phosphorus
requires either the addition of chemicals and subsequent disposal of inorganic
sludge or an increase of complexity and reactor volume for enhanced
biological phosphorus removal.
To achieve specific effluent goals for BOD, nitrogen and phosphorus,

different adaptations and modifications have been made to the basic activated
sludge design. Well known modifications include sequencing batch reactors
(SBR), oxidation ditches, deep shafts, extended aeration, moving beds and
membrane bioreactors.
i) Sequential Batch Reactors (SBRs)
Fig. 2.4 Primary storage reactor and sequential batch reactor for activated sludge
treatment. Source: SANIMAS (2005)
The process can be operated in batches, where the different conditions are all
achieved in the same reactor but at different times. The treatment consists of
a cycle of five stages: fill, react, settle, draw and idle. During the reaction
type, oxygen is added by an aeration system. During this phase, bacteria
oxidize the organic matter just as in activated sludge systems. Thereafter,
aeration is stopped to allow the sludge to settle. In the next step, the water
and the sludge are separated by decantation and the clear layer (supernatant)
is discharged from the reaction chamber. Depending on the rate of sludge
32 production, some sludge may also be purged. After a phase of idle the tank is
Environmental
filled with a new batch of wastewater. Biotechnology in Waste
Water Treatment
Fig. 2.5 Sequencing Batch Reactor process scheme including the five essential process
steps: (1) fill, (2) react, (3) settle, (4 and 5) draw and idle. Source: CESAME & UCL
(2005).
At least two tanks are needed for the batch mode of operation as
continuous influent needs to be stored during the operation phase. (Very)
small systems (e.g. serving small settlements) may apply only one tank.
In this case, the influent must either be retained in a pond or
continuously discharged to the bottom of the tank in order to not disturb
the settling, draw and idle phases. SBRs are suited to lower flows
because the size of each tank is determined by the volume of wastewater
produced during the treatment period in the other tank.
ii) Oxidation Ditches
Oxidation ditches are large round or oval ditches (channel reactors) with
one or more horizontal aerators to guarantee oxygen supply, and to mix
and move the content around the ditch. Screened influent enters the
oxidation ditch, is aerated and circulates at about 0.25 to 0.35 m/s.
Operation can be continuous or intermittent. Primary sedimentation is
usually not required, but secondary sedimentation tanks are generally
used. The required treatment volume per capita is about 1 m³. Oxidation
ditches are suitable for areas where land availability is high. They have
the advantage that they are relatively easy to maintain and are resilient to
shock loads that often occur in smaller communities (e.g. at breakfast
time and in the evening). Typical hydraulic retention time is between 24
to 48 hours with a sludge age of 12 to 20 days.
33
Overview of
Environmental Bi
Biotechnology
Fig. 2.6 Oxidation ditch activated sludge system.
iii) Deep Shafts
Where land is in short supply, sewage may be treated by injection of

oxygen into a pressured return sludge stream, which is injected into the
base of a deep column tank buried in the ground. This type of activated
sludge reactor is called deep shaft. Such shafts may be up to 100 m deep.
As the sewage rises the oxygen forced into solution by the pressure at the
base of the shaft breaks out as molecular oxygen. This provides a highly
efficient source of oxygen for the microorganisms contained in the
activated sludge. The rising oxygen and injected return sludge provide
the physical mechanism for mixing. Mixed sludge and wastewater
influent is decanted at the surface and separated into supernatant and
sludge components. The efficiency of deep shaft treatment can be high
but they require skilled professionals for construction, operation and
maintenance; and additionally a large amount of energy.
Fig. 2.7 Sewage treatment in a deep shaft activated sludge system. Source: Whole
34 Water Systems (2012)
Environmental
Health Aspects/Acceptance Biotechnology in Waste
Water Treatment
Operation and maintenance of activated sludge system is generally carried
out by skilled laborers, which should be sufficiently well trained regarding
any health risks. Because of space requirements and odours, Centralized
Treatment facilities are generally located in the periphery of densely
populated areas. Although the effluent produced is of high quality, it still
poses a health risk and should not be directly handled but undergo an
appropriate disinfection treatment before discharge (e.g. UV-light,
chlorination). In the excess sludge pathogens are substantially reduced, but
not eliminated.
Excess sludge contains even higher amounts of microorganisms, as well as

phosphorus and heavy metals if they are present in the influent wastewater.
This can be the case because the wastewater treated in such reactors generally
comes from an array of different sources (domestic, industrial and
stormwater), which makes it a hard-to-treat mixture and therefore also a
difficult resource to recycle. Hence, the sludge generally needs to be
thickened and incinerated with the ashes being stored in a controlled landfill.
Operation & Maintenance
Highly trained staff is required for maintenance and trouble-shooting. The

mechanical equipment (mixers, aerators and pumps) must be constantly
maintained. A continuous supply of oxygen and sludge is essential. Control
of concentrations of sludge and oxygen levels in the aeration tanks is required
and technical appliances (e.g. pH-meter, temperature, oxygen content etc.)
need to be maintained carefully. As well, the influent and effluent must be
constantly monitored and the control parameters adjusted, if necessary, to
avoid abnormalities that could kill the active biomass and the development of
detrimental organisms which could impair the process (e.g., filamentous
bacteria).
Two of the most serious problems with the activated-sludge process are:
1) A phenomenon known as bulking, in which the sludge from the aeration

tank will not settle, and
2) The development of biological surface foam. Bulking can be caused

either by organisms that grow in filamentous form instead of flocs and
will not settle, or the growth of microorganisms that incorporate large
volumes of water into their cell structure, making their density near that
of water. Foaming is caused most often by the excessive growth of an
organism called Nocardia. Filamentous organisms can be controlled by
the addition of chemicals (e.g. chlorine or hydrogen peroxide) to the
recycled activated sludge; the alteration of the dissolved-oxygen
concentration in the aeration tank; the addition of nutrients and growth
factors to favour other microorganisms etc. Nocardia can be controlled
by avoiding the recycling of the skimmed foam or the addition of a
35
Overview of chemical agent (e.g. polymers or chlorine) on the surface.
Environmental Bi
Biotechnology
In all likelihood
Working Principle Activated sludge consisting of suspended flocs of

active bacteria is mixed with the wastewater. The
organic pollutants are used for growth by bacteria and
thereby transformed to water, CO2 and new cell
material. Nitrogen is removed by nitrification/
denitrification and phosphorus is both removed
chemically or biologically and accumulated in the
excess sludge. Excess sludge requires a further
treatment chain.
Capacity/Adequacy High-tech centralized system, not adapted for small
communities. Almost every wastewater can be treated
as long as it is biodegradable. Usually applied in
densely populated areas for treatment of domestic
wastewater.
Performance 80 to almost 100% BOD and TSS removal. High
nitrogen removal. P accumulated in biomass and
sludge. Low pathogen removal. HRT of some hours up
to several days
Costs Very high construction and maintenance costs;
operation very expensive due to requirement of
permanent professional operation, high electricity
consumption and costly mechanical parts.
Self-help System parts not locally available; implementation only
Compatibility possible by experienced consultant firms.
O&M Activated sludge units require professional operation
and maintenance providers.
Reliability Fails in case of power failure or fall-out of technical
equipment.
Main strength High removal efficiency for large range of wastewaters.
Main weakness Highly mechanized system requiring expert design,
operation and maintenance as well as mechanical spare
parts. Large energy requirements (e.g. for aeration).
Applicability
An activated sludge process is only appropriate for a Centralized Treatment

facility with the construction of long distance sewage channels, a well-trained
staff, constant electricity , technical equipment (e.g. spare parts, monitoring
appliances), the funding for all of this, and a highly developed management
system that ensures that the facility is correctly operated and maintained.
Because of economies of scale and less fluctuating influent characteristics,

this technology is more effective for the treatment of large volumes of flows
of municipal wastewater from medium to large towns of 10000 up to 1
million population equivalent.
36
Environmental
An activated sludge process is appropriate in almost every climate for the Biotechnology in Waste
removal of both settable (physical primary treatment) and dissolved, colloidal Water Treatment
and particulate organic matter and nutrients (biological removal in the
activated sludge). However, treatment capacity is reduced in colder
environments.
The benefits of activated sludge treatment:
The activated sludge treatment process has numerous significant advantages

over other alternatives.
Benefits are:
• The quantity of unwanted sludge is reduced.

• Beneficial bacteria reseed themselves in sewage treatment plants.
• The activated sludge treatment process allows for longer emptying
intervals.
• The procedure is extremely dependable.
• The procedure is less complicated.
• Costs are being reduced.
• The method can be odorless.
Disadvantages of the activated sludge treatment process:
The activated sludge treatment process has some drawbacks and may not be
suitable for all applications. Some disadvantages of activated sludge include:
• Some facilities may be discouraged from using this method due to the
initial high capital and operating costs.
• The activated sludge system must be designed and built by professionals.
• Skilled personnel are required to operate and maintain the treatment of
activated sludge.
• Electricity must be used continuously, which increases the energy
consumption of wastewater treatment.
• Parts and materials may not be available locally.
• Sludge and effluent may necessitate additional treatment or discharge.
Notes: a) Use the Space given for your answer.
1. Explain about activated sludge process.
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Overview of 2.5.2 Trickling Filters Bi
Environmental
Biotechnology
Trickling filters are conventional aerobic biological wastewater treatment
units, such as active sludge systems or rotating biological contactors. The
advantage of all these systems is that they are compacting (i.e. applicable in
densely populated urban settings) and that they efficiently reduce organic
matter. However, they are high-tech and generally require skilled staff for
construction as well as for operation.
Trickling filters are a secondary treatment after a primary setting process.
Fig. 2.8 Schematic cross-section of a trickling filter. Source: TILLEY et al. (2014)
The trickling filter consists of a cylindrical tank and is filled with a high
specific surface area material, such as rocks, gravel, shredded PVC bottles, or
special pre-formed plastic filter media. A high specific surface provides a
large area for biofilm formation. Organisms that grow in the thin biofilm over
the surface of the media oxidize the organic load in the wastewater to carbon
dioxide and water, while generating new biomass. This happens mainly in the
outer part of the slime layer, which is generally of 0.1 to 0.2 mm thickness.
The incoming pre-treated wastewater is ‘trickled’ over the filter, e.g., with the
use of a rotating sprinkler. In this way, the filter media goes through cycles of
being dosed and exposed to air. However, oxygen is depleted within the
biomass and the inner layers may be anoxic or anaerobic.
The word filter is somehow misleading, as physical straining of solids is only

marginal. The removal of organic substances occurs by use of bacterial
action. Therefore trickling filters are also called bio-, or biological filters to
emphasize that the filtration. Fixed film biological treatments are also used in
other common treatment processes such rotating biological contactors of
fixed film activated sludge systems.
Design Considerations
The filter is usually 1 to 2.5 m deep, but filters packed with lighter plastic
filling can be up to 12 m deep.
Oxygen is obtained by direct diffusion from air into the filter and the
biological film from the bottom through a spontaneous airflow due to
temperature difference. Therefore, both ends of the filter should be ventilated,
38
Environmental
and sub-soil construction is not common. However, in cold climates, and Biotechnology in Waste
where energy for aeration and pumping is easily available, sub-soil Water Treatment
construction can give protection against temperatures shocks.
The primary factors that must be considered in the design of trickling filters
include:
• the type of filter media to be used

• the spraying system, and
• the configuration of the under-drain system
Filter media
The ideal filter material is low-cost and durable, has a high surface to volume
ratio, is light, and allows air to circulate. Whenever it is available, crushed
rock or gravel is the cheapest option. Specially manufactured plastic media,
such as corrugated plastic sheets or hollow plastic cylinders, which optimize
surface area for bacteria to attach free movement of air, are also available.
The particles should be uniform and 95% of them should have a diameter
between 7 and 10 cm. A material with a specific surface area between 45 and
60 m2/m3 for rocks and 90 to 150 m2/m3 for plastic packing is normally used.
Larger pores (as in plastic packing) are less prone to clogging and provide for
good air circulation. Primary treatment is also essential to prevent clogging
and to ensure efficient treatment.
Fig.2.9 Principles of a trickling filter. Source: SASSE & BORDA (1998)
39
Overview of Spraying System
Environmental Bi
Biotechnology
Adequate air flow is important to ensure sufficient treatment performance
and prevent odours. To evenly distribute the water on the filter, a “rotary
sprinkler/distributor” is most often used. The rotary distributor consists of a
hollow vertical centre column carrying two or more radial pipes or arms
some cm above the filter media (to spread out uniformly and prevent
interfering with ice accumulation during winter season in colder climates),
each of which contains a number of nozzles or orifices for discharging the
wastewater onto the bed.
Fig. 2.10Rotating sprinkler arm allows to evenly distribute the wastewater over the
filter. Source: TOPRAK (2000)
Under drain System
The under drain should provide a passageway for air at the maximum filling
rate. A perforated slab supports the bottom of the filter, allowing the effluent
and excess sludge to be collected. The trickling filter is usually designed with
a recirculation pattern for the effluent to improve wetting and flushing of the
filter material.
Fig. 2.11 The latest generation of constructed wetlands for cold climate with integrated
40 aerobic (trickling) filter in Norway. Source: JENSSEN (2004)
Environmental
Biotechnology in Waste
Water Treatment
Fig. 2.12 Typical flow-chart of a trickling filter system including a pre- and
posttreatment. Source: MOUNTAIN EMPIRE COMMUNITY COLLEGE (n.y.)
With time, the biomass will grow thick and the attached layer will be
deprived of oxygen; it will enter an endogenous state, will lose its ability to
stay attached and will slough off. High-rate loading conditions will also cause
sloughing.
With time, growth and reproduction of the bacteria results in an increase of

thickness of the biofilm layer, particularly at the top of the trickling filter. If
the biofilm grows too thick, oxygen is prevented to enter and anaerobic
organisms develop. The continuous growth, the metabolic waste products of
the anaerobic bacteria, and the maintenance of a hydraulic load to the filter
eventually lead to the fact that the microorganisms near the surface lose their
ability to stick to the filter. When microorganisms fall off the medium and are
carried with the effluent, this process is known as sloughing. The under-drain
system allows transporting these solid to a clarifier, where the solids settle
and separate from the treated effluent.
To keep sloughing minimal, the organic and the hydraulic load to the filters
should guarantee a balance between the growth of the biofilm and the amount
of rinsed-out dead bio-film. The collected effluent should be clarified in
a settling tank to remove any biomass that may have dislodged from the
filter. It can then be discharged to surface waters, percolated to groundwater
or used in irrigation. The hydraulic and nutrient loading rate (i.e., how much
wastewater can be applied to the filter) is determined based on the
characteristics of the wastewater, the type of filter media, the ambient
temperature, and the discharge requirements.
Trickling filters can be combined in decentralized wastewater treatment

systems (e.g. following septic tanks or anaerobic baffled reactors) but are also
often part of large centralized wastewater treatment plants (e.g.
following activated sludge treatment).
Although trickling filters are more easily operated and consume less energy
than activated sludge processes, they have lower removal efficiency for solids
and organic matter, they are more sensitive to low air temperatures, and can
become infested with flies and mosquitoes.
41
Overview of
Environmental Bi
Biotechnology
Fig. 2.13 Flow-chart of a centralised trickling filter system where the faecal sludge is
treated by anaerobic digestion. Source: TOPRAK (2000)
Treatment Capacity
Trickling filters are designed primarily for BOD removal. Treatment

performances depend on wastewater characteristics, hydraulic and organic
loading, medium type, maintenance of optimal dissolved oxygen levels, and
recirculation rates. A BOD reduction of 60 to 85 % can be expected with
loading rates of 1 kg BOD/m3/day. Bacterial reductions have been reported to
be 1 to 2 logs of faecal Coliforms, respectively 60 to 90 % of total Coliforms.
Physical adsorptions of virus on the biofilm or elimination by predation are
additional factors in pathogen elimination in trickling filters. Total suspended
solids (TSS) removal is expected to be very low (due to the down-flow
regime) and pre-settling as well as removal of the solids from the effluent is
recommended.
Because aerobic bacteria convert ammonia to nitrate, some nitrification can

also be achieved, depending on the organic loading rate to the filter, the
temperature and the aeration. Total nitrogen removal varies from 0 to 35 %,
while phosphorus removal of 10 to 15 % might be expected. However, the
capacity for nutrient removal of trickling filters depends strongly on the
operation conditions, and while some sources indicate a high removal of
ammonia other indicate no capacity of trickling filters for nutrients.
Health Aspects/Acceptance
Odour and fly problems require that the filter be built away from homes and
businesses. Appropriate measures must be taken for pre- and primary
treatment (settling), secondary treatment (eventually final clarifier), effluent
discharge and solids treatment, all of which can still pose health risks.
Costs Considerations
Capital costs are moderate to high depending on type of filter materials and
feeder pumps used. Operation and maintenance costs are moderate or high
depending on electricity consumption of feeder pumps. In any case expert
design and skilled labor is required for construction and maintenance (e.g.
prevents clogging, ensure adequate flushing, monitor hydraulic and organic
42
Environmental
loads, control filter flies, etc.). Another cost factor is energy consumption of Biotechnology in Waste
pumps (e.g. to bring the water to the top of the filter) and for the sprinkler Water Treatment
system. However, these requirements are low compared to actively aerated
systems such as activated sludge processes.
Operation and Maintenance
A skilled operator is required to monitor the filter and repair the pump in case
of problems. The sludge that accumulates on the filter must be periodically
washed away once in five to seven years or more to prevent clogging and
keep the biofilm thin and aerobic. High hydraulic loading rates (flushing
doses) (> 0.8 m3/m3h,) and temporal collection of the effluent can be used to
flush the filter. Optimum dosing rates and flushing frequency should be
determined from the field operation.
The rotary distributor may also require regular cleaning or technical

maintenance.
The packing must be kept moist. Constant hydraulic loading can be

maintained through suction level controlled pumps or dosing siphons. This
may be problematic at night when the water flow is reduced or when there
are power failures. Recirculation of effluent may also be required to avoid
low flow conditions, but a too strong flow overload would flush out the
microbes.
Besides drying out, excessive odour can also arise when anaerobic conditions
arise due to excessive organic loadings or insufficient aeration.
Snails grazing on the biofilm and filter flies are well known problems
associated with trickling filters and must be handled by backwashing and
periodic flooding.
In all likelihood
Working Principle Wastewater trickles vertically through a porous media

(e.g. a stone bed) with high specific surface. The
biofilm growing on the media removes organic matter
under aerobic conditions.
Capacity/Adequacy Semi-centralised to centralised. The system is usually
applied in urban areas for treatment of domestic
wastewater. It can be applied for bigger and smaller
communities.
Performance BOD: 65 to 90 %. Low TSS removal. Total
Coliforms: 1 to 2 log units.
N: 0 to 35%. P: 10 to 15 %.
Costs Medium; investment costs depend on type of filter
materials and feeder pumps used; operational costs
determined by electricity consumption of feeder
pumps.
43
Overview of Self-help Low. Design, planning and implementation by expert
Environmental Bi
Biotechnology Compatibility consultants; no community labour contribution
possible; feeder pumps required; permanent staff
required for operation.
O&M Civil engineer needed for construction, professional
service providers required.
Reliability Resistant to shock loadings but the systems does not
work during power failures.
Main strength High treatment efficiency with lower area requirement
compared to wetlands or ponds; resistant to shock
loading.
Main weakness Requires expert skills, pumps and continuous
electrical power, as well as ample and continuous
wastewater flow required.
Applicability
This technology can only be used following primary clarification since high
solids loading will cause the filter to clog. Since trickling filter only receive
liquid waste, they are not suitable where water is scarce or unreliable.
Moreover, trickling filters require some specific material (i.e. pumps and
replacement parts) and skilled design and maintenance. A low-energy
(gravity) trickling system can be designed, but in general, a continuous
supply of power and wastewater is required. However, energy requirement
for operating a trickling filter is less than for an activated sludge
process or aerated lagoons.
Compared to other technologies (e.g., Waste Stabilization Ponds), trickling

filters are compact, although they are still best suited for peri-urban or large,
rural settlements.
Trickling filters can treat domestic black water or brown water, greywater or
any other biodegradable effluent. They are typically applied as post-treatment
for up flow anaerobic sludge blanket reactors or for further treatment
after activated sludge treatment. In any case, primary sedimentation is
compulsory to avoid clogging of the filter bed and a secondary clarification
step and post-treatment of excess sludge (e.g. in sedimentation
ponds, unplanted drying beds, planted drying beds or anaerobic digesters) is
also compulsory.
Trickling filters can be built in almost all environments, but special

adaptations for cold climates are required. Proper insulation, reduced effluent
recirculation, and improved distribution techniques can lessen the impact of
cold temperatures.
Advantages:
• Simple, reliable, biological process.

• Suitable in areas where large tracts of land are not available for land
44
Environmental
intensive treatment systems. Biotechnology in Waste
Water Treatment
• May qualify for equivalent secondary discharge standards.
• Effective in treating high concentrations of organics depending on the
type of medium used.
• Appropriate for small- to medium-sized communities.
• Rapidly reduce soluble BOD5 in applied wastewater.
• Efficient nitrification units.
• Durable process elements.
• Low power requirements.
• Moderate level of skill and technical expertise needed to manage and
operate the system.
Disadvantages:
• Additional treatment may be needed to meet more stringent discharge

standards.
• Possible accumulation of excess biomass that cannot retain an aerobic
condition and can impair TF performance (maximum biomass thickness
is controlled by hydraulic dosage rate, type of media, type of organic
matter, temperature and nature of the biological growth).
• Requires regular operator attention.
• Incidence of clogging is relatively high.
• Requires low loadings depending on the medium.
• Flexibility and control are limited in comparison with activated-sludge
processes.
• Vector and odor problems.
• Snail problems.

Treatment Process and Basic Design Principles
Membrane Bioreactors combine conventional biological treatment

(e.g. activated sludge) processes with membrane filtration to provide an
advanced level of organic and suspended solids removal. When designed
accordingly, these systems can also provide an advanced level of nutrient
removal. In an MBR system, the membranes are submerged in an aerated
biological reactor. The membranes have porosities ranging from 0.035
microns to 0.4 microns (depending on the manufacturer), which is considered
between micro and ultra filtration.
This level of filtration allows for high quality effluent to be drawn through
the membranes and eliminates the sedimentation and filtration processes
typically used for wastewater treatment. Because the need for sedimentation
45
Overview of is eliminated, the biological process can operate at a much higher mixed
Environmental Bi
Biotechnology liquor concentration. This dramatically reduces the process tankage required
and allows many existing plants to be upgraded without adding new tanks.
To provide optimal aeration and scour around the membranes, the mixed
liquor is typically kept in the 1.0-1.2% solids range, which is 4 times that of a
conventional plant.
Fig. 2.14 Typical schematic for membrane bioreactor system. Source: FITZGERALD
(2008)
Membrane
During MBR wastewater treatment, solid–liquid separation is achieved

by Microfiltration (MF) or Ultra filtration (UF) membranes. A membrane is
simply a two-dimensional material used to separate components of fluids
usually on the basis of their relative size or electrical charge. The capability
of a membrane to allow transport of only specific compounds is called semi-
permeability (sometimes also perm selective). This is a physical process,
where separated components remain chemically unchanged. Components that
pass through membrane pores are called permeate, while rejected ones form
concentrate or retentate.
There are five types of membrane configuration which are currently in

operation:
• Hollow fiber (HF)

• Spiral-wound
• Plate-and-frame (i.e. flat sheet (FS)
• Pleated filter cartridge
• Tubular
Fig. 2.15 Hollow-fiber (HF) membrane module (Zenon, Canada) filtrating activated
46 sludge under vacuum. Source: RADJENOVIC et al. (2008)
Environmental
Pre-treatment Biotechnology in Waste
Water Treatment
To avoid unwanted solids in the waste stream, which enters the membrane
tank, fine screening is an essential pre-treatment step. This minimizes an
accumulation of solids and protects the membrane from damaging debris and
particles, extends the membrane life, reduces operating costs and guarantees
a higher sludge quality as well as a trouble free operation.
Cost Considerations
Although MBR capital and operational costs (membranes, oxygen utilisation,

expert design, etc.) exceed the costs of conventional process, it seems that the
upgrade of conventional process occurs even in cases when conventional
treatment works well. This can be related to increase of water prices and the
need for water reuse as well as with more stringent regulations on the effluent
quality.
Operation and Maintenance
Most MBRs employ chemical maintenance cleaning on a weekly basis,

which lasts 30–60 min, and recovery cleaning when filtration is no longer
durable, which occurs once or twice a year. A deposit that cannot be removed
by available methods of cleaning is called “irrecoverable fouling”. This
fouling builds up over the years of operation and eventually determines the
membrane life-time. All O&M tasks have to be done by skilled workers.
Fouling
Modern systems are maintained with chemicals, i.e. it is not necessary to

remove the membranes from the membrane tank. Organic fouling can be
cleaned with as sodium hypochlorite and inorganic fouling with oxalic acid.
Fig. 2.16 Fouling mechanisms. Source: RADJENOVIC et al. (2008)
Fouling occurs as a consequence of interactions between the membrane and

the mixed liquor, and is one of the principal limitations of the MBR process.
Fouling of membranes in MBRs is a very complex phenomenon with diverse
interlinkages among its causes, and it is very difficult to localize and define
membrane fouling clearly. The main causes of membrane fouling are:
• Adsorption of macromolecular
• Growth of biofilms on the membrane surface
• Precipitation of inorganic matter
47
Overview of • Aging of the membrane
Environmental Bi
Biotechnology
Health Aspects
Operation and maintenance of MBR systems is generally carried out by

skilled laborers, which should be sufficiently trained regarding any health
risks. Sludge from the biological system should be dewatered
(e.g. mechanical dewatering or drying bed) and incinerated with the ashes
being stored in a controlled landfill.
In all likelihood
Working Principle Membrane Bioreactors (MBRs) combine conventional

biological treatment (e.g. activated sludge) processes
with membrane filtration to provide an advanced level
of organic and suspended solids removal.
Capacity/Adequacy Applicable in conventional wastewater plants.
Performance High
Costs High capital and operational costs.
Self-help Low
Compatibility
O&M Membranes need to be cleaned regularly.
Reliability High if membranes are maintained correctly.
Main strength Secondary clarifiers and tertiary filtration processes
are eliminated, thereby reducing plant footprint.
Main weakness High operation and capital costs (membranes).
Applicability
Membrane Bioreactor systems are widely used in municipal and industrial

wastewater treatment plants. Furthermore, MBRs are also suitable
for landfill leachate treatment. It is a high-tech system that needs expert
design and professional operators.
Advantages
1) Smaller footprint (new WWTPs) or higher hydraulic throughput

(existing WWTPs) Large clarifiers no longer are needed. A smaller often
rectangular shaped chamber, fitted with the membrane cassettes replaces
the secondary clarifier whose size is governed by hydraulic and solids
loading. On top, because of the higher biomass concentrations that can
be sustained within the bioreactors, the same total mass of solids is
stored in a smaller tank, resulting in up to 50% smaller footprint.
2) High-quality effluent, free of bacteria and pathogens
In comparison to the activated sludge (CAS) process, the effluent is free
of suspended solids and reduced bacterial and viral content. Therefore,
48
Environmental
minimum disinfection is required. Biotechnology in Waste
Water Treatment
Therefore, the MBR process easily allows the treated effluent to be
discharged to sensitive receiving bodies or to be reclaimed for
applications such as urban irrigation, utilities or toilet flushing.
Meanwhile, it is also of high quality for feeding directly to a reverse
osmosis (RO) process.
This is becoming increasingly crucial in the view of the strict effluent
quality requirements imposed by local regulations taking effect during
the recent years and in the near future.
3) Higher automation capabilities
The operation of the MBR system can be fully automated, minimising
operators intervention that are typically required for conventional
treatment plants. This means that the MBR process can be easily
implemented also in decentralized sites.
Disadvantages
High operation and capital costs (membranes)
Membrane complexity and fouling
Energy costs
1. Write advantages and disadvantages of trickling filter and membrane

bioreactors.
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2.5.4 Anaerobic Wastewater Treatment

Anaerobic wastewater treatment is a biological process where
microorganisms degrade organic contaminants in the absence of oxygen. In a
basic anaerobic treatment cycle, wastewater enters a bioreactor receptacle.
The bioreactor contains a thick, semi-solid substance known as sludge, which
is comprised of anaerobic bacteria and other microorganisms. These
anaerobic microorganisms, or “anaerobes,” digest the biodegradable matter
present in the wastewater, resulting in an effluent with lower biological
49
Overview of oxygen demand (BOD), chemical oxygen demand (COD), and/or total
Environmental Bi
Biotechnology suspended solids (TSS), as well as biogas byproducts.
Anaerobic wastewater treatment is used to treat a variety of industrial effluent

streams from agricultural, food and beverage, dairy, pulp and paper, and
textile industries, as well as municipal sewage sludge and wastewater.
Anaerobic technologies are typically deployed for streams with high
concentrations of organic material (measured as high BOD, COD, or TSS),
often prior to aerobic treatment. Anaerobic treatment is also used for
specialized applications, such as treatment of waste streams with inorganic or
chlorinated organic, and is well-suited for treating warm industrial
wastewater.
Anaerobic contact reactor Upflow reactor

Fig. 2.17: Anaerobic wastewater treatment and Upflow reactor
Working of anaerobic wastewater treatment
Anaerobic wastewater treatment is a type of biological treatment where

anaerobic microorganisms are used to break down and remove organic
contaminants from wastewater. While anaerobic treatment systems may take
a variety of forms, they generally include some form of bioreactor or
repository capable of maintaining the oxygen-free environment needed to
support the process of anaerobic digestion.
The anaerobic wastewater treatment process consists of two stages: an

acidification phase followed by a methane production phase, with both
processes occurring in dynamic equilibrium. In the initial acid-forming
phase, anaerobes break down complex organic compounds into simpler,
short-chain volatile organic acids. The second phase, known as the methane-
production phase, consists of two steps: acetogenesis, where anaerobes
synthesize organic acids to form acetate, hydrogen gas, and carbon dioxide;
and methanogenesis, where the anaerobic microorganisms then act upon
these newly-formed molecules to form methane gas and carbon dioxide.
These byproducts can be reclaimed for use as fuel, while the wastewater can
be routed for further treatment and/or discharge.
Depending upon specific application needs and facility requirements,

anaerobic digester systems can be designed as single- or multi-stage
units, meaning that they can be configured with a separate acidification
50 tank and bioreactor unit. Common types of anaerobic wastewater
Environmental
treatment systems include the following: Biotechnology in Waste
Water Treatment
i) Anaerobic lagoons
Anaerobic lagoons are large man-made ponds, typically ranging between

1-2 acres in size, and up to 20 feet deep. They are used widely for
treatment of agricultural wastewater resulting from meat production, as
well as treatment of other industrial wastewater streams, and as a
primary treatment step in municipal wastewater treatment. Wastewater is
typically piped into the bottom of the lagoon, where it settles out to form
an upper liquid layer, and a semi-solid sludge layer. The liquid layer
prevents oxygen from reaching the sludge layer, allowing a process of
anaerobic digestion to break down the organic materials in the
wastewater. On average, this process can take as little as a few weeks, or
up to six months to bring BOD/COD levels to the target range.
Anaerobic bacteria favor certain environmental conditions, such as warm
water temperatures (85-95° F) and a near-neutral pH, therefore,
maintaining optimal conditions will enhance the rate of anaerobic
microorganism activity, resulting in a shorter wastewater detention time.
The rate of anaerobic respiration can also be limited by a number of
factors, including fluctuations in BOD/COD concentration, and presence
of substances such as sodium, potassium, calcium, and magnesium.
Fig. 2.18 Anaerobic lagoons
ii) Anaerobic sludge blanket reactors
Sludge blanket reactors are a type of anaerobic treatment where

wastewater is passed through a free-floating “blanket” of suspended
sludge particles. As the anaerobes in the sludge digest the organic
constituents in the wastewater, they multiply and collect into larger
granules that settle to the bottom of the reactor tank, and can be recycled
for future cycles. The treated effluent flows upward and out of the unit.
Biogases resulting from the degradation process are collected by
collection hoods throughout the treatment cycle.
Anaerobic sludge blanket reactors are available in a few different forms,

including:
• Up flow anaerobic sludge blanket (UASBs): In UASB treatment, 51

Overview of wastewater is pumped into the bottom of a UASB bioreactor with
Environmental Bi
Biotechnology upward flow applied. This causes the sludge blanket to float as the
wastewater flows through it.
• Expanded granular sludge beds (EGSBs): EGSBs are very similar
to UASB technology, with the key distinguishing factor being that
the wastewater is recalculated through the system to promote greater
contact with the sludge. They are also typically taller than UASBs,
and influent flows are sustained at a higher velocity. As a result,
EGSBs are able to treat streams with higher loads of organics
comparative to UASB systems.
• Anaerobic baffled reactors (ABRs): ABRs are constructed with
semi-enclosed compartments that are separated by alternating
baffles. The baffles interrupt the smooth flow of the wastewater
stream, encouraging greater contact with the sludge blanket as it
travels from the reactor inlet to outlet.
iii) Anaerobic filter reactors
Anaerobic filter reactors are comprised of a reactor tank outfitted with a

fixed filter medium of some kind. Anaerobic microorganisms are
allowed to establish themselves on the filter media, forming what is
known as a biofilm. Filter media vary from one system to the next, with
common materials including plastic films and particles, as well as gravel,
pumice, bricks and other materials. New filter media must be inoculated
with anaerobes, and the biofilm may take several months to become
established to the point that it is ready for treatment at full capacity.
During treatment cycles, the wastewater stream is passed through the

filter media, which serves to capture particles from the stream, while also
providing ample surface area for exposing anaerobes in the biofilm to the
organic materials present in the stream. Filter reactor performance must
be carefully monitored over time, as the filter media will eventually
become clogged with excess biofilm and particulate buildup, requiring
maintenance steps such as backwashing and cleaning to maintain optimal
performance.
Advantages:
• Formation of biogas:
The encountered organic pollutant is converted into biogas with a high
energetic value. This, for example, allows the energy needed to operate
the water purification system to be fully or partly recuperated.
• High loads
The volumetric load (COD load per m³ active volume per day) in an
anaerobic reactor is typically 5 to 10 times higher than aerobic
wastewater purification.
52
Environmental
• Very little sludge production Biotechnology in Waste
Water Treatment
Sludge growth in an anaerobic reactor is 4-5 times lower than in an
aerobic system.
Working in campaigns is possible. If the anaerobic sludge is not fed, it will

hibernate, which means longer periods without food can be spanned without
excessive sludge mortality. The system will almost immediately become
active after re-start.
Disadvantages:
• Incomplete break-down of organic compounds: Need for post-

purification via, for example, aerobic purification;
• No thorough nutrient removal: Again, later aerobic purification with
nutrient removal is often needed;
• Most efficient purification in the mesophilic range, i.e. between 30-37°C,
whereby the influent must be heated in most cases;
• Less robust system with regards to toxicity and inhibition;
• Risk of odour problems.
2.6 SOME RECENT DEVELOPMENT OF

ADVANCED BIOTECHNOLOGY FOR
Highly efficient materials and technologies for wastewater treatment are
urgently needed due to the aggravation of the water crisis in the green
twenty-first century. So some recent advanced and emerging biotechnology
methods are-
a. Moving bed biofilm reactor

b. Anaerobic ammonia oxidation process
c. Denitrifying anaerobic methane oxidation
d. Integrated biotreatment processes
 Simultaneous partial nitrification, anammox, and denitrification
process in integrated fixed film activated sludge
 Combined up-flow anaerobic sludge blanket biological aerated filter
e. Combined advanced oxidation-biotreatment process
 Fenton’s reaction coupled with a biotreatment Process
 Ozonation coupled with biotreatment process
f. Biosensors for environmental monitoring
 Optical biosensor
 Electrochemical biosensor
53
Overview of g. Other biosensors
Environmental Bi
Biotechnology
Besides optical and electrochemical biosensors, there are many other
biosensors also applied in environmental monitoring. For example a
paper-based multi-anode microbial fuel cell combined with power
management systems for wastewater, which was a self-support real time
biosensor and it achieved 28 times data transmission per charging cycle,
posing great potentials in in-situ monitoring. Reliable, highly sensitive,
and practical online monitoring of water quality biosensors based on
single-chambered microbial fuel cells.

b) Check your answer with the one given at the end of this unit..
1. Write about some recent advances of biotechnology in wastewater
treatment. (Answer in about 200 words)
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……………………………………………………………………………
2.7 LET US SUM UP

A scarcity of water supply and inadequate wastewater treatment combined
with intensified industrial activity have led to increased contamination in
lakes, rivers, and other water bodies in developing countries. Nevertheless,
some common techniques for wastewater treatment are not practicable for
developing countries. The biotechnological approach is considered an
important tool for waste water treatment. The biological method is the
method of choice for nutrient removal from wastewater because of its low
cost and environmentally friendly. Also, it can be respected as a dynamic
force for integrated environmental protection that leads to sustainable
development. In this chapter, we show the useful tools of biotechnology to
wastewater treatment such as activated sludge, membrane bioreactors, media
filters, anaerobic treatment, biosorption, biocatalysts, soil biotechnology, and
hydroponic system that are feasible in the context of the developing countries
in addition to the role of the genetic engineering in this context
2.8 KEY WORDS

Activated sludge : A biological treatment process for domestic
or industrial wastewater which converts
soluble, organic matter to solid biomass.
54 Advanced oxidation : An advanced oxidation process (AOP) is one
process that uses the hydroxyl radical to achieve Environmental
Biotechnology in Waste
oxidation. There are several different process Water Treatment
that generate hydroxyl radicals.
Aeration : The process of increasing oxygen saturation
by introducing air. This is commonly used in
secondary wastewater treatment during the
activated sludge process.
Aeration tank : A tank used in the aeration process to inject
air into the water.
Aerobic : An environment that contains sufficient
oxygen for the micro-organisms to use for the
oxidation of pollutants.
Anaerobic : An environment where there is an absence of
oxygen and no oxidised species (for example
nitrates, sulphates etc).
Bacterial water : Contamination of water with unwanted
contamination bacteria.
Bag filter : A disposable sediment filter designed to be
used as a barrier or sift method for removing
particulates from water.
Bed load : Sediment particles resting on the bottom of
water channel which are pushed along by the
flow of water.
Biochemical (Biological) : A measure of the amount of dissolved oxygen
Oxygen Demand (BOD) (measured in mg/L) required for the
decomposition of organic matter by micro-
organisms. BOD is most frequently used as a
test method to gauge the effectiveness of
water treatment processes. This is usually
specified as a BOD5 which is the oxygen
required to oxidise the biodegradable organics
in 5 days.
Biodegradable : Pollutants that can decompose naturally.
pollutants
Biological contaminants : Living organisms that can have negative
health effects on humans.
Bioreactors : Systems that support biological processes
under controlled conditions. Activated sludge
bioreactors are commonly used in water
treatment.
Bioremediation : The biological treatment of wastewater and
sludge by inducing the breakdown of organics
and hydrocarbons to carbon dioxide and
water.
Chemical Oxygen : A measure of the organic pollutants in water.
Demand (COD) Measure in mg/L or ppm
Membrane : A thin, semi-permeable skin which allows
water some particles to pass through but not
others.
55
Overview of Membrane BioReactors : A combination of biological treatment
Environmental Bi
Biotechnology (MBR) processes and membrane filtration processes
for wastewater treatment.
Membrane filtration : Separating dissolved and suspended solids in
a wastewater solution by using a membrane.
Oxidation : A chemical reaction involving the transfer of
electron, specifically the loss of electrons.
This is often achieved by the addition of
oxygen to a chemical.
Primary water : Removal of solids in sewage through
treatment sedimentation/clarification
Screening : Use of screens to remove floating substances
and solids from sewage.
Secondary treatment : Biological treatment of contaminants from
primary wastewater treatment.
Sedimentation : Particles and substances being deposited in
water due to a natural process, usually
gravity.
Sludge : A residue, containing microorganisms and
products.
Tertiary treatment : The third level of wastewater treatment that is
used to remove nutrients and completes
advanced treatment or disinfection that is
needed.
Trickling filter : A treatment unit that contains material with a
bacteria film over which water is trickled. The
bacteria break down the organic wastes.
Wastewater : The used water from a place that contains
dissolved or suspended matter.
Wastewater Treatment : Facilities designed for the treatment of
Plants wastewater.
Water Pollution : Material that has enough concentration in
water to be harmful.

Biotechnology finds application fields in the treatment of wastewaters by

biological methods and disposal of solid wastes by composting technique in
environmental engineering. The methods based on biotechnology in
wastewater treatment are activated sludge, trickling filters, oxidation ponds,
biofilters and anaerobic treatment. Furthermore, solid waste composting
techniques, biotrickling filters and biosorption are the examples of
biotechnology applications in environmental engineering. In all these
methods, it is essential to find suitable microorganisms that will degrade
organic substances and to complete the treatment process in favorable
56 conditions.
Environmental
Check Your Progress 2 Biotechnology in Waste
Water Treatment
The activated sludge process is one of the biological methods of industrial
waste waste treatment. The activated sludge process consists of converting
the suspended and particulate organic matter in the raw waste water into
harmless end products and new cells growth with the help of Anaerobic and
Aerobic Microorganisms.
In this process Activated Sludge is developed when these micro-organisms

are allowed to coagulate with the organic matter in the waste water to form
“Flocs’.
Under the activated sludge treatment process, the biodegradable organic

matter is consumed by a mass of micro-organisms under aerobic conditions,
resulting in:
1. Micro organisms consuming oxygen to satisfy their energy demand.

2. Formation of excess sludge generated from a surplus of living and inert
matter in the wastewater.
3. BOD and COD of the effluent is reduced maximum to the extent of 80 %
and 65 % respectively.
Trickling filter-
Advantages:
 Simple, reliable, biological process.
 Suitable in areas where large tracts of land are not available for land
intensive treatment systems.
 May qualify for equivalent secondary discharge standards.
 Effective in treating high concentrations of organics depending on the
type of medium used.
 Appropriate for small- to medium-sized communities.
 Rapidly reduce soluble BOD5 in applied wastewater.
 Efficient nitrification units.
 Durable process elements.
 Low power requirements.
 Moderate level of skill and technical expertise needed to manage and
operate the system.
Disadvantages:
 Additional treatment may be needed to meet more stringent discharge
standards.
 Possible accumulation of excess biomass that cannot retain an aerobic
condition and can impair TF performance (maximum biomass thickness 57
Overview of is controlled by hydraulic dosage rate, type of media, type of organic
Environmental Bi
Biotechnology matter, temperature and nature of the biological growth).
 Requires regular operator attention.
 Incidence of clogging is relatively high.
 Requires low loadings depending on the medium.
 Flexibility and control are limited in comparison with activated-sludge
processes.
 Vector and odor problems.
 Snail problems.
Membrane bioreactors-
Advantages
 Smaller footprint (new WWTPs) or higher hydraulic throughput
(existing WWTPs)
 High-quality effluent, free of bacteria and pathogens
 Higher automation capabilities
Disadvantages
 High operation and capital costs (membranes)
 Membrane complexity and fouling
 Energy costs
Highly efficient materials and technologies for wastewater treatment are

urgently needed due to the aggravation of the water crisis in the green
twenty-first century. So some recent advanced and emerging biotechnology
methods are-
a. Moving bed biofilm reactor

b. Anaerobic ammonia oxidation process
c. Denitrifying anaerobic methane oxidation
d. Integrated biotreatment processes
 Simultaneous partial nitrification, anammox, and denitrification
process in integrated fixed film activated sludge
 Combined up-flow anaerobic sludge blanket biological aerated filter
e. Combined advanced oxidation-biotreatment process
 Fenton’s reaction coupled with a biotreatment Process
 Ozonation coupled with biotreatment process
f. Biosensors for environmental monitoring
 Optical biosensor
58
Environmental
 Electrochemical biosensor Biotechnology in Waste
Water Treatment
g. Other biosensors
2.10 SUGGESTED FURTHER

READING/REFERENCES
• Alleman, J. E. (2000). The history of fixed-film wastewater treatment
systems.
• Buyukgungor, H., & Gurel, L. (2009). The role of biotechnology on the
treatment of wastes. African Journal of Biotechnology, 8(25).
• Darweesh, M., Zedan, A. M., El-Banna, A., Elbasiuny, H., & Elbehiry,
F. (2021). Biotechnology for Green Future of Wastewater Treatment.
• Gunnar Stensen, I. M. T., Hanserud, U. O. S., & NORAGRIC, U. THT
281 Term paper Appropriate sanitation under stress.
• Heeb, J. Jenssen, P. Gnanakan Conradin, K. (2008): Ecosan Curriculum
2.3. Switzerland, India and Norway: seecon international, International
Ecological Engineering Society, Norwegian University of Life Sciences,
ACTS Bangalore [Accessed: 10.06.2019]
• Mountain Empire Community College (n.y): Trickling Filters - Filter
Classification. Virginia: Mountain Empire Community
College [Accessed: 17.05.2012]
• Ridderstolpe, P. (2004). Introduction to greywater management.
EcoSanRes Programme.
• Samer, M. (2015). Biological and Chemical Wastewater Treatment
Processes. In (Ed.), Wastewater Treatment Engineering. IntechOpen.
https://doi.org/10.5772/61250
• Sasse, L. (1998). DEWATS: Decentralised wastewater treatment in
developing countries. BORDA, Bremen Overseas Research and
Development Association.
• Shahot, K., Idris, A., Omar, R., & Yusoff, H. M. (2014). Review on
biofilm processes for wastewater treatment. Life Sci J, 11(11), 1-13.
• Strauss, M. (1996, July). Health (pathogen) considerations regarding the
use of human waste in aquaculture. In Environmental Research
Forum (Vol. 5, No. 6, pp. 83-98).
• Tchobanoglus, G., Burton, F., & Stensel, H. D. (2003). Wastewater
engineering: treatment and reuse. American Water Works Association.
Journal, 95(5), 201.
• Technologien, N., & Wirtschaftsberatung, B. U. (2001). Anaerobic
treatment of municipal wastewater in UASB-reactors. TBW GmbH:
Frankfurt, Germany.
• Tilley, E. (2014). Compendium of sanitation systems and technologies.
Eawag. 59
Overview of • Tilley, E., Lüthi, C., Morel, A., Zurbrügg, C., & Schertenleib, R. (2008).
Environmental Bi
Biotechnology Compendium of Sanitation Systems and Technologies
Dübendorf. Zwitzerland, Swiss Federal Institute of Aquatic Science and
Technology (EAWAG).
• https://academicjournals.org/article/article1380817563_Buyukgungor%2
0and%20Gurel.pdf
• https://organicabiotech.com/how-is-biotechnology-used-in-sewage-
treatment/
• https://sswm.info/search/all?keys=activated+sludge
• https://www3.epa.gov/npdes/pubs/trickling_filter.pdf
60
Environmental
UNIT 3 ENVIRONMENTAL Biotechnology for Solid
Waste Management
BIOTECHNOLOGY FOR SOLID
WASTE MANAGEMENT
Structure
3.1 Introduction
3.2 What is Solid Waste?
3.3 Municipal Solid Waste (Msw)
3.3.1 Hazardous Waste
3.3.2 Non- Hazardous Waste
3.4 Sources of Solid Waste
3.5 Classification of Waste
3.5.1 Based on Hazardous Potential
3.5.2 Based on Content
3.5.3 Based on Origin
3.6 Solid Waste Management (SWM)
3.6.1 Collection
3.6.2 Segregation and Storage
3.6.3 Transportation
3.6.4 Treatment and Disposal
3.6.4.1 Landfilling
3.6.4.2 Bioreactor Landfill
3.6.4.3 Composting
3.6.4.4 Vermi-Composting
3.7 Biotechnological Advancements in Solid Waste Management
3.7.2 Objective of Bioremediation
3.7.3 Principle of Bioremediation
3.7.4 Categories of Bioremediation
3.7.4.1 Phytoremediation
3.7.4.2 Microbial Remediation
3.7.5 Types of Bioremediation Methods
3.7.5.1 In situ bioremediation
3.7.5.2 Ex situ bioremediation
3.7.6 Advantages of Bioremediation
3.7.7 Limitations of Bioremediation
3.8 Role of Biotechnology in Solid Waste Management
3.9 Resource recovery
3.9.1 Biomethanation
3.10 Summary
3.11 Key Words
61
Overview of 3.12 Suggested Further Reading/References
Environmental Bi
Biotechnology 3.13 Answers to Check Your Progress
3.2 INTRODUCTION
With the growing civilization and improvement in the living standard of
human beings, a direct or indirect impact has always been faced by nature. A
tremendous amount of waste has been generated by the ever-increasing
population that can harm the environment if not properly disposed of or
treated. Most developed countries have effective waste management
technologies to deal with the waste hence; they prevent its entry into the
environment. But most developing countries do not have such expensive
technologies, so the litter generated impacts the environment. Every day solid
waste production is 0.1 kg in small towns, 0.3-0.4kg in medium towns, and
0.5 kg in large cities per capita. Every country has stipulated rules and
regulations for the collection, handling, and disposal of solid wastes
generated from mines and industries still, there was no specific set of rules
for handling solid waste generated due to community activities as a result of
urbanization, population growth, changes in the pattern of dwelling and
lifestyle (Municipal or Urban solid waste) until the last decade resulting in a
tremendous production of Municipal Solid Waste, which is now controlled
mainly by Urban Local Bodies. Scientific input in solid waste management
has controlled waste to a great extent. Further, biotechnological approaches
have been researched to improve wastewater treatment methods.
3.3 WHAT IS SOLID WASTE?

The unwanted material that is left behind after everyday human activities,
residues, and refuses from plants and animals is said to be waste. It has no
further user value and is required to be disposed of. Wastes may be solids like
food waste, plastics, and glass; liquids like sewage, oil, and fats, or gaseous
like greenhouse gases, hydrocarbons, CFC, etc. Solid wastes are refused that
are non-liquid in nature and arise from households, trade, industries,
agricultural and commercial activities, and public service sectors. The waste
from Water Treatment Plant, Water Supply Treatment Plant, and Air
Pollution Control Facility as sludge or in some other form along with semi-
solids, compact amount of liquid, industrial or commercial gaseous materials,
or any kind of solids that have been discarded are also solid waste.
3.4 MUNICIPAL SOLID WASTE (MSW)

MSW is a heterogeneous mixture of diverse-abandoned materials like food
waste, kitchen waste, any kind of paper, metal, glass, or plastic packaging,
discarded clothing, garden waste, furnishing, ash as well as dust, which may
be compostable or non-compostable. MSW can be classified as-
62
Environmental
3.3.1 Hazardous Waste Biotechnology for Solid
Waste Management
Hazardous wastes pose a potential or substantial hazard to human health or
the environment when not managed properly. These are products such as
paints, cleaners, oils, batteries, and other materials that carry ingredients that
are potentially hazardous and require special attention and care while
disposing of, like pesticides, laboratory chemicals, radioactive substances,
etc. The waste generally possesses at least one of the four characteristics:
reactivity, ignitability, corrosivity, and toxicity.
Reactivity: Substances that are unstable and can easily react under normal
conditions to create different threats to living beings.
Ignitability: Substances that have the potential to burn easily at a rapid rate.
Corrosivity: Substances that have the potential to corrode metal surfaces

thereby degrading them. Toxicity: Substances that have the capability to
create toxic conditions for living organisms even in a small amount.
3.3.2 Non-Hazardous Waste

Any solid waste, biodegradable or non-biodegradable that is not toxic,
corrosive, ignition or reactive is considered non-hazardous solid waste.
3.4 SOURCES OF SOLID WASTE

There are various sources of solid waste generation. Some of these sources
are listed below:
1. Residential
2. Commercial establishments
3. Industrial
4. Street sweeping
5. Institutional premise (school, colleges, etc.)
6. Hospitals
7. Construction and demolition material
8. Agricultural activities
3.5 CLASSIFICATION OF WASTE:

Waste is classified on the basis of content, origin and hazardous potential
(FIG 1):
63
Overview of
Environmental Bi
Biotechnology
Fig 3.1: Different categories of waste.
3.5.1 Based On Hazardous Potential

Radioactive: These wastes may be the byproducts of any of the following-
nuclear reactors, hospitals and research facilities, processing plants for fuel.
Infectious:Wastes containing bodily fluids and blood, discarded samples,

stocks and cultures of infectious agents from the laboratory, or waste from
infected patients like bandages, swabs, used medical supplies.
Toxic:any kind of discarded material in any form that can be harmful (for
example, if it is swallowed, ingested, or absorbed via the skin).
3.5.2 Based On Content

Plastic: Plastic bags, plastic containers, toys, styro foam, etc.
Metals: Metal wires, parts of automobiles or electrical appliances, iron or

mate steel, etc.
Glass: Bottles, jars, broken glass, etc.
Organic: Discards and peelings of vegetables and fruits, egg shells, spoilt
food, bones from meat and fish, tree branches, grass clippings, flowers,
plants, leaves, etc.
3.5.3 Based On Origin

Industrial: Manufacturing and material processing trade generated waste.
Domestic: Kitchen materials, house cleaning waste, papers, crockery waste,

furnishing remains, packaging, bottles, plant waste and garden trimmings,
etc.
Commercial: Waste produced at departmental stores, business premises,

offices, shops, markets, organic as well as inorganic hazardous and
64 chemically reactive waste
Environmental
Construction: Frequent construction of buildings, digging and construction Biotechnology for Solid
of roads and other activities leaving behind earth, brick stones, wooden logs, Waste Management
etc.
3.6 SOLID WASTE MANAGEMENT (SWM)

SWM is the way to reduce a load of solid waste before it enters the
environment. This is achieved by following the four key Steps: Collection,
Segregation and Storage, Transportation and Treatment and Disposal
3.6.1 Collection
In this step, the waste collection is conducted in various ways. The best way
of collection that is considered for effective waste management is the Door-
to-Door collection method. Many NGOs work with government bodies to
implement this method in developing countries.
3.6.2 Segregation and Storage

This is a crucial stage for the success of solid waste management in any
country. Waste is collected in a segregated form from the owner. Different
dustbins are provided to the owner to store the different types of waste. This
technique helps to keep the waste segregated as biodegradable, non-
biodegradable, and recyclable.
3.6.3 Transportation
After waste collection, it is essential to take the waste to the treatment plant
safely. Different transportation facilities are used for this purpose like; hand
carts, animal carts, trucks, etc. Care is taken that waste should not be spilled
while transporting.
3.6.4 Treatment and Disposal

With different types of wastes, different treatment methods are applied.
These treatment processes are mention below-
3.6.4.1 Landfilling
Traditionally, this method was used to dump solid waste in large pits and
cover them with soil. The waste gets degraded due to an anaerobic reaction
hence the organic matter is reduced to organic acids and gases like methane,
carbon dioxide, hydrogen sulphide, etc. Sometimes the water-soluble organic
acids get leached down with runoff and contaminate groundwater. Further, it
is sprayed with insecticides to avoid incidents of mosquito breeding and flies.
Less than 60% moisture should be maintained for better breakdown, and
degradation of refuse leads to stabilization which takes about 2 to 12 months
to complete. The organic fraction with the soil then becomes humus which
helps to improve soil structure and quality.
65
Overview of Modern sanitary landfills are the improved version of the primitive types of
Environmental Bi
Biotechnology landfills. A liner system is developed in modern landfills that separate solid
waste from groundwater. It also contains a landfill cap that prevents the entry
of rainwater into the landfill. The liner system and landfill cap work together
to avoid leaching harmful substances into groundwater by keeping the
leachate inside the landfill and minimizing the leachate generation. When the
landfill is in an early (young) phase of its life, it stays in the acidogenic phase
means the fermentation process is much faster and produces a large number
of volatile fatty acids (VFA). In young landfills containing large amounts of
biodegradable organic matter, rapid anaerobic fermentation occurs, resulting
in VFA that is equivalent to almost 95% of the total organic content of the
landfill in terms of quantity. As the landfill ages, the acidogenic phase
changes to the methanogenic phase leading to the development of
methanogenic bacteria in the landfill. These methanogenic microorganisms
convert VFA to landfill gases, methane, and carbon dioxide while organic
fraction transforms to humus.
Advantages
• Landfilling is an easy and economical method.

• It does not require any kind of specialized equipment or plants.
• No special skilled labor is required for handling the landfill.
• Segregation of waste is not required.
• No waste byproduct is formed after the process.
Disadvantages
• A large land is required.

• Very important to cover the top layer.
• Foul smell is generated.
• Leaching of different constituents of waste may contaminate
groundwater.
3.6.4.2 Bioreactor Landfill

A bioreactor landfill is a method for municipal solid waste disposal and
treatment. In this method, the liquid is controlled, which helps
microorganisms to decompose the waste. The waste degradation and
stabilization process are accelerated by enhancing microbial activity. This is
achieved by adding the required amount of liquid and air to the reactor
(Fig.2). Bioreactor landfills work to quickly decompose and transform
organic wastes by recirculating leachate and/or adding more liquid wastes.
66
Environmental
Biotechnology for Solid
Waste Management
Fig.3.2 A schematic diagram of Bioreactor.
Types of Bioreactor Landfills
Aerobic - In an aerobic bioreactor landfill, other than re-circulating leachate

in a controlled manner, the air is also injected into the waste unit through
vertical or horizontal wells that help to accelerate aerobic microbial activity
and thus result in quick waste stabilization.
Anaerobic- In anaerobic bioreactor landfill, moisture is maintained by re-

circulating leachate from time to time as per requirement of waste mass. The
liquid is also sprinkled from the top to sustain the moisture content in the
waste. The whole process of biodegradation takes place in the absence of
oxygen. Anaerobic microbial degradation of waste results in landfill gas like
methane and carbon dioxide that can be pumped and stored to use for
different energy projects.
Hybrid (Aerobic-Anaerobic) -Both aerobic and anaerobic conditions are

maintained in hybrid landfill bioreactors. Aerobic conditions can facilitate the
degradation of organic matter and rapidly improve leachate quality. Further,
the anaerobic condition can produce gases that can be used for different
energy projects. The hybrid bioreactor landfill enhances waste degradation by
this sequential aerobic-anaerobic treatment to rapidly degrade waste in the
upper sections of the landfill and collect gas from the lower sections.
Pros
• Waste is stabilized very early in this process.

• Minimal leachate production leads to the reduction in the disposal cost of
leachate
• Increased capacity to handle waste.
• Increases the recovery percentage of landfill gases that could be used for
different purpose.
Cons
• The landfill is well engineered and hence, requires initial investment.

67
Overview of • It requires highly skilled labour for the operation and efficiency of the
Environmental Bi
Biotechnology landfill.
• It requires very controlled temperature and hence, requires maintenance.
• Odour control.
3.6.4.3 Composting
The decomposition of degradable/organic waste under aerobic or anaerobic

conditions can be called composting. Certain industries produce maximum
organic waste, i.e., food processing, paper, and agriculture. This type of
organic matter can be degraded and decomposed to form compost manure of
better quality. Compost manure is formed along carbon dioxide, methane,
energy, and water vapor as the end product when organic solid waste is
exposed to aerobic or anaerobic decomposition. Organic compounds are
decomposed via oxidation using aerobic microbes as oxidizing agents in
aerobic decomposition. The carbon dioxide formed during this process is
used as an energy source while nitrogen produced in the form of nitrogen
dioxide and nitrogen trioxide is recycled.
There are four general types of composting
1. Windrow composting- In this process, the organic solid waste is placed

in the form of a pile whose cross section is triangular, and the width and
height of the pile are less than their length. The height of this pile is
usually half of its width. It enables the generation of an adequate amount
of heat and allows sufficient oxygen diffusion to the center of the pile.
The ideal height is between 1.25- 2.75 m and width from 4.75-5.25 m.
Piles may be turned every week letting the composting take place.
2. Aerated static pile- It requires composting mixture to be placed in a pile

placed over a network of pile that are mechanically aerated with a
blower. The piles are broken up for the first time only when the process
of composting is almost complete. The land use in the Aerated static pile
method is comparatively less than that in windrow composting.
3. In-vessel composting- In this method, the organic waste matter is

enclosed in a closed vessel that can provide a sufficient amount of
controlled aeration, moisture, and mixing.
This method has more advantages over other composting methods as the
conditions required for composting can be easily regulated along with
adequate oxygen content leading to the formation of homogenized and good-
quality compost. The process requires one to four weeks, and if well
operated, there is very little to no amount of leachate and minimum odor.
Different factors affect the progress of composting. The major’s factors are
listed in the table 1 with the optimal range.
68
Environmental
Table 3.1: Factors affecting the composting process. Biotechnology for Solid
Waste Management
SNo. Factor Optimum range
1. Temperature 50o to 60oC
2. pH 5.8-7.2
3. Moisture 50 to 60 %
4. C/N ratio 25:1 and 30:1
5. Particle size 5-20cm
6. Surface area Large
7. Aeration Proper ventilation
Advantages of composting
• The product formed after the composting process i.e., compost, is a fully
organic fertilizer that enhances soil structure and soil quality.
• It improves the water retention capacity of the soil.
• Composting can be done in one’s own household or at the mass level.
• It is a feasible, eco-friendly, and an economical method to deal with
organic waste.
• Helps in reducing the quantity of waste and is very easy to produce with
just a few garbage pickups.
• In-vessel processing of compost requires lesser area and can control
odor.
• Nitrogen in compost is released at a slower rate hence available to plants
for a prolonged time.
Disadvantages of composting
• It may attract snakes, rats, and bugs.

• Unpleasant smell and physical appearance.
• The quality of compost depends on the type of organic waste. It is not
suitable for all kinds of organic waste.
• It requires an initial investment, space, and some monitoring.
• It is a time-consuming process.
1. Discuss the basic steps of solid waste management?
……………………………………………………………………………
……………………………………………………………………………
…………………………………………………………………………… 69
Overview of 3.6.4.4 Vermi-Composting
Environmental Bi
Biotechnology
It is the biotechnological approach for composting organic matter using
efficient earthworm species capable to improve the waste conversion process
and make good quality compost fertilizer. The vermicomposting process is
faster-composting than the microbial composting. Segregated biodegradable
waste is fed to the bed prepared in alternate layers of waste and cow dung
slurry each having a thickness of 15-20cm. The heap is watered and mixed
for initial 25 days to maintain the moisture content and earthworm
distribution. After 45 days the manure shall be kept heaped for a few days for
maturity. Three species of earthworm, Eisenia fetida, Eudrilus eugeniae, and
Perionyx excavates are widely employed for the composting of urban solid
waste and also, extract heavy metals like cadmium, lead, copper, manganese,
zinc, etc.
Advantages of vermi composting
• Vermicomposting is the fastest method for composting and requires less

than half of the time needed for other composting methods.
• Vermicompost boosts water holding capacity, porosity, drainage and
aeration in the soil.
• It improves the texture and structure of soil resulting in the reduction in
soil erosion.
• It regulates plant growth and increases the productivity.
• The compost enhances soil microbial activity and keeps the pests and
pathogens away.
• It is easily applicable, economical, odorless, and do not require any
expertise for handling.
• It does not contain any kind of harmful materials as well as pathogens.
Disadvantages of vermi composting
• If vermicomposting is not carried out properly, it may release bad odor.

• It requires a lot of attention and care and the bin must be properly
covered to avoid the attack of rodents and flies.
• The process needs 2-3 months for finalization which is quiet long.
• A lot of maintenance is required in respect of temperature for better
degradation by earthworms.
3.7 ADVANCEMENTS IN SOLID WASTE

MANAGEMENT
This section describes the few advancements in solid waste management.
70
Environmental
3.7.1 Bioremediation Biotechnology for Solid
Waste Management
Bioremediation is the use of microorganisms (Bacteria, Fungi or
Actinomyces) to degrade organic waste materials under partial or fully
controlled conditions. Through bioremediation toxic elements can be
removed from organic waste like agricultural waste, domestic waste, etc.
3.7.2 Objective of Bioremediation

• Bioremediation direct towards completely destroying and or to bring
down the level of toxicity of organic pollutants upto the permissible
limit.
3.7.3 Principle of Bioremediation

 Bioremediation employs those microorganisms that use waste as a source
of food and thereby oxidize or reduce it into different harmless gases and
water.
 For effective bioremediation different parameters like light, temperature,
nutrients, microorganism growth, and food play an important role.
Therefore, sometimes these parameters are optimized for better
efficiency.
 The bioremediation treatment is time taking process. It basically depends
upon the type and amount of waste to be treated.
 Sometimes few external amendments are also added to enhance the
process. These amendments favor the growth of microorganisms and
thereby enhance the degradation rate.
 Various other factors like, temperature, light, pH, the concentration of
waste, etc., affect the bioremediation process.
3.7.4 Categories of Bioremediation

Biological remediation can be categorized into two types: phytoremediation
and microbial remediation
3.7.4.1 Phytoremediation
The process which employs various category of plants for removal,

degradation and stabilization of waste from soil and water. There are multiple
types of phytoremediation mechanisms.
1. Rhizo-degradation. The organisms associated with plant roots stimulate

soil microbial activity to speed up the breakdown of organic pollutants.
The region of soil that envelops the roots, known as the rhizosphere, is
where this process takes place. The organic pollutants are biodegraded as
a result of the carbohydrates and acids that is released by plants, which
encourage the activity of microorganisms.
2. Phyto-stabilization. In this process instead of being degraded the soil
pollutants are immobilized (fixed) by chemical compounds produced by 71
Overview of the plant roots. In order to prevent pollutants from leaking into the soil,
Environmental Bi
Biotechnology Phyto stabilization lowers the mobility of compounds in the
environment.
3. Phytoextraction. The contaminants are absorbed through the roots along
with water and other nutrients enabling the contaminant to reach
different parts of the plant. The contaminant can be recovered afterward
from the plant biomass. This method is mostly used for the recovery of
metals from waste.
4. Phyto-volatilization. Contaminants are absorbed by the plant during this
process and then evaporate into the atmosphere as a result of
transpiration.
5. Phyto-degradation. Phytodegradation is the process of utilizing plants
or microbes to break down organic contaminants in the soil or within the
plant itself. The plant roots secrete enzymes that break down organic
molecules. The plant then absorbs the degraded substances and releases
them through.
Plants, trees, weeds have natural ability to remove or degrade few

selectedcontaminants present that are present in soil, sludge, waste, water and
wastewater. A list of few plants species and the phytoremediation process
used for the contaminants removal is mentioned as table 2
Table 2 List of plants species and the phytoremediation process used for
the contaminants removal
S.No Plant/tree Contaminant Process

1. Brassica, Petroleum hydrocarbons Phytodegradation
Typha,
Jatropha,
Cassia
2. Helianthus Heavy metals (Cr, Cd, Pb, Phytostabilization
annuus, Zn)
Chenopodium
3. Poplar, scirpus, Organic/inorganic Phytovolatilization
Phragmites, contaminants (Se, Hg, As)
4. Eichhornia, Heavy metals and Rhizofiltration
Lemna radionuclides
5. Brassica, Inorganic components like Phytoextraction
Helianthus radionuclides and metals
annuus, Thapsi, (Cr, Cu, Ni, Zn, Cd, Ag)
3.7.4.2 Microbial Remediation
Microorganisms have the potential to degrade the different types of waste

material into products that are either non-toxic or less toxic in nature. This
tendency of microbes to degrade the waste is because it acts as a food source
72
Environmental
for them. This process of cleaning up waste with the help of microorganisms Biotechnology for Solid
is called bioremediation. Microorganism’s consortiums are used for cleaning Waste Management
different types of material like heavy metals, radioactive compounds, solid
waste, oil, petroleum hydrocarbon, etc. Microorganisms like bacteria, fungi,
yeasts, and actinomycetes or their mixed culture are implemented for the
removal of contaminants from the waste, soil, and water. Few of the species
are listed
1) Bacteria: Arthrobacter spp., Pseudomonas veronii, Sporosarcina

ginsengisoli, Kocuria flava, Bacillus cereus, and Burkholderia spp.
2) Fungi: Aspergillus versicolor, Penicillium canescens, and Aspergillus
fumigatus;
3) Algae: Cladophora fascicularis, Spirogyra spp. and Cladophora spp.,
and Spirogyra spp. and Spirullina spp.
4) Yeast: Candida utilis and Saccharomyces cerevisiae
3.7.4 Types of Bioremediation Methods

• Natural attenuation or intrinsic bioremediation: This type of
bioremediation takes place on its own and does not require addition of
anything.
• Bio-stimulation: This type of bioremediation occurs by adding
fertilizers to increase the bioavailability within the medium.
Technologies can be generally classified as in situ or ex-situ.
3.7.4.1 In Situ Bioremediation
In situ bioremediation is the removal of waste or contaminants directly at the

place of their origin.In situ bioremediation have been successfully employed
for the treatment of heavy metals, chlorinated solvents, oil, dyes, and other
hydrocarbons from the polluted sites. To enhance the in situ bioremediation
process following techniques are used.
• Bio-sparging: Biosparing is the process of injecting high-pressure air

below the water table to increase the dissolved oxygen level in
groundwater which will further trigger the activity of microorganism
thereby resulting in more degradation of contaminants.
• Bioaugmentation: The process of bioremediation involves introducing

waste-degrading microorganisms, either native or exogenous, to
contaminated locations.
• Bioventing: It entails feeding nutrients and air through wells to

contaminated soil to encourage local microorganisms. In order to
minimize volatilization and the release of contaminants into the
environment, bioventing uses modest airflow rates and only supplies the
amount of oxygen required for the biodegradation.
73
Overview of 3.7.4.2 Ex Situ Bioremediation Bi
Environmental
Biotechnology
It is the process of removing contamination away from its place of origin.
Different techniques are used for Ex-situ bioremediation.
• Landfarming:It is a simple procedure in which polluted soil is dug up,

put over a bed, and repeatedly tilled until toxins are broken down. The
intention is to promote the aerobic breakdown of pollutants by local
biodegradative bacteria.
• Composting: To convert organic solid waste into manure by the

microbial population.
• Bio pile: It is a hybrid method of landfarming and composting.Excavated

soils are combined with soil amendments, shaped into compost pile, and
sealed off for treatment in a bio-pile technique. The bio-pile system
consists of a leachate collection system, an aeration system, an irrigation
system, and a treatment bed. Bio pile provides an optimum environment
condition for the growth of indigenous aerobic and anaerobic
microorganisms.
• Bioreactors: Bioremediation in reactors involves the processing of

contaminated solid material (soil, sediment, sludge) or water through an
engineered containment system.

• It is an eco-friendly technique as it causes less harm to environment.
• This process is cheaper than the other methods of treatments.
• It also improves the structure of soil making it healthier.
• It is a natural way of treatment in which plants and microbes utilize
sunlight and oxygen from the environment for the degradation of waste.
• It helps in the permanent removal of waste by converting them into
water and carbon dioxide which is not toxic in nature.

• The process is time taking and depends on environmental variables.
• This process only works on biodegradable compounds.
• We may not determine the bioavailability and toxicity of biodegradation
products.
• In phytoremediation, sometimes the concentration of contaminant is very
high which can cause plant death.
• It requires a large land area for treatment specially in phytoremediation.
74
Environmental
3.8 ROLE OF BIOTECHNOLOGY IN SOLID Biotechnology for Solid
WASTE MANAGEMENT Waste Management
Biotechnology plays a crucial role in managing solid wastes by increasing the

pace of degradation of waste along with its efficiency. A few applications of
biotechnology in SWM are listed below-
• Sewage farms, which use sewage for fertilization and irrigation of

agricultural land, convert the suspended solids into humus providing the
plant's nutrients as well as the water in sewage provides irrigation to the
land.
• Biotechnological approaches like phytoremediation/ bioremediation can

help in converting industrial and chemical wastes into useful
products. This process uses plants and some other biota the remove or
degrades contaminants from soils, sediments, sludges, surfaces, and
groundwater.
• Biotechnology has low capital and operating costs; it reduces water and
energy usage and petrochemical solvents contributing to the reduction of
environmental footprints of any activity.
• The xenobiotic compounds can also be degraded using microorganisms

that have an inbuilt natural capacity to convert most of the xenobiotic
compounds. For example, Bacillus sp. S6 and S35 are used to degrade
petroleum hydrocarbons, Pseudomonas putida, P. aeruginosa, and
Acinetobacter faecalis are used to degrade pesticides glyphosate.
• Prof. Ananda Mohan Chakraborty and his team in 1970 developed a

“superbug” that was made to biodegrade a number of toxic organic
chemicals like octane, hexane, xylene, toluene, camphor and
naphthalene. Pseudomonas putida was engineered into superbug to clean
the sites contaminated with the oil.
• Bio-sparging is a biotechnological approach that can be used for the

treatment of wastes at sites that are polluted with petroleum products i.e.,
diesel, lubricating oil, gasoline, etc.
• During petroleum and gasoline extraction substantial solid waste is

generated. The waste generated is later on treated with nitrogen, oxygen
and phosphorous applying the process of bio-venting that increases the
rate of the removal of these solids
• Bioremediation of rubber waste using fungi such as Recinicium

bicolor which removes toxic compounds of rubber making it available
for devulcanization by oxidizing bacteria or sulfur. This rubber after the
removal of toxic compounds can be recycled easily.
75
Overview of
Environmental 3.9 RESOURCE RECOVERY Bi
Biotechnology
Resource recovery is the activity of minimizing waste and using it to its
immense potential. Resource recovery is done in the following steps-
• Reduction or minimal production of waste at the source

• Reusing waste materials
• Waste products that can be recycled should be separated for recycling
• Using the waste material as a source of renewable energy to recover
energy
• Treating the remaining waste after energy recovery
• Disposing of the waste through proper channels after treatment
The crucial step in resource recovery is avoiding waste and producing as

minimum waste as possible, but complete waste minimization is not possible
so resource recovery becomes important. The first step in resource recovery
is to separate the waste based on its origin and degree of degradation.
Separation of the waste can be done by either handpicking or by processing
through separating machines, or using separate bins at the source itself. After
waste separation, materials like glass, plastics, and metals are recycled;
stones, bricks, concrete, and soil are crushed; and screened for waste while
the remaining soil is added to the land or is used for landscaping.
Biodegradable materials like kitchen waste, plant remains, furniture, etc., are
degraded using techniques like landfilling, digestion, etc., to produce energy
and other products like compost manure, vermicompost, and biogas. Non-
biodegradable materials are transported and filled in government-certified
landfills.
3.9.1 Biomethanation
Bio methanation is the process of improving methane content in the biogas
produced during anaerobic digestion by introducing Hydrogen gas. The
injected Hydrogen gas and the carbon dioxide produced during the anaerobic
digestion are subjected to methanogenic bacteria and the archaeal community
leading to an increase in the methane content of biogas. Normally, biogas
produced during anaerobic digestion contains 40 to 75% methane and 25 to
60% carbon dioxide and other trace components. The biogas thus produced
has a low calorific value in comparison to natural gas which has more than
95% methane content. Hence, to improve the methane content in the biogas,
various purification methods are used. Methanation is one of the methods of
purification of biogas which uses hydrogenotrophic methanogens as reducing
agents. These hydrogenotrophic methanogens reduce the carbon dioxide
generated during anaerobic digestion using the injected hydrogen gas. Bio
methanation can be performed in two ways.
76
Environmental
In-situ bio-methanation is the process where Hydrogen gas is directly Biotechnology for Solid
introduced into an anaerobic digestor along with methanogens to produce Waste Management
biogas with high calorific value and manure.
In Ex-situ bio-methanation, the biogas produced during anaerobic

digestion is collected in a separate bioreactor. Then Hydrogen gas is
introduced into this bioreactor along with methanogens. The methanogens
reduce the carbon dioxide present in the biogas using artificially introduced
Hydrogen to produce methane-rich biogas.
3.10 LET US SUM UP

Escalating population and development, the generation of waste have also
increased abruptly. Conventionally many techniques are used for waste
treatment, but those were not eco-friendly. Hence with the advent of
biotechnology, these conventional Technologies are modified to make them
more efficient and less environmentally hazardous. Biotechnology played an
important role not only in waste treatment, but also to recover resources from
this waste that may be used for different purposes. Technologies like
bioreactor, phytoremediation, bioremediation have employed various
organisms like plant microbes for efficient treatment of waste. Genetic
engineering of organisms has helped us to reduce a large amount of specific
waste into less harmful products or useful resources.

1. Write the marits and demarits of Vermicompost
……………………………………………………………………………
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2. Discuss the major differences between insitu and exsitubiore mediation
basic steps of solid waste management?
……………………………………………………………………………
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77
Overview of
Environmental 3.11 KEYWORDS Bi
Biotechnology
Biodegradation : The breakdown of substances by micro-organisms.
Biogas : A mixture of methane and carbon dioxide resulting
from the anaerobic decomposition of waste such as
domestic, industrial and agricultural sewage.
Bioremediation : The use of plants and micro-organisms to consume or
otherwise help remove materials (such as toxic
chemical wastes and metals) from contaminated sites
(especially from soil and water). A natural process in
which environmental problems are treated by the use
of bacteria or other micro-organisms that break down a
problem substance, such as oil, into less harmful
molecules.

READING/REFERENCES
• Solid Waste Engineering and Management", Springer Science and
Business Media LLC, 2021
• Solid waste Management, K Sasikumar, S.G. Krishna. PHI Learning
Private Limited. 2009.
• Charu Gupta, Dhan Prakash. "chapter 7. Novel Bioremediation Methods
in Waste Management", IGI Global, 2016
• Neha Badola and Jaspal Singh Chauhan, chapter 7. Waste Management:
Challenges and Opportunities. In Bioremediation of Environmental
Pollutants Emerging Trends and Strategies. 2022.

Check your Progress 1
1. Refer Section Number 3.6.
2. Refer section Number 3.7.4.1 and 3.7.4.2
78
Biotechnological
UNIT 4 BIOTECHNOLOGICAL PROCESSES Processes
Structure
4.1 Introduction
4.2 Objectives
4.3 Biodegradation of Macromolecules and Biodegradation of Xenobiotics
4.4 Biotechnological Innovations for Recovery of Food
4.5 Energy and Feed from Natural Bio-soilds
4.6 Bioreactors
4.7 Process Parameters Optimization, Cell Immobilization
4.8 Application of Nanotechnology in Bioremediation and Recovery of
Products
4.9 Let Us Sum Up
4.10 Key Words
4.1 INTRODUCTION
The tern biodegradation deals with the process of decomposition of organic
matter by microbial activities. This process maintains the reusability and
recycling of the wastes and its components. The essential conditions for
biodegradation are the availability of nutrients and physical conditions which
can sustain the microbial growth. Microorganisms responsible for
biodegradation are bacteria, fungi (yeast and moulds also). It occurs under
aerobic as well as anaerobic conditions. There are various factors which
affects the degradation of organic compound i.e. water, oxygen, light and
temperature.
4.2 OBJECTIVES
• Describe biodegradation of macromolecules and of xenobiotics;

• Explain Biotechnological innovations for recovery of food;
• Describe application of nanotechnology in Bioremediation and recovery
of products;
• Explain Biorectors, Process parameters optimization, cell immobilization
biotechnologies role in providing alternate solutions for sustainable
Development.
79
Overview of
Environmental 4.3 BIODEGRADATION OF
Biotechnology MACROMOLECULES
Biodegradation of polymers: These are highly stable, covalently bonded
macromolecules and their degradation process is restricted. Therefore, small
change in chemical structure can enhance biodegradability of polymers. The
degradation of, polyesters, polyethers, polycaprolactones, polylactic acid,
nylon polylactides and PVA, is initiated by interaction of organism on to the
polymer followed by hydrolytic reactions.
Major types biodegradable polymers
1. Polyethers: polytetramethylene glycols and polypropylene glycols are

the polyethers which releases one glycol unit during each oxidation
cycle. These are used in lubricants, inks, cosmetics, pharmaceuticals, and
surfactants. The bacterial strains such as Flavobacterium and
Pseudomonas are able to catalyze the decomposition of these polymers.
PEGs are degraded by under aerobic and conditions e.g. anaerobic
bacterium Pelobactervenetianus can degrade the PEG derivatives.
2. Polyesters: These polymers are bonded by ester linkage that hydrolyzes
the biodegradable synthetic polymers.
3. Polyvinyl Alcohol: PVA is soluble in aqueous media and it is
biodegradable using various microbial geenra. It has been widely used in
the manufacture of deliverance method for pesticides, fertilizers, and
herbicides. Pesudomonas can completely degrade the PVA as a major
source of carbon in its metabolic pathways.
4. Polyethylene: Low densities of polyethylene are widely used in
packaging industries. But their slow rate of degradation can cause serious
change in environment, fresh water and animals. To overcome on those
problems extracellular Streptomyces sp. is widely used to degrade such
starch blended polyethylene.
5. Polyacrylamide: Polyacrylamides are composed of acrylic acid and
acrylamide which has immense applications in food processing, paper,
fiber and pulp industries. Acrylamide can be degraded by soil inhabiting
microorganisms by conversion of acrylic acid to ammonia under aerobic
conditions.
Xenobiotics
Xenobiotics are the chemical entities present in the organism that is not
produced or expected to be found within the organism. Xenobiotics also
covers the substances with increased concentration inside the body of living
organisms.
80
Biotechnological
Sources of macromolecules and Xenobiotics Processes
Macromolecules Xenobiotics
Carbohydrates Plastic industry
Lipids Petrochemical and Paint
Protein Pesticide
Nucleic acid Electronic, Textile
Hazards from Xenobiotic Compounds

Toxicity
Many xenobiotics are toxic to the soil bacteria and greatly influence the rate
of bioremediation.
Carcinogenicity
Xenobiotics containing halogenated alknes are carcinogenic in nature.
• Many xenobiotics may get accumulated in the environment and become
hazardous in nature.
• DDT and PCB’s are recalcitrant which show bioaccumulation or bio-
magnification by a factor of 104 – 106.
Mechanism involved in biodegradation of xenobiotics:
Various enzymes of microbial animals and plant origin play very important
role in the degradation of xenobiotic compounds e.g. oxygenases. The site
and mode of attack depends on the action of enzyme, its concentration and
the favourable conditions.
Methods of degradation
1. Enzymatic degradation
Some biotic and abiotic microbes can be used to degrade high molecular
weight polymers into monomers this release of smaller molecules during
macromolecules degradation could occur either by excreted or cell-
bound enzymes (breakdown of hydrogen bonds) in any case, rate of
biodegradation is exceedingly impacted by the crystallinity index,
spherulite measure and lamellar organization. A polymer having a lower
melting temperature is highly influenced the biodegradation that can be
analyzed by some techniques to monitor the changes that took place.
These techniques are as follows:
• Atomic force microscopy.
The surface structures of biodegradable polymers are studied by the
means of atomic force microscopy.
• Dynamic mechanical analysis.
It used to examine the visco-elastic properties of the materials.
81
Overview of • Differential Scanning Calorimetry.
Environmental
Biotechnology This technique monitors the physical and the chemical changes
associated while applying heat or at high temperature.
2. Photo-catalytic degradation
Sewage treatment can be done with this respective method by passing the
contaminated water with the chamber and having the exposure of UV
light over there, the small chain polymers having the property of photo
degradation can be easily eluted out from the water/ solution.
3. Ultrasound based degradation
The polymers can also be degraded by ultrasonic rays that help in
hydrolyzing the long chain of polymers into smaller chains because of
getting cleaved off from the weaker bonds present in the polymer
structure.
Biodegradation of other xenobiotics
Biodegradation of pesticides make it a non-toxicant compound, by removing

the toxicant moiety from the chemical structure. Degradation is the next step
to transform the complex into simpler products by different processes such as
decarboxylation, dehalogenation, hydroxylation, hydrolysis. Plastics are the
polymers with high molecular weight and can be degraded by different
chemical processes i.e. Oxidation and Hydrolysis.
Acidogenesis is the process that results in further breakdown of the remaining

residual matter by acidogenic bacteria such as- Streptococcus acidophilus.
Hydrocarbons consists of carbon and hydrogen molecules. Generally occur in
the form of oil e.g. alkanes, alkenes, alkynes, cycloalkanes etc. Microbes
such as Bacteria, yeast fungi and Algae have the ability to metabolize both
lower and higher molecular weight; most of the microbes have been found to
oxygenate the hydrocarbons and can be further oxidized to catechols.
Harmful effects of xenobiotics

These chemicals are associated with life threatening risks such as-
• Carcinogenic: Cancer causing compounds present in the chemicals.
• Neural System damage.
• Causes Reproductive disabilities.
4.4 BIOTECHNOLOGICAL INNOVATIONS FOR

RECOVERY OF FOOD
There are various symbolic representations of biotechnology such as- Red
biotechnology for medical industries, green biotechnology refers to the
environment and agriculture, white biotechnology refers to industrial
development, and blue biotechnology is for marine and water based
82 production sectors.
Biotechnological
Green biotechnology and its importance Processes
Food is the basic demand of every living organism present on this planet
earth. As the human population is increasing worldwide and mostly (80%)
living in developing countries, so it is important in such scenario to nourish
the population with good and healthy food. Also due to shrinkage of
agricultural land for providing infrastructure to growing population, it is
needed to increase crop yield in smaller land area. Improvement in food,
crops and nutrients comes under the category of Green biotechnology. With
the emergence of modern techniques and biological sciences, different ways
and methods have been developed that can be used in various areas to
enhance the benefits coming out from use of biotechnology.
Biotechnological innovations in food
Biotechnological innovations in the field of food and agriculture begun with

the cultivation of genetically modified crops during 1996 in USA. Later on in
2009, the useful effects of the same were seen as 14 million farmers from 25
different countries used GM crops and significant majority of them (13
million) were small-scale farmers in developing and emerging countries. In
the current scenario, the annual global coverage of area with GM crops has
increased to more than 134 million hectares worldwide. Although use of GM
crops isn’t cost effective as GM seed are more expensive but their high costs
pays off in several other ways like reduction of expenses in costs of
pesticides, machines and labor. Above all, there is a considerable increase in
the yield and there is also an effective management of weeds which in turn
reduce harvest losses which was inevitable at earlier time.
Besides GM crops, food and agriculture biotechnology is offering promising

benefits by helping in feeding our growing planet and bringing additional
benefits in various terms such as engineering plants to grow in such areas
where they can’t be grown before. Such category of potential products with
added health benefits necessitates the use of more related term known as
‘Functional Foods’ which generally refers to foods that provide health
benefits beyond basic nutrition. Likewise, it is made easier now to combat
nutrient deficiencies worldwide by increasing mineral and vitamin levels.
Food and agriculture biotechnology is also paying attention towards adapting
such methods and ways to reduce anti-nutritional and toxic substances from
food. Another step towards betterment in food involves improvement in the
digestibility by reducing gossypol,phytic acid, glycoalkaloids.
Importance of technology
In order to have food products with desirable nutritional and other qualities,
various methods and techniques must be used. With the advancement in
technologies, it is easy to know about genes and biochemical pathways that
directly control attributes that could offer more direct consumer benefits. So,
techniques that directly target the specific genes and their products are much
preferred in biotechnology. 83
Overview of Techniques used in food biotechnology
Environmental
Biotechnology
The achievements and innovations in food biotechnology are being delivered
through number of different techniques such as recombinant DNA,
genetically modified organisms (GMOs), genetic modification(GM),
advanced breeding, new breeding technologies, gene editing. Delivery of
desirable traits in plants started with use of bacteria Agrobacterium
tumefaciens, known for transfer of tumor-inducing plasmids into the cells of
host organisms in nature. This plasmid is engineered for elimination of
undesirable tumor inducing genes to encode genes with desirable traits to be
inserted in plants. These days methods such as microprojectile bombardment,
gene gun, or biolistics are widely used for gene transfer. Recently, much
advanced technique CRISPR-CAS9 is emerging as a potential technique for
gene editing. Although there are not much reports of its use in food
biotechnology but it can be a useful tool for doing desirable changes at
specific site of a gene. All such technologies are used to engineer plants to
induce changes at gene level that either enhance the production of some
important compound or remove an undesirable trait that might be harmful.
Innovative food technologies like production of recombinant enzyme and
alternative protein based ingredients make use of common technology tools
such as synthetic biology and microbial fermentation. To increase crop yield
keeping the other factors same, techniques like hybrid technology and seed
technology are used. Besides that cry gene in cotton is a wonderful example
of biotechnological innovation. These days various food crops and vegetables
such as BT brinjal, BT potato, enhanced varieties of wheat, golden rice etc.
are available in the market which are useful outcomes of biotechnological
innovations. In order to make plants and crop resistant to climate change and
natural nutrient scarcity, hybrid engineered seeds are used so that wastage of
crop due to changing weather conditions can be minimized.
Future perspective
Food and agriculture biotechnology is a growing area as the demand of food

and nutritional products are increasing day by day in such expanding
population. Although, there are number of inventive steps taken so far yet
more needs to be done in the same area. Extensive research is being carried
out in various institutes and industries. So, it must be concluded simply as
green biotechnology is efficiently paying off economically.

1. What are major types biodegradable polymers?
……………………………………………………………………………
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84
Biotechnological
4.5 ENERGY AND FEED FROM NATURAL BIO- Processes
SOILDS
Nutrient rich organic material formed from waste water treatment facility
through domestic sewage treatment is considered as biosolid. Being a rich
source of essential nutrients, biosolid is a valuable resource and recycled as
fertilizer and soil amendment. Biosoilds stimulate the growth of crops and
plants by providing the essential nutrients, act as fertilizer to improve the
productivity of soil. Physical characteristics of biosolid differ from
suspension of black liquid (< 4 % solid) to moist earthlike material with 20-
50 % soild to 90% soild material. Physical characteristics of biosolids are
affected by many factors. i.e waste water source, process to form biosoilds,
sewage sludge treatment process etc. 50- 70 % of biosolid is constituted by
organic material shows increased benefits in agricultural field.
Potential use of biosoilds

1. Fertilizer for crop production.
2. Land restoration and land reclamation.
3. Reclaiming mining sites.
4. Energy production and recovery.
Benefits of bio-soilds in agricultural land
Biosolids contain both macronutrients as well as micronutrients including

nitrogen, phosphorus, potassium, boron, chlorine, copper, iron, and zinc etc.
Phosphorus is found to be higher in biosolid as compare to animal manure
whereas amount of nitrogen is equal to animal manure. Being a rich source of
organic matters there are increased benefits of biosolids which are already
documented such as:
1. Enhance nutrient retention in the soil.

2. Increase water holding and retention capacity.
3. Enhance the aggregate formation and soil structure.
4. Enhance the cation exchhange in soil.
5. Increase microbial diversity and metabolic activities in soil.
Deficiency of trace elements in the soil is amended by biosoilds and results

in increased crop yield. According to the survey there are no such fertilizers
in the market which provide more complex array of essential trace elements
required by the soil as that of biosolids. One of the long term benefits of
biosolid is to improve the soil structure. It is observed that when biosoild
gets decomposed, form the substances that bind soil particles together to form
the blocks which results in the formation of stable soil with improved
physical properties. This leads to increase the water availability root
penetration and porosity of the soil.
85
Overview of Bio-solid feed to cattle
Environmental
Biotechnology
According to some research studies, bio-solid is a good feed source for dairy
and cattle animals. There are no adverse health results with 10 to 20 % of
biosoilds in their diet.
Energy production and recovery
Biosolids is found to be one of the great options to offset energy usage and
reduces carbon foot print by energy recovery from the domestic residues.
There are different methods documented in the literature for energy recovery
from biosolids. Recovery of biogas from anaerobic digestion and produce
energy by using combined heat and power generation (CHP) technologies is
one of the established method for energy recovery. Another method of
recovering energy from biosolids is utilization of dried biosolids as fuel
source for energy generation by using gasification process. Gasification is an
arebic digestion based newer technology for the biosolids energy recovery
systems.
4.6 BIOREACTORS DESIGNING

The bioreactor can be described with simple words that a device which can
provide the biologically active environment and can be used for a specific
purpose. The design and organization of a bioreactor has a significant role on
the overall performance of machine. The preliminary knowledge like kinetics
of bioprocess, nature of process etc. is the key requirement during the
designing of the bioreactor. The biological process can be aerobic or
anaerobic. It is widely used for the industrial purpose to produce the desired
material or perform the desired reactions. It is the heart of any biochemical
process in which enzymes, microbial, mammalian or plant cell systems are
used for the manufacture of a wide range of useful biological products. Many
factors play a key role in the performance of the bioreactor which is listed
below in detail.
• Total concentration or load of biomass

• Removal of heat generated
• Adequate sheer and agitation conditions
• Metabolisms/microbial activities
• Sufficient supply of nutrients
• Sterile conditions
• Removal of products and byproducts
• Aeration an sparging
• Effective agitations
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Biotechnological
Parts of the Bioreactor Processes
The major parts of a bioreactors are as given

a. Agitator: An Agitator is used to mix the contents of the reaction mixture
and ensure the continuous supply of nutrients to the homogenuous
mixture of cells.
b. Baffle: The major function of baffle is to prevent the vortex formation in
the bioreactor tank. This process is of less utility because it changes the
gravity centre of the system and consumes additional power.
3. Sparger: It supplies the required amount of oxygen to the microbial cells
during the cultivation process.
4. Jacket: It helps to maintain the constant temperature of the bioreactor.
Types of Bioreactor
Various types of bioreactors are used for research and industrial purpose.
a. Airlift Bioreactors
The airlift bioreactors are existed in many forms and widely used for
tissue culture. The tissues are very shear sensitive and for tissue culture,
the normal mixing cannot be done because it generates the shear forces.
The airlift bioreactors remove these constraints of the tissue culture.
b. Airlift Pressure Cycle Bioreactors
In this type of bioreactor the delivery of air is ensured by supplying gas
under high pressure. In airlift bioreactors, an injection is used to supply
air in a central tube, with fluid recirculation through the annulus between
the tube and the tower or vice versa.
c. Loop Bioreactor
Gas and circulated liquid are injected into the tower through a nozzle. It
is an extended version of the airlift system with gas and liquid flow
patterns. In this bioreactor, the pump transports the air and liquid through
the vessel. For the removal of liquid, it uses a mechanical pump with an
external loop.
d. Stirred Tank Bioreactor
It is a widely useful and most important bioreactor for industrial purpose.
It has a wide application in the conventional mixing due to the dual
advantage, first is low capital and second is the low operating costs. The
vessel for laboratory experiments is made up of glass and its volume up
to 20 liters. If the volume is larger so the vessel is made up by stainless
steel.
d. Bubble Column Fermenter
The bubble column fermenter is widely used for baker’s yeast, vinegar,
and beer. The bubble column fermenter is also useful for the treatment of
87
Overview of wastage water. The mass transfer ratio and hydrodynamic parameters inn
Environmental
Biotechnology bubble column generally depends upons the rate of air release from the
sparger.
e. Photobioreactor
The photobioreactor provides the light source which will be natural
sunlight or artificial illumination. The photobioreactor term is more
commonly used for the describe the closed system and it cannot be an
open system like tanker pond. It is used for the cyanobacteria or moss
plant culture because it can provide the light for necessary reaction. This
light is used by the phototrophic organisms as an energy source for
performing the photosynthesis reaction instead of carbon and lipid
source. In this type of photobioreactors, the risk of contamination of
bacteria or fungi is lower than the bioreactors which are used for
heterotrophic organisms.
f. NASA tissue cloning bioreactor
These are the new type of bioreactors and widely used for thereaupitic
and experimental purposes. The design of these types of bioreactors is
different from industrial bioreactors. Mostly tissues and cells especially
in the case of mammalian cells or tissue, they grow with the help of
surface or other structural support while the agitated environments are
often destructive to these cell types and tissues. A new type of bioreactor
is specially developed by NASA, in which the tissue and human cell
cultures can be done. These tissues can be of any type like ligaments,
skeletal tissue, cancer tissues for study, heart tissue or any other type of
tissue.
Applications of Bioreactors
The bioreactors have a wide variety of applications which are described

below.
1. It is used for cell culture or tissue culture for therapeutic or experimental

purposes.
2. The bioreactors are widely used for the fermentation process because
they contain the favorableenvironment for the fermentation. The stirred
tank bioreactors are commonly used in the fermentation process.
3. Fermentation of ethanol is widely done by using Saccharomyces
Cerevisiae in the bioreactor.
4. Various organic acids and organic products like butyric acid, citric acid
and acetic acid can be synthesized in a bioreactor using Eubacterium
limosum.
5. The antibiotics like penicillin, cephlomycin and thienamycin is also
produced in the bioreactor.
6. Various industrially important enzymes such as lipase, cellulase,
88
Biotechnological
protease amylase can also be produced by various bacterial and fungal Processes
genera in the bioreactor.
4.7 PROCESS PARAMETERS OPTIMIZATION,

CELL IMMOBILIZATION
The present demands of the world’s biotechnological industries are superior
cell/enzyme productivity and improvement of novel techniques for escalating
the shelf life of cell/biocatalyst. These desires are foreseeable to make
possible large-scale economical formulation. Cell/enzyme immobilization
provides a tremendous approach for increasing the accessibility of enzyme to
the substrate with better turnover over in a substantial phase of time.
Numerous natural and synthetic supports have been assessed for their
efficiencies for cell/enzyme immobilization. Nowadays, immobilized
cells/enzymes are ideal over their free counterparts due to their long-lasting
ease of access that curtails redundant downstream and purification processes.
Immobilization process involves optimization of the operational performance
of a cell/enzyme for industrial applications. Many matrices have been
described in the literature to improve the performance of the immobilized
cells/enzymes. Although the basic methods of cell/enzyme immobilization
may be divided into different methods, but based on combinations of these
original methods, number of variations have been developed.
Correspondingly, different physical and chemical carriers of different origins
have been proposed for a number of bio-separations and bio-immobilization.
Due to economical consideration, application of lipases on an industrial scale
requires their immobilization and thus re-usability.
Besides adsorption, there are several other techniques for cell/enzyme

immobilization which include cross-linking, covalent bonding, entrapment
and encapsulation. A comparison of these immobilization methods has been
summarized (Table 1and Figure 1). Recently, various types of nanomaterials
and bioinspired materials have been used for the cell immobilization due to
their peculiar physical characteristics to prevent the cells from direct effect
ofheat shock organic solvents. These nano-materials equilibrate the principal
factors which determine the surface area, mass transfer resistance and
biocatalysts efficiency, of microbial cells in catalytic reactions.
89
Overview of
Environmental
Biotechnology
Figure 4.1: Different enzyme/cell immobilization methods.
Table 4.1: Different cell/enzyme immobilization types with supportive

material
Immobilization Support material

types
Adsorption Silica, Celite, Gelatin, porous glass, cotton fibers
and DEAE cellulose
Cross-linking Gluteraldehyde
Covalent bonding Cellulose, cyanuric chloride, titanium oxide and
metal-NPs
Entrapment or Alginate, polyester, hydrogel and alginate
encapsulation polylysine.
Generally various biomass based particle and bio-inspired material have been
studied to stabilize and immobilize the living cells. Enzymes as purified
protein have been widely studied to to immobilize using covalent cross-
linking and physical adsorption techniques. On the other hand the microbial
cells immobilization is being generally preferred by entrapment and
encapsulation or physical adsorption.

1. What is Biorectors?
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
90
1. Describe the functioning of bioreactors? Biotechnological
Processes
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
4.8 APPLICATION OF NANOTECHNOLOGY IN

BIOREMEDIATION AND RECOVERY OF
PRODUCTS
Owing to the rapid growth of industrialization, urbanization, and modern
agricultural practices, pollution of stream water or groundwater and soil is
on the rise.To ful fill the human desire for energy generation and other
needs, natural resources have been exploited resulting in the degradation of
water quality and environmental pollution leading to ecological imbalance.
Present treatment technologies, though efficient, cause several problems
which make remediation processes complex. Among these technologies,
bioremediation has been prominently practiced as an efficient cost-
effective technology for controlling hazardous pollutants like heavy metals
in soil and water. A novel method of nano-bioremediation is effective and
more significant for heavy metal removal in all aspects in which the
drawbacks of bioremediation can be possibly avoided by the application of
nanotechnology.
Figure 4.2: Effect of nano-particles on contaminate sites and their possible mechanism
to reduce the contaminants
Nanotechnology and nanomaterials
Nanomaterials (NMs) are defined as the materials having size ranging from 1
through 100 nm with a minimum of one dimension. In this small scale, NMs
possess unique properties compared to the other materials. Many of these
91
Overview of materials have been explored for application in wastewater treatment. They
Environmental
Biotechnology utilize the size-dependent properties of NMs, such as high surface area, high
reactivity, strong sorption, and faster dissolution. Although many nano-based
technologies are successful on the laboratory scale, only few technologies
have been used for small-scale testing or commercialization. Such nano-
based technologies include nanotechnology-associated membranes, nano-
adsorbents, and nano-photocatalysts.
Need for New Technology
Amid all the water contaminants, heavy metals play a major role in causing
severe health-associated complications in human beings and animals. This is
due to their nonbiodegradable characteristics and extreme toxicity. Itai-Itai is
a well-known Japanese disease caused due to the prolonged exposure of the
heavy metal chromium. The severe and prolonged disorders caused by
chromium are hypertension, skeletal malformation in the fetus, testicular
atrophy, renal damage, and emphysema. Therefore, it is essential that new
innovative approaches are required in order to eradicate these hazardous
metals from water.
Biosynthesis of nanoparticles
Green technology is most widely used method for bioremediation as well as

synthesis of nano-particles from organic material. There are various
conventional and chemical methods of the synthesis of nano-particles such as
hydrothermal process, spray pyrolysis, sol-gel method, chemical precipitaion
but the biological method which is based on the reduction reaction using
phenolics contents present in plant extract is most eco-friendly technique.
The microbial enzymes also catalyze the reduction of metal and various
elements into their respective nano-particles. The particle synthesized by
microbial reduction of enzymatic reduction has high surface area to volume
ratio and high catalytic activity. These particles can be widely used for the
various industrial application such as drug delivery, enzyme iimobilizaqtion ,
biotransformation and biocatlysis reactions. In the environmental
biotechnology theese particles have great potential in waste water treatment,
heavy metal ion sequestration gas sensing, and pollutant or contaminant
detection. The major fields of bioremediation of organically synthesized
particles are contaminated sites near river , industrial area, mud soil, and crop
fields.
Applications of nanotechnology
1. Application in Heavy Metal Remediation
In the recent past, applications of nanotechnology for feasible solutions
in wastewater treatment have been adopted. The different nanoparticles
with desirable properties and their application in wastewater treatment
for the removal of heavy metals and other contaminants are discussed.
NMs having unique properties combined with conventional treatment
92
techniques provide wide opportunities to make dramatic changes in Biotechnological
Processes
wastewater treatment methodologies. The combination of both
nanoscience and engineering offers better opportunities in the restoration
of heavy metal contaminated groundwater.
NMs have wide applications in various fields. Recently they are being
applied in the area of water purification in order to reduce the bulk
concentrations of toxic substances, such as radionuclides, metal ions, and
organic and inorganic compounds to the ppb levels. Magnetite
nanoparticles (Fe3O4) impregnated with a silica compound is used for
the removal of a large amount of toxic substances usually existing in the
environment, also for the biological separation of cells and remediation
purposes. Apart from this, nanostructured silica alone can also be applied
in wastewater treatment in order to eliminate heavy metal ions.
2. Nano-particles as Adsorbents
NMs exhibit good adsorbent properties due to greater surface area to
volume ratio and high affinity towards specific chemicals and pesticides.
For the removal of heavy metals, the adsorption process is considered to
be the better remediation option compared to conventional treatment
technologies. The adsorption process has several advantages, such as
cost effectiveness ,operational simplicity, and greater ease or efficiency.
a. Polymers as nano-adsorbents:
Polymers such as dendrimers are good adsorbents capable of removing
both heavy metals and organic compounds. The sorption mechanism of
polymers includes electrostatic interactions, hydrophobic effects, bond
formation between hydrogen atoms, and complexation. The interior
portion of dendrimers is hydrophobic,in nature for adsorption of organic
compounds and the exterior portion is linked either with a hydroxyl or
amine group for heavy metal adsorption. The dendrimer is associated
with an ultrafiltration system in order to recover Cu2+ ions from aqueous
solutions and remove the metal ions at an initial concentration of 10 ppm
(parts per million). After the adsorption process, the dendrimers along
with heavy metal ions were recovered andregenerated by a filtration
system with a pH as low as 4.
b. Other nano-sorbents
The adsorption of Pb(II), Cd(II), and Ni(II) ions from aqueous solution
using chitosan methacrylic acid (MAA) nanoparticles was studied (100).
Two types of akaganeite materials were prepared and tested for the
sorption of heavy metals such as antimony (Sb) and arsenic derivatives.
It has also been reported that the nanocrystalline akaganeite (−FeOOH)-
coated quartz sand (CACQS) can be used for bromate removal from
aqueous solutions. Some of the nano-adsorbents used for the deduction
of arsenic are already in commercial use.
c. Metal-based nano-adsorbents
Metallic nanoparticles are being investigated with the aim of removing
heavy metals, such as mercury, nickel, copper, arsenic, cadmium,
chromium, lead, etc. Calcium-doped zinc oxide nanoparticles as a 93
Overview of selective adsorbent for the extraction of lead ion were explored. Many
Environmental
Biotechnology metal oxidized NMs along with nanosized magnetite and titanium
dioxide outcompetes the adsorption capacity of activated carbon.
d. Applications of Iron-based NMs
The selection of a suitable process for wastewater treatment is an
intricate assignment, because the selection is based on several factors,
such as standard quality, cost, and efficiency. Iron nanoparticles and
polymer coated nanoparticles plays a significant role in the removal of
heavy metals such as Cr(VI) and As(III). The common iron-based NMs
used for remediation are nanosized zero-valent ion (NZVI), iron sulphide
nanoparticles, bimetallic Fe nanoparticles, and nanosized FeO.
Applications of iron-oxide NMs in water treatment are divided into two
groups, that is, nano-adsorbent or immobilized carriers to increase
removal efficiency and as photocatalysts to break down the hazardous
toxic contaminants into a less toxic material.
e. Photocatalytic NMs
Photocatalysis is an advanced oxidation process (AOP) which is used in
the deterioration of organic contaminants in a modest and effective way.
Oxidation process through photocatalysis is an innovative method to
remove trace amounts of pathogens and pollutants. It is considered to be
the significant pretreatment method for the eradication of
nonbiodegradable and toxic pollutants and thereby increases their
decontamination activity. In these photocatalytic materials, nanosized
semiconductor materials, such as zinc oxide (ZnO), titanium dioxide
(TiO2), tungsten oxide (WO3), and cadmium sulfide (CdS) are
categorized under various processes, such as conjugated adsorption along
the electrical double layers, high-adsorption surface area, and
photochemical activity. Also these materials are immediately available,
low cost, and have low toxicity.
f. Nanobiomaterials for heavy metal remediation
Studies were conducted on some bacteria to produce an iron sulfide
compound which acts as an adsorbent for several toxic metal ions. Apart
from bacteria, Noaea mucronata is a plant species used for the
accumulation of heavy metals, such as lead, copper, cadmium, zinc, iron,
and nickel. The nanoparticles obtained from this plant are used for the
bioremediation of heavy metal contaminants from groundwater, streams,
and rivers. The study conducted on plant species such as Centaurea
virgata, Scariola orientalis, Noaea mucronata, Chenopodium album,
Cydonia oblonga, Resedalutea, and Salix excelsa revealed that these
plants are very good heavy metal accumulators. Specifically Noaea
mucronata is a suitable accumulator for lead to a level more than 1000
ppm.
g. Carbon-based nanoparticles
Carbon-based NMs are extensively used for the removal of heavy metals
because of its nontoxicity and greater adsorption capacity. The first used
adsorbent commonly used for metal ion removal is activated carbon, but
94
it is difficult for activated carbon to reduce up to ppb levels. Biotechnological
Processes
h. Nanofibers
Electrospinning is a plain, proficient, and cheaper method for the
production of ultra-fine nanofibers by means of resources such as metals,
polymers, or ceramics. These nanofibers form mats with complex pore
structures due to their porosity and higher surface area. The physical
properties such as morphology, composition, diameter, spatial
arrangement, and secondary derivatives of these electrospun nanofibers
are fabricated depending upon particular fields of application.
4.9 STRATEGIES OF NANO-PARTICLES TO

CONTROL THE POLLUTION
Control of pollutants from the source of origin is promising step of
controlling pollution. The pollution control strategies can be regulating by
different ways:
Advantages of nano-particles
These are as listed below
• Higher absorption capacity due to large surface area to volume ratio and
greater surface energy.
• The rate of reaction in nanoaprticle catalysed reaction is high.
• The nanoparticles promote in-situ remediation rather than ex-situ
remediation.
• The coated nano-particles with tuned physical properties and surface area
to volume ratio are useful to design bio-sensors with high detection limit
and sensitivity.
4.10 LET US SUM UP

In this unit you have studied about biodegradation of macromolecules and of
xenobiotics; Biotechnological innovations for recovery of food; application
of nanotechnology in Bioremediation and recovery of products; Biorectors,
Process parameters optimization, cell immobilization biotechnologies role in
providing alternate solutions for sustainable Development
4.11 KEY WORDS

Nanotechnology : From the Latin nanus = "dwarf", so it literally means
"dwarf technology". The word was originally coined by
Norio Taniguchi in 1974, to refer to high precision
machining. However, Richard Feynman and K. Eric
Drexler later popularized the concept of nanotechnology
as a new and developing technology in which humans
manipulate objects whose dimensions are approximately
95
Overview of 1 to 100 nanometers.
Environmental
Biotechnology Xenobiotic : A chemical compound that is not produced by, and often
cannot be degraded by, living organisms

READING/REFERENCES
• Tahri, N., Bahafid, W., Sayel, H., & El Ghachtouli, N. (2013).
Biodegradation: Involved Microorganisms and Genetically Engineered
Microorganisms. InTech. doi: 10.5772/56194
• Vroman I, Tighzert L. Biodegradable Polymers. Materials (Basel). 2009
Apr 1;2(2):307–44. doi: 10.3390/ma2020307. PMCID: PMC5445709.
• Syed A,. (2016). Types of Biodegradable Polymers. 10.1016/B978-0-
323-39396-6.00005-1.
• Thakur, I. S. (2008). Xenobiotics: Pollutants and their degradation-
methane, benzene,pesticides, bioabsorption of metals.
• Houde, M., Muir, D. C., Kidd, K. A., Guildford, S., Drouillard, K.,
Evans, M. S., &; Kling, H. (2008). Influence of lake characteristics on
the biomagnification of persistent organicpollutants in lake trout food
webs. Environmental Toxicology and Chemistry: An International
Journal, 27(10), 2169-2178.
• Fenchel, T., Blackburn, H., Blackburn, T. H., & King, G. M.
(2012). Bacterialbiogeochemistry: the ecophysiology of mineral cycling.
Academic press.
• Laura, Ma., Snchez-Salinas, E., Dantn Gonzlez, E., & Luisa, M.
(2013). PesticideBiodegradation: Mechanisms, Genetics and Strategies
to Enhance the Process. InTech. doi:10.5772/56098
• Andualem, B., & Seid, A. (2021). The role of green biotechnology
through geneticengineering for climate change mitigation and adaptation,
and for food security: currentchallenges and future perspectives. Journal
of Advances in Biology & Biotechnology, 24(1),1-11.
• Lucht JM. Public Acceptance of Plant Biotechnology and GM Crops.
Viruses. 2015 Jul 30;7(8):4254-81. doi: 10.3390/v7082819. PMID:
26264020; PMCID: PMC4576180.
• Lacroix B, Citovsky V. Biolistic Approach for Transient Gene
Expression Studies in Plants. Methods Mol Biol. 2020;2124:125-139.
doi: 10.1007/978-1-0716-0356-7_6. PMID:32277451; PMCID:
PMC7217558.
• Raman R. The impact of Genetically Modified (GM) crops in modern
agriculture: A review.GM Crops Food. 2017 Oct 2;8(4):195-208. doi:
10.1080/21645698.2017.1413522. PMID:29235937; PMCID:
PMC5790416.
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• Estahbanati, M. K., Kumar, S., Khajvand, M., Drogui, P., & Tyagi, Processes
R. D. (2021).Environmental impacts of recovery of resources from
industrial wastewater. In Biomass,biofuels, biochemicals (pp. 121-162).
Elsevier.
• McBride MB. Long-Term Biosolids Application on Land: Beneficial
Recycling of Nutrientsor Eutrophication of Agroecosystems? Soil
Systems. 2022; 6(1):9.https://doi.org/10.3390/soilsystems6010009
• Nunes N, Ragonezi C, Gouveia CSS, Pinheiro de Carvalho MÂA.
Review of Sewage Sludgeas a Soil Amendment in Relation to Current
International Guidelines: A Heavy MetalPerspective. Sustainability.
2021; 13(4):2317. https://doi.org/10.3390/su13042317
• Spier, M. R., Vandenberghe, L. P. D. S., Medeiros, A. B. P., &
Soccol, C. R. (2011).Application of different types of bioreactors in
bioprocesses. Bioreactors: design, properties and applications, 53-87.
• Merchuk, J. C. (2003). Airlift bioreactors: review of recent
advances. The Canadian Journalof Chemical Engineering, 81(3‐4), 324-
337.
• Lidén, G. (2002). Understanding the bioreactor. Bioprocess and
biosystems engineering, 24,
• 273-279.
• Dutt, K., Harris-Hooker, S., Ellerson, D., Layne, D., Kumar, R., &
Hunt, R. (2003). Generation of 3D retina-like structures from a human
retinal cell line in a NASA bioreactor. Cell transplantation, 12(7), 717-
731.
• Hassan ME, Yang Q, Xiao Z, Liu L, Wang N, Cui X, Yang L. Impact of
immobilization technology in industrial and pharmaceutical applications.
3 Biotech. 2019 Dec;9(12):440. doi: 10.1007/s13205-019-1969-0. Epub
2019 Nov 8. PMID: 31750038; PMCID: PMC6841786.
• Vázquez-Núñez, E., Molina-Guerrero, C. E., Peña-Castro, J. M.,
Fernández-Luqueño, F., &de la Rosa-Álvarez, M. G. (2020). Use of
nanotechnology for the bioremediation of contaminants: A
review. Processes, 8(7), 826.
• Shams Forruque Ahmed, M. Mofijur, Nazifa Rafa, Anika Tasnim
Chowdhury, Sidratun Chowdhury, Muntasha Nahrin, A.B.M. Saiful
Islam, Hwai Chyuan Ong,
• Ahmed, S. F., Mofijur, M., Rafa, N., Chowdhury, A. T., Chowdhury, S.,
Nahrin, M., and Ong, H. C. (2022). Green approaches in synthesising
nanomaterials for environmental nanobioremediation: Technological
advancements, applications, benefits and challenges. Environmental
Research, 204, 111967.
• Khin, M. M., Nair, A. S., Babu, V. J., Murugan, R., &
Ramakrishna, S. (2012). A review on nanomaterials for environmental
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Overview of remediation. Energy & Environmental Science, 5(8), 8075- 8109.
Environmental
Biotechnology • Mohsenzadeh, F., & Rad, A. C. (2012). Bioremediation of heavy
metal pollution by nanoparticles of noaea mucronata. International
Journal of Bioscience, Biochemistry and Bioinformatics, 2(2), 85.
• Subbiah, T., Bhat, G. S., Tock, R. W., Parameswaran, S., &
Ramkumar, S. S. (2005). Electrospinning of nanofibers. Journal of
applied polymer science, 96(2), 557-569.

• Refer Section Number 4.3
• Refer Section Number 4.6
• Refer section Number 4.6
98
Biotechnological
Processes
BLOCK 2
BIODEGRADATION OF NATURAL
XENOBIOTIC COMPOUNDS
99
Overview of
Environmental BLOCK 2 BIODEGRADATION OF
Biotechnology
NATURAL AND XENOBIOTIC
COMPOUNDS
Biodegradation is biological catalyzed reduction in complexity of chemical

compounds. It is the process by which organic substances are broken down
into smaller compounds by living microbial organisms. Biodegradation is the
degradation of the materials into environmentally acceptable products such as
water, carbon dioxide, and biomass by the action of naturally available
microorganisms under normal environmental conditions. This block covers
the biodegradation of natural and xenobiotics compound.
Unit 5 “Degradation of Natural Compound” gives an overview of

degradation of cellulose, hemicelluloses, chitin and lignin compounds,
environmental factors influencing biodegradation, composting and
vermicomposting of agroresidues, use of agro waste in mushroom cultivation
etc.
Unit 6 “In Silage Production from Waste” elaborates about silage production
from wastes, basic principles and role of saccharolytic and proteolytic
organisms, enzymology of silage production etc.
Unit 7 “Microbes in Greenhouse Gases Mitigation” covers the green house

gases in soil, industrial effluent and land fill leachate, forest and ocean, It
gives a brief account of sequestration of greenhouse gases (CO2, methane,
and nitrous oxide), transformation of greenhouse gases, role of carboxylatic
enzymes in mitigation of CO2 for recovery of bioproductetc.
Unit 8 “Degradation of Xenobiotic Compounds” gives an account of

xenobiotic and recalcitrant compounds in the waste, degradation of
xenobiotic compounds viz. hydrocarbons, chlorinated, nitrogen containing
compounds and polyaromatic hydrocarbons, petroleum products and plastic
waste etc.
degradation of natural and xenobiotic compounds.
100
Degradation of
UNIT 5 DEGRADATION OF NATURAL Natural Compound
COMPOUND
Structure
5.1 Introduction
5.2 Objectives
5.3 Degradation of Cellulose,
5.4 Hemicelluloses,
5.5 Chitin
5.6 Lignin Compounds
5.7 Environmental Factors Influences in Biodegradation
5.8 Lignocellulolytic Enzymes,
5.9 Composting and Vermicomposting of Agro-residues
5.10 Use of Agro Waste in Mushroom Cultivation,
5.11 Process and Newly Emerging Technologies,
5.12 Advantages and Cost Considerations etc.
5.13 Let Us Sum Up
5.14 Key Words
5.15 Suggested Reading/ Refreneces
5.16 Answers to Check your Progress exercise
5.1 INTRODUCTION
Biodegradation is biological catalyzed reduction in complexity of chemical
compounds. It is the process by which organic substances are broken down
into smaller compounds by living microbial organisms (Rani et al., 2008).
When biodegradation is complete, the process is called "mineralization".
However, in most cases the term biodegradation is generally used to describe
almost any biologically mediated change in a substrate (Brunner, 2014).
The microbial organisms transform the substance through metabolic or

enzymatic processes. Several microorganisms, including fungi, bacteria and
yeasts are involved in biodegradation process. Final product of the
degradation is carbon dioxide. Organic material can be degraded aerobically,
with oxygen, or anaerobically, without oxygen. Certain environmental factors
such as temperature, pH, and available nitrogen and phosphorus sources,
then, seem to determine the rate and the extent of degradation (Gupta et al.,
2013).
101
Biodegradation of
Natural Xenobiotic 5.2 OBJECTIVES
Compounds
After reading this unit you should be able to:
• Explain Biodegradation.
• Describe the degradation of cellulose.
• Describe the many aspects of hemicellulose degradation.
• Describe the Lignin and its degradation.
• Explain the Lignocellulolytic enzymes
• Explain Composting and Vermicomposting
• Explain mushroom Cultivation by using agro residues.
5.3 DEGRADATION OF CELLULOSE

Definition of cellulose
Cellulose is an organic polysaccharide composed of a linear chain of

hundreds of β-linked D-glucose units. Cellulose, a linear polymer of D-
glucose units (two are shown) linked by β(1→4)-glycosidic bonds (figure 1)
Figure 1: Structure of cellulose (Source: Xiros et al., 2011)
Cellulose is the most abundant extracellular structural polysaccharide or

organic polymer of all biomolecules in the biosphere. Cellulose is present in
all land plants but is completely lacking in meat, egg, fish, and milk. It is,
however, not metabolized by the human system. It is the most widely
distributed carbohydrate of the plant kingdom that comprises about 50% of
all the carbon in vegetation (Brunner, 2014).
Cellulose is a homopolymer of a glucose derivative, and thus, it acts as a

great source of fermentable sugar. Cellulose is cultivated in the form of
energy crops for the production of ethanol, ethers, acetic acid, etc. The
abundance of cellulose is due to the constant photosynthetic cycles in higher
plants, synthesizing about 1000 tons of cellulose. Cellulose is a fibrous, rigid,
white solid, insoluble in water but soluble in ammonical cupric hydroxide
solution. Although insoluble in water, cellulose absorbs water and adds to the
bulk of the fecal matter, and facilitates its removal (Xiros et al., 2011).
Structure of cellulose is shown in figure 1.
102
Degradation of
Microbial degradation of cellulose Natural Compound
The molecular weight of cellulose ranges between 200,000 and 2,000,000.

Cellulose consists of a D-glucose unit at one end with a C4-OH group as the
non-reducing end, and the terminating group is C1-OH as the reducing end.
The bond is formed by taking out a molecule of water from the glycosidic
OH group on carbon atom 1 of one β-D-glucose molecule and the alcoholic
OH group on carbon atom 4 of the adjacent β-D-glucose molecule.
Anhydrocello biose is the repeating unit of cellulose. Cellulose has great
tensile strength and low accessibility. Most of the cellulose found in wood is
highly crystalline with about 65% crystalline regions (Brunner, 2014).
Microorganisms involved in cellulose degradation (cellulolytic

microorganisms)
A broad spectrum of cellulolytic microorganisms, mainly fungi, and bacteria,

have been identified over the years (Brunner, 2014). The structure and mode
of action of the cellulases produced by different microorganisms are also
different.
Cellulolytic Fungi
Cellulase-producing fungi are widespread among fungi and include species

from the ascomycetes (Trichoderma reesei), basidiomycetes (Fomitopsis
palustris) with few anaerobic species.
• Soft rot: Trichoderma Aspergillus niger, Fusarium oxysporum,

Neurospora crassa, etc.
• Brown rot and:Poria placenta, Lenzites trabea, Coniophora puteana,
and Tyromyces palustris.
• White rot fungi:Phanerochaete chrysosporium, Sporotrichum
thermophile, and Trametes versicolor.
Cellulolytic Bacteria
Most of the bacterial cellulolytic enzymes are Bacillus, Acinetobacter,

Cellulomonas, Fibrobacter succinogenes, StaphylococcPseudomonas,
Proteus Ruminococcus albus and Clostridium. Some thermophilic bacteria
like Anoxybacillus sp, Geobacillus sp, and Bacteroides also exhibit cellulase
activity.
Process (Simple Steps) of cellulose degradation

Cellulose degradation occurs in three simple steps;
1. Hydrolysis by endoglucanases
The first step in the degradation of cellulose is the action of
endoglucanases that randomly attack the cellulose fibrils. This step
results in a decrease in the size of cellulose chains as it degrades the
polymer into smaller fragments. The enzyme acts internally at random
103
Biodegradation of points of the polymer.
Natural Xenobiotic
Compounds 2. Hydrolysis by exoglucanases
Exoglucanases act on the smaller fragments resulting in even smaller
units of tetrasaccharides or disaccharides. Exoglucanases act on the
reducing end of the fragments to form either dimeric units or cellobiose.
3. Hydrolysis by β-glucosidase
β-glucosidase or cellobiose act on the dimeric units of glucose of
cellobiose to form monomeric units, glucose. This is the final step of
cellulose degradation that results in the formation of free individual units
of the glucose molecule. (López-Mondéjar, 2016)
Schematic diagram has been shown in figure 2
Figure 2: Simple Steps of cellulose degradation (Source: Xiros et al., 2011)
5.4 DEGRADATION OF HEMICELLULOSE

Hemicellulose is a group of complex polysaccharides that are found in the
fibers of plants along with other polysaccharides like cellulose and pectin.
Hemicelluloses are mostly mixed polymers, whereas cellulose is a
homopolymer of glucose. Apart from arabinogalactan, all other
hemicelluloses have short side-chains and low molecular weights. The
hemicelluloses consist of either pentose (xylose, arabinose) or hexoses
(glucose, mannose, galactose) as well as uronic acids. These polysaccharides
adsorb water and function as storage and supporting substances in plants.
Hemicelluloses are grouped into groups that include xyloglucan, xylans,

mannans and glucomannans, and β-(1→3, 1→4)-glucans. These glycans all
have the same equatorial configuration at C1 and C4, and hence the
backbones have significant structural similarity. Xylan is the representative
polysaccharide of this group that is composed of a backbone of β-(1→4)-
linked xylose residues. Xylans are also the most abundant carbohydrates after
cellulose (Scheller and Ulvskov, 2010).
Structure of hemicellulose
Hemicelluloses consist of 50–3000 sugar units as opposed to 7000–15,000
104 glucose molecules per polymer in cellulose. Hemicellulose is amorphous in
structure, not crystalline as cellulose, and therefore more susceptible to Degradation of
Natural Compound
hydrothermal extraction and hydrolysis. Hemicelluloses are classified into
different groups as xylans, mannans, and glucans on the basis of the primary
sugar residue in the backbone. Structure of hemicellulose is represented in
figure 3.
Figure 3: Structure of hemicellulose (Source:Scheller and Ulvskov, 2010)
1. Xylans
Xylans are a group of polysaccharides consisting of β-(1→4)-linked
xylose sugar residues with side branches of α-arabinofructose and α-
glucuronic acids that contribute to the cross-linking of cellulose. Xylans
are categorized into three classes; glucoronoxylan, arabinoxylan, and
glucoronoarabinoxylans.
2. Mannans
Mannans are a group of β-(1→4)-linked polysaccharides where the
backbones consist entirely of mannose units.
3. Glucans
Glucans are polysaccharides composed of glucose units linked by
glycosidic bonds. Glycans in hemicellulose are either xyloglucans or β-
(1→3,1→4)-glucans.
Hemicellulases
Hemicellulases are categorized into four different groups depending on the

substrate and linkages they act on; L-arabinanases, D-galactanases, D-
mannanases, and D-xylanases. Some of these enzymes might even differ in
structure and their mode of action. Different hemicellulases might even be
produced by a different group of organisms like bacteria, fungi, protozoans,
algae, and plants.
Microorganisms involved in hemicellulose degradation

1. Hemicellulolytic fungi
• Alternaria solani, Botryosphaeria ribis, Botrytis allii, Corticium
centrifugum, Monilia fructigena, Neurospora, Penicillium digitatum,
Rhizopus nigricans, Sclerotinia fructigena, etc. are known to produce L-
arabinanases and D-mannanases.
105
Biodegradation of • Similarly, fungi like Gibberella saubinetti, Helminthosporium oryzae,
Natural Xenobiotic
Compounds Phytophthora infestans, Trametes gibbosa, etc. produce D-galactanases.
2. Hemicellulolytic bacteria
Most of the bacterial cellulolytic enzymes are Clostridium felsineum,
Bacillus subtilis, Acetenobacter mannanolyticus, Bacillus aroideae,
Sporocytophaga myxococcoides, etc.
Factors affecting hemicellulose degradation
Degradation of hemicelluloses is affected by a number of factors, some of

which are temperature and pH (pH 6 and 40°C), organic matter,dosage of
enzymes, Substrate conversion
Process (Simple Steps) of hemicellulose degradation
• The degradation begins with the attack by exoglycocides on the

hemicelluloses in order to remove the side-chain substituents, hereby,
‘opening up’ or exposing the backbone glycan chain.
• This first step allows the glycan chain to be exposed so that it can be
easily attacked by the hemicellulases, as a steric hindrance by the side
chain residues is reduced.
• Alternatively, the degradation begins with the attack of
endohemicellulases on the regions of glycan chain that are unbranched or
relatively moderately branched by substituents.
• It is then followed by the action of endohemicellulases which yields an
array of oligosaccharides of a mixed constitution.
• Both exoglycosidases and endohemicellulases further degrade the
resulting fragments. (López-Mondéjar, 2016)
Schematic diagram of hemicellulose is shown in figure 4.
Figure 4: Hemicellulose Degradation (Source:Scheller and Ulvskov, 2010)
5.5 DEGRADATION OF CHITIN

Chitin is a complex homopolysaccharide consisting of units of amino sugar
106 glucosamine that accounts for the second most abundant polysaccharide of
Degradation of
nature after cellulose. The term ‘chitin’ is derived from the Greek word Natural Compound
‘chiton’ which means a coat of mail. Depending on the source, chitin occurs
in two forms; α and β conformation. A third less discussed γ form is also
known. These allomorphs differ from one another in their orientation of the
micro-fibrils. Chitin is considered an essential polymeric structure due to its
characteristics like high porosity, biodegradability, predictable degradation
rate, and structural integrity. Chitin degradation in soil or on artificial media
can be affected by several factors, some of which areMoisture content, added
glucose, Aeration and Organic matter.
Figure 5: Structure of Chitin (Moussian, 2019)
Structure of chitin
Chitin exists as a linear polymer of N-acetyl-D-glucosamine units linked

together by β-1,4-glucosidic linkages. This structure results in a three-
dimensional α-helix configuration. The stability of the α-helix chitin structure
is brought about by the hydrogen bonding of the N-acetyl side chains. In
nature, however, the chitin polymers bind extracellularly by intermolecular
hydrogen bonding that forms a crystalline microfibril structure. Structure of
chitin is shown in figure 5.
Microorganisms involved in chitin degradation
1. Chitinolytic bacteria
Bacterial species of Vibrio, Photobacterium, Aeromonas, Cytophaga,
Streptomyces, Photobacterium, Bacillus, Clostridium, and
Chromobacterium are well-known chitinolytic bacteria.
2. Chitinolytic fungi
The most common fungal species involved in chitinolysis include
Mucorales like Mortierella spp, and Deuteromycetes and Ascomycetes
like Aspergillus, Verticillium, Thielavia, Trichoderma, Penicillium, and
Humicola.
3. Slime mold, protozoa, and algae
Myxomycetes (true slime molds) like Physarum polycephalum are a rich
source of lytic enzymes that produce a complex of extracellular
chitinases. Soil protozoa like Hartmanella and Schizopyrenus, along with
107
Biodegradation of slime mold Plasmodium are also known to produce chitinases that
Natural Xenobiotic
Compounds participate in the digestion of chitinous food particles engulfed by these
invertebrates (Moussian, 2019).
Process (Simple Steps) of chitin degradation
The hydrolysis of chitin occurs in a two-step process;
1. Depolymerization
Depolymerization is the process of reduction of chitin polymer length by

the breakdown of β-1,4 linkages between the N-acetyl glucosamine
units. This process results in the release of N-acetylglucosamine units by
the action of chitinases of chitosanases.
2. Deacetylation
Depolymerization is followed by acetylation which causes the release of

glucosamine units and acetic acid. Chitin deacetylases act on the N-
acetyl glucosamine dimer or trimers, resulting in the catalytic
degradation of the larger molecule into smaller ones. The end products of
this step are glucosamine and acetic acid, which are then utilized by the
microorganism for various purposes.
Microbial chitin degradation occurs by one of the two mechanisms;

chitinoclastic mechanism and deacetylation mechanism.
1. Chitinoclastic
In this mechanism, the substrate is acted upon by the chitinolytic system,

consisting of chitinases. Exochitinase breakdown acetylchitobiose units
from the non-reducing end of the polysaccharide chain. Endochitinase
cleaves glycosidic linkages randomly along the chain, eventually
resulting in the formation of diacetylchitibiose as the major product,
along with some tri-acetyl chitotriose (Casadidi et al., 2019).
2. Deacetylation
The group of enzymes involved in the deacetylation mechanism is

termed deacetylases. These enzymes catalyze the process of
deacetylation of N-acetylglucosamine polymer. The hydrolysis of
chitosan occurs in the presence of chitosanases that breakdown the
linkages between the β-glucosamine units linked together by β-1, 4-
glycosidic linkages. This cleavage results in the release of chitobiose
(glucosaminyl-(1-4)- β-glucosaminide). Degradation of Chitin is shown
n figure 6.
108
Degradation of
Natural Compound
Figure 6: Degradation of Chitin (Moussian, 2019)

Note: a) Use the space given below for your answers.
b) Check your answers with those given at the end of the unit
1. What is Biodegradation?
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2. Explain degradation of cellulose.
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3. Define Chitin and its degradation?
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5.6 DEGRADATION OF LIGNIN

Lignin is a It is the most abundant aromatic biopolymer that consists of about
30% of the organic carbon on Earth. Lignin is an essential component of the
lignocellulosic biomass where it accounts for about 15-30% of the total
weight (Rani et al., 2008).
109
Biodegradation of The chemical species like hydroxycinnamyl alcohols (or monolignols)
Natural Xenobiotic
Compounds coniferyl alcohol and sinapyl alcohol, with typically minor amounts of p-
coumaryl alcohol, are the primary building blocks of lignin.
Structure of lignin
Lignin is composed of phenylpropane units linked together by the chemical

linkages of alkyl-alkyl, alkyl-aryl, and aryl-aryl groups. Ecological factors,
like plant growth, nutrition, climate, and illumination, also affect the
chemical structure of lignin.
Natural lignin consists of three important elements: carbon, hydrogen, and

oxygen, of which the carbon content is much higher than in homogenous
carbohydrates. Besides, important structural characteristics of lignin include
the functional groups, including alcohol hydroxyl group, carbonyl group,
carboxyl group, phenolic hydroxyl group, methoxyl, and sulfonic acid.
Structure of lignin is shown in figure 7.
Figure 7: Structure of Lignin (Source: Vinardell et al., 2017)
Microorganisms involved in lignin degradation
Different microbial population dominates at various stages and has distinct

roles in the degradation of organic matter.
1. Lignin-degrading bacteria
The occurrence of lignin-degrading enzymes has been observed in
Mycobacterium tuberculosum, M. avium, Pseudomonas syringae, P.
aeruginosa, P. putida, Bordetella pertussis, Xanthomonas campestris,
Escherichia coli, Caulobacter crescentus Rhodobacter capsulatus,
Yersinia pestis, Campylobacter jejuni, and Aquifex aeolicus.
2. Lignin-degrading Actinomycetes
Streptomyces and other actinomycetes have been identified as lignin-
degrading species and can be isolated from a wide variety of sources,
including a range of soils, high-temperature environments, and termite
guts. Lignin-degrading enzymes have been observed in five different
species of Streptomyces; Streptomyces antibioticus, S. griseus, S.
110
Degradation of
coelicolor, S. cyaneus, and S. lavendulae. Natural Compound
3. Lignin-degrading Fungi
Among different wood-decaying fungi, only the white rots have the
potential to degrade all three major components of wood entirely. These
fungi mainly belong to the Ascomycetes, Deuteromycetes, or
Basidiomycetes group. Typical examples of white-rot fungi are
Ganoderma applanatum and Heterobasidion annosum that preferentially
remove lignin without a substantial loss of cellulose and cause white-
pocket or white-mottled type of rot. Other Ascomycetes like Rhizoctonia
solani, Aspergillus nidulans, Podospora anserina, Neurospora crassa,
Gaeumannomyces graminis var. tritici and Trichoderma reesei have also
been described to produce laccase and other lignocellulolytic enzymes.
Lignolytic Enzymes
Primarily, three different enzymes are involved in lignin degradation;
manganese peroxidase (MnP), lignin peroxidase (LiP), and laccases..
1. Lignin peroxidases (LiP)
Lignin peroxidase is an extracellular heme-containing peroxidase that is

dependent on H2O2 and degrades a variety of lignin-related compounds.
These enzymes have an unusually high redox potential and low optimum
pH, typically showing little specificity toward substrates (Rani et al.,
2008).
Lignin peroxidase is well known as part of the ligninolytic system both

of aphyllophoralic and agaricalic fungi. These peroxidases preferably
oxidize methoxylated aromatic ring without a free phenolic group.
Methoxylated benzenes and benzyl alcohols are the simplest aromatic
substrates for lignin peroxidase. The role of lignin peroxidase in
ligninolysis could be the further transformation of lignin fragments
which are initially released by manganese peroxidase.
LiP is used commercially to mineralize a variety of recalcitrant aromatic

compounds, like three- and four-ring polyaromatic hydrocarbons,
polychlorinated biphenyls, and natural dyes.
2. Manganese peroxidases (MnP)
Manganese peroxidase is an extracellular heme-containing peroxidase

with a requirement for Mn2+ as its reducing substrate that has lignin-
reducing properties. Manganese peroxidase is one of the most common
lignin-degrading peroxidases produced by the majority of wood-
decaying fungi and many litter-decomposing fungi. Structurally, these
enzymes are glycosylated proteins with an iron protoporphyrin IX
(heme) prosthetic group.
The enzyme oxidizes Mn2+ to Mn3+,which in turn oxidizes phenolic

structures to phenoxyl radicals. Mn3+ formed is highly reactive and 111
Biodegradation of complexes with chelating organic acids such as oxalate or malate. As the
Natural Xenobiotic
Compounds redox potential of the MnP-Mn complex is lower than that of lignin
peroxidase, it preferably oxidizes phenolic substrates. The phenoxyl
radicals formed might further react, resulting in the eventual release of
CO2. The phenoxyl radicals formed subsequently cleave Cα -Cα or alkyl-
phenyl bonds causing depolymerization to smaller intermediates
including quinones and hydroxyl quinines.
3. Laccases
Laccases are a group of lignin-degrading enzymes consisting of N-

glycosylated extracellular blue oxidases and four copper atoms in the
active site that are distributed among different binding sites. Laccases
catalyze the oxidation of several aromatic hydrogen donors with
subsequent reduction of oxygen to water.
Moreover, laccases oxidize not only the phenolic and methoxyphenolic

acids but also decarboxylate them and attack their methoxy side chains
or groups. Several fungal laccases have been considered for the oxidation
of compounds like 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)
propan-1,3-diol (I), and phenolic lignin model compounds like phenol
red in the presence of the redox mediators. Laccases have been reported
to oxidize many recalcitrant substances, such as chlorophenols
polycyclic aromatic hydrocarbons (PAHs), lignin-related structures, and
organophosphorus compounds (Rani et al., 2008).
Process (Simple Steps) of lignin degradation

The overall process of lignin degradation can be explained in two simple
steps;
1. Depolymerization
The first step of lignin degradation is the depolymerization of aryl and
biaryl compounds such as β-aryl ethers. This step is a non-specific step
that occurs extracellularly by different bacterial and fungal enzymes.
Depolymerization of lignin occurs as a result of cleavage of β-O-4 ether
bond that represents about 50% or more of the total linkages in lignin.
This step decreases the length of the lignin polymer so as to obtain
dimeric or oligomeric units, which can then further be degraded into
smaller molecules. Depolymerization in microorganisms is catalyzed by
different lignin-degrading enzymes found in many microorganisms like
peroxidases and phenol oxidases. These enzymes attack the lignin
randomly, then convert the phenolic group to free-radicals, and these
radicals lead to lignin depolymerization(Vinardell et al., 2017).
2. Solubilization and Mineralization
The smaller molecules of lignin formed after depolymerization are now
taken by different microorganisms that catalyze a series of conversion by
various in vivo enzymes. Most of the linkages within the lignin
112
Degradation of
molecules have their specific metabolic pathways to cleave these specific Natural Compound
linkages. The mineralization and solubilization of oligomers and
monomers result in the formation of CO2 and other essential molecules
that can be utilized by the organisms. Degradation of lignin is shown in
figure 8.
Figure 8: Degradation of Lignin (Source: Vinardell et al., 2017)
5.7 ENVIRONMENTAL FACTORS INFLUENCES

IN BIODEGRADATION
Soil type and soil organic matter content affect the potential for adsorption of
an organic compound to the surface of a solid. Absorption is an analogous
process wherein a contaminant penetrates into the bulk mass of the soil
matrix. Both adsorption and absorption reduce the availability of the
contaminant to most microorganisms and the rate at which the chemical is
metabolized is proportionately reduced. Variations in porosity of the
unsaturated and saturated zones of the aquifer matrix may influence the
movement of fluids and contaminant migration in groundwater. The ability of
the matrix to transmit gases, such as oxygen, methane and carbon dioxide, is
reduced in fine grained sediments and also when soils become more saturated
with water. This can affect the rate and type of biodegradation taking place.
The oxidation-reduction potential of a soil provides a measurement of the
electron density of the system. Biological energy is obtained from the
oxidation of compounds in which electrons are transferred to various more
oxidized compounds referred to as electron acceptors. A low electron density
(Eh greater than 50 mV) indicates oxidizing, aerobic conditions, whereas
high electron density (Eh less than 50 mV) indicates reducing, anaerobic
conditions
113
Biodegradation of
Natural Xenobiotic 5.8 COMPOSTING
Compounds
Composting is the natural process of turning organic matter in waste into a
beneficial fertilizer that can benefit both soil and plants. Composting converts
organic waste such as food waste, manure, leaves, grass trimmings, paper,
wood, feathers, agricultural residue, etc. into beneficial organic fertilizer by
using various microorganisms such as bacteria and fungus (Ipek et al., 2002).
Compostable materials include anything that can be consumed or cultivated

in a field or garden. Compostable materials include fruits, vegetables, dairy
products, cereals, bread, unbleached paper napkins, coffee filters, eggshells,
meats, and newspapers. Plastics, grease, glass, and metals, such as plastic
utensils, condiment packages, plastic wrap, plastic bags, foil, silverware,
drinking straws, bottles, polystyrene, and chemicals, cannot be composted.
Red meat, bones, and small bits of paper can be composed, but they
decompose more slowly.
Composting goes through three main phases under optimal conditions:
Mesophilic Phase: It is an initial phase where mesophilic bacteria such as

Listeria monocytogenes, Staphylococcus aureus, and Escherichia coli
decompose the material at moderate temperatures.
Thermophilic Phase: As the temperature rises, a second, thermophilic phase
begins, in which diverse thermophilic bacteria such as lactobacilli,
Streptococcus thermophilus and bifidobacteria carry out breakdown at higher
temperatures (50 to 60 °C).
Maturation Phase: In the maturation phase, when the supply of high-energy
molecules diminishes, the temperature begins to drop, and mesophilic
bacteria once again take the lead.
Procedures
Sorting and Shredding
Sorting and separating procedures isolate decomposable materials from glass,
metal, and other inorganic components in trash. These are done mechanically,
based on variances in the refuse's physical features such size, density, and
magnetic properties. The size of the waste items is reduced by shredding or
crushing, resulting in a homogenous mass of material. Hammer mills and
rotary shredders are used to accomplish this.
Digesting and Processing
Composting crushed waste can be done in an open windrow or in an enclosed
mechanical facility. It may take five to eight weeks for the waste to be
completely digested, depending on moisture conditions. Temperatures in an
active compost pile reach around 65 °C (150 °F) due to the metabolic action
of aerobic bacteria, destroying harmful organisms that may be present in the
waste material (Ipek et al., 2002).
114
Degradation of
Schematic Layout of Composting is shown in figure 9. Natural Compound
Figure 9: Schematic Layout of Composting (Source: Kumar 2011)
Various Methods
Passive Composting or Piling includes simply stacking the materials and

allowing them to degrade naturally is passive composting or piling. This
process is simple and inexpensive, but it is slow and may produce unpleasant
odors. Various methods are described below
• Aerated Static Piles: Air is added to the stacked pile via perforated
pipes and blowers in aerated static piles. This approach does not involve
any labor to change the compost, but it is weather-dependent and can
result in unpredictable pathogen elimination due to poor mixing.
• Windrows: Windrows are long, narrow piles that are turned when
temperature and oxygen requirements dictate. This process generates a
consistent output that can be remotely tracked. Turning the compost, on
the other hand, might be time-consuming or expensive. Windrows are
commonly utilized for huge volumes that take up a lot of space.
Windrows can also cause odor issues and leachate concerns if they are
exposed to rain.
• Bins: Small amounts of food waste are usually composed in bins. Bins
with wire mesh or wooden frames allow for adequate air circulation and
require little labor.
• In-vessel System: In-vessel systems, which use perforated barrels,
drums, or specifically manufactured containers, are easy to use, turn,
require little effort. They are not weather sensitive, and can be used in
urban and public settings. The initial investment can be expensive, yet
handling volumes are usually minimal. (Ipek et al., 2002).
Advantages
• Reduction in Methane
115
Biodegradation of • Reduction in Chemical Fertilizers
Natural Xenobiotic
Compounds • Compost encourages agricultural crop yields to increase.
• By rehabilitating contaminated, compacted, and marginal soils, compost
can help with reforestation, wetlands restoration, and habitat
rejuvenation projects.
• Compost can be used to rehabilitate soils that have been contaminated by
hazardous waste at a low cost.
• Compost can save money over traditional soil, water, and air pollution
remediation solutions.
• Improves Moisture Dispersion
• Aids Carbon Sequestration
5.9 VERMICOMPOSTING
The term vermiculture refers to the cultivation or production of earthworms.
Vermicomposting is the method by which worms are used to turn organic
materials (usually waste) into a humus-like substance known as Vermicast.
The term vermicast is also termed as worm castings, worm manure, worm
feces and worm humus. Vermicompost includes not only worm castings, but
also bedding materials and organic waste in different phases of
decomposition. It also includes worms that are at different stages of
development and microorganisms involved in the composting process.
In the home garden, earthworm castings often contain between 5 and 11

times more nitrogen, phosphorous, and potassium than the surrounding soil.
Types of vermicomposting worms:
On the basis of their feeding habits, they are classified as detrivores and
geophages. Detrivores feed on plant litter or dead roots, and other plant
debris or on mammalian dung on or near the soil surface. These worms are
referred to as humus formers and comprise the epigeic and anecic forms.
Some examples of detrivorous worms are Perionyx excavatus, Eisenia fetida,
Eudrilus euginae, Lampito mauritii, Polypheretima elongata, Octochaetona
serrata and Octochaetona curensis. Geophagous worms, feed below the
surface and intake greater quantities of organically rich soil. Red worm
species in both Eisenia fetida and Lumbricus rubellus are composters, living
naturally in soils that contain a lot of organic matter. For this reason, they are
often used together, with Eisenia fetida on the surface and Lumbricus
rubellus farther down, in vermicomposting systems (Soobhany et al., 2015).
Process of vermicomposting:
1. Feeding materials:
Worms can eat dung from animals, agricultural waste, residues from
vegetables, waste from the food market, waste from the flower market,
116
Degradation of
agro-industrial waste, waste from the fruit market and all other bio- Natural Compound
degradable waste. Before being used for vermicompost production, cattle
dung should be dried in open sunlight. Depending on the feedstock being
used, temperature, moisture levels and the density of the worm
population, the exact loading rate (at which raw feedstock will be applied
to a worm bed) can differ. Proper loading rates require no inclusion of
new feedstock until the bulk of the feedstock previously introduced has
been decomposed. A high protein feedstock such as grains, mash, or
cottonseed meal is added if worms are not growing.
2. Bedding materials
As bedding products, certain agricultural residues may be used, such as

plant waste and solid composted manure. In general, because of bedding
content’s effect on increasing soil pH, which is harmful to worms, the
bedding content should maintain moisture, stay loose and aerated, and be
low in protein and nitrogen. The bedding content should be varied to
provide the earthworms with a variety of nutrients and to create richer
compost.
Suitable bedding materials includecoir waste, Cardboard, Shredded fall

leaves, sawdust, chopped straw, mulched paper such as newspaper, semi-
composted solid manure, By shredding raw materials into small pieces,
decomposition can be accelerated.
3. Blending
To achieve a near optimal C/N ratio of 30:1-40:1, carbonaceous

substances such as sawdust, paper and straw can be combined with
nitrogen-rich products such as sewage sludge, biogas slurry, and fish
scraps. Good quality compost, rich in main and micro nutrients, is
produced by a varied mixture of substances.
4. Pre-composting/Half digestion:
In order to avoid worm systems from feeling so much sun, manure

feedstocks and bedding should be pre-composted. When introduced into
the worm systems, fresh manures produce a lot of energy that transfers
into additional heat. Strong heat in the beds of worms can be deadly. The
bedding and feeding materials are then combined, watered and allowed
to ferment for approximately two to three weeks. It is necessary to hold
the raw materials in piles to allow the temperature to exceed 50-55o C.
The substance is overturned 3 to 4 times during this phase to get the

temperature down and to aid in uniform decomposition. It is passed to
the vermicompost production method as the material becomes very
fragile, and worms are inserted into it ranging from a few days to a few
weeks old.
117
Biodegradation of
Natural Xenobiotic
Compounds
Figure 10: Process of Vermicomposting (Souce: Sahariah et al, 2015)
5. Moisture, temperature and pH:

50-60 percent is the optimal moisture level for maintaining aerobic
conditions. The temperature should be within 25-30° C of the stacks. The
activity of microflora and earthworms can be decreased by higher or
lower temperatures. The height of bed can help to regulate the increase in
temperature. The raw materials pH should not be greater than 6.5 to 7.
Methods of vermicomposting:
• A 1 meter by 1 meter by 0.3 meter container carries about 30-40 kg of
bedding and feeding materials. It is possible to prepare a vermiculture
bed or worm bed (3 cm) by putting dust or husk or coir waste or sugar
cane garbage in the bottom of the tub/container.
• The culture bed can be spread with a sheet of fine sand (3 cm) followed
by a layer of garden soil (3 cm). A15-20 cm sheet of organic waste
material (pre-composted/half digested) can be spread on the worm bed.
• It is sprinkled with rock phosphate powder (to increase the content of
phosphate) if required. Soil or cow dung is used to cover the organic
layer with (sprinkle cow dung slurry).
• The selected earthworms are released through the cracks created (1000-
1500). In order to prevent birds from eating the earthworms, cover the
ring with wire mesh or gunny sack. Water is sprinkled to maintain
adequate humidity and temperature regularly/daily. l.
Harvesting of vermicompost:
• In about 3 months, the vermicompost is ready (may vary depending on
organic waste used as substrate). It will be black, granular, lightweight
118 and humus-rich. Vermicompost harvesting requires manual isolation of
Degradation of
worms from the castings Natural Compound
• Watering is stopped for two to three days before emptying the beds to
facilitate the removal of the worms from the compost. The worms will be
pushed to the bottom of the bed by this. For new culture beds, the worms
are collected. To retrieve the cocoons, young worms, and unconsumed
organic waste, the gathered vermicompost is dried and passed through a
3 mm sieve. For seeding the new culture beds, cocoons and young
worms are used.
Storage and packing of vermicompost:
• The harvested vermicompost should be stored in dark, cool location.
• It should have moisture of at least 40 percent.
• Sunlight should not fall on the content being composted.
• At the point of sale, packaging can be done.
• Periodic sprinkling of water can be done to retain the level of moisture
and also to maintain a beneficial microbial population if it is kept in an
open location.
• Vermicompost may be preserved for duration of one year without loss of
quality if the moisture level is kept at 40%.
5.10 USE OF AGRO WASTE IN MUSHROOM

CULTIVATION
Agricultural wastes are rich in lignocellulosic components which are difficult
to breakdown, but can effectively be done mushroom cultivation. Mushrooms
are fleshly fungi, spore bearing fruiting bodies which are produced above
ground on soil. They often refer to fruiting body of the gill fungi, which do
not contain chlorophyll like green plants and as a result cannot manufacture
food by their own. They are very nutritious products that can be generated
from lignocellulosic waste materials. The bioconversion of agricultural
wastes into a value added products is a good mean of their use. The property
of edible mushroom fungi to convert complex organic compounds into
simpler one’s is used to transform the useless agricultural waste into valuable
product. Various edible mushroom strains are cultivated worldwide
(Udayasimha L, Vijayalakshmi YC 2012). Some of them are given below:
• Button -Agaricus
• Oyster -Pleurotous
• Shiitake -Lentinula Edodes
• Straw -Volvallella volvacea
• Chinese mushroom - Ganoderma
Besides having many nutrional values they are also useful in waste
management. The choice of species to cultivate depends on the availability of
growth media. Oyster mushroom is the third most cultivated edible
119
Biodegradation of mushroom in the world. Oyster mushrooms are easiest to grow as they can
Natural Xenobiotic
Compounds grow on many substrates but their cultivation has one drawback as some
people are allergic to their spores. In these cases, air-cleaning equipment or
respirators are necessary in order to safely work in the production facility.
Because of the short shelf life this species offer a special advantage to the
local grower who markets directly and can continuously deliver a fresh, high-
quality product (Reis et al., 2012).
Two mycorrhizal mushrooms, morels and truffles are commercially
cultivated. Mushroom cultivation offers benefits to market gardens when it is
integrated into the existing production system. Mushrooms are rich in various
nutrients such as:
• Protein- Protein content of dry weight is between 18% and 37%.
• Fat- Fat is present at low rate, content between 1-8%. The high content
of linollic acids is one of the reasons why mushrooms are considered
healthy food.
• Vitamins and minerals - Mushrooms are a good source of vitamins
such as thiamine (Vitamin B), Riboflavin (vitamin B2) and ascorbic acid
(vitamin C), folic acid. They also contain significant amounts of
phosphorus, sodium, potassium, calcium, magnesium, iron and Zinc.
Protein content of mushroom in paddy was significantly higher than in wheat
straw while lipid content of mushrooms was higher in wheat straw than
paddy straw. Mushrooms have medicinal values as they contain substances
which lower the cholesterol level in serum and liver which in turn makes it
good for those suffering from heart diseases. Some of them contain
substances, which suppress the growth rate of tumors (Reis et al., 2012)
Free radicals can damage body cells and induce cancers. Free radicals are the
result of specific transformation process. Many bio active compounds protect
the body against these radicals. These substances are often called anti-
oxidants and are present in many mushrooms. In other words, the body
immunity is boosted. This will be a relief to those suffering from HIV/AIDS
(Reis et al., 2012).
Agricultural Wastes
Agricultural wastes are the good source for the cultivation of mushrooms.
Some of them are most commonly used such as wheat straw, paddy straw,
rice straw, rice bran, molasses, coffee straw, banana leaves, tea leaves, cotton
straw, saw dust etc.
For the cultivation of Pleurotus rice straw, wheat straw and cotton straw are
the substrates that are commonly used while for Agaricus, it is wheat straw
which is usually used. A disadvantage of straw is that it should be prepared
first, especially if mushrooms are to be grown indoors. Straw is laden with
other microbes, and it is necessary to get rid of those tiny competitors, as
there will be no scope of mushroom mycelium to grow in their presence.
Rice bran, coffee pulps are the main substrates used for the cultivation of
Lentinula edodes. Banana leaves and tea leaves are used for Volvallella and
120 Pleurotus respectively. Reis et al., (2012) proposed using banana leaves for
the cultivation of Pleurotus spp. Degradation of
Natural Compound
Ganoderma can be cultivated using sawdust. Sawdust itself is often not
nutritious enough and needs to be supplemented with a nitrogen source such
as bran, urea, sunflower seed and horse manure.
Cultivation of oyster mushroom is of most concern as its spores are allergic
to some people, so related preventive measures should be done in working
facility. Besides this, oyster mushrooms have a short life span, so they are
beneficial to those growers who can sell them fresh in market (Tables 1-3).
Table 5.1: Various types of agricultural wastes used for mushroom

cultivation. (Source: Nicolcioiu et al., 2016)
Sl no. Agriculture waste Strains

1 • Agricultural waste Pleurotus sp.
• .Rice straw
• Wheat straw
• Cotton straw
• Tea leaves
• Banana leaves
2. • Wheat straw Agaricus bisporus
3 • Rice bran Lentinula edodes
• Coffee pulp
4. • Tea leaves Volvallella
5. • Sawdust Ganoderma
Table 5.2: Composition of agricultural wastes used for mushroom

cultivation. (Source: Nicolcioiu et al., 2016)
1. 1% protein
13% lignin
Wheat straw
39% hemicelluloses
40% cellulose
2. Rice straw 41% cellulose
14% lignin
0.8% total nitrogen
0.25% P2O5
0.3% K2O
6% SiO2
pH 6.9
3. Sugarcane bagasse Cellulose 35-40%
Hemicellulose 20-25%
Lignin 18-24%
Ash 1-4%
Waxes <1%
Nitrogen 0.7%
121
Biodegradation of Table 5.3: Combination of substrates reported on various strains and
Natural Xenobiotic
Compounds their effect. (Source: Nicolcioiu et al., 2016)
S. No Substrate Composition strain Effects

1. Barley straw+wheat bran Pleurotus 4.64% protein
and wood chips+soybean eryngii content
powder+rice bran
treatments
2. Wheat straw+wheat
Pleurotus 13.66% protein
bran+soybean powder
eryngii content
treatment
3. soybean straw+wheat Pleurotus 87.3% Biological
straw sajorcaju efficiency
4. soybean straw+saw dust Pleurotus 43.8% Biological
sajorcaju efficiency
5. corncob (CC)+sugarcane Pleurotus corncob
bagasse ostreatus (CC)+sugarcane
bagasse
Correlation of Agricultural Wastes Composition with Mushroom

Cultivation
For high yield of mushroom cultivation, it is necessary that the entire

nutritional requirement should be fulfilled in optimum concentration as
various researches has reported low yield when nutrients in a medium are
either in low or high concentration. Banana stalk and Bahia grass are used for
the cultivation of Pleurotus sajor-caju with biological efficiency of 74.4%
and 74.12% respectively but there is a low yield when they are supplemented
with other components. This can be due to high nitrogen concentration which
hinders its yield.
Growth of Pleurotus ostreaus resulted similar in paddy straw and wheat

straw while in sugarcane bagasse it resulted in low yield. Reason behind this
selective high yield must be appropriate concentration of lignin,
hemicelluloses, cellulose in substrate.
There is a Positive correlation of cellulose: lignin with mycelia growth and

high yield in Pleurotus ostreatus and carbon: nitrogen ratio with mushroom
yield in Pleurotus eryngii and Agaricus aegerita while in V. volvacea strains
high yield is related to cellulose content (Thongklang and Luangharn, 2016).
Combination of Agricultural Substrates Used For Cultivation
In addition to the use of supplements with agricultural wastes as a substrate,

various combination of agricultural wastes are also used for the cultivation
and are reported to be optimal substrate.
122
Degradation of
Vegetable waste when used in combination with paddy straw resulted in high Natural Compound
yield of oyster mushroom. To cultivate P. ostreatus sawdust in addition to
rice husks is reported as an optimal substrate. The quality of P. eryngii was
significantly affected by substrate ingredients. On barley straw and sugar beet
pulp substrate complemented with rice bran, highest mushroom fresh weight
and moisture content were achieved.
For Pleurotus sajor-caju, combination of soybean straw, wheat straw showed

significantly highest yield while soybean straw and saw dust combination
showed significantly lesser yield.
Supplements Used With Agricultural Wastes
Agricultural wastes are used in addition to various supplements such as

gypsum, lime and urea. Gypsum contributes as a calcium source and
regulates the acidity level. Water holding capacity of gypsum is high which
prevent excess wetting of the substrate. Lime is used to adjust pH. Mushroom
cultivation needs appropriate nitrogen content for high yield, which can be
fulfil by various components such as urea, bran, sunflower seed, molasses,
horse manure.
Optimum Conditions for Cultivation
Besides having appropriate composition for significantly high yield of

mushroom, optimum conditions of the environment during cultivation should
also be maintained. Given below are the usual optimum conditions that
should be maintained during cultivation.
• Temperatures of 15-35°C
• pH of about 6.5
• Carbon dioxide (CO2) level to be between 15-20%
• Humidity to be between 86-90%
• CO2 should be between 0-0.6percent.
• Temperatures and humidity levels should be regulated at 86% and 10-
28°C respectively.
5.11 ADVANTAGES AND COST CONSIDERATION

India, being a second major producer of vegetables in the world; contributes
14% of total world vegetable production. Taking estimated production of
fruits and vegetables in India at 150 million tons, the total waste generation
comes to about 50 million tons per annum. Due to their chemical
composition fruits and vegetables wastes are more prone to spoilage than
cereals, which create unhygienic condition leading to spread of diseases and
loss of resources. The vegetable wastes are a rich in nitrogen and
carbohydrate but are not fit for consumption. These wastes can be utilized for
the production of various types of mushroom such as the oyster variety.
123
Biodegradation of In the recent times the waste management is of most concern. Proper
Natural Xenobiotic
Compounds management and execution of waste disposal practices have become today’s
need. The inappropriate management of waste gives rise to many problems
such as rapid spread of infectious diseases, development of new varieties of
diseases. The exponential increase in the present amount of waste produced
brings to notice an immediate requirement of solution to overcome this
problem.
An agricultural waste consists of lignin and cellulose, which are difficult to

breakdown. They are insoluble and bind to inert substances in soil and get out
of reach of bacterial culture present in soil. While mushroom’s mycelium
releases extracellular enzymes, which are responsible for the lignin
degradation. Pleurotus and Lentinus have their own enzymes systems based
on endoglucanase, laccase and phenoloxidases. The large amount of
agricultural wastes and appropriate climatic conditions provide massive
scope for oyster mushroom cultivation in Sagar, M.P.
An agricultural waste provides the opportunity for cost effective farming.

Even after being used for mushroom cultivation, it can be used later on as
manure for agricultural field as now the nutrient contents are at acceptable
range. Cultivation of mushroom on these residual wastes is one of the most
eco-friendly practices to fight the malnutrition and environmental pollution
caused by these wastes. Various researches is still going on to exploit the
potential of agricultural wastes either by using them in combination or by
giving them pre-treatment.
5.12 PROCESS AND NEWLY EMERGING

TECHNOLOGIES
Mushroom growing involves spawn production, composting, cultivation.
Steps are shown in figure 11.
Figure 5.11: Three steps in mushroom cultivation (Source: Ralph and Kurtzman, 1994)
Cultivation technology is different for different mushrooms. Proper

knowledge on mushroom life cycle and good training of all the steps is a
must before starting cultivation of any mushroom. However the basic steps
are the same for majority of mushrooms (figure11).
• The first step before starting cultivation is to procure or produce spawn

of good quality.
• Second step is to prepare the substrate of good quality. Method of

spawning, that is mixing of spawn in compost, and amount of spawn
required will also vary in different mushrooms. In some cases spawn
124
Degradation of
may be mixed thoroughly whereas in other cases it may be put layer Natural Compound
wise. Spawning in some cases can be done in open under hygienic
conditions whereas in other cases, particularly where the substrate has
been autoclaved, the spawning can be done only under sterile conditions.
We need only half kg to one kg of spawn for 100 kg of compost in
button mushroom, where as in oyster we need 2.5 kg and in milky
mushroom we may require up to 5 kg spawn for 100 kg of substrate.
• The third step is cropping. After spawn run, that is allowing the fungus to
spread throughout the substrate, we take steps to induce formation of
mushrooms. In some cases it is required to put a layer of casing material
whereas in other cases fruiting can be obtained as such. In all cases, to
induce fruiting some sort of change is required. For example in case of
button mushroom temperature is lowered from 25 to 17°C and carbon
dioxide levels are lowered by giving fresh air. In Oyster, to induce
fruiting both fresh air and diffused light is necessary.
In India, mushroom cultivation in rural areas has emerged as an important

activity for educated, school dropouts, women, landless people, etc.
Considering the demand for quality foods, mushroom cultivation has
emerged as an important avocation. Many commercial units that grow
mushrooms under controlled conditions have also been set up in different
parts of our country. However, before taking up this venture a thorough
knowledge of the subject and scientific aptitude towards agriculture is
necessary.

1. What is Composting?
…………………………………………………………………………….
…………………………………………………………………………….
…………………………………………………………………………….
…………………………………………………………………………….
…………………………………………………………………………….
2. What is Vermicomposting?
…………………………………………………………………………….
…………………………………………………………………………….
…………………………………………………………………………….
…………………………………………………………………………….
…………………………………………………………………………….
…………………………………………………………………………….
125
Biodegradation of 3. What is Mushroom cultivation?
Natural Xenobiotic
Compounds …………………………………………………………………………….
…………………………………………………………………………….
…………………………………………………………………………….
…………………………………………………………………………….
…………………………………………………………………………….
5.13 LET US SUM UP

We have studied the degradation of cellulose, hemicellulose, chitinn and
lignin. We have studied the many aspects of composting, the assessment of
the importance of vermicomposting and mushroom cultivation by utilizing
agro residues. .
5.14 KEY WORDS

Biodegradation: Biodegradation is the degradation of the materials into
environmentally acceptable products such as water, carbon dioxide, and
biomass by the action of naturally available microorganisms under normal
environmental conditions.
Cellulose: Cellulose, a complex carbohydrate, or polysaccharide, consisting
of 3000 or more glucose units.
Lignin: Lignin is an important organic polymer which is abundant in cell
walls of some specific cells. It has many biological functions such as water
transport, mechanical support and resistance to various stresses.
Hemicelulose: Hemicellulose is a natural polymer like cellulose, consisting
of carbohydrate monomers.
Chitin: A fibrous substance consisting of polysaccharides, which is the
major constituent in the exoskeleton of arthropods and the cell walls of fungi.
Case Study:
Exploring the microbiota dynamics related to vegetable biomasses

degradation and study of lignocellulose-degrading bacteria for industrial
biotechnological application
The aims of this study were to evaluate the microbial diversity of different
lignocellulosic biomasses during degradation under natural conditions and to
isolate, select, characterise new well-adapted bacterial strains to detect
potentially improved enzyme-producing bacteria. The microbiota of biomass
piles of Arundo donax, Eucalyptus camaldulensis and Populus nigra were
evaluated by high-throughput sequencing. A highly complex bacterial
community was found, composed of ubiquitous bacteria, with the highest
representation by the Actinobacteria, Proteobacteria, Bacteroidetes and
126
Degradation of
Firmicutes phyla. The abundances of the major and minor taxa retrieved Natural Compound
during the process were determined by the selective pressure produced by the
lignocellulosic plant species and degradation conditions. Moreover,
cellulolytic bacteria were isolated using differential substrates and screened
for cellulase, cellobiase, xylanase, pectinase and ligninase activities. Forty
strains that showed multienzymatic activity were selected and identified. The
highest endo-cellulase activity was seen in Promicromonospora sukumoe
CE86 and Isoptericola variabilis CA84, which were able to degrade
cellulose, cellobiose and xylan. Sixty-two percent of bacterial strains tested
exhibited high extracellular endo-1,4-ß-glucanase activity in liquid media.
These approaches show that the microbiota of lignocellulosic biomasses can
be considered an important source of bacterial strains to upgrade the
feasibility of lignocellulose conversion for the ‘greener’ technology of
second-generation biofuels.

REFERENCES
• Scheller H. V and Ulvskov P. (2010). Hemicelluloses. Annual Review of
Plant Biology, 61(1), 263–289. doi:10.1146/annurev-arplant-042809-
112315
• Xiros C, Katapodis P, Christakopoulos P. Factors affecting cellulose and

hemicellulose hydrolysis of alkali-treated brewers spent grain by
Fusarium oxysporum enzyme extract. Bioresour Technol. 2011
Jan;102(2):1688-96. DOI: 10.1016/j.biortech.2010.09.108.
• López-Mondéjar R, Zühlke D, Becher D, Riedel K, Baldrian P. (2016).
Cellulose and hemicellulose decomposition by forest soil bacteria
proceeds by the action of structurally variable enzymatic systems. Sci
Rep. Published 2016 Apr 29. doi:10.1038/srep25279
• Brunner, G. (2014). Processing of Biomass with Hydrothermal and

Supercritical Water. Supercritical Fluid Science and Technology, 395–
509.doi:10.1016/b978-0-444-59413-6.00008-x
• Gupta, R., Mehta, G., Deswal, D., Sharma, S., Jain, K. K., Kuhad, R. C.,
& Singh, A. (2013). Cellulases and Their Biotechnological Applications.
Biotechnology for Environmental Management and Resource Recovery,
89–106.DOI:10.1007/978-81-322-0876-1_6
• Moussian B. (2019) Chitin: Structure, Chemistry and Biology. In: Yang

Q., Fukamizo T. (eds) Targeting Chitin-containing Organisms. Advances
in Experimental Medicine and Biology, vol 1142. Springer, Singapore.
https://doi.org/10.1007/978-981-13-7318-3_2
• Casadidio C, Peregrina DV, Gigliobianco MR, Deng S, Censi R, Di

Martino P. Chitin and Chitosans: Characteristics, Eco-Friendly
Processes, and Applications in Cosmetic Science. Mar Drugs.
2019;17(6):369. Published 2019 Jun 21. doi:10.3390/md17060369
127
Biodegradation of • Vinardell, Maria Pilar, and Montserrat Mitjans. “Lignins and Their
Natural Xenobiotic
Compounds Derivatives with Beneficial Effects on Human Health.” International
journal of molecular sciences 18,6 1219. 7 Jun. 2017,
doi:10.3390/ijms18061219
• Chapter 2 – Structure and Characteristics of Lignin. Lignin Chemistry
and Applications. Elsevier. 2019. Pages 25-50.
https://doi.org/10.1016/B978-0-12-813941-7.00002-3.
• Udayasimha L, Vijayalakshmi YC (2012) Sustainable Waste
Management By Growing Mushroom (Pleurotusflorida) On
Anaerobically Digested Waste And Agro Residues. Int J Eng Res Tech
1: 12.
• Nicolcioiu MB, Popa G, Matei F (2016) Mushroom Mycelia Cultivation
on Different Agricultural Waste Substrates. Romania Scientific Bulletin
Series F. Biotechnologies Vol. Xx.
• Thongklang N, Luangharn T (2016) Testing agricultural wastes for the

production of Pleurotus ostreatus. Mycosphere 7: 766-772.
• Ralph H, Kurtzman J (1994) Nutritional needs of mushroom and

substrate supplements. In: Nair MC (editor), Advances in mushroom
biotechnology: 106-110.
• Rani P, Kalyani N, Prathiba K (2008) Evaluation of Lignocellulosic

Wastes for Production of Edible Mushrooms. Applied Biochemistry And
Biotechnology 151: 151-159.
• Reis FS, Barros L, Martins A, Ferreira I (2012) Chemical Composition

And Nutritional Value Of The Most Widely Appreciated Cultivated
Mushrooms: An Inter-Species Comparative Study. Food and Chemical
Toxicology 50: 191-197.
• Vyas D, Chaubey A, Dehariya P, Wagay AJ, Bajpai A, at al. (2009)
Perspectives on Agripreneurship And Rural Development, ICARD.
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• Kumar S., 2011. Review Article: Composting of municipal solid waste.

Critical Reviews in Biotechnology. 31(2), 112-136.
• Ipek, U., Obek, E., Akca, L., Arslan, E.I., Hasar, H., Dogru, M.,
Baykara, O., 2002. Determination of degradation of radioactivity and its
kinetics in aerobic composting. Bioresource Technology 84(3), 283-286.
• N. Soobhany et al. Comparative assessment of heavy metal content
during the composting and vermicomposting of municipal solid waste
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• B. Sahariah et al. Efficacy of bioconversion of paper mill bamboo sludge
and lime waste by composting and vermiconversion technologies
Chemosphere (2014).
128
Degradation of
5.16 ANSWERS TO CHECK YOUR PROGRESS Natural Compound

1. Biodegradation is biological catalyzed reduction in complexity of
chemical compounds. It is the process by which organic substances are
broken down into smaller compounds by living microbial organisms.
When biodegradation is complete, the process is called "mineralization".
However, in most cases the term biodegradation is generally used to
describe almost any biologically mediated change in a substrate.
2. Cellulose degradation occurs in three simple steps;
1. Hydrolysis by endoglucanases
The first step in the degradation of cellulose is the action of
endoglucanases that randomly attack the cellulose fibrils. This step
results in a decrease in the size of cellulose chains as it degrades the
polymer into smaller fragments. The enzyme acts internally at random
points of the polymer.
2. Hydrolysis by exoglucanases
Exoglucanases act on the smaller fragments resulting in even smaller
units of tetrasaccharides or disaccharides. Exoglucanases act on the
reducing end of the fragments to form either dimeric units or cellobiose.
3. Hydrolysis by β-glucosidase
β-glucosidase or cellobiose act on the dimeric units of glucose of
cellobiose to form monomeric units, glucose. This is the final step of
cellulose degradation that results in the formation of free individual units
of the glucose molecule.
Simple Steps of cellulose degradation
3. Chitin is a complex homopolysaccharide consisting of units of amino

sugar glucosamine that accounts for the second most abundant
polysaccharide of nature after cellulose.
Microbial chitin degradation occurs by one of the two mechanisms;
chitinoclastic mechanism and deacetylation mechanism.
129
Biodegradation of 1. Chitinoclastic
Natural Xenobiotic
Compounds In this mechanism, the substrate is acted upon by the chitinolytic system,
consisting of chitinases. Exochitinase breakdown acetylchitobiose units
from the non-reducing end of the polysaccharide chain. Endochitinase
cleaves glycosidic linkages randomly along the chain, eventually
resulting in the formation of diacetylchitibiose as the major product,
along with some tri-acetyl chitotriose.
2. Deacetylation
The group of enzymes involved in the deacetylation mechanism is
termed deacetylases. These enzymes catalyze the process of
deacetylation of N-acetylglucosamine polymer. The hydrolysis of
chitosan occurs in the presence of chitosanases that breakdown the
linkages between the β-glucosamine units linked together by β-1,4-
glycosidic linkages. This cleavage results in the release of chitobiose
(glucosaminyl-(1-4)- β-glucosaminide) which is then

1. Composting is the natural process of turning organic matter in waste into
a beneficial fertilizer that can benefit both soil and plants. Composting
converts organic waste such as food waste, manure, leaves, grass
trimmings, paper, wood, feathers, agricultural residue, etc. into beneficial
organic fertilizer by using various microorganisms such as bacteria and
fungus.
2. The term vermiculture refers to the cultivation or production of
earthworms. Vermicomposting is the method by which worms are used
to turn organic materials (usually waste) into a humus-like substance
known as Vermicast. The term vermicast is also termed as worm
castings, worm manure, worm feces and worm humus. Vermicompost
includes not only worm castings, but also bedding materials and organic
waste in different phases of decomposition. It also includes worms that
are at different stages of development and microorganisms involved in
the composting process.
3. Mushroom growing involves spawn production, composting, cultivation.
130 Three steps in mushroom cultivation

Degradation of
Cultivation technology is different for different mushrooms. Proper Natural Compound
knowledge on mushroom life cycle and good training of all the steps is a
must before starting cultivation of any mushroom. However the basic
steps are the same for majority of mushrooms (Fig. 1.10).
• The first step before starting cultivation is to procure or produce spawn
of good quality.
• Second step is to prepare the substrate of good quality. Method of
spawning, that is mixing of spawn in compost, and amount of spawn
required will also vary in different mushrooms. In some cases spawn
may be mixed thoroughly whereas in other cases it may be put layer
wise. Spawning in some cases can be done in open under hygienic
conditions whereas in other cases, particularly where the substrate has
been autoclaved, the spawning can be done only under sterile conditions.
We need only half kg to one kg of spawn for 100 kg of compost in
button mushroom, where as in oyster we need 2.5 kg and in milky
mushroom we may require up to 5 kg spawn for 100 kg of substrate.
• The third step is cropping. After spawn run, that is allowing the fungus to
spread throughout the substrate, we take steps to induce formation of
mushrooms. In some cases it is required to put a layer of casing material
whereas in other cases fruiting can be obtained as such. In all cases, to
induce fruiting some sort of change is required. For example in case of
button mushroom temperature is lowered from 25 to 17°C and carbon
dioxide levels are lowered by giving fresh air. In Oyster, to induce
fruiting both fresh air and diffused light is necessary.
In India, mushroom cultivation in rural areas has emerged as an
important activity for educated, school dropouts, women, landless
people, etc. Considering the demand for quality foods, mushroom
cultivation has emerged as an important avocation. Many commercial
units that grow mushrooms under controlled conditions have also been
set up in different parts of our country. However, before taking up this
venture a thorough knowledge of the subject and scientific aptitude
towards agriculture is necessary.
131
Biodegradation of
Natural Xenobiotic UNIT 6 IN SILAGE PRODUCTION FROM
Compounds
WASTE
Structure
6.1 Introduction
6.2 Objectives
6.3 Silage production from wastes
6.4 Benefit of Silage
6.4.1 Advantages
6.4.2 Disadvantages
6.5 The ensiling process
6.6 Basic principles
6.7 Role of saccharolytic and proteolytic organisms
6.7.1 Desirable microorganisms
6.7.2 Undesirable microorganisms and their metabolites
6.8 Preserving technique
6.9 Preventive Measures to Control Silage Spoilage
6.10 Preparation of Silage
6.11 Process in silage making
6.12 Planning for silage making
6.13 Use of silage
6.14 Quality of Silage
6.15 Strategies to limit silage degradation by undesirable microorganisms
6.16 Silage additives
6.17 Enzymology of silage production
6.18 Let Us Sum Up
6.19 Key Words
6.21 Answers to Check Your Progress Exercise
6.1 INTRODUCTION
During the cold, continental winter, the major fodders available are wheat or
maize straw, together with hay and concentrated feeds. As a minimum, it is
essential to provide a green fodder supplement to enhance rumen function for
bovine animals. For smallholder farmers with limited production capacity,
finding enough feed in the winter months to maintain good milk production is
always a problem. Many are forced to buy hay, concentrates or silage just to
keep their animals alive and are unable to benefit due to the higher prices
132 paid for animal feed in the winter months (Woolford et al., 1984).
In Silage Production
Fresh forage crops such as maize, grasses, legumes, wheat and lucerne can be from Waste
preserved by ensiling. In many countries ensiled forages are highly valued as
animal feed. In European countries such as The Netherlands, Germany and
Denmark more than 90% of the forages locally produced are stored as silage.
Even in countries with generally good weather conditions for hay making
such as France and Italy approx. 50% of the forages are ensiled (Wilkinson et
al.1996). It is essential to have a good microbial fermentation process to
produce high quality silage. A good fermentation process is not only
dependent on the type and quality of the forage crop, but also on the
harvesting and ensiling technique. In this paper our current knowledge on
general silage microbiology is reviewed with the aim to aid with the choice of
the best ensiling strategy to produce high quality silage (Auerbach et al.,
1998).
6.2 OBJECTIVES
• Define silage;
• Describe the advantages and disadvantages of silage;
• Describe the Basic principle of Silage Production;
• Describe the role of saccharolytic and proteolytic organisms in Silage
Production;
• Explain Phases of Silage Production;
• Explain the Enzymology of silage production.
6.3 SILAGE PRODUCTION FROM WASTES

Forage which has been grown while still green and nutritious can be
conserved through a natural 'pickling' process. Lactic acid is produced when
the sugars in the forage plants are fermented by bacteria in a sealed container
('silo') with no air. Forage conserved this way is known as 'ensiled forage' or
'silage' and will keep for up to three years without deteriorating. Silage is
very palatable to livestock and can be fed at any time.
Silage is the fodder which is conserved by reducing pH through natural

anaerobic fermentation and is used for feeding during scarcity period,
drought or floods and for utilizing surplus forage. The suitable crops are
sorghum, maize and oat etc. During lean period feeding of silage acts as a
green fodder and maintains livestock productivity ( Wiedmann, et al., 1994)
6.4 BENEFIT OF SILAGE

Forages can be made into hay to conserve the nutrients, especially protein,
before they decline in the plant. However it is often too wet to dry the
successfully and special machinery, has to be used to assist the forage to dry 133
Biodegradation of quickly. Forage crops such as maize, are too thick-stemmed to dry
Natural Xenobiotic
Compounds successfully as hay.
Silage is considered the better way to conserve forage crops. A forage crop
can be cut early and only has to have 30% dry matter to be ensiled
successfully. There is no need to dry out the plant material any more than
that, so wet weather is not such a constraint as it is with making hay.
Silage making is long practiced by the larger agricultural sector, but the
production method relies on heavy equipment and large production, in order
to dig or build storage pits and to compress the green mass, putting it beyond
the reach of smallholder farmers.
Silage is storage system of green fodder which keeps all parts of fodder in
appropriate condition for feeding than any other system of storage of fodder.
Silage requires less space for storage as it is pressed in pit/tank than hay
making. For daily cutting, transporting & chaffing of fodder in traditional
way requires more labour & time but in case of silage, fodder cutting,
transport, chaffing is done at one time only, so it is less labour & time
consuming practice.
Land under fodder cultivation is emptied, and immediately it is used for

plantation of other crops. So farmers’ can take more crops in same land in a
year against traditional way where land is reserved for fodder until all crops
is harvested. Silage is prepared in closed & air tight condition so there is no
danger of fire. Due to lactic acid in silage, it is easily digestible to animals, so
energy required for digestion is used for other purposes like milk production
etc. Silage is tasty & flavoured, so it increases appetite of dairy animals.
Important thing behind to adopt silage is in scarcity it provide supply of
fodder to dairy animals. Situations like drought, high rainfall & scarcity of
fodder, farmers may use silage for feeding to dairy animals. (Rain fed area
where shortage of green fodder is for March to June & in high rainy area or
water logged lands, it is impossible to cultivate or harvest fodder) Due to
treatment of additive for silage, farmers can supply energy, mineral &
vitamins to dairy animals (Hengeveld, 1983).
6.4.1 Advantages
• Stabile composition of the feed (silage) for a longer period (up to 5
years);
• Plants can be harvested at optimal phase of development and are
efficiently used by live-stock.
• Reduction of nutrient loses which in standard hay production may
amount to 30% of the dry matter (in silage is usually below 10%);
• More economical use of plants with high yield of green mass;
• Better use of the land with 2-3 crops annually;
• Silage is produced in both cold and cloudy weather
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6.4.2 Disadvantages from Waste
• Silage is not interesting for marketing as its value is difficult to be

determined.
• It does not allow longer transportation;
• The weight increases manipulation costs;
• Has considerably lower vitamin D content compared to hay.
• The fermentation in silage reduces harmful nitrates accumulated in plants
during droughts and in over-fertilized crops.
• Allows by-products (from sugar beet processing, maize straw, etc.) to be
optimally used;
• Requires 10 times less storage space compared to hay;
• Maize silage has 30-50% higher nutritive value compared to maize grain
and maize straw;
• 2 kg of silage (70% moisture) has the equal nutritive value of 1 kg of
hay.
6.5 THE ENSILING PROCESS

Ensiling is a forage preservation method based on spontaneous lactic acid
fermentation under anaerobic conditions. The epiphytic lactic acid bacteria
ferment the water-soluble carbohydrates (WSC) in the crop to lactic acid, and
to a lesser extent to acetic acid. Due to the production of these acids the pH of
the ensiled material decreases and spoilage microorganisms are inhibited.
Once the fresh material has been stacked and covered to exclude air, the
ensiling process can be divided into 4 stages (Weinberg and Muck 1996;
Merry et al. 1997). System for manufacturing of silage has been shown in
Figure 1.
Figure: 1 System for the manufacturing of Silage
Aerobic phase (Phase 1): This phase normally only takes a few hours in
which the atmospheric oxygen present between the plant particles is reduced,
due to the respiration of the plant material and aerobic and facultative aerobic
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Biodegradation of microorganisms such as yeasts and enterobacteria. Furthermore, plant
Natural Xenobiotic
Compounds enzymes such as proteases and carbohydrases are active during this phase,
provided the pH is still within the normal range for fresh forage juice (pH
6.5-6.0).
Fermentation phase (Phase 2): This phase starts when the silage becomes
anaerobic, and it continues for several days to several weeks, depending on
the properties of the ensiled forage crop and the ensiling conditions. If the
fermentation proceeds successfully lactic acid bacteria develop, and become
the predominant population during this phase. Due to the production of lactic
and other acids the pH decreases to 3.8-5.0.
Stable phase (Phase 3): As long as air is prevented from entering the silo,
relatively little occurs. Most microorganisms of phase 2 slowly decrease in
numbers. Some acid tolerant microorganisms survive this period in an almost
inactive state, others such as clostridia and bacilli survive as spores. Only
some acid tolerant proteases and carbohydrases and some specialized
microorganisms, such as Lactobacillusbuchneri continue to be active at a low
level.
Feed-out phase or aerobic spoilage phase (Phase 4): This phase starts as
soon as the silage gets exposed to air. During feed out this is unavoidable, but
it can already start earlier due to damage of the silage covering (e.g. by
rodents or birds). The process of spoilage can be divided into two stages. The
onset of deterioration is due to the degradation of preserving organic acids by
yeasts and occasionally acetic acid bacteria. This will cause a rise in pH, and
thus the second spoilage stage is started, which is associated with increasing
temperature, and activity of spoilage microorganisms such as bacilli. The last
stage also includes the activity of many other (facultative) aerobic
microorganisms such as moulds and enterobacteria. Aerobic spoilage occurs
in almost all silages that are opened and exposed to air. However the rate of
spoilage is highly dependent on the numbers and activity of the spoilage
organisms in the silage. Spoilage losses of 1.5-4.5 % dry matter loss/day can
be observed in affected areas. These losses are in the same range as losses
that can occur in airtight silos during several months of storage (Honig and
Woolford 1980).
To avoid failures it is important to control and optimize each phase of the

ensiling process. In phase 1 good silo filling techniques will help to minimize
the amount of oxygen present between the plant particles in the silo. Good
harvesting techniques combined with good silo filling techniques will thus
minimize WSC losses through aerobic respiration in the field and in the silo,
and in turn will leave more WSC available for lactic acid fermentation in
phase 2. During phases 2 and 3 the farmer cannot actively control the ensiling
process. Methods to optimize phases 2 and 3 are therefore based on the use of
silage additives that are already applied at the time of ensiling.
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Phase 4 will start as soon as oxygen is available. To minimize spoilage losses from Waste
during storage an airtight silo is required, and any Silage fermentation
processes and their manipulation damage to the silo covering should be
repaired as soon as possible.
During feed-out spoilage by air ingress can be minimized by a sufficiently

high feed-out rate. In addition, at the time of ensiling silage additives can be
applied that are able to decrease spoilage losses.
6.6 BASIC PRINCIPLES

At harvest, plant cells do not immediately "die"; they continue to respire as
long as they remain adequately hydrated and oxygen is available. The oxygen
is necessary for the physiological process of respiration, which provides
energy for functioning cells. In this process, carbohydrates (plant sugars) are
consumed (oxidized) by plant cells in the presence of oxygen to yield carbon
dioxide, water and heat.
Sugar + oxygen=carbon dioxide + water + heat
Once in the silo, certain yeasts, molds and bacteria that occur naturally on
forage plants can also reach populations large enough to be significant
sources of respiration. In the silage mass, the heat generated during
respiration is not readily dissipated, and therefore the temperature of the
silage rises.
Although a slight rise in temperature from 80° to 90°F is acceptable, the goal
is to limit respiration by eliminating air (oxygen) trapped in the forage mass.
Some air will be incorporated into any silo during the filling process. These
temperature increases can clearly be limited by harvesting at the proper
moisture content and by increasing the bulk density of the silage. Generally,
it is desirable to limit respiration during the fermentation process by using
common sense techniques that include close inspection of the silo walls prior
to filling, harvesting the forage at the proper moisture content, adjusting the
chopper properly (fineness of chop), rapid filling, thorough packing, prompt
sealing and close inspection of plastics for holes (Honig and Woolford 1980).

1. What is Silage?
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Biodegradation of 2. What are advantage and disadvantages of Silage Production?
Natural Xenobiotic
Compounds …………………………………………………………………………….
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3. State Basic Principle of Silage Production?
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6.7 ROLE OF SACCHAROLYTIC AND

PROTEOLYTIC ORGANISMS
The silage microflora plays a key role in the successful outcome of the
conservation process. The flora can basically be divided into two groups
namely the desirable and the undesirable microorganisms. The desirable
microorganisms are the lactic acid bacteria. The undesirable ones are the
organisms that can cause anaerobic spoilage (e.g. clostridia and
enterobacteria) or aerobic spoilage (e.g. yeasts, bacilli, listeria and moulds).
Many of these spoilage organisms do not only decrease the feed value of the
silage, but also have a detrimental effect on animal health and/or milk quality
(e.g. listeria, clostridia, moulds and bacilli).
6.7.1 Desirable Microorganisms

Lactic acid bacteria. (LAB): Lactic acid bacteria belong to the epiphytic
microflora of plant material. Often the population of LAB increases
substantially between harvesting and ensiling. This is probably mainly due to
the resuscitation of dormant and non-culturable cells, and not by inoculation
by the harvesting machinery or growth of the indigenous population. Crop
characteristics like sugar content, dry matter content, and sugar composition,
combined with lactic acid bacterial properties such as acid and osmo-
tolerance, and substrate utilization will decisively influence the
competitiveness of the lactic acid bacterial flora during silage fermentation
(Woolford 1984;McDonald et al. 1991).
Lactic acid bacteria that are regularly associated with silage are members of
the genera Lactobacillus, Pediococcus, Leuconostoc, Enterococcus,
Lactococcus and Streptococcus. The majority of the silage lactic acid bacteria
are mesophilic, i.e. they can grow at temperatures between 5 and 50oC, with
138
an optimum between 25 oC and 40oC. They are able to decrease the silage pH from Waste
to pH 4-5, depending on the species and the type of forage crop. All lactic
acid bacteria are facultative aerobes, but some have a preference for
anaerobic conditions (Holzapfel and Schillinger 1992; Hammes et al.
1992;Weiss 1992). Based on their sugar metabolism lactic acid bacteria
canbe classified as obligate homofermenters, facultative heterofermenters or
obligate heterofermenters. Obligate homofermenters produce more than 85%
lactic acid from hexoses (C-6 sugars) such as glucose, but cannot degrade
pentoses (C-5 sugars) such as xylose. Facultative heterofermenters also
produce mainly lactic acid from hexoses, but in addition they also at least
degrade some pentoses to lactic acid, and acetic acid and/or ethanol. Obligate
heterofermenters degrade both hexoses and pentoses, but unlike
homofermenters they degrade hexoses to equimolar mounts of lactic acid,
CO2, and acetic acid and/or ethanol (Hammes et al.1992; Schleifer and
Ludwig 1995).
Obligate homofermenters are species such as Pediococcus damnosus and

Lactobacillus ruminis,Facultative heterofermenters are for example
Lactobacillus plantarum , Lactobacillus pentosus, Pediococcus acidilactici,
Pediococcus pentosaceus and Enterococcus faecium . To the obligate
heterofermenters belong members of the genus Leuconostoc and some
Lactobacillus sp. such as Lactobacillus brevis and Lactobacillus buchneri
(Weiss 1992; Holzapfel and Schillinger 1992; Hammes et al. 1992).
The ensiling process completes in the following steps: harvesting the crop
(30-35%DM), chopping, and loading into a silo, compacting and sealing to
exclude air, storing and feed out phase (unloading for animal feeding).
Biochemical and microbiological incidents can ariseduring the different
stages of ensiling which may affect the silage quality. At first step of ensiling
is the enzymatic activity of intact plant cells whenresidual respiration occurs.
The intact cellsuse glucose and fructose as carbohydrate source and consume
oxygen entrapped in the silage. Early consumption of carbohydrates is
detrimental for the subsequent anaerobic lactic acid fermentation. In initial
phase of ensiling epiphytic aerobic flora such as Enterobacteria, yeasts and
molds develop until oxygen has been entirely consumed or acidification is
sufficient to stop their metabolism. At ensiling, the facultative anaerobic
bacteria carry out a heterolactic fermentation which slightly decreases the pH
of the silage.
As the conditions become anaerobic in silage pit, fermentation phase starts.

This phase continue for several days or weeks, during which different groups
of facultative aerobic or anaerobic microorganisms naturally found in plants
compete for available nutrients. With gradual acidification, acid tolerant
bacterial development start; convert water soluble carbohydrates into lactic
acid. In well-processed silage, LAB dominates the fermentation, rapidly
producing the low pH conditions that help to preserve the silage. In case of
accidental soil incorporation in ensiled material, a long aerobic phase or slow
139
Biodegradation of acidification, the microbial communities in the silage will be dominated by
Natural Xenobiotic
Compounds Clostridia, yeasts, molds and accidentally incorporated pathogenic
microorganisms such as Listeria sp.
As long as the pH is sufficiently low and anaerobiosis is maintained, storage

phase lasts and few changes occur. Numbers of LAB and other viable
microorganisms decline over time, except for some specialized species such
as L. buchneri with continues to be active at low population densities. Some
acid-tolerant microorganisms can survive this storage period in an almost
inactive state (e.g. acid-tolerant yeasts) or as spores (e.g. butyric acid
bacteria). Homofermentative lactobacilli such as L. plantarum and
Lactobacillus curvatus tend to predominate in well preserved silage until the
final stage of fermentation, when they are invariably replaced by
heterofermentative species such as L. brevis and L. buchneri.
The fourth phase is the unloading or feedout phase. On opening of silos air
penetrates into the silage depending on the density and porosity of the plant
material and the rate of silage removal. This causes the growth of undesirable
aerobic microorganisms initially present in the silage, such as yeasts and
molds and an increase in pH.
6.7.2 Undesirable Microorganisms and their Metabolites

Several undesirable microorganisms can grow during ensiling process which
can affect silage quality and thus affect animal performance or both animal
and human health. These microorganisms are known as spoilage
microorganisms; responsible for silage degradation mainly induce economic
losses.
Yeasts and molds: Yeasts are considered to be the most important

groupamong the undesirable microorganisms of silage because they are
involved in aerobic spoilage either during the aerobic phase at the beginning
of ensiling or during the unloading phase. The organic acid metabolism
pathways (succinic, citric and lactic acids) of yeasts restarts on exposure of
silage to air; inducing a pH increase and allowing the growth of less acid-
tolerant microorganisms. Yeasts present in silage convert WSC into CO2 and
alcohols; impair silage quality and lead to a decrease in feed intake. The
alcohol production also has negative effect on milk taste.
Moulds are eukaryotic micro-organisms and develop in part of silage where

oxygen is present. Many mould species produce the large filamentous
structures and coloured spores in silage. Penicillium (70%), Fusarium (47%)
and Aspergillus (34%) are the most frequent mycotoxin-producing fungi
isolated from corn silage. There metabolites remain in the silage even after
the fungus has disappeared. More than 20 mycotoxins can be produced by
Fusarium sp., mainly dioxynivalenol (DON), zearalenone (ZEN) and
fumonisin (FB).
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Chronic exposure to mycotoxins produce non-specific symptoms such as from Waste
immune system impairment, increased infections and metabolic and
hormonal imbalances. Ruminants are better protected than other animals
against many mycotoxins but fumonisin B1 is only poorly metabolized in the
rumen.In high producing dairy animals fed on silage-based diets with high
levels of concentrates; the consequent acidification of the rumen environment
may increase the animal’s sensitivity to mycotoxins.
Acetic acid bacteria: Acetic acid bacteria are obligate aerobic, acid-tolerant
bacteria. Thus far all acetic acid bacteria that have been isolated from silage
belonging to the genus Acetobacter (Spoelstra et al. 1988). The activity of
Acetobacter ssp. in silage is undesirable because they can initiate aerobic
deterioration, due to the fact that they are able to oxidize lactate and acetate
to carbon dioxide and water. Generally, yeasts are the main initiators of
aerobic spoilage, and acetic acid bacteria are absent, or play only a minor
role. However, for whole crop corn silages there is evidence that acetic acid
bacteria alone can initiate aerobic deterioration (Spoelstra et al. 1988).
Furthermore, selective inhibition of yeast also can increase proliferation of
acetic acid bacteria in silage.
Bacilli: Bacilli are like clostridia endospore-forming rod shaped bacteria.

Nevertheless, they can easily be distinguished from clostridia due to the fact
that they are (facultative) aerobes, whereas all clostridia are obligate
anaerobes. Facultative aerobic bacilli ferment a wide range of carbohydrates
to compounds such as organic acids (e.g., acetate, lactate, and butyrate) or
ethanol, 2,3-butanediol, and glycerol. Some specific Bacillus sp. are able to
produce antifungal substances, and have been used to inhibit aerobic spoilage
of silage. Except for these specific strains, the proliferation of bacilli in silage
is generally considered undesirable. Not only are bacilli less efficient lactic
and acetic acid producers than lactic acid bacteria (McDonald et al. 1991),
they can also enhance (later stages of) aerobic deterioration. Furthermore,
high numbers of Bacillus spores in raw milk have been associated with high
spore numbers in fresh cow feces. In seems very plausible that bacillus spores
are transferred from silage to milk via feces similar to clostridial spores.
Butyric acid bacteria (BAB): Soil accidentally included with the plant
material during silo filling is the source of Butyric acid bacterial
contamination of silage. Endospore-forming bacteria of the genera
Clostridium, especially C. tyrobutyricum and C. butyricum and Bacillus are
main BAB found in silage.At a relatively low ph,BABconvert lactic acid into
butyric acid, hydrogen and carbon dioxide. A typical “clostridial silage” is
characterized by a high butyric acid content of more than 5 g/kg DM, a high
pH (over pH 5 in low DM silages), and a high ammonia and amine content.
Excess butyric acid from feeding butyric silages results in higher levels of
plasma Ketones. Daily doses of over 50 – 100g of butyric acid can cause
ketosis. Feeding butyric silage has long term negative effects on production,
fertility and health, thus economy of the farm business.
141
Biodegradation of Listeria: The presence of L. monocytogenes in silage, feces or both increases
Natural Xenobiotic
Compounds the risk of its presence in milk and hence of its transmission to humans. pH of
silage over 4.5 increases the risk of presence of Listeria sp. In ruminants it
could cause encephalitis, absorption or septicemia, even death.
E. coli (STEC):E. coli and higher prevalence of E. coli O157 or E. coli

O157:H7 has been found in herds fed corn silage. At ensiling, insufficient
anaerobiosis could delay the establishment of lactic acid fermentation,
slowing the pH decrease andincreasing the survival of pathogenic E. coli,
which is a food born pathogen.
Biogenic amines (BA): Putrescine, cadaverine and tyramine are main

biogenic amines found in silage; derived from arginine, lysine and tyrosine,
respectively. These are produced due to amino acid decarboxylation by
enzymes of several lactic acid bacteria. Species of many genera present in
silage such as Clostridia, Bacillus, Klebsiella, Escherichia and Pseudomonas
can also cause biogenic amine production. Several factors such as
temperature, oxygen availability and rapidity of pH decrease during the initial
stage of fermentation results inthe formation of BA. Feeding of silage having
BA acts as causative factors in ketonemia. BA presence in silage decreases
the palatability and reduces Dry Matter Intake (DMI) and cattle performance.
6.8 PRESERVING TECHNIQUE

After the fermentation process is done and once all of the oxygen is used up,
lactic acid bacteria start to multiply. These are the bacteria that are needed to
make the silage. They play a key role in turning the plant sugars into lactic
acid causing the pH to drop (mixture becomes more acidic). Once the pH is
around 4-5, the sugars stop breaking down and the grass is preserved until the
silage is opened and exposed to oxygen.
If the pH isn’t low enough, a different kind of bacteria will start fermenting
the silage, producing by-products (like ammonia) that taste bad to cows and
sheep. Thus, the latter situation needs to be avoided at all costs.
6.9 PREVENTIVE MEASURES TO CONTROL

SILAGE SPOILAGE
Preservation of surplus fodder as silage makes it possible to have the green
fodder availability throughout the year. Lactic acid fermentation is important
for good silage production, but some undesirable bacteria can make entry
during ensiling, which can degrade the quality of silage and cause economic
losses to the farmer. Safety control measures and additives may be helpful
used in producing quality silage.
In developing countries including India, livestock sector is one of the fastest

growing segment of the agricultural economy. The supply of the green forage
142 throughout the year is an economic priority to the farmers, in order to
maintain the production from the ruminant stock. But forage production is from Waste
mostly seasonal in many parts of the world, with surplus availability in
harvesting season and shortage of fodder during dry season. Silage making is
an option to preserve the green fodder to make the greens available
throughout the year. Ensiling is based on natural lactic acid fermentation
under anaerobic conditions. The most important fodder crops for ensiling are
corn, sorghum, barley and various other grasses. Beside these crops various
moist “by-products” of the food industry, such as apple pomace, beet pulp
and brewer’s mash can also be used for silage preparation. Ensiling process
has many steps which should be timed and controlled carefully to ensure
successful ensiling.
6.10 PREPARATION OF SILAGE

During the silage making process, the pasture is cut when the grasses contain
the highest nutrient levels. This level is attained just before they are fully
mature. The reason why it is cut just before they are fully mature is that all
forms of preserved grass, such as hay and silage, will have lower amounts of
nutrients than fresh pasture, so everything must be done to make the end
product be as nutritious as possible. During Silage preparation, the grass is
allowed to wilt in the field for a few hours to reduce the moisture content to
around 60-75% as this is the optimum level. If the grass is left out longer, it
may get too dry, or it may get rained on and both these will reduce the
efficiency of the fermentation (Hoogkamp,1999).
6.11 PROCESS IN SILAGE MAKING

1. Selection of forage crops and their maturity stage
The optimum dry matter for crop harvesting for silage depends on the
stage of harvesting (Table 1). Most of crops are harvested at 50%
flowering to dough stage when the moisture content varies between 18-
22%. After overnight wilting the dry matter content become 30-35%
which is proper dry matter content for ensiling. Table 1: Optimum stage
for crop harvesting Common forage crops Stage of harvest Maize 50%
flowering to dough stage Sorghum 50% flowering to dough stage Bajra
50% flowering to dough stage Oat Boot to dough stage
2. Steps in silage making
Silage making involves four major steps viz., harvesting and
transportation, chaffing, filling and compaction and covering of silo.
1. Pit making:
Firstly, a silage pit has to be dug for storing silage. The pit size may be
determined based on the amount of silage to be stored. A pit with a
dimension of 1 metre wide X 1 metre length X 1 metre depth can store
500 kilograms of silage. The location of pit should be free from water
stagnation. The pit should be surrounded on all sides with thick plastic 143
Biodegradation of sheet. Pit can also be constructed using bricks and cement.
Natural Xenobiotic
Compounds 2. Preparation of fermentation mixture:
For preparing 1 ton silage, the following materials are required.
• Jaggery or Molasses – 1 Kg
• Salt – 1 Kg
• Mineral Mixture – 1 Kg
• DCP (Di-Calcium Phosphate) – 1 Kg
• LAB (Lactic Acid Bacteria)
• Urea – 1 Kg
Mix all of the above into a drum by adding water
3. Harvesting and transportation of crop (ensiling):
Harvesting at proper stage but delay in transportation may lead to loss of
excess moisture results in haylage (DM 70-80%) 4. Chaffing:
It has to be chaffed into small pieces preferably 2-4 cm length using a
chaff cutter. This improves the packing density which favours the growth
of lactic acid bacteria, naturally present in crops. Add the fermentation
mixture in small quantities as the fodder is loaded to chaff cutter.
Position the chaff cutter so the chaffed fodder directly falls into the
silage pit. Level the chaffed pieces evenly and press it hard so that all air
comes out. Pressing and removing air is very important.
5. Filling of silo and compaction:
Chaffed material should be spread evenly over entire surface of silo (the
structure) and then compacted through trampling (in case of small silo).
In case of large silo (trenches) the compaction can be done using tractor.
It helps in rapid evacuation of air from the silo, thus checks the aerobic
respiration and nutrient loss.
6. Properly sealing and covering of silo pit:
It should be done in such a way that neither air enters in to the silo nor
the gas comes out from the silo. It is better to use polythene sheet but
care should be taken that entire surface of polythene sheet should be
covered with straw or any other dried material up to 6-8 inch thickness to
avoid the damage of polythene sheet by dog, cat or other animals. Make
sure water does not enter the pit during rains. The silage will be ready in
45 to 60 days, depending on the types of material used. The silage of thin
stem crop like oat becomes ready in 45 days while thick stem crops like
maize, sorghum and bajra become ready in 60 days. Ideal silage is
golden yellowish green colour with good aroma. After completion of
incubation period the silo is opened for feeding. The whole silo should
not be disturbed and it should be opened from one place/corner to avoid
the loss of moisture and nutrients. Depending on the type of animal,
stage of production and availability of silage it can be supplemented in
144
the ration (5-25 kg per animal) of animal during lean period. After from Waste
opening the pit, silage should be used within 30 days.
6.12 PLANNING FOR SILAGE MAKING

There are two methods for silage making which are vastly used i.e. Pit
method or tank method. These two methods are economically viable for dairy
farmers.
• Pit/tank method for silage making
For pit method, select location for making pit at higher level on ground so
that rain waer may not percolate in to pit. In rectangular pit, corner edges
should be making round so that while filling & pressing chaffed fodder, air
will not remain inside in the corners of pit or tank. Wall of pit/tank should be
air proof to avoid air too come inside in pit /tank through cracks or crevices
.To avoid this situation, plaster wall of silo pit or tank with cement or
moistened soil.
• Treatment for Silage
For making best quality & balanced silage, needs proper treatment of
additives like Per ton of chaffed green fodder requires 1 kg Urea, 2 kg
jaggary, 1 kg common salt, 1 kg mineral mixture & 1 litre of Whey.
• Procedure for filling silo pit/tank
When fodder crop is in cob stage or Tussling stage, harvest it for preparation
of silage. Very mature stage is not good for preparing silage as its sugar
content is decreased as well as fibre percentage in increased; this kind of
fodder is less suitable for silage making.
After harvesting fodder crops, let it dry for 5-6 hours in shed so that moisture
content of fodder will decreases from 80% to 65-70%.Care to be taken to
avoid silage making in rainy days or crops containing dew drops in winter
season because moisture is more in this situation so there may be chances for
development of mould in silo pit during storage period.
Following steps to be taken while filling silo pit-
• Prior to filling silo pit / tank, clean·& dry it.

• Cover with plastic film inside pit/tank in such way that it will cover all
sides of pit/tank.·
• For making silage, chaffing of fodder is essential component. With the
help of chaff cutter machine, make pieces of 1.5c.m. to 2 c.m. length of
green maize.sorghum,sugarcane tops,marwel,Fodder bajara etc for filling
silo pit.·
• Prepare separate solution in 15 to 20 litres of water for Urea, Jaggary,
Mineral mixture· & common salt in separate pots/buckets & then spread
145
Biodegradation of it on layer of pressed chaffed green fodder while filling silo pit/tank.
Natural Xenobiotic
Compounds • Start to fill chaffed green fodder in pit or tank.
• After making 4” thick layer of chaffed green fodder, press it with
wooden plank(Like Mortar) in such a way that air will not entangled in
chaffed fodder. Then sprinkle it with prepared solution of Jaggary,
Mineral mixture, Urea, Common salt· & whey.
• Follow the same procedure until filling of pit/tank 1 to 1.5 feet above the
ground level(In pit).Then covet it from plastic film from all side
carefully.·
• Covet it with Trash, Wheat straw, Soil· & dry hay to protect it from
entering rain water in to it. If possible to temporary arrangement of shed
above the silo pit/tank.
• It will require 45 to 60 days to make good quality of silage (Hengeveld
1983).
6.13 USE OF SILAGE

After 8-10 weeks, silage is ready as feed for animals. Open pit/tank initially
from one side of for use. If it is not in use, then cover it carefully with plastic
film so that air will not go inside in silage. Initially animals should be fed
with 5-6 kg silage by adding it with chaffed green fodder to develop taste to
animals. Once animal likes sweet-sour taste of silage; it will eat it with good
liking.
6.14 QUALITY OF SILAGE

• Mould- If silage while filling pit/tank, not well pressed; there will be
growth of mould.
• Odour- Good quality silage has sweet & sour taste.
• Colour- Good quality silage has faint green or brownish colour. Rotten
silage has black colour.
• pH- Good quality silage has pH of 3.5 to 4.2.
6.15 STRATEGIES TO LIMIT SILAGE

DEGRADATION BY UNDESIRABLE
MICROORGANISMS
Spoiled silage has to be thrown out because till now no any measure has been
develop to improve the quality of spoiled silage. Main factors which affect
the quality during silage processing are the use of poor quality or immature
plant material, insufficiently rapid filling (causes delay in establishment of
anaerobiosis leading to weak silage acidification), and contamination by
pathogenic or spoilage microorganisms. Preventive measures which should
146 be taken care for quality silage production are discussed below.
Promoting acidification: Silage preservation is mainly based on from Waste
acidification which depends on anaerobiosis (promoting LAB fermentation),
buffering capacity and DM of the crop. Soil incorporation in silage increases
its buffering capacity, encourages the growth of aerobic microorganisms,
which reduce the quantity of hexoses and pentoses available for further LAB
fermentation. This all leads to delay in silageacidification during which lactic
acid is converted to butyric acid, followed by pH increase and further
spoilage due to secondary fermentation by Clostridia. Mineral acids such as
sulfuric and chlorhydric acids has been used to promote silage acidification
and to limit the pathogenic microorganism growth.
Establishment of anaerobiosis during ensiling: For good silage production

anaerobic conditions should be established as soon as possible in the silo.
This can be achieved by rapid filling of silo and compaction of the silo to
exclude the trapped air. Compaction of silage is easy with small particle size
of crops. So chopping length plays an important role in good silage
production, while every small particle size may impair the rumen function.
For grass silage optimum chopping length is 4-6 cm. The corn silage
prepared for dairy cow feeding should contain less than 1% of large particles
(>2 cm), 8–12% of medium particles from 1 to 2 cm and less than 50% of
very short particles (<6 mm). Harvesting of crop at appropriate dry matter is
helpful to reduce effluent losses, which may impair anaerobiosis.
Prevention of air ingression during storage of silage: Silo should be sealed

properly to avoid air ingression during storage, which can lead silage
spoilage. A barrier made up of plastic sheets is helpful to prevent air
ingression and give protection against damage by birds, rodents and UV rays.
To reduce spoilage of silage due to exposure during feed-out phase,silage
requirement and silo pit dimensions should be calculated prior to silage
making.
Improving the aerobic stability of silage: Chemical additives like formic

acid, silage additives containing partially neutralized acids in salt form
(nitrites, sulfates) in association with formol-based preservatives can be used
to improve aerobic stability of silage. Bacterial additives like Lactobacillus
buchneri and Lactobacillus plantarum, Pediococcus acidilactici,
Pediococcus cerevisiae and Propionibacterium acidipropionici can also be
used during ensiling to improve the silage quality. Bacterial inoculants also
limit pathogen development andproduce substances which may have
antimicrobial potential (H2O2, ethanol, diacetyl, exopolysaccharides) and
antibacterial peptides such as bacteriocins.
Prevention of pathogen introduction during harvesting and ensiling of

crop: Entry of soil in the silage is the source of butyric acid bacterial/
pathogen contamination. Crops should be harvested 4 weeks after manure
application taking care to prevent contamination by soil (Woods et al., 1981).
147
Biodegradation of
Natural Xenobiotic 6.16 SILAGE ADDITIVES
Compounds
In the past decade it has become increasingly common to use silage additives
to improve the ensiling process. The choice of additives appears to be sheer
limitless if one looks at the large number of chemical and biological silage
additives that are commercially available. (Table 1).
Table 1: Category of additives
Additive category Selection of Active Remarks

ingredients
Fermentation stimulants Lactic acid bacteria May impair aerobic
Sugars (molasses) stability
Enzymes
Fermentation Formic acid Inhibition of clostridia
inhibitors Lactic acid
Mineral acids
Nitrite salts
Sulfite salts
Sodium chloride
Aerobic Lactic acid bacteria
deterioration Propionic acid
inhibitors Benzoic acid
Sorbic acid
Nutrients Urea Can improve aerobic
Ammonia stability
Minerals
Absorbents Dried sugar beet
pulp
Straw
6.17 ENZYMOLOGY OF SILAGE PRODUCTION

Enzyme addition to silages has received considerable attention over the past
decade. Their primary function is to break down forage fiber during
fermentation, rendering the silage more digestible during feedout. The
breakdown of fiber into soluble sugars also helps bacteria produce lactic acid,
which helps to lower silage pH. An enzyme is a naturally occurring protein
that catalyzes chemical reactions in biological systems. Enzymes promote the
breakdown of complex feed molecules into smaller chemical fractions such
as glucose or amino acids that are digestible by the ruminant animal. As an
example, the enzyme cellulase initiates the breakdown of cellulose (fiber)
into sugars. Common enzyme-based silage additives contain cellulases,
hemicellulases, xylanases, amylases, and pectinases. Cellulases,
hemicellulases, and pectinases are enzymes that degrade the fiber portion of
forages. Amylase breaks down starch (amylose) therefore its use would be
148
directed towards starch containing silages such as corn silage. from Waste
A number of studies have reported reductions in neutral detergent fiber

(NDF) by using enzyme-based silage additives. Observed NDF reductions
have been more consistent in grass silages as compared to alfalfa silage.
Research data also suggests that hemicellulases and pectinases are more
effective than cellulases at reducing fiber content. Unfortunately,
hemicellulases and pectinases break down fiber fractions (hemicellulose,
pectin) that are more easily digested by ruminants. Consequently, these
enzymes reduce the concentration of digestible NDF fractions rather than the
indigestible NDF fraction cellulose.
Enzymes can improve silage fermentation when the substrate (e.g., sugars) is
limiting. Soluble sugars are required to help bacteria produce lactic acid,
which is required to lower silage pH for proper fermentation. Generally,
enzyme addition to silages has a small positive effect on fermentation.
Interestingly, current trends in enzyme technology involve incorporating

enzymes directly into total mixed rations (TMRs) or silages prior to feeding.
Initial research has observed some positive effects using this method. The
economics of this practice have yet to be determined.
Enzyme technology continues to improve. New products designed for

ruminants and for specific types of feed will increase the potential for
profitable use of enzymes with silages.

1. Explain process of silage making.
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
2. Describe uses of Silage?
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
149
Biodegradation of 3. Name the enzymes involved in silage production?
Natural Xenobiotic
Compounds ……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
6.18 LET US SUM UP

We have studied the Silage with its advantages and disadvantages. We have
studied the principle of silage production with well define role of proteolytic
and cellulolytic organisms. We have studied the enzymes required for silage
production.
6.19 KEY WORDS

Silage: Silage, also called ensilage, forage plants such as corn (maize),
legumes, and grasses that have been chopped and stored in tower silos, pits,
or trenches for use as animal feed.
Additives: A substance added to something in small quantities to improve or

preserve it.
Enzyme: Enzymes are proteins that help speed up metabolism, or the

chemical reactions in our bodies. They build some substances and break
others down. All living things have enzymes.
Fungi:A fungus (plural: fungi or funguses) is any member of the group of

eukaryotic organisms that includes microorganisms such as yeasts and molds,
as well as the more familiar mushrooms.
Bacteria: Bacteria are small single-celled organisms. Bacteria are found

almost everywhere on Earth and are vital to the planet's ecosystems.
Cellulose: Cellulose is a polysaccharide composed of a linear chain of β-1,4

linked d-glucose units with a degree of polymerization ranged from several
hundreds to over ten thousands, which is the most abundant organic polymer
on the earth.

READING/REFERENCES
• Wilkins, R.J., L. Syrjälä-Qvist, and K.K. Bolsen1999. p. 23-35. In: T.
Pauly (ed.) Proc. 12th Int. Silage Conference, Uppsala, Sweden, 5-7
July. 1999.
• Weinberg, Z.G., and R.E.Muck 1996. New trends and opportunities in
150
the development and use of inoculants for silage. FEMS Microbiol. from Waste
Rev. 19, 53-68.
• Merry, R.J., K.F. Lowes, and A. Winters 1997. Current and future
approaches to biocontrol in silage. p. 17-27. In:V. Jambor, L. Klapil,
P.Chromec, and P. Prochazka. (ed.) Proc. 8th Int. Symposium Forage
Conservation, Brno, Czech Republic. 29 Sept.-1 Oct. 1997. Research
Institute of Animal Nutrition, Pohorelice, Czech Republic.
• Honig, H., and M K.Woolford1980. Changes in silage on exposure to
air. p. 76-87. In: C. Thomas (ed.) Forage Conservation in the 80s .
Occasional Symposium No. 11 . British Grassland Society, Hurley,
Berkshire, UK.
• Hengeveld, A.G. 1983. Sporen van boterzuurbacteriën in kuilvoer.
Report 88. Proefstation voorde Rundveehouderij, schapenhouderijen
paardenhouderij, Lelystad, The Netherlands.
• Holzapfel, W.H., and U. Schillinger1992. The Genus Leuconostoc . p.
1508-1534. In: Balows, A., H.G. Trüper, M. Dworkin, W. Harder, K.-
H. Schleifer (ed.) The Prokaryotes. 2nd ed. Springer Verlag, New
York, USA.
• Hoogkamp, W. 1999. Koeien smullen van kuilgras met
bacteriemengsels. Boerderij- Veehouderij 84,32-33.
• Weiss, N. 1992. The Genera Pediococcus and Aerococcus . p. 1502-
1507. In: Balows, A., H.G. rüper, M. Dworkin, W. Harder, K.-H.
Schleifer (ed.) The Prokaryotes. 2nd ed. Springer Verlag, New York,
USA.
• Weissbach, F., and H. Honig1996. Über die Vorhersage und
Steuerung des Gärungsverlaufs bei der Silierung von Grünfutter aus
extensivem Anbau. Landbauforschung Völkenrode, Heft 1,10-17,
Germany.
• Wiedmann, M., J. Czajka, N. Bsat, M. Bodis, M.C. Smith, T.J. Divers,
and C.A. Batt1994. Diagnosis and epidemiological association of
Listeria monocytogenes strains in two outbreaks of listerial encephalitis
in small ruminants. J. Clin. Microbiol. 32, 991-996.
• Wieringa, G.W. 1958. The effect of wilting on butyric acid fermentation
in silage. Neth. J. Agr. Sci. 6, 204-210. Swedish University of
Agricultural Sciences, Uppsala, Sweden.
• Wilkinson, J.M., F. Wadephul, and J. Hill 1996. Silage in Europe, a
survey of 33 countries. ChalcombePublications, Welton, UK.
• Woods, L.F.J., J.M. Wood, and P.A. Gibbs1981. The involvement of
nitric oxide in the inhibition of the phosphoriclasticsystem in
Clostridium sporogenes by sodium nitrite. J. Gen. Microbiol. 125, 339-
406.
• Woolford, M.K. 1975a. Microbiological screening of the straight chain
151
Biodegradation of fatty acids (C1-C12) as potential silage additives. J. Sci. Food Agr. 26,
Natural Xenobiotic
Compounds 219-228.
• Woolford, M.K. 1975b. Microbiological screening of food preservatives,
cold sterilants and specific antimicrobial agents as potential silage
additives. J. Sci. Food Agr. 26, 229-237.
• Woolford, M.K. 1984. The Silage Fermentation. Microbiological Series,
14, Marcel Dekker, Inc., New York and Basel.
• McDonald P., A.R.Henderson, and S.J.E. Heron 1991. The
Biochemistry of Silage. 2nd edition. ChalcombePublications, Marlow,
Bucks, UK.
• Hammes, W.P., N. Weiss, and W. Holzapfel1992. The Genera
Lactobacillus and Carnobacterium . p. 1535-1594. In: Balows, A., H.G.
Trüper, M. Dworkin, W. Harder, K.-H. Schleifer (ed.) The Prokaryotes.
2nd ed. Springer Verlag, New York, USA.
• Schleifer (ed.) The Prokaryotes. 2nd ed. Springer Verlag, New
York,USA.
• Spoelstra, S.F. 1983. Inhibition of clostridialgrowth by nitrate during
the early phase of silage fermentation. J. Sci. Food Agr. 34, 145-152.
• Spoelstra, S.F. 1985. Nitrate in silage. A review. Grass Forage Sci. 40,
1-11. Spoelstra, S.F. 1987. Degradation of nitrate by
enterobacteriaduring silage fermentation of grass. Neth. J. Agr. Sci. 35,
43-54.
• Spoelstra, S.F, M.G. Courtin, and J.A.C. van Beers 1988. Acetic acid
bacteria can initiate aerobic deterioration of whole crop maize silage. J.
Agr. Sci.Camb. 111, 127-132.
• Auerbach, H., E. Oldenburg, and F. Weissbach1998. Incidence of
Penicillium roqueforti and roquefortinC in silages. J. Sci. Food Agr.
76,565-572.

1. Forage which has been grown while still green and nutritious can be
conserved through a natural 'pickling' process. Lactic acid is produced
when the sugars in the forage plants are fermented by bacteria in a sealed
container ('silo') with no air. Forage conserved this way is known as
'ensiled forage' or 'silage' and will keep for up to three years without
deteriorating. Silage is very palatable to livestock and can be fed at any
time.
2. Advantages:
• Stabile composition of the feed (silage) for a longer period (up to 5
years);
152
• Plants can be harvested at optimal phase of development and are from Waste
efficiently used by live-stock.
• Reduction of nutrient loses which in standard hay production may
amount to 30% of the dry matter (in silage is usually below 10%);
• More economical use of plants with high yield of green mass;
• Better use of the land with 2-3 crops annually;
• Silage is produced in both cold and cloudy weather
Disadvantages:
• Silage is not interesting for marketing as its value is difficult to be
determined.
• It does not allow longer transportation;
• The weight increases manipulation costs;
• Has considerably lower vitamin D content compared to hay.
• The fermentation in silage reduces harmful nitrates accumulated in
plants during droughts and in over-fertilized crops.
• Allows by-products (from sugar beat processing, maize straw, etc.)
to be optimally used;
• Requires 10 times less storage space compared to hay;
• Maize silage has 30-50% higher nutritive value compared to maize
grain and maize straw;
• 2 kg of silage (70% moisture) has the equal nutritive value of 1 kg of
hay.
3. At harvest, plant cells do not immediately "die"; they continue to respire
as long as they remain adequately hydrated and oxygen is available. The
oxygen is necessary for the physiological process of respiration, which
provides energy for functioning cells. In this process, carbohydrates
(plant sugars) are consumed (oxidized) by plant cells in the presence of
oxygen to yield carbon dioxide, water and heat.
Sugar + oxygen=carbon dioxide + water + heat
Once in the silo, certain yeasts, molds and bacteria that occur naturally
on forage plants can also reach populations large enough to be significant
sources of respiration. In the silage mass, the heat generated during
respiration is not readily dissipated, and therefore the temperature of the
silage rises.
Although a slight rise in temperature from 80° to 90°F is acceptable, the
goal is to limit respiration by eliminating air (oxygen) trapped in the
forage mass. Some air will be incorporated into any silo during the filling
process. These temperature increases can clearly be limited by harvesting
at the proper moisture content and by increasing the bulk density of the
153
Biodegradation of silage. Generally, it is desirable to limit respiration during the
Natural Xenobiotic
Compounds fermentation process by using common sense techniques that include
close inspection of the silo walls prior to filling, harvesting the forage at
the proper moisture content, adjusting the chopper properly (fineness of
chop), rapid filling, thorough packing, prompt sealing and close
inspection of plastics for holes.
1. Selection of forage crops and their maturity stage
The optimum dry matter for crop harvesting for silage depends on the
stage of harvesting (Table 1). Most of crops are harvested at 50%
flowering to dough stage when the moisture content varies between 18-
22%. After overnight wilting the dry matter content become 30-35%
which is proper dry matter content for ensiling. Table 1: Optimum stage
for crop harvesting Common forage crops Stage of harvest Maize 50%
flowering to dough stage Sorghum 50% flowering to dough stage Bajra
50% flowering to dough stage Oat Boot to dough stage
2. Steps in silage making
Silage making involves four major steps viz., harvesting and
transportation, chaffing, filling and compaction and covering of silo.
1. Pit making:
Firstly, a silage pit has to be dug for storing silage. The pit size may be
determined based on the amount of silage to be stored. A pit with a
dimension of 1 metre wide X 1 metre length X 1 metre depth can store
500 kilograms of silage. The location of pit should be free from water
stagnation. The pit should be surrounded on all sides with thick plastic
sheet. Pit can also be constructed using bricks and cement.
2. Preparation of fermentation mixture:
For preparing 1 ton silage, the following materials are required.
• Jaggery or Molasses – 1 Kg
• Salt – 1 Kg
• Mineral Mixture – 1 Kg
• DCP (Di-Calcium Phosphate) – 1 Kg
• LAB (Lactic Acid Bacteria)
• Urea – 1 Kg
Mix all of the above into a drum by adding water
3. Harvesting and transportation of crop (ensiling):
Harvesting at proper stage but delay in transportation may lead to loss of
excess moisture results in haylage (DM 70-80%) 4. Chaffing:
It has to be chaffed into small pieces preferably 2-4 cm length using a
154
chaff cutter. This improves the packing density which favours the growth from Waste
of lactic acid bacteria, naturally present in crops. Add the fermentation
mixture in small quantities as the fodder is loaded to chaff cutter.
Position the chaff cutter so the chaffed fodder directly falls into the
silage pit. Level the chaffed pieces evenly and press it hard so that all air
comes out. Pressing and removing air is very important.
5. Filling of silo and compaction:
Chaffed material should be spread evenly over entire surface of silo (the
structure) and then compacted through trampling (in case of small silo).
In case of large silo (trenches) the compaction can be done using tractor.
It helps in rapid evacuation of air from the silo, thus checks the aerobic
respiration and nutrient loss.
6. Properly sealing and covering of silo pit:
It should be done in such a way that neither air enters in to the silo nor
the gas comes out from the silo. It is better to use polythene sheet but
care should be taken that entire surface of polythene sheet should be
covered with straw or any other dried material up to 6-8 inch thickness to
avoid the damage of polythene sheet by dog, cat or other animals. Make
sure water does not enter the pit during rains. The silage will be ready in
45 to 60 days, depending on the types of material used. The silage of thin
stem crop like oat becomes ready in 45 days while thick stem crops like
maize, sorghum and bajra become ready in 60 days. Ideal silage is
golden yellowish green colour with good aroma. After completion of
incubation period the silo is opened for feeding. The whole silo should
not be disturbed and it should be opened from one place/corner to avoid
the loss of moisture and nutrients. Depending on the type of animal,
stage of production and availability of silage it can be supplemented in
the ration (5-25 kg per animal) of animal during lean period. After
opening the pit, silage should be used within 30 days.
2. After 8-10 weeks, silage is ready as feed for animals. Open pit/tank
initially from one side of for use. If it is not in use, then cover it carefully
with plastic film so that air will not go inside in silage. Initially animals
should be fed with 5-6 kg silage by adding it with chaffed green fodder
to develop taste to animals. Once animal likes sweet-sour taste of silage;
it will eat it with good liking.
3. Common enzyme-based silage additives contain cellulases,
hemicellulases, xylanases, amylases, and pectinases. Cellulases,
hemicellulases, and pectinases are enzymes that degrade the fiber portion
of forages. Amylase breaks down starch (amylose) therefore its use
would be directed towards starch containing silages such as corn silage.
155
Biodegradation of
Natural Xenobiotic UNIT 7 MICROBES IN GREENHOUSE
Compounds
GASES MITIGATION
Structure
7.1 Introduction
7.2 Objectives
7.3 Climate change
7.4 Cause of Global warming
7.4.1 Source of Greenhouse Gases
7.4.2 Can microbes help save the planet?
7.5 Mechanisms to solve climate change
7.5.1 Microbial Communities and Carbon Cycle
7.5.2 Microbial Communities and Methane Cycle
7.5.3 Microbial Communities and Nitrogen Cycle
7.6 How are Microbes Contributing to Global Warming?
7.7 Greenhouse Gases in Soil
7.8 Microbes as Carbon Sink
7.9 What is Sequestration of Greenhouse Gases
7.10 Reduction of CO2 Using Photosynthetic Cyanobacteria
7.11 Combating Global Warming Through Biofuels
7.12 Microbes and Global Warming
7.13 Microbes as Carbon Sink
7.14 Industrial Effluent and Land Fill Leachate
7.15 What is Sequestration of Greenhouse Gases?
7.16 Forest and Ocean, Sequestration of Greenhouse Gases
7.16.1 The Role of Forests in Climate Change
7.16.2 Ocean, Sequestration of Greenhouse Gases
7.17 Transformation of Greenhouse Gases
7.18 CO2 Sequestration/Assimilation Using Algae
7.19 Let Us Sum Up
7.20 Key Words
7.21 Suggested Further Readings/References
7.22 Answers To Check Your Progress Exercise
7.1 INTRODUCTION
The Earth is surrounded by a thick layer of gases which work like a blanket.
Without this blanket the Earth would be 20–30°C colder and much less
suitable for life. Climate change is happening because there has been an
156
increase in temperature across the world. This is causing the earth to heat up, Microbes in
Greenhouse Gases
which is called global warming. Global warming can be defined as the Mitigation
increase in the temperature of the earth atmosphere due to human activities.
(Panikov, 1999)
The major cause of global warming is the greenhouse gases which traps the
heat energy reflected by the earth’s surface. The major greenhouse gases are
carbon dioxide, methane, nitrous oxide and ozone. Some microbes are
solving the problem of global warming gases. The microbes, found in
geothermal areas in acidic and hot environment, utilizes methane gas. These
microbes can consume huge amount of methane about 11 kg/year and can be
helpful in reducing methane emission from methane producing factories and
landfills. Methylobacillus are one of earth’s most important carbon recycler
and they recycle carbon compounds as methane, methanol and methylated
amines. Besides, there are some naturally occurring microbes that convert
carbon dioxide into calcium carbonate that can fetch minerals of economic
value. Thus microbes have great potential to fight against global warming
and can serve as a powerful tool to combat pollution (IPCC. 2007).
Microbes play an important role in the production and consumption of
powerful greenhouse gases including CO2 and methane, have negative and
positive feedback responses to temperature changes, and play a vital function
in the regulation of ocean acidity. All of these can suffer perturbations due to
anthropogenic climate change. They also have important functions in
agriculture and the food web (Briones et al, 2004)
Microbes plays significant roles in the cycling of three major greenhouse

gases: carbon dioxide, methane, and nitrous oxide. Land management
practices that increase the amount of carbon (organic matter) stored in soil
are reducing the amount of CO2 released to the atmosphere.The capture,
sequestration, and utilization of CO2 is an effective way to decrease the
atmospheric CO2 concentration (Sanford et al, 2012)
7.2 OBJECTIVES
• Define Climate change.
• Describe different sources of greenhouse of gases.
• Describe Greenhouse gases in soil.
• Describe different natural resources which sequestrationof Greenhouse
Gases.
• Explain Role of enzymes in mitigation of CO2.
7.3 CLIMATE CHANGE

Most scientists now agree that climate change is taking place. This is being
demonstrated globally by the melting of the polar ice sheets and locally by
the milder winters we are having, coupled with more extreme weather such as 157
Biodegradation of heavy rain and flooding.
Natural Xenobiotic
Compounds
The Earth is surrounded by a thick layer of gases which keeps the planet
warm and allows plants, animals and microbes to live. These gases work like
a blanket. Without this blanket the Earth would be 20–30°C colder and much
less suitable for life. Climate change is happening because there has been an
increase in temperature across the world. This is causing the Earth to heat up,
which is called global warming.
7.4 CAUSES OF GLOBAL WARMING

The blanket of gases that surrounds the Earth is getting much thicker. These
gases are trapping more heat in the atmosphere causing the planet to warm
up.
7.4.1 Source of Greenhouse Gases

These gases are called greenhouse gases. The three most important
greenhouse gases are carbon dioxide, methane and nitrous oxide and these
have increased dramatically in recent years due to human activity.
The Earth is known as a ‘closed system’ which means that it produces

everything it needs to ensure the survival and growth of its residents. In
nature there are chemical cycles such as the carbon cycle to control and
balance these gases that surround the Earth. The carbon cycle is a complex
series of processes through which all of the carbon atoms in existence rotate.
This means that the carbon atoms in your body today have been used in many
other molecules since time began e.g. as the carbon found in carbon dioxide
in the air. Microbes play an important role as either generators or users of
these gases in the environment as they are able to recycle and transform the
essential elements such as carbon and nitrogen that make up cells (Willey et
al, 2009) . Global Greenhouse Gas Emission by Gas is given in Figure 7.1.
Figure7. 1: Global Greenhouse Gas Emission by Gas (Source: Willey et al, 2009)
Bacteria and archaea are involved in the ‘cycles’ of all the essential elements.
For example:-In the carbon cycle methanogens convert carbon dioxide to
methane in a process called methanogenesis. In the nitrogen cycle nitrogen-
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Microbes in
fixing bacteria such as Rhizobium fix nitrogen, which means they convert Greenhouse Gases
nitrogen in the atmosphere into biological nitrogen that can be used by plants Mitigation
to build plant proteins. Other microbes are also involved in these cycles. For
example:-Photosynthetic algae and cyanobacteria form a major component of
marine plankton. They play a key role in the carbon cycle as they carry out
photosynthesis and form the basis of food chains in the oceans (Jenkinson et
al, 1991). Fungi and soil bacteria the decomposers play a major role in the
carbon cycle as they break down organic matter and release carbon dioxide
back into the atmosphere.
7.4.2 Can Microbes Help Save the Planet

Microbes play an important role as either generators or users of these gases in
the environment as they are able to recycle and transform the essential
elements such as carbon and nitrogen that make up cells. Bacteria and
archaea are involved in the ‘cycles’ of all the essential elements e.g. in the
carbon cycle methanogens convert carbon dioxide to methane in a process
called methanogenesis; in nitrogen cycle nitrogen-fixing bacteria such as
Rhizobium fix nitrogen there by converting nitrogen in the atmosphere into
biological nitrogen that can be used by plants to build plant proteins. Other
microbes are also involved in these cycles’ e.g. photosynthetic algae and
cyanobacteria form a major component of marine plankton. They play a key
role in the carbon cycle as they carry out photosynthesis and form the basis of
food chains in the ocean. Fungi and soil bacteria are the decomposers that
play a major role in the carbon cycle as they break down organic matter and
release carbon dioxide back into the atmosphere (Melillo et al, 2002).
Greenhouse gas fluxes in terrestrial ecosystems, atmospheric carbon

dioxide (CO2) is fixed into sugars by the autotrophic (mainly plant)
communities in the presence of daylight. Plants release a great portion of
fixed carbon back to the atmosphere through autotrophic respiration. Along
with the release of a substantial portion of newly fixed carbon through their
roots, plant litters form a main source of energy for soil heterotrophs,
including microorganisms and animals; this carbon pool is respired back to
the atmosphere through heterotrophic respiration. A smaller amount of
organic carbon remains unused and is stored in the soil. Some organic carbon
is also used by some microorganisms for energy, but at a slower rate. CO2 is
also released into the atmosphere by anthropogenic activites such as fossil
fuel burning (not shown). The methane (CH4) cycle involves the conversion
of organic residues (sugars) into CH4 by methanogenesis, which is mainly
carried out by a specialized group of archaea, called methanogens, under
anoxic conditions. However, most CH4 produced in soils is immediately
oxidized by methanotrophs, which use CH4 as a source of energy. This is
mainly an aerobic process, and the availability of oxygen is a rate-limiting
step. Methanotrophs also oxidize some atmospheric CH4. The CO2 produced
by methane oxidation then enters into the CO2 cycle (part a). c | The
substrates for nitrous oxide (N2O) production, ammonium (NH4+) and nitrate
159
Biodegradation of (NO3–), enter soils in various forms. Atmospheric dinitrogen (N2) can be
Natural Xenobiotic
Compounds deposited in the soil following fixation by soil microorganisms and is
subsequently converted to NH4+; alternatively, reactive forms (mainly NO3
and NH3) can be deposited in precipitation or as dry deposition. Sources of
N2O, including fixed N2, can also be released from organic residues from
plants and animal waste and nitrogen fertilizers. The major source of
anthropogenic substrate is agricultural application of nitrogen fertilizers and
manure. In soil, a considerable amount of NH4+ is used by plants and
microorganisms, and the remaining portion is transformed into NO3– by NH3
oxidizing bacteria and archaea through nitrification. Most NO3– is converted
into N2 via various nitrogen oxides (including N2O) by denitrification
processes (carried out by denitrifying bacteria), and these then escape in the
atmosphere. Some nitrate is leached into the groundwater, and some is used
by plants.
7.5 MECHANISMS TO SOLVE CLIMATE

CHANGE
Microbial processes have a central role in the global fluxes of the key
biogenic greenhouse gases (carbon dioxide, methane and nitrous oxide) and
are likely to respond rapidly to climate change. Microorganisms regulate
terrestrial greenhouse gas flux. This involves consideration of the complex
interactions that occur between microorganisms and other biotic and abiotic
factors. The potential to mitigate climate change by reducing greenhouse gas
emissions through managing terrestrial microbial processes is a tantalizing
prospect for the future ( Anisimov et al,1999)
7.5.1 Microbial Communities and Carbon Cycle

Microorganisms play key role in determining the longevity and stability of
this carbon and whether or not it is released into the atmosphere as
greenhouse gas which means mediate the processes of carbon cycle.
Microorganisms are slow down global warming and implications for crucial
ecological processes such as nutrient cycling which rely on microbial
activity. Microorganisms are critical in the process of breaking down and
transforming dead organic material into forms that can be reused by other
organisms. This is why the microbial enzyme systems involved are viewed as
key ‘engines’ that drive the Earth’s biogeochemical cycles. The terrestrial
carbon cycle is dominated by the balance between photosynthesis and
respiration. Carbon is transferred from the atmosphere to soil via ‘carbon-
fixing’ autotrophic organisms such as photosynthesizing plants, photo and
chemoautotrophic microorganisms these are synthesis atmospheric carbon
dioxide in to organic material. Practically, microorganisms use carbon for
their metabolism substrate due to these highly consume atmospheric carbon
dioxide (Schlesinger & Lichter, 2001). Carbon Cycle is shown in Figure 7.2.
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Mitigation
Figure7.2:carbon cycle (Source:Schlesinger & Lichter, 2001)
The cycling of carbon by variety of bacteria and fungi species occurs in

aquatic habitats. Even relatively oxygen-free zones such as in the deep mud
of lakes, ponds and other water bodies can be regions where the anaerobic
conversion of carbon takes place. Both types of conversion take place in the
presence and the absence of oxygen. Algal involvement is an aerobic process.
In anaerobic environments, microorganisms can cycle the carbon compounds
to yield energy in a process known as fermentation. Other microorganisms
are able to participate in the cycling of carbon. For example, green and purple
sulfur bacteria are able to use the energy they gain from the degradation of a
compound called hydrogen sulfide to degrade carbon compounds (Benhelal,
2017). Other bacteria such as Thiobacillus ferrooxidans uses the energy
gained from the removal of an electron from iron containing compounds to
convert carbon. The anerobic degradation of carbon is done only by
microorganisms. This degradation is a collaborative effort involving
numerous bacteria such as Bacteroides succinogenes, Clostridium butyricum,
and Syntrophomonas sp. This bacterial collaboration is termed interspecies
hydrogen transfer and finally responsible for bulk of carbon dioxide and
methane is released in to atmosphere (Schlesinger & Lichter, 2001).
7.5.2 Microbial Communities and Methane Cycle

Cycling of carbon between carbon dioxide and organic compounds are
considered as ecologically significant. Both eukaryotes (plants and algae) and
autotrophic bacteria (cyanobacteria) are contribute a great significance role in
the fixation of carbon dioxide into organic compounds. As well as consumers
are used organic compounds and release carbon dioxide. Methane (CH4) is a
greenhouse gas most of the time enters to atmosphere because of microbial
action. Methane consuming microorganisms are critical to maintaining a
healthy climate on Earth (Bajracharya et al, 2015). Bacteria use methane for
metabolism as energy source. Methanotrophic bacteria is consume methane
as their only source of energy and convert it to carbon dioxide during their
digestive process. These bacteria can consume huge concentration of
methane which is helpful in reducing methane emission from methane
producing factories and landfills. Microorganisms are used high amount of
methane compounds which is found at everywhere.
161
Biodegradation of In anaerobic conditions just like deep compacted mud, carbon dioxide easily
Natural Xenobiotic
Compounds changed in to methane this is accomplished by methanogenic bacteria. The
conversion process needs hydrogen, yields water and energy for the
methanogens. To accomplish the recycling pattern another group of methane
bacteria called methaneoxidizing bacteria or methanotrophs (literally
"methane eaters") can convert methane to carbon dioxide. This conversion,
which is an aerobic process, also yields water and energy. In the presence of
oxygen, CH4 is oxidized to CO2 by methanotrophic bacteria. The oxidation of
CH4 to CO2 completes the carbon cycle. Methanotrophs tend to live at the
boundary between aerobic and anaerobic environments. They have access to
the methane produced by the anaerobic methanogenic bacteria, but also
access to the oxygen needed for their conversion of the methane (Alexis,
2017).
7.5.3 Microbial Communities and Nitrogen Cycle

Nitrogen is existed in an elemental form. It is the major component of the air
constituting about 78% of the gases in the earth atmosphere. There are also
different nitrogen gaseous compounds that exist in the atmosphere including
NH3, NO and N2O. Nitrogen is in the form of a very stable molecule (N2)
which is unusable by plants and animals without fixation. Nitrogen Fixation
is the process of changing atmospheric nitrogen into chemical forms which is
usable by living things (Staddon et al, 2004). N2 enters in to biosphere via
biological fixation. Biological nitrogen fixation will ever totally replace
industrial fixation for intensive agriculture. Rhizobium bacteria which cause
formation of nodules on the roots of legumes such as soybeans and alfalfa.
The bacteria are fairly specific for certain plants for example, the species
which infects soybeans will not infect alfalfa. The bacterium attaches to a
root hair of the plant and in response the plant forms a hollow thread leading
into the root. Bacteria grow through this infection thread and eventually
initiate formation of a nodule on the root. As much as 30 percent of the
weight of a nodule may be bacteria. The plant supplies energy and nutrients
for bacteria; bacteria and fungus supply nitrogen from the air in a form the
plant can use through fixation. This is an example of symbiotic nitrogen
fixation. Specific bacteria (Rhizobium trifolium) possess nitrogeniase
enzymes that can fix atmospheric nitrogen into a form (ammonium ion) that
is chemically useful to higher organisms as part of the symbiotic relationship,
the plant convert the 'fixed' ammonium ion to nitrogen oxides and amino
acids to form proteins and other molecules like alkaloids (Berman-Frank et
al, 2003). Nitrogen Cycle is shown in Figure 7.3.
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Mitigation
Figure 7.3. Nitrogen Cycle (Source:Berman-Frank et al, 2003)
7.6 HOW ARE MICROBES CONTRIBUTING TO

GLOBAL WARMING?
A group of animals called the ruminants such as sheep, cattle, goats, camels
and water buffalo have a special four chambered stomach. The largest
compartment is called the rumen. This pouch is teeming with billions of
bacteria, protozoa, moulds and yeasts. These microbes digest the cellulose
found in the grass, hay and grain that the animal consumes, breaking it down
into simpler substances that the animal is able to absorb. Cellulose is the
tough insoluble fibre that makes up the cell walls of plants; it gives the plant
structure.
Animals can’t break down cellulose directly as they don’t produce the
necessary digestive enzymes. The methanogens, a group of archaea that live
in the rumen, specialise in breaking down the animal’s food into the gas
methane. The ruminant then belches this gas out at both ends of its digestive
system. Methane is a very powerful greenhouse gas because it traps about 20
times as much heat as the same volume of carbon dioxide. As a result, it
warms the planet up to 20 times more than carbon dioxide. Around 20% of
global methane production is from farm animals.
Scientists are looking at ways to reduce the amount of methane emissions

from ruminants. One group in Australia has developed a vaccine which can
be given to the animal. The vaccine works by preventing the microbes that
live in the animals’ rumen from producing methane. The vaccine was tested
on sheep which belched 8% less methane in a 13-hour test.
At present the vaccine is only effective against 20% of the microbial species
that produce methane. To reduce methane production further scientists, need
to produce a vaccine that is active against more of the archaea that produce
methane (Bartdorff et al, 2008).
163
Biodegradation of The scientists were concerned that stopping the methanogens from working
Natural Xenobiotic
Compounds might affect the digestion of the ruminants and result in the animal being
smaller. However, what they did find is that cutting down the amount of
methane an animal produces actually boosts its growth. This is because the
process of methanogenesis uses energy which can result in a small but
significant loss of energy available to an animal. Another group of
researchers is looking to see if adding certain food additives to the diet of
Genome: It is a map of the complete genetic make-up of an organism. The

basic units of genetic information are called genes. The genome, which is
made up of genes, contains all of the biological information needed to build
and maintain a living example of that organism.

1. What is climate Change?
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2. What are different Sources of Greenhouse Gases?
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3. What are important causes of global warming?
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7.7 GREENHOUSE GASES IN SOIL

The role of soil microbes in climate change
• Soil is not a sterile substance. It is home to a vast array of life ranging

from moles to microbes which makes it a very active substance. As the
climate heats up it is predicted that the activity of microbes responsible
for the breakdown of carbon-based materials in the soil will speed up. If
this happens then even more carbon dioxide will be released into the
164
Microbes in
environment. This is because increased microbial activity results in an Greenhouse Gases
increase in respiration, which produces more carbon dioxide as a waste Mitigation
product.
• Group of microorganisms called denitrifying bacteria convert nitrous
oxide into harmless nitrogen gas. other groups of microorganisms, all of
which consume nitrous oxide and potentially mitigate emissions.
• Prochlorococcus and Synechococcus are single-celled cyanobacteria.
They are the smallest yet most abundant photosynthetic microbes in the
ocean. Researchers esti-mate that Prochlorococcus and Synechococcus
remove about 10 billion tons of carbon from the air each year; this is
about two-third of the total carbon fixation that occurs in the oceans.
• Microbial power could slow down increases in levels of carbon di-oxide
and other greenhouse gases and eventually reduce global climate change.
The researchers screened available microbial genomes encoding the
enzyme systems that catalyze the reduction of the nitrous oxide to
harmless nitrogen gas. They discovered an unexpected broad
distribution of this class of enzymes across different groups of microbes
with the power to transform nitrous oxide to innocuous nitrogen gas.
• Methylobacillus is a group of methylotrophic anaerobic bacteria, found
in large numbers in marine and fresh wa-ter ecosystems. These
organisms are one of earth’s most important carbon recycler, and they
recycle carbon com-pounds as methane, methanol and methylated
amines. In general, methylotrophs can use green-house gases such as
carbon dioxide and methane as substrates to fulfill their energy and
carbon needs.
• Microbes that convert carbon dioxide, a green-house gas, into calcium
carbonate. When the bacteria are used as an enzyme, it has been found to
transform CO2 into calcium carbonate (CaCO3)( Bardgett et al, 2008)
7.8 REDUCTION OF CO2 USING

PHOTOSYNTHETIC CYANOBACTERIA
• Most types of photosynthetic bacteria derive energy from ATP, which
helps in the conversion of CO2 to biomass and other products.
• Cyanobacteria are capable of fixing atmospheric nitrogen and carbon.
Similar to algae, they are distinct and broadly distributed and exist as
biofilms or as suspended planktonic cells.
• cyanobacteria played a decisive role in atmospheric formation by
decreasing CO2 concentration and increasing oxygen.
• cyanobacteria are the most efficient in atmospheric carbon utilization
over algae,cyanobacteria are found to be a key player by accounting for
20∼30% of Earth’s photosynthetic activity (Drigo et al, 2004).
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Biodegradation of
Natural Xenobiotic 7.9 COMBATING GLOBAL WARMING
Compounds THROUGH BIOFUELS
• Genetically modified it to overproduce the cellulase enzyme that is
capable of converting the cellulose and hemicellulose into ethanol. The
remaining lignin by-product can be burned to produce energy.
• The plant material used is the edible part of the plant such as sugar cane
and sugar beet or corn kernels because it can be easily being broken
down to sugar (glucose). The sugar can then be fermented (broken down)
to ethanol by mi-crobes such as the yeast Saccharomyces cerevisiae.
• The scientists are also trying to identify and isolate the genes from wood
termites that control cellulase enzyme along with the genes responsible
for the breakdown of sugar into ethanol and transfer them into another
bacte-rium that can be easily cultivated in the laboratory. This
genetically modified bacterium, with its new set of genes, will be able to
produce biofuel from cellulose.
• The fungus is genetically modified to produce large quantities of
cellulase. A staggering 75% of the straw fibre is converted into sugar.
The left over woody matter, lignin, is dried and then pressed into
burnable cakes. The glucose is then fermented with yeast to produce the
biofuel ethanol.
• So microbes could be the key to the future of powering cars in an eco-
friendly way with cellulose ethanol and Combating Global Warming
Through Biofuels (Drigo et al, 2004).
7.10 MICROBES AND GLOBAL WARMING

Most studies to date on the effects of climate change on biological systems
and soil microbes have examined single factors, such as elevated atmospheric
CO2 concentration, warming, or drought. However, there is considerable
potential for interactions between these factors to have additive or
antagonistic effects on soil microorganisms and their activities related to
greenhouse gas production. Very little is known about the effects of multiple
and interacting climate drivers on soil microbes and their contribution to
climate change, and, being so complex, they are likely to be very difficult, if
not impossible to predict.
Soil microbial communities and their activities to combined effects of
elevated temperature and atmospheric CO2, the combined and positive effect
of elevated temperature and atmospheric CO2on microbial decomposition of
peat was found to be greater than when these factors operated alone creating
an even stronger positive feedback on carbon loss from soil as DOC and
respiration.
Diversity of different components of the soil microbial community, including
bacteria, saprophytic fungi and mycorrhizal fungi are also affected directly
and indirectly by climate change.
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Microbes in
7.11 MICROBES AS CARBON SINK Greenhouse Gases
Mitigation
A group of oceanic micro-organisms play a vital role in the fight against

climate change and the role of oceans in absorbing carbon dioxide is crucial
to understand. There is a lot of carbon floating in the oceans that is known as
refractory dissolved organic matter and it has been put there by a hitherto
little-regarded group of creatures called aerobic anoxygenic
photoheterotrophic bacteria (AAPB).
A whole new “sink” for carbon dioxide is atmosphere.The main way that
carbon dioxide is absorbed by the ocean is through photosynthesis by
planktonic algae. These algae are the basis of most food chains in the sea that
are being eaten by tiny animals that are, in turn, eaten by larger ones. After
the death of these creatures, their remains sink to the sea floor, where some
are eaten and some are buried indefinitely. These remains are known in the
jargon as particulate organic matter. The dead creatures contain some of the
organic com-pounds that dissolve out of them and into the water.This
dissolved organic matter was not thought to be an important component of
the total carbon dioxide
7.12 INDUSTRIAL EFFLUENT AND LAND FILL

LEACHATE
Methane (CH4), a potent greenhouse gas (GHG) with a global warming
potential (GWP) ~23 times higher than that of carbon dioxide (CO2) is
produced by Landfills. There is urgent need to prevent methane emissions
from large landfills. Methanotrophs are a group of bacteria that consume
methane. In cover soil methanotrophs prevent fugitive methane emissions. In
present scenario two general research techniques are performed to prevent
methane emissions. First, a dimensionless number method developed based
on Michaelis-Menten kinetics. Second, effects of nutrient amendments on
methane oxidation and nitrous oxide production were examined by
constructing soil microcosms using landfill cover soils. Mehanotrophic
activity and community structure can be differentially affected by both
landfill gas composition and amendments thus provide insights for best
manipulation of methanotrophic processes to better mitigation of GHG
emission.
7.13 WHAT IS SEQUESTRATION OF

GREENHOUSE GASES?
Carbon dioxide is the most commonly produced greenhouse gas. Carbon
sequestration is the process of capturing and storing atmospheric carbon
dioxide. It is one method of reducing the amount of carbon dioxide in the
atmosphere with the goal of reducing global climate change.
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Biodegradation of
Natural Xenobiotic 7.14 FOREST AND OCEAN, SEQUESTRATION
Compounds OF GREENHOUSE GASES
Forests are important carbon pools which continuously exchange CO2 with
the atmosphere, due to both natural processes and human action.
Understanding forests' participation in the greenhouse effect requires a better
understanding of the carbon cycle at the forest level.
7.14.1 The Role of Forests in Climate Change

Organic matter contains carbon susceptible to be oxidized and returned to the
atmosphere in the form of CO2. Carbon is found in several pools in the
forest.
The vegetation: living plant biomass consisting of wood and non-wood

materials. Although the exposed part of the plant is the most visible, the
below-ground biomass (the root system) must also be considered. The
amount of carbon in the biomass varies from between 35 to 65 percent of the
dry weight (50 percent is often taken as a default value). Dead wood and
litter: dead plant biomass, made up of plant debris. Litter in particular is an
important source of nutrients for plant growth (Morgan, 2002). (Figure 7.4)
Figure7.4: Flow chart for Carbon Sequestration (Source: Morgan, 2002)
Soil organic matter, the humus. Humus originates from litter decomposition.
Organic soil carbon represents an extremely important pool. At the global
level, 19 percent of the carbon in the earth's biosphere is stored in plants, and
81 percent in the soil. In all forests, tropical, temperate and boreal together,
approximately 31 percent of the carbon is stored in the biomass and 69
percent in the soil. In tropical forests, approximately 50 percent of the carbon
is stored in the biomass and 50 percent in the soil (IPCC, 2007).
The process of photosynthesis explains why forests function as CO2 sinks,

removing CO2 from the atmosphere. Atmospheric CO2 is fixed in the plant's
chlorophyll parts and the carbon is integrated to complex organic molecules
which are then used by the whole plant. (Figure 7.5)
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Mitigation
Figure 7.5: Carbon Sequestration (Source: Morgan, 2002)
7.14.2 Ocean, Sequestration of Greenhouse Gases

Oceans absorb roughly 25 percent of carbon dioxide emitted from human
activities annually. Carbon goes in both directions in the ocean. Colder and
nutrient rich parts of the ocean are able to absorb more carbon dioxide than
warmer parts. Therefore, the polar regions typically serve as carbon sinks. By
2100, much of the global ocean is expected to be a large sink of carbon
dioxide, potentially altering the ocean chemistry and lowering the pH of the
water, making it more acidic.
The capacity of oceans sequestration of CO2 worldwide lies in between 1400

and 20,000 Gt of CO2 which is the highest among other sedimentary basins.
When CO2 is stored into the ocean, the dissolved CO2 can react with the
oceanic water and form carbonic acid which will increase the acidity of the
oceanic water and decrease in pH.
7.15 TRANSFORMATION OF GREENHOUSE

GASES
Greenhouse gases such as methane and carbon dioxide are emitted from
landfills, as well as from agricultural facilities due to concentrated animal
waste, contributing to climate change. Safe and environmentally sound
disposal of animal wastes is a major challenge for larger scale agricultural
producers. Using fossil fuels for energy contributes to global warming, and
energy needs are growing in many countries. Capturing waste gases and
converting them to energy can help to mitigate climate change by lowering
consumption of fossil fuels, at the same time reducing the release of harmful
gases. According to the United Nations Environment Programme (UNEP), in
order to ensure the average global temperature does not rise by more than
2°C by 2020, the percentage of primary energy that is produced from non-
fossil fuel energy sources, such as biogas, will need to increase by as much as
28% by 2020.
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Biodegradation of
Natural Xenobiotic 7.16 CO2 SEQUESTRATION/ASSIMILATION
Compounds USING ALGAE
CO2 mitigation with algae is found to be a sustainable process with
simultaneous generation of high calorific products, such as biodiesel,
pigments, fatty acids, etc. The potential utilization of algae is mainly
attributed to its wide distribution, high biomass production, capability to
adjust in adverse conditions, swift carbon uptake and utilization. Both macro
and micro algae have the capability to metabolize inorganic carbon by a
photoautotrophic mechanism using carbonic anhydrase enzyme (CAE). The
generated NADH2 from the electron transport chain combines with the
RuBisCo (Ribulose- 1,5-bisphosphate carboxylase/oxygenase, provided by
CAE) and helps in carbohydrate generation from CO2 and provides the
reducing power in the Calvin cycle for glucose synthesis (Green et al,2004).
(Figure 7.7)
Figure 7.7:Various biological processes that have potential for CO2 sequestration along
with sustainable bioenergy/chemical production. (Source: Green et al, 2004)
7.17 OCEAN CARBON ABSORPTION

When carbon-dioxide-rich air meets seawater containing less carbon dioxide,
the greenhouse gas diffuses from the atmosphere into the ocean. Today, about
a quarter of human-produced carbon dioxide emissions get absorbed into the
ocean. Once the carbon is in the water, it can stay there for hundreds of years.
Warm, CO2-rich surface water flows in ocean currents to colder parts of the
globe, releasing its heat along the way. In the polar regions, the now-cool
water sinks several miles deep, carrying its carbon burden to the depths.
Eventually, that same water wells up far away and returns carbon to the
surface; but the entire trip is thought to take about a thousand years. In other
words, water upwelling today dates from the Middle Ages – long before
fossil fuel emissions.
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Microbes in
That's good for the atmosphere, but the ocean pays a heavy price for Greenhouse Gases
absorbing so much carbon: acidification. Carbon dioxide reacts chemically Mitigation
with seawater to make the water more acidic. This fundamental change
threatens many marine creatures. The chain of chemical reactions ends up
reducing the amount of a particular form of carbon the carbonate ion that
these organisms need to make shells and skeletons. Dubbed the “other carbon
dioxide problem,” ocean acidification has potential impacts on millions of
people who depend on the ocean for food and resources.

1. Explain the role of soil microbes in climate change.
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2. What is Sequestration of Greenhouse Gases?
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3. Explain Reduction of CO2 Using Photosynthetic Cyanobacteria?
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7.18 LET US SUM UP

We have studied the Climate change. We have studied the climate change
mitigation by microorganisms and sequestration of carbon by natural
resources. We have studied the enzymes required for mitigation of
greenhouse Gases.
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Biodegradation of
Natural Xenobiotic 7.19 KEY WORDS
Compounds
Global warming: Global warming is the long-term warming of the planet's
overall temperature. Though this warming trend has been going on for a long
time, its pace has significantly increased in the last hundred years due to the
burning of fossil fuels.
Pollution: Pollution is the introduction of harmful materials into the

environment. These harmful materials are called pollutants.
Greenhouse gas: A gas that absorbs and emits radiant energy within the
thermal infrared range, causing the greenhouse effect. The primary
greenhouse gases in Earth's atmosphere are water vapour (H2O), carbon
dioxide (CO2), methane (CH4), nitrous oxide (N2O), and ozone (O3).
Methanotrophs: Methanotrophs are prokaryotes that metabolize methane as
their source of carbon and chemical energy. They are bacteria or archaea, can
grow aerobically or anaerobically, and require single-carbon compounds to
survive.
Enzyme: Enzymes are proteins that help speed up metabolism, or the
chemical reactions in our bodies. They build some substances and break
others down. All living things have enzymes.
Fungi:A fungus (plural: fungi or funguses) is any member of the group of
eukaryotic organisms that includes microorganisms such as yeasts and molds,
as well as the more familiar mushrooms.
Bacteria: Bacteria are small single-celled organisms. Bacteria are found
almost everywhere on Earth and are vital to the planet's ecosystems.
Biofuel:“liquid, solid, or gaseous fuel produced by conversion of biomass
such as bioethanol from sugar cane or corn, charcoal or woodchips, and
biogas from anaerobic decomposition of wastes”

READING/REFERENCES
• Alexis Bazzanella, D. K. (2017). Technologies for Sustainability and
Climate Protection –Chemical Processes and Use of CO2. Available
online at: https:// dechema.de/en/energyandclimate_CO2_Buch_engl.pdf
(accessed February 15, 2020).
• Anisimov OA, Nelson FE, Pavlov AV. Predictive scenarios of
permafrost development under condi-tions of global climate change in
the XXI century. Earth Cryology. 1999; 3: 15-25.
• Bajracharya, S., Ter Heijne, A., Dominguez Benetton, X.,
Vanbroekhoven, K., Buisman, C. J. N., Strik, D. P. B. T. B., et al.
(2015). Carbon dioxide reduction by mixed and pure cultures in
microbial electro synthesis using an assembly of graphite felt and
stainless steel as a cathode. Bioresour. Technol. 195, 14–24. doi:
172 10.1016/j.biortech.2015.05.081
Microbes in
• Bardgett, R. D., Freeman, C. & Ostle, N. J. Microbial contributions to Greenhouse Gases
climate change through carbon cycle feedbacks. ISME J. 2, 2805–2814 Mitigation
(2008). This paper highlights the central role of soil microorganisms in
land–atmosphere carbon exchange and its consequences for climate
change.
• Bartdorff, O. Wallmann, K., Latif, M. and Semenov, V. Phanerozoic
evolution of atmospheric methane. Global Biogeochem. Cycles 22,
GB1008 (2008).
• Benhelal, E., Zahedi, G., Shamsaei, E., and Bahadori, A. (2013). Global
strategies and potentials to curb CO2 emissions in cement industry. J.
Clean. Prod. 51, 142–161. doi: 10.1016/j.jclepro.2012.10.049
• Berman-Frank, I., Lundgren, P., and Falkowski, P. (2003). Nitrogen
fixation and photosynthetic oxygen evolution in cyanobacteria.Res.
Microbiol. 154, 157–164. doi: 10.1016/S0923-2508(03) 00029-9
• Briones MJI, Poskitt J, Ostle N. Influence of warming and enchytraeid
activities on soil CO2 and CH4 fluxes. Soil Biol. Biochem. 2004; 36:
1851-1859.
• Drigo, B., Kowalchuk, G. A. & van Veen, J. A. Climate change goes
underground: effects of elevated atmospheric CO2 on microbial
community structure and activities in the rhizosphere. Biol. Fertil. Soils
44, 667–679 (2008)
• Green, J. L. et al. Spatial scaling of microbial eukaryote diversity. Nature
432, 747–750 (2004).
• IPCC. 2007. Climate Change 2007: Synthesis Re-port. Available at
<http://www.ipcc.ch/publications_and_data/publications _ipcc_
fourth_assessment_report _synthesis_report.htm>.
• Jenkinson DS, Adams DE, Wild A. Model estimates of CO2 emissions
from soil in response to global warming. Nature, 1991; 351: 304–306.
• Melillo JM, Steudler PA, Aber JD, Newkirk K, Lux H, Bowles FP,
Catricala C, Magill A, Ahrens T, Morrisseau S. Soil Warming and
Carbon-Cycle Feedbacks to the Climate System, Science, 2002; 298:
2173-2176.
• Morgan, J. A. Looking beneath the surface. Science 298, 1903–1904
(2002).
• Panikov NS. Understanding and prediction of soil microbial community
dynamics under global change. Appl. Soil Ecol. 1999; 11: 161-176.
• Sanford RA, Wagner DD, Cu QW, Chee-Sanford J, Thomas SH, Cruz-
Garcia C, Rodriguez G, Mas-sol-Deya A, Krishnani KK, Ritalahti KM,
Nissen S, Konstantinidis KT, Loffler FE. Unexpected non-denitrifier
nitrous oxide reductase gene diversity and abundance in soils. Proceed.
Natl. Acad. Sc. 2012; 109(48): 19709-19714. DOI: 10.1073/
pnas.1211238109
173
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Compounds experimental forest plots under in-creased CO2. Nature. 2001; 411: 466-
469.
• Staddon, P. L., Jakobsen, I. & Blum, H. Nitrogen input mediates the
effect of free-air CO2 enrichment on mycorrhizal fungal abundance.
Glob. Chang. Biol. 10, 1678–1688 (2004).
• Willey JM, Sherwood LM, Woolverton CJ. Pres-cott’s Principles of
Microbiology, McGraw-Hill, New York, 2009.

1. Climate change refers to long-term shifts in temperatures and weather

patterns. These shifts may be natural, such as through variations in the
solar cycle. But since the 1800s, human activities have been the main
driver of climate change, primarily due to burning fossil fuels like coal,
oil and gas. Burning fossil fuels generates greenhouse gas emissions that
act like a blanket wrapped around the Earth, trapping the sun’s heat and
raising temperatures.
Climate change describes a change in the average conditions — such as
temperature and rainfall — in a region over a long period of time. For
example, 20,000 years ago, much of the United States was covered in
glaciers. In the United States today, we have a warmer climate and fewer
glaciers.
Global climate change refers to the average long-term changes over the
entire Earth. These include warming temperatures and changes in
precipitation, as well as the effects of Earth’s warming, such as:
• Rising sea levels
• Shrinking mountain glaciers
• Ice melting at a faster rate than usual in Greenland, Antarctica and
the Arctic
• Changes in flower and plant blooming times.
2. Different sources of Greenhouse Gases:Greenhouse gases trap heat
and make the planet warmer. Human activities are responsible for almost
all of the increase in greenhouse gases in the atmosphere over the last
150 years.The three most important greenhouse gases are carbon dioxide,
methane and nitrous oxide and these have increased dramatically in
recent years due to human activity.
GHG that are emitted as a result of human activity:
carbon dioxide (CO2), which comes from Industries, Thermal power
plants, Transportation and fossil fuels combustion.
174 Methane (CH4), which comes from landfills, coal mines, agriculture,
Microbes in
and oil and natural gas operations Greenhouse Gases
Mitigation
Nitrous oxide (N2O), which comes from using nitrogen fertilizers and
certain industrial and waste management processes and burning fossil
fuels
High global warming potential (GWP) gases, which are human-made
industrial gases
Hydrofluorocarbons (HFCs)
Perfluorocarbons (PFCs)
Sulfur hexafluoride (SF6)
Nitrogen trifluoride (NF3)
3. Global warming occurs when carbon dioxide (CO2) and other air
pollutants collect in the atmosphere and absorb sunlight and solar
radiation that have bounced off the earth’s surface. Normally this
radiation would escape into space, but these pollutants, which can last for
years to centuries in the atmosphere, trap the heat and cause the planet to
get hotter. These heat-trapping pollutants—specifically carbon dioxide,
methane, nitrous oxide, water vapor, and synthetic fluorinated gases—
are known as greenhouse gases, and their impact is called the greenhouse
effect.
Causes for rising emissions
Burning coal, oil and gas produces carbon dioxide and nitrous oxide.
Cutting down forests (deforestation)
Increasing livestock farming.
Fertilisers containing nitrogen produce nitrous oxide emissions.
Fluorinated gases are emitted from equipment and products that use
these gases.
1. The role of soil microbes in climate change
 Soil is not a sterile substance. It is home to a vast array of life ranging
from moles to microbes which makes it a very active substance. As the
climate heats up it is predicted that the activity of microbes responsible
for the breakdown of carbon-based materials in the soil will speed up. If
this happens then even more carbon dioxide will be released into the
environment. This is because increased microbial activity results in an
increase in respiration, which produces more carbon dioxide as a waste
product.
 Group of microorganisms called denitrifying bacteria convert nitrous
oxide into harmless nitrogen gas. other groups of microorganisms, all of
which consume nitrous oxide and potentially mitigate emissions.
 Prochlorococcus and Synechococcus are single-celled cyanobacteria.
175
Biodegradation of They are the smallest yet most abundant photosynthetic microbes in the
Natural Xenobiotic
Compounds ocean. Researchers esti-mate that Prochlorococcus and Synechococcus
remove about 10 billion tons of carbon from the air each year; this is
about two-third of the total carbon fixation that occurs in the oceans.
 Microbial power could slow down increases in levels of carbon di-oxide
and other greenhouse gases and eventually reduce global climate change.
The researchers screened available microbial genomes encoding the
enzyme systems that catalyze the reduction of the nitrous oxide to
harmless nitrogen gas. They discovered an unexpected broad
distribution of this class of enzymes across different groups of microbes
with the power to transform nitrous oxide to innocuous nitrogen gas.
 Methylobacillus is a group of methylotrophic anaerobic bacteria, found
in large numbers in marine and fresh wa-ter ecosystems. These
organisms are one of earth’s most important carbon recycler, and they
recycle carbon com-pounds as methane, methanol and methylated
amines. In general, methylotrophs can use green-house gases such as
carbon dioxide and methane as substrates to fulfill their energy and
carbon needs.
 Microbes that convert carbon dioxide, a green-house gas, into calcium
carbonate. When the bacteria are used as an enzyme, it has been found to
transform CO2 into calcium carbonate (CaCO3).
2. Sequestration of Greenhouse Gases: Carbon dioxide is the most
commonly produced greenhouse gas. Carbon sequestration is the process
of capturing and storing atmospheric carbon dioxide. It is one method of
reducing the amount of carbon dioxide in the atmosphere with the goal
of reducing global climate change.
Biological carbon sequestration is the storage of carbon dioxide in
vegetation such as grasslands or forests, as well as in soils and oceans.
• Oceans absorb roughly 25 percent of carbon dioxide emitted from human
activities annually. Carbon goes in both directions in the ocean. When
carbon dioxide releases into the atmosphere from the ocean, it creates
what is called a positive atmospheric flux. A negative flux refers to the
ocean absorbing carbon dioxide. Think of these fluxes as an inhale and
an exhale, where the net effect of these opposing directions determines
the overall effect. Colder and nutrient rich parts of the ocean are able to
absorb more carbon dioxide than warmer parts. Therefore, the polar
regions typically serve as carbon sinks. By 2100, much of the global
ocean is expected to be a large sink of carbon dioxide, potentially
altering the ocean chemistry and lowering the pH of the water, making it
more acidic.
• Carbon is sequestered in soil by plants through photosynthesis and can
be stored as soil organic carbon (SOC). Agroecosystems can degrade and
deplete the SOC levels but this carbon deficit opens up the opportunity to
176 store carbon through new land management practices. Soil can also store
Microbes in
carbon as carbonates. Such carbonates are created over thousands of Greenhouse Gases
years when carbon dioxide dissolves in water and percolates the soil, Mitigation
combining with calcium and magnesium minerals, forming “caliche” in
desert and arid soil.
• Carbonates are inorganic and have the ability to store carbon for more
than 70,000 years, while soil organic matter typically stores carbon for
several decades. Scientists are working on ways to accelerate the
carbonate forming process by adding finely crushed silicates to the soil
in order to store carbon for longer periods of time.
• About 25 percent of global carbon emissions are captured by plant-rich
landscapes such as forests, grasslands and rangelands. When leaves and
branches fall off plants or when plants die, the carbon stored either
releases into the atmosphere or is transferred into the soil. Wildfires and
human activities like deforestation can contribute to the diminishment of
forests as a carbon sink.
• While forests are commonly credited as important carbon sinks,
California’s majestic green giants are serving more as carbon sources
due to rising temperatures and impact of drought and wildfires in recent
years. Grasslands and rangelands are more reliable than forests in
modern-day California mainly because they don’t get hit as hard as
forests by droughts and wildfires, according to research from the
University of California, Davis. Unlike trees, grasslands sequester most
of their carbon underground. When they burn, the carbon stays fixed in
the roots and soil instead of in leaves and woody biomass. Forests have
the ability to store more carbon, but in unstable conditions due to climate
change, grasslands stand more resilient.
3. Reduction of CO2 Using Photosynthetic Cyanobacteria
• Most types of photosynthetic bacteria derive energy from ATP,
which helps in the conversion of CO2 to biomass and other products.
• Cyanobacteria are capable of fixing atmospheric nitrogen and
carbon. Similar to algae, they are distinct and broadly distributed
and exist as biofilms or as suspended planktonic cells.
• cyanobacteria played a decisive role in atmospheric formation by
decreasing CO2 concentration and increasing oxygen.
• cyanobacteria are the most efficient in atmospheric carbon
utilization over algae, cyanobacteria are found to be a key player by
accounting for 20∼30% of Earth’s photosynthetic activity.
177
Biodegradation of
Natural Xenobiotic UNIT 8 BIODEGRADATION OF
Compounds
XENOBIOTIC COMPOUNDS
Structure
8.1 Introduction
8.2 Objectives
8.3 Main sources of xenobiotics in the environment
8.4 Examples of Xenobiotic compounds
8.3.1 Pharmaceuticals
8.3.2 Pesticides
8.3.3 Halogenated Organic Compounds
8.3.4 Synthetic Polymers
8.3.5 Heavy Metals
8.3.6 Polycyclic aromatic hydrocarbons (PAHs)
8.3.7 Azo dyes
8.5 Degradation of xenobiotics
8.4.1 Abiotic Conversion
8.4.2 Biotic Conversion
8.4.2.1 Primary/ Partial biodegradation
8.4.2.2 Mineralization/ Complete biodegradation
8.6 Microbial Enzymes in Bioremediation
8.5.1 Oxygenases
8.5.2 Microbial Laccases
8.5.3 Microbial Peroxidases
8.5.2 Microbial lipases
8.5.3 Esterase
8.7 Factors influencing biodegradation of xenobiotics
8.6.1 Specific Chemical factors
8.6.1.1 State/Solubility/hydrophobicityof the compound
8.6.1.2 Adsorbability
8.6.1.3 Size and Shape
8.6.1.4 Charge
8.6.1.5 Toxicity
8.6.1.6 Concentration
8.6.1.7 Molecular Structure
8.6.2 Environmental factors
8.6.2.1 Presence of potent organisms
8.6.2.2 Physical factors
8.6.2.3 Availability of nutrients
8.6.2.4 Oxygen availability
8.6.2.5 pH
8.6.2.6 Inhibitory materials
178
8.6.2.7 Soil type Biodegradation of
Xenobiotic
8.8 Limitations of microbial remediation Compounds
8.9 Mode of action and toxicity of xenobiotics

8.8.1 Effects on Aquatic Organisms
8.8.2 Effects on Animals
8.8.3 Effects on Humans
8.10 Let Us Sum Up
8.11 Key Words
8.12 Suggested Further Reading/ References
8.13 Answer to Check Your Progress Exercise
8.1 INTRODUCTION
There is an intricate relationship between quality of life on Earth and
environmental quality. Unfortunately, advancements in science and
technology have led to generation of large amount of waste containing toxic
compounds which are a serious threat to the biological organisms. The book
Silent Spring written by famous author Rachel Carson in the 1960s resulted
in creating a widespread public awareness regarding the xenobiotics present
in the environment. A variety of adverse effects were attributed to the
indiscriminate use of synthetic pesticides, particularly Dichlorodipheny-
ltrichloroethane (DDT) in this book. This book was a stepping stone for
creating awareness and concern amongst the public about the xenobiotics and
their effects.
Xenobiotic word has been derived from a combination of two Greek words
"xenos", meaning foreign, and "bios", meaning life. Thus, manmade
synthetically derived compounds foreign to the environment are referred to as
xenobiotics and include pharmaceuticals, petrochemicals, pesticides, plastics
to name a few. These xenobiotic compounds are progressively becoming a
huge problem as they are comparatively new in origin, thus extremely hard to
be eradicated from the environment.
The major issue with the xenobiotics is their ability to biomagnify resulting
in the buildup of the xenobiotic in the organism at greater levels than present
in its food. Xenobiotic compounds being recalcitrant in nature are difficult to
be degraded by micro-organisms and thus have higher bioaccumulation as
well as biomagnification potencial as they enter into biotic entities. The
increase in concentration for some xenobiotic compounds, as in case of DDT
may be enormous, and therefore dangerous for the biological organism. Due
to the omnipresent nature of xenobiotics, exposure to them is unavoidable as
they are used voluntarily by humans as drugs, antibiotics, pesticides, dyes,
cosmetics etc. Nevertheless, the advantages of the xenobiotics for the
progress of human civilization cannot be neglected. However, it is important
to stop the indiscriminate use of them and take their degradation and toxicity
seriously. 179
Biodegradation of A large number of bacterial and fungal organisms may be utilized to degrade
Natural Xenobiotic
Compounds this group of pollutants by biodegradation. Biodegradation is the process of
breakdown of complex compounds into simpler ones with the help of
biological organisms and their enzymes. It consists of two processes: (a)
growth and (b) co-metabolism. In case of the former process, organic
pollutants are utilized as the sole source of carbon and energy resulting in a
mineralization of organic contaminants. In case of co-metabolism, the
microbe metabolizes the organic compound only when another compound is
present which it uses as a substrate for meeting its carbon and energy needs.
The microbes can degrade the xenobiotics through its well developed
enzymatic system.
The aim of the unit is to provide an insight into the major sources of
xenobiotics, examples of xenobiotics, their fate in the environment,
biological effects, biodegradation pathways using various bacteria and fungi,
factors affecting biodegradation and the various enzymes involved in
biodegradation of xenobiotics.
8.2 OBJECTIVES
After studying this unit you should be able to:
• Describe major sources and types of xenobiotics;

• Explain the fate of xenobiotics in the environment;
• Describe biodegradation pathways using various bacteria and fungi;
• Explain factors affecting biodegradation;
• Elaborate various enzymes involved in biodegradation of xenobiotics.
8.3 MAIN SOURCES OF XENOBIOTICS IN THE

ENVIRONMENT
The major cause of xenobiotic pollution in the environment includes:
1) Chemical and pharmaceutical industry which release various

xenobiotics;
2) Pulp and paper industries release manmade chlorinated organic
compounds;
3) Mining releases heavy metals;
4) Agriculture sector releases enormous amounts of pesticides, fertilizers
and herbicides.
5) Hydrocarbons are released from fossil fuels into the environment during
transportation to processing sites through accidents, such as oil spillage
or as a result of combustion.
180
Biodegradation of
8.4 EXAMPLES OF XENOBIOTIC COMPOUNDS Xenobiotic
Compounds
Figure 1 shows the various types of xenobiotic compounds commonly found

in the environment.
Biogenic/Natural
compounds
Pharmaceuticals
Chemical
compounds Pesticides
Anthropogenic Flame retardants

compounds / Xenobiotics
Plasticizers
Halogenated
Compounds from
pulp and paper
industry
Heavy metals
from mining
Polycyclic
Aromatic
Hydrocarbons
Azo dyes
Fig.8.1 Examples of xenobiotic compounds found in the environment
8.4.1 Pharmaceuticals
Pharmaceutical active compounds (PhACs) are xenobiotics commonly used
in agriculture and industries and are considered as potentially toxic
compounds. However, their toxicity and environmental implications has been
neglected for a long time. Environmental pharmaceutical persistent pollutants
(EPPPs) are the recalcitrant xenobiotics found in waterbodies throughout the
world and their impacts on humans and environment has not been studied in
depth. However, few reports suggest that too much exposure to EPPPs can
lead to species extinction as they largely affect the reproductive systems of
aquatic organisms. The sewage generated from the pharmaceutical industries
is also loaded with large amounts of antibiotics and other pharmaceutical
compounds (analgesics, anti-inflammatory drugs etc.). There can be many
possible routes for entry of these pharmaceutical compounds into the
environment which includes patient excretion, release of contaminated
wastewater from pharmaceutical manufacturing units, hospitals, application
of sludge to land, leaching of contaminated soil and leachate from landfill
sites, contamination from agricultural wastes and from plants treated with
various pharmaceutical drugs.
181
Biodegradation of 8.4.2 Pesticides
Natural Xenobiotic
Compounds
Majority of the pesticides in use currently are synthetic in nature, though
there has been an increase in efforts towards development of organic and
environmentally friendly pesticides. Though the pesticides were created to
protect human beings by killing microbes, insects or plants that are harmful,
however, a large number of them have been found to have toxic effects on
humans and animals. Organophosphate and organochlorine insecticides are
man-made pesticides and linked to a broad range of diseases ranging from
lung disorders to cancer to neurological diseases in humans. The chlorinated
hydrocarbons found in these man-made pesticides like endosulfan get
accumulated in the fatty tissue of the organism and cause adverse effects.
Thus, their proper disposal is of utmost importance to protect the biological
organisms. These days biological pesticides are becoming more and more
popular considering the fact that they are safer than the traditional pesticides.
8.3.3 Halogenated Organic Compounds

A variety of halogenated organics like Halocarbons, Polychlorinated
Biphenyls (PCBs) and other Chlorinated/Brominated/Florinated aromatic
compounds are widely used in various applications such as cosmetic,
pharmaceutical, paper and paint industry, as plasticizers, flame retardants
and are unintentionally released into the environment. Their recalcitrance is
mainly related to chemical inertness and hydrophobicity making them widely
distributed in the environment.
8.3.4 Synthetic Polymers

These include polyethylene, polystyrene, polyvinyl chloride, nylons and
others. These are highly recalcitrant due to their molecular size and
insolubility in water.
8.3.5 Heavy Metals

Mining and industrial processing leads to contaminating the environment
with heavy metals and disturbing the biogeochemical cycles. Due to the
persistence of these, they accumulate in the environment, contaminating the
food chains and pose a severe threat to health of humans. Though, some of
the heavy metals are considered essential (Mn, Fe, Cu, and Zn) and are
required in traces for their role in biological systems, many of them are non-
essential with no known biological role in organisms. Chromium, Cadmium,
Lead, Mercury and the metalloid Arsenic have been identified as the high
priority metal pollutants.
8.3.6 Polycyclic Aromatic Hydrocarbons (PAHs)

PAHs are released into the environment from the fossil fuel industries, fossil
fuel and biomass combustion and forest fires. These groups of contaminants
are well known for their carcinogenicity in humans.
182
Biodegradation of
8.3.7 Azo Dyes Xenobiotic
Compounds
Azo dyes are a class of synthetic dyes, used extensively globally in paints,
textiles, printing, cosmetics as well as pharmaceutical industries. Structurally,
they contain two adjacent nitrogen atoms between carbon atoms and are
known for their toxicity and mutagenicity to biological systems.
8.4 DEGRADATION OF XENOBIOTICS

The xenobiotic compounds can be degraded via 3 routes namely, physical,
chemical and biological. The major disadvantage of the physical and
chemical routes for degradation of xenobiotic compounds is that they are not
cost-effective and often produce undesirable intermediates and end-products
which may be more deadly than the parent compounds. The natural degraders
i.e. micro-organisms such as bacteria and fungi are more preferred for
degradation of xenobiotics due to their eco-friendly nature and being
comparatively less expensive process. These organisms are characterized by
rapid rate of multiplication, can act on wide variety of substrates and have
vast metabolic potential. However, selection of potent micro-organism
having the ability to degrade a particular xenobiotic compound or group of
compounds in a brief time without the production of any toxic intermediates
is an essential step for successful detoxification. Either naturally occurring
microbes can be used for the purpose or they can be genetically engineered
for degradation of xenobiotics. Since, xenobiotics may sometimes be
inaccessible to microbial degradation; engineering micro-organisms can help
in increasing their mobility towards these compounds, a process commonly
known as chemotaxis.
Xenobiotics in the presence of microbes can undergo biodegradation

depending on the microbe and type of xenobiotic compound. A majority of
aromatic xenobiotics contain a variety of substituents such as methyl groups,
halogen atoms, nitro-, and nitroso-groups which reduces the potential of
microbial degradation. However, the structural similarity of aromatic
xenobiotics to diverse metabolic intermediates of microorganisms creates a
base for their adaptation to xenobiotic assimilation. Figure 2 depicts the
likely environmental fate of a xenobiotic compound.
183
Biodegradation of
Natural Xenobiotic XENOBIOTICS
Compounds
NO CONVERSION ABIOTIC CONVERSION BIOTIC CONVERSION
Photo-chemical/
Chemical Partial Mineralization
transformation degradation
UNCHANGED CO2, H2O

MODIFIED XENOBIOTIC
XENOBIOTIC COMPOUND
COMPOUND
UTILIZED BY
DEPOSITION IN SOIL, SEDIMENTS, WATER/TRANSFER TO THE PLANTS AND
ATMOSPHERE THROUGH VOLATILIZATION/ TRANSFER TO THE MICRO-ORGANISMS
FOOD CHAIN THROUGH BIOMAGNIFICATION
Fig. 8.2 Possible environmental fates of xenobiotics
As shown in the figure, there can be either no conversion of the xenobiotic or

it may be converted by means of abiotic or biotic factors. The details of both
are given below:
8.4.1 Abiotic Conversion

Xenobiotics may be modified photochemically under natural conditions
through sun irradiation or due to the presence of chemicals such as O3, H2O2
acting as photocatalysts. For example, the pesticides, napropamide and
carbaryl degrade photochemically easily due to the presence of naphthoxy
chromophore group. However, some compounds due to sorption to
suspended particles, might evade photochemical degradation. This is the case
with tetracyclines which show high affinity to soil particles.
8.4.2 Biotic Conversion

8.4.2.1 Primary/ Partial Biodegradation
In case of primary/partial biodegradation, the compound is slightly changed

in its structural complexity and loses its characteristic properties. For
example, reductive decolorization of an Azo dye results in color change and
removal, but leads to the generation of two aromatic amines, which in some
cases may be more toxic to the organisms than the original dye as they are
known to be potential carcinogens.
8.4.2.2 Mineralization/ Complete Biodegradation
Mineralization refers to complete biodegradation into the mineral

components (CO2 and H2O). On the basis of composition of the compound,
184 other minerals (sulphide, sulphate, nitrite or nitrate; phosphate or phosphate)
Biodegradation of
may also be released. In case anaerobic mineralization occurs, methane may Xenobiotic
also be a produced. Compounds
8.5 MICROBIAL ENZYMES IN BIOREMEDIATION

An array of enzymes is involved in bacterial/fungal degradation of xenobiotic
compounds. Details about the different microbial enzymes involved in
degradation of xenobiotics are provided below:
8.5.1 Microbial Oxido-Reductases

Oxido-reductases are an important group of enzymes which helps in
elimination of lethal organic compounds such as phenolics or anilinic
compounds via oxidative coupling. During these reactions, the contaminants
get oxidized to less toxic compounds. They are known to be involved in
detoxification of lethal xenobiotics through different mechanisms which
includes polymerization/ co-polymerization with other substrates.
8.5.1.1 Oxygenases
Oxygenases are a group of oxido-reductase enzymes involved in oxidation of
reduced substrates by utilizing a co-substrate such as NADH/FAD/NADPH.
They are majorly of two types, namely monooxygenases and dioxygenases,
based on the number of oxygen atoms they use for oxygenation reaction i.e
one oxygen atom in case of monooxygenases and two in case of
dioxygenases. They increase water solubility as well as reactivity of organic
compounds as well as cleavage of the aromatic rings which ultimately
increases their metabolism rate. They have the potential to utilize a broad
range of substrates. Generally cleavage of the aromatic rings is through the
introduction of oxygen atoms into the molecule by the oxygenase enzyme.
Halogenated organic compounds are categorized under one of the major
environmental pollutants due to their widespread use in herbicides,
insecticides, fungicides. These groups of compounds are majorly degraded by
oxygenases. Oxygenases also act upon halogenated alkanes and alkynes by
means of catalyzing dehalogenation reactions with the help of multifunctional
enzymes.
8.5.1.2 Microbial Laccases

Laccases, belong to multi copper oxidase family, known for catalyzing the
oxidation reaction of a large number of reduced phenolic compounds,
methoxy-substituted monophenols and aromatic substrates. Major proportion
of Laccases is produced by Fungi and plants but many microorganisms also
produce it. Some microorganisms producing both intra and extracellular
laccases cause catalysis of various phenolic compounds, amines, lignin and
some inorganic ions. Laccases are known to act not only through oxidation
but also through decarboxylation reaction. Laccases also produce various
phenolic compounds by depolymerization of lignin.
185
Biodegradation of 8.5.1.3 Microbial Peroxidases
Natural Xenobiotic
Compounds
Peroxidases catalyze lignin oxidation as well as other phenolic compounds
oxidation by utilizing hydrogen peroxide (H2O2) in the presence of a
mediator. There are three major classes of microbial peroxidases namely,
Manganese Peroxidases (MnP), lignin peroxidases (LiP) and versatile
peroxidases (VP). MnP are extracellular heme enzymes utilizing Mn3+ as a
mediator for the oxidation of various phenolic compounds. LiPs are type of
secreted hemeproteins, which act in the presence of co-substrate H2O2 and a
mediator- veratryl alcohol for degrading lignin and other phenolic
compounds. VP directly oxidizes Mn2+, methoxy-benzenes and other
phenolic substrates just like MnP, LiP do. Due to its broad substrate
specificity, it has oxidizes the substrates in the absence of Mn in contrast to
other peroxidases. VPs are also reported to oxidize both phenolic and non-
phenolic lignin model dimers, which increases their demand for
bioremediation of recalcitrant pollutants.
8.5.2 Microbial Lipases

Lipase degrades lipids and their activity is responsible for extreme reduction
in total hydrocarbons from polluted soil such as in cases of oils spills.
8.5.3 Esterase
Esterase enzyme comes under hydrolase group of enzymes and hydrolyzes a
broad range of ester-containing compounds, like the organophosphates,
carbamates and pyrethroids.

1. Explain the process of microbial degradation of xenobiotics?
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8.6 FACTORS INFLUENCING

BIODEGRADATION OF XENOBIOTICS
There are broadly 2 types of factors, namely chemical specific factor and
environment-related factor that influence the biodegradation of xenobiotics
and contribute towards its recalcitrance, slow degradation or degradation at a
fast pace. Sometimes it may even be a combination of both. The factors are
discussed in detail below:
186
Biodegradation of
8.6.1 Specific Chemical Factors Xenobiotic
Compounds
8.6.1.1 State/Solubility/Hydrophobicity of the Compound
Degradation of a compound occurs in close association with an organism and

its enzymes. The accessibility of a compound varies due to its different
physical states like solids, liquids and gases. Gases permeate easily.
Degradation is also affected by its solubility, readily soluble compounds are
easily available to microbes but insoluble compounds may be available to
microbes at very low concentration, which may retard degradation.
Hydrophobicity is another important factor towards the availability of
compounds and inversely related to water solubility. The hydrophobic
materials are prone to form droplets by coalescing, reducing the surface area,
and restricting the availability. Also, another problem associated with organic
compounds that are hydrophobic in nature is their high affinity for
bioaccumulation in thefatty tissues of higher organisms. The bioaccumulative
compounds like organochlorines are hydrocarbons for e.g: polychlorinated
biphenyls, DDT, lindane, Aldrin etc. The bioaccumulation capacity can be
determined by octanol water partition coefficient. Higher co-efficient
indicates higher degree of bioaccumulation.
8.6.1.2 Adsorbability
A compound's availability to microbes and their enzymatic system decreases
with increased adsorption of compounds to other materials. This property
directly restricts the process of biodegradation. A classical case of this are the
herbicides parquet and piquet, which are adsorbed very strongly to clay
fractions in soils. Biodegradation cannot occur under such circumstances
even in the presence of microorganisms. Presence of herbicide concentration
and its absorbability directly affects the rate of degradation.
8.6.1.3 Size And Shape

For degradation of a chemical compound, direct contact with an enzyme or
microbe is essential but size and surface of both matters as well. When the
degradative enzymes are intracellular and not secreted out of a microbial cell,
compounds need to cross the permeability barrier that is selective in nature
for degradation process. Large size chemical compounds cannot cross the cell
membrane and thus they cannot contact the enzymes for their degradation. In
such a case, initially compounds have to be acted upon by extracellular
enzymes to break them to small sizes which then access their permeability
inside the cell. Some compounds may even be unable to complex with the
active site of the enzyme either due to their size, or due to their shape.
8.6.1.4 Charge
In order to penetrate through microbial cell membrane, external compounds
should possess specific charge. Mostly cations are preferable to cross cell
membrane for intracellular enzymatic activity, as biological membranes
187
Biodegradation of possess negative charge. Therefore, some highly positive charged molecules
Natural Xenobiotic
Compounds such as azo dyes and various surfactants are difficult to cross the membrane.
8.6.1.5 Toxicity
It is a major problem in bioremediation of toxic compounds where pollutants
are lethal to degradative microbes and enzymes. The optimal concentration of
toxic compounds needs to be measured before degradation process by
microorganism is initiated. In these cases, biodegradation is proportional to
their concentration and the resistance of the microbes. Some cases are also
reported where compounds are not toxic to microbes but particularly
inhibitory to a key enzyme of the degradation pathway. Various recalcitrant
compounds exhibit toxicity to microorganisms; for examples mostly phenols,
nitro derivatives and chloro-derivatives are harmful to microbial growth.
8.6.1.6 Concentration
Concentration of the compound is a chief factor in determining the
environmental fate of the compounds that are recalcitrant in nature. In case
the concentration of the compound is very high, problems related to toxicity
may occur. Conversely, when concentration is too low, then also there can be
lack of biodegradation due to two reasons. Initially low concentrations of the
compounds are insufficient for inducing enzyme production required for
biodegradation. Secondly, in case of evolution of new microbial community
with high metabolic capacities, presence of recalcitrant compounds in low
concentrations will not supply enough C and energy to provide the newly
evolved microbe a selective advantage.
8.6.1.7 Molecular Structure

Complexity of molecular structure directly affects the rate of degradation.
Complex compounds are more difficult to undergo degradation by microbial
metabolism whereas simple primary structured compounds are prone to
degradation by simple microorganisms. Complex functional groups like ester
of carboxylic, phosphate groups, sulphate bonds, that are widespread in
nature are hardly degraded by simpler microbial action. Rare functional
groups are difficult to metabolize because they cannot be processed by
normal metabolism. Complex carbon structures are difficult to undergo
biodegradation because of their molecular stability and strong hydrogen
bonding. Sometimes quaternary carbon structures limit the biodegradability,
for example alkyl benzene sulphonate, hydrocarbons, surfactants, and
xenobiotic compounds. Most of the xenobiotic compounds possess carbon-
halogen bonds that are not common in natural compounds. Presence of
organo-halogens, polychlorinated carbon compounds and nitro groups of
xenobiotic compounds retard the process of biodegradation. Above complex
structures play a major role in biodegradation process, degree of branching
has a greater influence on biodegradability. In view of molecular structure,
effect of substituents cannot be ignored. The substituents can have either
188 direct or indirect effects. Direct effects include change in electron distribution
Biodegradation of
of compound, hydrophobicity alter the ability of the compound to participate Xenobiotic
in enzymatic reactions. Substituents may act indirectly by increasing Compounds
hydrophobicity, increasing toxicity, and decreasing solubility, increasing
absorbability to or by altering charge, ultimately decreasing biodegradability.
8.6.2 Environmental Factors

A complete range of environmental factors can determine a compound's
environmental fate. These may either act individually or in synergism as
explained below:
8.6.2.1 Presence of Potent Organisms
For successful degradation of a contaminant, the presence of appropriate

micro-organisms is necessary. The presence of organisms acclimatized to the
target compound due to earlier exposure is known to greatly enhance its
biodegradation.
8.6.2.2 Physical Factors
Optimal physical parameters are necessary for biodegradation action of

microorganisms as every individual organism have their own optimum for
enhanced growth and degradative reactions. Physical factors influence the
microflora and their rates of reaction. Any change may either prove to be
beneficial or adverse to degradation. Microorganisms are difficult to establish
and perform biodegradation activity in abyssal depths of the oceans due to
the presence of extremes temperature conditions.
8.6.2.3 Availability of Nutrients
Establishment of well grown microbiota is based upon the availability of

wide range of nutrients they need. Most microbes capable of degrading
organic contaminants are heterotrophic organisms and their carbon and
energy source may be the target recalcitrant compound. The various
components of micronutrients (iron, magnesium, selenium, zinc) and
macronutrients (C, N, P, H, S) are major elements required for particular
enzymatic reactions.
8.6.2.4 Oxygen Availability
Oxygen is an important substrate for biodegradation process. Mostly

biodegradation processes are applied for organic compounds but some
inorganic compounds are also transformed by biodegradation as well. In the
presence of oxygen, transformation of organic compound occurs whereas
inorganic compound do not need oxygen for their degradation. Aerobic
degradation occurs in case of lignin compounds and the complex
lignosulphonates where oxygen requirement is obligate. Oxygen may
sometimes react directly as a substrate in a few enzymatic oxidation
reactions. In absence of oxygen or anaerobic conditions, microbes, which are
strictly anaerobic, will restrict their degradation activity, which can be
189
Biodegradation of supplemented with facultative anaerobes. Initially it was thought that
Natural Xenobiotic
Compounds recalcitrant xenobiotic compounds require oxygen essentially for their
degradation, but now it is known that many compounds are degraded
preferentially under anaerobic conditions. However, lignin degradation and
that of other complex lignosulphonates occurs under obligate requirement of
oxygen. There are some catalysis restricted to strict anaerobes; still use of
facultative anaerobes can do the job in the absence of obligate anaerobic
organisms.
8.6.2.5 pH
Microbial diversity is well adaptive to different saline environments with

variable alkalinity or acidity. Every organism has an optimum pH for their
growth and activity. For any kind of biological degradation or enzymatic
catalysis, organisms do need an optimum pH in their environment. Mostly,
research has found it to be near the neutral pH (6-8) and there is very few
reports on biodegradative reactions occurring at very high or very low pH.
Mostly fungi metabolize better in acidic environment, for example
cellulolytic and saccharolytic fungi decrease the pH of growth medium to 2
and 3. Fungi use comparatively less amount of recalcitrant organics for their
growth than bacteria.
8.6.2.6 Inhibitory Materials
Natural as well as artificial environments contain a wide array of toxic as

well as inhibitory compounds, which may either kill the microorganisms or
be inhibitory to their degradative pathways. Waste products released from the
treatment plants contain toxic chemicals such as cyanide, toluene which may
inhibit microbial activity. Some chemicals have the potential to wipe off
particular taxa, groups of microbes or even species.
8.6.2.7 Soil Type
Soil composition profoundly affects the compounds degradation in the soil.

Some fractions of soil are known for their ability to adsorb as well as
sequester the recalcitrant compounds. Also, there is possibility for soil
fractions such as clays to attach to the extracellular enzymes, consequently
inactivating them and allowing their substrates to endure for a longer time in
the soil than otherwise would have happened.
8.7 LIMITATIONS OF MICROBIAL

REMEDIATION
Following are the limitations of microbial remediation process:
1. It is a very slow process in comparison to physical and chemical

treatment methods.
2. A major limitation of bioremediation is that for efficient removal of

190
Biodegradation of
contaminants by the microbes, optimal conditions are needed to be Xenobiotic
maintained which is difficult in an environmental setting. Thus, most of Compounds
the times it is observed that the microbes may be effective in degradation
of xenobiotics under laboratory conditions, but not in the actual field
conditions.
3. A single micro-organism may not be capable of degrading all the

xenobiotic compounds. In that case, a microbial consortium needs to be
employed where a group of microbes work in conjunction leading to
degradation of all the xenobiotics present. Since, fungal and bacterial
communities are found together, so it is quite possible that their mixed
consortia can be more suitable for xenobiotic degradation and
environmental detoxification.
8.8 MODE OF ACTION AND TOXICITY OF

XENOBIOTICS
The toxicity of xenobiotic compound to the target organism mainly depends
on the properties of the compound which includes its basic characteristics
(structure, number of C-atoms, surface area) and physico-chemical properties
(water solubility, chemical reactivity etc.). Based on this there occurs
adsorption and bioaccumulation of the xenobiotic compound within the
organism leading to its toxicological action. Following are some of the
examples of effects on different groups of organisms.
8.8.1 Effects on Aquatic Organisms

Aquatic organisms are important biological indicators of xenobiotic
contamination. Among all ecosystems, the aquatic ecosystem is highly
hampered by the pollutants due to all the chemicals originally released on
land or into the atmosphere or directly dumped into the rivers will eventually
end up in the aquatic ecosystems as their final depository. Hence, the
extensive range of pollutants such as factory waste, effluents from sewage,
agrochemicals and pesticides, fossil fuel combustion are found in aquatic
ecosystem. The xenobiotics are also responsible to affect the reproductive
system of fish (sperm motility, fertilization rate) and larval survival. The
reproductive impairment in birds like eggshell thinning via DDE (dichloro
diphenyl dichloroethylene) causing a decline in population of a number of
raptor species throughout the world. Also, ovotestis in male western gulls is
found due to gradual exposure to the DDT. Similarly, a variety of fish species
that are exposed to environment containing EDCs (endocrine disruptors) get
negatively affected by them leading to reproductive problems. Turtles are
also victims of the above.
8.8.2 Effects on Animals

Xenobiotic pesticides, polychlorinated biphenyls (PCBs) have undesirable
side effects to the non-target organisms (polar bear, rabbit, guinea pig,
191
Biodegradation of alligators) which include effects on the reproduction and immune function.
Natural Xenobiotic
Compounds The xenobiotics have been increasingly reported to affect the immune system
either directly or indirectly through its metabolites. Due to the major function
of immune system in regulating organs, any dysfunction in the immune
system leads to adverse health impacts in animals.
8.8.3 Effects on Humans

Similar immuno-toxic impacts have also been reported in humans due to
xenobiotic exposure. The likely pathway for exposure of endocrine disruptors
(EDCs) in humans is through fast food, plastic packaging, paints, detergents,
cosmetics and direct exposure at the workplace as well as indirect exposure
by the environment, viz. air, water and soil. The major health effects of
pesticides toxicity includes damage to the nervous system, kidney and
increased risk of cancer, irritation to nose throat and eye, whereas chronic
exposure can result in damage to liver, kidney, endocrine and nervous
system. Cadmium (Cd) is a known nephrotoxic agent whereas lead is
regarded as being hepatotoxic agent. EDCs affects the male reproductive
system which leads to reduced semen quality, low sperm counts, and elevated
number of spermatozoa with abnormal motility, cancer of the testes and
malformed reproductive tissues. Bisphenol A is a component used in plastic
products, leads to block the human cardiac sodium channel. EDCs also lead
to diseases in females such as polycystic ovarian syndrome, breast and
reproductive organ tissue cancers and uterine fibroids. Phthalates most
commonly used in various cosmetics are known to affect the endocrine
system and are lined to infertility in women. EDCs have been linked to lower
immune health which results in affecting mental, immune and thyroid
functions in developing children along with impaired behavior.

1. Explain the limitations of microbial remediation limitations.
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8.9 LET US SUM UP

Increasing population and urbanization has led to increased environmental
contamination with xenobiotics. Xenobiotic compounds are ubiquitous,
found everywhere from personal care products to agricultural products. They
192 have adverse effects to human health leading to liver and kidney damage,
Biodegradation of
cardiac issues and eventually cancer due to prolonged consumption of these Xenobiotic
via ingestion of contaminated foods and drinks. Considering the severity of Compounds
xenobiotic contamination and its associated effects, it is important to find a
solution to mitigate it. One of the prospective remedy to do so is through
bioremediation using micro-organisms, as it is cost-effective as well as can
selectively biodegrade contaminants without causing damage to indigenous
flora and fauna. Despite of its advantages, they have had restricted
applications due to the challenges associated with substrate and
environmental variability. Also, more research should be directed towards
developing a suitable microbial consortium for effective degradation of
xenobiotic compounds.
8.10 KEY WORDS

Biodegradation:Process where the carbon-based material goes through a
change from complex modules into simpler ones before returning to the
environment.
Xenobiotics: A chemical compound (as a drug, pesticide, or carcinogen) that

is foreign to a living organism
8.11 SUGGETSED FURTHER

READINGS/REFERENCES
• Thakur, I.S., 2011. Environmental Biotechnology: Basic Concepts and
Applications, 2/e. IK International Pvt Ltd.
• Gangola, S., Joshi, S., Kumar, S. and Pandey, S.C., 2019. Comparative
analysis of fungal and bacterial enzymes in biodegradation of xenobiotic
compounds. In Smart bioremediation technologies (pp. 169-189).
Academic Press.
• Gaur, N., Narasimhulu, K. and PydiSetty, Y., 2018. Recent advances in
the bio-remediation of persistent organic pollutants and its effect on
environment. Journal of cleaner production, 198, pp.1602-1631.
• Petryszak, P., Kaszycki, P. and Szklarczyk, M., 2016. Bacterial
consortium for biodegradation of aromatic compounds. New
Biotechnology, (33), p.S137.
• Zdarta, J., Jankowska, K., Wyszowska, M., Kijeńska-Gawrońska, E.,
Zgoła- Grześkowiak, A., Pinelo, M., Meyer, A.S., Moszyński, D. and
Jesionowski, T., 2019.
• Robust biodegradation of naproxen and diclofenac by laccase
immobilized using electrospun nanofibers with enhanced stability and
reusability. Materials Science and Engineering: C, 103, p.109789.
• Dordio, A.V. and Carvalho, A.J.P., 2013. Organic xenobiotics removal
in constructed wetlands, with emphasis on the importance of the support
matrix. Journal of Hazardous materials, 252, pp.272-292. 193
Biodegradation of • Kadri, T., Rouissi, T., Brar, S.K., Cledon, M., Sarma, S. and Verma, M.,
Natural Xenobiotic
Compounds 2017. Biodegradation of polycyclic aromatic hydrocarbons (PAHs) by
fungal enzymes: A review. Journal of environmental sciences, 51, pp.52-
74.
• Kolvenbach, B.A., Helbling, D.E., Kohler, H.P.E. and Corvini, P.F.,
2014. Emerging chemicals and the evolution of biodegradation capacities
and pathways in bacteria. Current opinion in biotechnology, 27, pp.8-14.
• Bilal, M., Adeel, M., Rasheed, T., Zhao, Y. and Iqbal, H.M., 2019.
Emerging contaminants of high concern and their enzyme-assisted
biodegradation–a review. Environment international, 124, pp.336-353.
• Srivastava, V., Srivastava, T. and Kumar, M.S., 2019. Fate of the
persistent organic pollutant (POP) Hexachlorocyclohexane (HCH) and
remediation challenges. International Biodeterioration & amp;
Biodegradation, 140, pp.43-56.
• Caracciolo, A.B., Topp, E. and Grenni, P., 2015. Pharmaceuticals in the
environment: biodegradation and effects on natural microbial
communities. A review. Journal of pharmaceutical and biomedical
analysis, 106, pp.25-36.
• Pollard, K.M., Christy, J.M., Cauvi, D.M. and Kono, D.H., 2018.
Environmental xenobiotic exposure and autoimmunity. Current opinion
in toxicology, 10, pp.15-22.

EXERCISE
Refer Section Number 8.5

194
Biodegradation of
Xenobiotic
Compounds
BLOCK 3
BIOREMEDIATION
195
Biodegradation of
Natural Xenobiotic BLOCK 3 BIOREMEDIATION
Compounds
Bioremediation technology uses microorganisms to transform, reduce, and
destroy contaminants present in soil to harmless products. It depends on the
presence of suitable microorganisms present in appropriate amount in the soil
and proper environmental conditions. The indigenous microbes present in the
contaminated soil are well adapted to the contaminated conditions of the soil
(toxic components, temperature, pH etc.). Bioremediation process comprises
biotransformation and biodegradation by microbes. Microbes alter the
molecular or atomic structure of a compound in the process of
biotransformation. Biodegradation involves break down of organic or
bioaccumulation and biotransformation of inorganic compound into non-
hazardous products.
Unit 9 “Principles of Bioremediation” gives an account of bioremediation

methods, scope and advantages of bioremediation, in situ and ex situ
bioremediation, Factors affecting the process of bioremediation, Risk
assessment etc.
Unit 10 “Bioremediation for Soil Environment” covers in situ bioremediation

of contaminants in soil, bioremediation technologies viz. bioslurping,
biostabilization, soil mound, biotechnologies for ex-Situ remediation of soil,
Unit 11 “Bioremediation for Air Environment” covers biological filtration

processes for decontamination of air Stream, Bioscrubbers etc.
Unit 12 “Phytoremediation” gives different techniques for phyto-remediation

and elaborates upon the processes for phyto remediation
bioremediation of and phytoremediation.
196
Principles of
UNIT 9 PRINCIPLES OF BIOREMEDIATION Bioremediation
Structure
9.1 Introduction to Bioremediation

9.2 Bioremediation Methods
9.2.1 Bacterial assisted Bioremediation Method
9.2.2 Fungi assisted Bioremediation Method
9.2.3 Plant assisted Bioremediation Method
9.2.4 Combination of techniques Method
9.3 Scope of Bioremediation

9.4 Bioremediation Strategies - In Situ and Ex Situ Bioremediation and
Bioreactors
9.4.1 In-Situ Bioremediation
9.4.1.1 Biosparging
9.4.1.2 Bioventing
9.4.1.3 Bio-augmentation
9.4.2 Ex-Situ Bioremediation
9.4.2.1 Composting
9.4.2.2 Solid phase treatment systems
9.4.2.3 Biopiling
9.4.3 Bioreactors or controlled biosystems (Slurry and aqueous reactors)
9.5 Factors affecting the process of Bioremediation

9.5.1 Environmental factors
9.5.1.1 Nutrients
9.5.1.2 Soil
9.6 Risk Assessment (Advantages and limitations of Bioremediation)

9.7 Bioremediation, Sustainable Development and Future Prospects

9.8 Key Words
9.9 Suggested Further Readings/ Reference
9.10 Answers to Check you Progress Execise
9.1 INTRODUCTION TO BIOREMEDIATION

Space projects like SPUTNIK, APOLLO or MANGAL YAN etc. gave
humans a time depended comparative promising view of planet earth from
the space and thanks to the T.V. millions to billions peoples got a precious
opportunity to observe their home planet from the space or another place.
This observation also initiated a rough idea to almost every viewer that the
earth is very sensitive, its resources are finite, the space is limited and most
importantly humans can’t throw or dump their wastes anywhere else. The 197
Bioremediation industrialization, urbanization and population is rapidly increasing and the
same time the amount of these wastes are also increasing that reflecting in the
form of environmental pollution.
Pollution is a critical problem and its solution is one of the most important
goal to achieve because pollution is causing negative effects to biosphere,
permanent changes in ecosystems, damage to biodiversity,lethal effects on
public health and triggering several other socio-economic consequences. In
this regards as environment pollution mitigation approach, the bioremediation
is a useful technique.
The term Bioremediation consists two words Bio means biological and
remediation means to remedy or to solve any problem thus Bioremediation is
a method in which the biological entities (mainly microorganism) are used to
accelerate the natural process of the degradation of pollutants. The more
specifically microorganisms/plants or more directly their metabolic
enzymes/exudates are used as bio resources or acting agents to degrade or
neutralize unnecessary materials/xenobiotics and convert them in to less toxic
or more easily degradable forms. The selected classes of enzymes such as
oxydoreductase, laccase, hydrolytic enzymes and peroxidases are the classes
that directly involved in the remediation applications of very- very broad
range of pollutants targeting and very important in the microbial and/or plant
assisted bioremediation processes.
Despite the presence of various traditional methods (chemical, physical or

thermal) of dumping, transportation or excavating of the waste
entities/materials etc. the bioremediation is a far better option with the
attributes of cost effectiveness, less energy inputs, destroy pollutant or
transform it in to less toxic forms and use of more sustainable ecofriendly
approachto do all this. For these eye catching reasons bioremediation is
becoming a preferential choice over other clean up technologies or processes.
9.2 BIOREMEDIATION METHODS

Bioremediation is an environmental clean-up biotechnology in which
microorganism or plants are used according to site specific or pollutants
specific fashion and accelerate their degradation. Mostly the bioremediation
term has been applied for microbial remediation and in the case of plant, the
phytoremediation term often used. As the microorganisms are extremely
diverse and enormous physiological versatility to eat and live on various
substrates in any environment so the bioremediation method often target this
microbial ability to make environmental monitoring, assessment and clean
ups.
As the effective bioremediation always depends on the potentials

bioremediators or more precisely the appropriate enzymes attack/
biochemical exudate on the pollutants or combination of contaminants to be
remediated thus according to the biological entity used, the bioremediation
198
Principles of
methods can be broadly classify in to following types – Bioremediation
9.2.1 Bacterialassisted Bioremediation Method

Bacteria are the main focus of bioremediation process because they can use
pollutants as energy source of nutrient for their metabolism and can crack
them in to not just less toxic forms but also in to the organic matter forms.
The attention in this area targets the characterization, identification and
isolation of selected bacteria that can degrade several pollutants in parallel
fashion under adverse environments. The microbes such as aerobic and
anaerobic both are able to perform bioremediation but mostly aerobic
bacteriaor facultative aerobic bacteria often useful for bioremediation of
many compounds present in the surface/subsurface of soil/water such as
industrial dyes, pesticides, chlorinated solvents or other organic solvents and
oily/grease compounds while anaerobic microbes potentially useful to
degrade the chemicals that are found to be more recalcitrant in the deeper
soils or anaerobic zones including polychlorinated biphenyls (PCBs) and
chlorinated solvents present in the river sediments.
The various species of bacterial are excellent bioremediation potential such

as Bacillus species (the most common and most efficient), Pseudomonas
species, various Aeromonas species, Rhodococcus, mycobacterium,
Spingomonas, Alcaligens and several other microorganisms, among them
most of often used as commercial bioremediation solutions for site specific
pollution remediation.
Some aerobic microbes such as methylotrophs are also an attractive choice

for bioremediation of sites where excess of methane is present because these
microbes can use methane as carbon/energy source and grow very well along
with high level of monooxygenase enzyme production and as a result a
promising source of wide range of compounds remediation such as aliphatics,
organic solvents etc.
Table.9.1: Specific microbial enzymes, their targeted chemical classes

and bioremediation application.
Microbial enzyme Target chemical classes Bioremediation

class applications
Oxygenase Fatty acids, aromatic Bioremediation of
(Dioxiganase and chemicals/compounds, pharmaceuticals,
monooxiganase) steroids, sterols and synthetic chemicals,
selected alkanes soil/water surface
contaminants etc.
Peroxidase Polyphenolic compounds, Textile, paper, pulp,
(manganese and lignin lignin, polycyclic and/or pharma -food
and versatile) aromatic compounds, industrial waste
long chain aromatics, remediation, industrial
benzenes and halogenated Biocatalysis etc.
199
Bioremediation compounds etc.
Laccase Polyphenols, Cosmetics, paper, pulp,
aminophenol, diamines, textile, synthetic
diphenols etc. chemical industrial
waste remediation etc.
Hydrolase (protease, Protein, complex Baking industry,
lipase and callulase) carbohydrates, cellulose, leather industry, paper,
oils, fat, organic pulp, detergent,
pollutants etc. personal care product
industrial waste
remediation
9.2.2 Fungi Assisted Bioremediation Method

Often called mycoremediation method of bioremediation.The fungi are the
biochemical factories for producing huge amounts of enzyme having
bioremediation potentials thusfungi are useful in the bioremediation because
their digestive enzymes are capable to degrade or crack down various class of
xenobiotic such as heavy metals, complex hydrocarbons, industrial solvents
and many hazardous pesticides/dyes etc. The various aerobic filamentous
fungi such asAspergillus, Phanaerochaetechrysosporium(ligninolyticwhite
rot fungus) etc. are already showed promising potential and as an ideal model
in the bioremediation to destroy a tremendously varied range of persistent or
toxic environmental pollutants.Some other fungus also important for
xenobiotic remediation such as Pleurotus species (P. pumonarius or P.
ostreatus or P. tuber-regiumetc.),Agaricusbisporus, Trametesversicolor and
Irpexlacteus etc.
As bacteria needs the contact or pre-exposure of pollutant for bioremediation

application to induce the enzyme mediated metabolic response that in turn
help in the remediation purpose therefore pollutant’s very low levels often
become a limitation of bacterial assisted bioremediation methods. The
mycoremediation is good option in this sense to make a bioremediation
strategy.
The Mycoremediation not just eliminate the toxic compounds from soil and
water bodies but also provide other value added options such as synergies
with plants (mycorrhiza - especially arbuscularmycorrhizal fungi or AMF)
and edible mushrooms/medicinal nutraceuticals/enzymes byproducts etc.
9.2.3 Plant Assisted Bioremediation Method

Not all chemicals or pollutants are the bioremediation targets for the
microbes such toxic heavy metals like cadmium and lead etc., for this reason
plant assisted bioremediation or phytoremediation often useful. Plants are
always helpful in the bioremediation processes by different techniques
towards the pollutants such as binding, extraction, absorption, accumulating
or converting etc. Plants are having well developed enzymatic machinery that
200
Principles of
can make the enzymes or plant exudates effective in the bioremediation point Bioremediation
of views and can absorb the selected contaminants in their bodies such as
some heavy metals and remediation the soils. Several other chemicals such as
simple to complex hydrocarbons, various metals, industrial solvents,
petrochemical products or by products etc. are susceptible towards plant
assist remediation processes.
Figure.1. Phytoremediation process.
9.2.4 Combination Of Techniques Method

Many new bioremediation strategies of clean up often use a combined
approach to get desired outputs. The combination of soil microbes along with
several fungus and/or actinomycetes or their consortium or augmented whole
microbial community often useful as better remediation candidates for soil
remediation. The root of rhizospheric structure/assemblies or more simply
rhizospheric bioremediation is also a combination remediation technique in
which the rhizospheric plant root structures such as several grasses or weeds
or plants are useful as a source of plant exudates in the contaminated soils.
These root assemblies also helpful for microbial communities to grow well
thus provides a more favorable environment to microbial community
associate with rhizospheric zones. As a result of rhizospheric plant exudates
trigger the initial remediation and microbes elongate this process towards the
better results. Sometimes it is also observed that many plants exudates or
fungus digested enzymes are capable to change the initial chemical structure
or toxicity of contaminant towards the bacterial thus bacterial take the
advantage of this reinforcements from fungus/plants and do remediation
along with better outputs in less time framework.

1. Define the process of bioremediation and explain its methods?
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201
Bioremediation
9.3 SCOPE OF BIOREMEDIATION
The scope of bioremediation is very vast because it is cheap, promotes
natural process and sustainable technique to solve the pollution. The use of
various plants such as marine, terrestrial as well as coastal plants enabled this
technique for a wider range of ecosystems or their interconnected places
remediation. With the advances in microbiology alongwith biotechnology
and nanotechnology, biologists are employing microorganisms on a diverse
degree of chemical environmental remediation as well as a wider range of
earth’s very critical environments and places for clean ups. Very recent
advancements like Aero-microbiology, genetic engineering and
nanobiotechnology opened a huge future opportunities in the scope of
bioremediation.
Moreover, well intended bioremediation plans will produce direct and

indirect employment openings along with learning a more sustainable
management of natural resources against pollutants. That in turn facilitate the
integration of new sustainable socio-economic green actions/activities/
thinking in the human acts and its ecofriendly practices.
Unquestionably, this technique is representing a potential and untapped

resource for new environmental cleanup strategies and very promising future
outputs.
9.4 BIOREMEDIATION STRATEGIES - IN SITU

AND EX SITU BIOREMEDIATION AND
BIOREACTORS
In and ex situ bioremediation are two main classes of bioremediation based
on the site depended use of technique as well as the dependence of saturation
and/or degree of aeration of the site such as in the in situ class the
remediation of pollutants involves on –site practices with minimal on site
disturbances (soil, water) while on other hand the ex situ class of remediation
involves the treatment practices generally way from its original site by using
excavation of soil or pumping of ground water or pumping of surface oil
from sea water etc.
The contact between microorganisms and the targeted pollutant is the key
interaction in the bioremediation processes but many times the contact has
not been possible because of many important reasons such as the hug bulk of
impurities difficult to find core attachment of microbes, irregular or un-
uniformed distribution of xenobiotic, the specific nature of selected microbes
like their mobility (some bacterial are mobile and more sensitive towards
targeted compounds while some are not so there is an activation always
needed on other hand in the case of some fungus such as filamentous fungus
need the filamentous locomotion to reach towards the target an act etc.) or the
interaction/attraction level or mobilization with chemicals or surfactants etc.
202
Principles of
Bioremediation
Figure.2. Types of bioremediation.
Thus simple venting or sparging or spreading type of environmental cleanup

belongs to at the site only clean up treatment or in situ bioremediation kind of
techniques, while ex situ involves mining of polluted soil or miscellaneous
sediments or switching of groundwater to treat it elsewhere, soil that polluted
with oily sludge or micro plastics or poly nuclear aromatic hydrocarbons etc.
thus the ex situ processes commonly involved some techniques such as
bioreactors or slurry processing bioreactors, bio pumping, biopiling, and
land-farming etc.
9.4.1 In-Situ Bioremediation

In situ bioremediation is more frequent, low cost and desirable option to at
the site remediation prospects. As no other excavation or pumping is required
thus less energy or money cost necessity is a great advantage of this strategy.
Mainly easily to handle pollutants or subsurface hazardous chemicals are
treated by in situ applications.
9.4.1.1 BIOSPARGING
Biosparging involves application of air under pressure by the injections to

develop the high oxygen levels that in turn can improve the rate of pollutants
degradation. As this technique can enhance the mixing in the saturation
region by sparging technique thus it provides a better contact scenario
between the ground water and soil. Biosparging is very active technique in
the soil – water remediation as it provides flexible designs of working
systems along with cheap and easy to operate air injection assemblies as a
result it makes this process noninvasive and most efficient process.
9.4.1.2 Bioventing
Bioventing is a most common in situ strategy of normal in-situ

biodegradation of any aerobic method for contaminants removalby encourage
the standing as well as indigenous soil microorganisms by providing oxygen.
It is a very desirable technique when oil or grease or petrochemicals lubricant
contaminated sites are as a case study. The typical process includes providing
a very low flow oxygen to avoid unnecessary release of volatiles in to the air
203
Bioremediation by making an oxygen concentration just enough to sustain the microbes and
degrade the pollutant and not any excess of oxygen. Thus it helps to manage
atmosphere clean in this process as the volatile compounds are already
minimized by the technique. Bioventing often use direct injection of oxygen
with the help of wells in to the soil and ground water where the pollutants are
present more dipper from the surface.
9.4.1.3 Bio-Augmentation
Bio-augmentation is a process in which an extra microbial augmentation or

addition is required for bioremediation purposes. The introduction of a group
of naturally indigenous/exogenous microbial strains or consortium or
community or a genetically engineered variant is used in the augmentation
process to solve contaminated soil or water pollution. It is commonly used in
municipal wastewater treatment to restart activated sludge bioreactors for full
removal of contaminate. Most microbial solutions in this category available
for practice oftenuse of specific consortium of microbial community or well
researched populations that are having capabilities to perform an in situ
remediation in a site specific manner byall necessary microorganisms. Bio
augmentation process desires complete or close to complete degradation by
in situ microorganisms and converts them in to the nontoxic forms. The main
drawback of this process is a very difficult monitoring of the process.
9.4.2 Ex-Situ Bioremediation

In this section we will discuss ex situ bioremediation methods.
9.4.2.1 Composting
The organic waste or biodegradable compounds are main targets in the
composting applications by microorganisms, typically at elevated
temperatures. Typical compost temperatures are in the range of 55° to 65° C.
Composting are very simple process of ex-situ remediation along with the
advantage of value added by products extraction. A decent ex situ
composting application consists a flow chart of excavation followed by
removal of rocks and medium debris and finally contaminants depended
composting to get organic compounds and removal of mixture of
contaminants.
Figure.9.1. A typical composting process.

204
Principles of
9.4.2.2 Solid Phase Treatment Systems Bioremediation
Solid phase treatments are basically landfarming process to facilitate the

aerobic microbial degradation of pollutants by excavating the polluted soil
from somewhere else. This excavated soil is subjected to place on the
surface like a thin sheet or uniform bed like structure to gain a most possible
contact between indigenous microorganisms with the xenobiotic and
pollutants present in the excavated soil. The landfarming is a preferential
choice among other landfilling or old dumping practices because of low cost
and very affordable maintenance. The main laydown of this technique is the
pollutant should be on the surface and no more than 30 cm think surface layer
should be present in the system.
9.4.2.3 Biopiling
Biopiles are soil amendments process along with forced aeration supplements
thus it is a full scale method for ex situ excavated soil treatment. A typical
biopiling system includes a handling thin layered bed of excavated soil, a
much operated aeration arrangement, an irrigation/nutrient scheme and a
leach ate assembly system. To enhance the process attributes the major
factors such as heat, oxygen, moist air, nutrients and pH etc. are subjected to
optimize for application specific manner. The irrigation/nutrient system is
submerged under the soil to provide sufficient air flow, oxygen content and
necessary nutrient supply with the help of positive pressure or vacuum
pumps.Better materials such as heat resistant plastics are now a days making
Biopiles more strong/ long (up to 25 feet) and preferential over other
techniques when solar heating or evaporation or vitalization is also an
important concern in the specific ex situ application.
9.4.3 Bioreactors or Controlled Biosystems (Slurry and

Aqueous Reactors)
Slurry reactors or aqueous reactors are engineered systems to not just perform
ex situ mediated design of bioremediation applications but to get a better
controlled evaluation as well. A well-developed reactor system includes a
systematic process or sequence of chambers like initial processing of polluted
samples such as removal of big particles or solid/sediments or sludge
containing materials soil or water followed by specific vessel structure or
group of structures that can deal with the polluted samples in order to provide
them necessary exposure towards the bioremediators for better attachment.
The additional parameters such as rate of air or oxygen or pH of medium or
temperature or rotation speed etc. are also monitored to get desired outcomes.
The various types of reactor present commercially as well as non-
commercially depend on the need of bioremediation experiment such as
single pot bioreactors, single slurry reactor, sequential sludge reactor, two
phase reactors for complex mixtures of water/oil or other immiscible
solvents, three phase reactors for different phase mixture conditions etc. The
control over process, predictability of expected process output, easy
205
Bioremediation maintenance are some great advantages of reactor mediated ex situ
bioremediation while extra affords or cost attached with the elimination of
alternative pollutants are some major dis advantage of this technique.
9.5 FACTORS AFFECTING THE PROCESS OF

BIOREMEDIATION
As the biological entities are involved as acting agents in the bioremediation
process so factors and environments associated with their growth, activity,
life span and responsible for their death etc. are very important. For this
reason bioremediation becomes a very complex system of various factors that
need to be controlled for a successful bioremediation process application and
desirable outputs.
For a specific microbial assisted bioremediation process some important

factors becomes critical for a typical application such as presence of specific
microbes or microbial consortium or microbial community, capable enough
to use the pollutant as energy source or as carbon source and can degrade the
pollutant, the toxicity of pollutant towards the microbes, the specific pollutant
or groups of pollutants, the degree of the interaction between the availability
of pollutants to the microbes, the influence of surrounding environmentsuch
as soil texture and type of particles in soil, temperature variations, pH,
presence or absence of oxygen or level of oxygen, presence or absence of
electron acceptors along with nutrients etc.
Table.2: The specific influential factors that can affect a typical

bioremediation process.
Factors Condition required

Microorganisms Aerobic or Anaerobic
Natural Biological processes of Catabolism and Anabolism
microorganism
Nutrients Elements like C, N, O etc.
Soil Moisture Between 25-28% moisture along with
specific measurement of water
holding capacity
Type of soil Easy to handle, clay or silt gratified
Environmental Factors Temperature, pH ,Oxygen content,
Electron acceptor/donor
Microorganisms are the masters of this world and present almost every place
on this planet because they only want an effective carbon source and a
potential energy source thus can be easily isolated from almost any
environmental surroundings where these two main components are present. A
huge range of critical conditions are acceptable for the adaptation or growth
of microorganisms such as extremely high or low temperatures, exceptional
206
Principles of
heat, sea or desert conditions, presence or absence of oxygen, more diverse Bioremediation
environments with very toxic containments etc. In a more classified way the
microorganisms can be subdivided in to aerobic, anaerobic, facultative forms
of both types are useful in this sense.
9.5.1 Environmental Factors

9.5.1.1 Nutrients
The major nutrient is carbon for this reason carbon is needed in a very higher
amount as compared with other elements. The carbon along with oxygen,
nitrogen as well as hydrogen a more than 95 percent of quantity is an
important factor.The type of bioremediation also depends on the ratio of
nutrients like carbon: nitrogen ration or /and carbon: phosphorous ratio. In a
typical desired experiment 10:1 (C: N) and 30:1 (C: P) ratio often required.
Sometimes phosphorus and sulfur levels are also very important.
Table.9.2: Specific environmental conditions for the optimal growth of

microbes.
Environmental Optimum conditions Condition required for

Factor microbial
Available soil 25-85% water holding 25-28% of water holding
moisture capacity capacity
Oxygen >0.2 mg/L DO, >10% Aerobic, minimum air-filled

air-filled pore space for pore space of 10%
aerobic degradation
Redox potential Eh > 50 mill volts
Nutrients C:N:P= 120:10:1 molar N and P for microbial
ratio growth
pH 6.5-8.0 5.5 to 8.5
Temperature 20-30 ºC 15-45ºC
Pollutants Hydrocarbon 5-10% of Not too much toxic levels
dry weight of soil
Heavy metals 700ppm Total content 2000ppm
9.5.1.2 Soil
1. High concentrations of contaminants (roughly 5% or more): The soil is

agitated in a purifying water solution containing interface active agent,
then separated from the oils. After that, bioremediation is started to
efficiently clean the soil. At the experimental stage, bioremediation alone
has been able to turn contaminated soil into soil suited for landscaping,
and work is continuing to make this process even more efficient and
effective.
207
Bioremediation 2. Low concentrations of contaminants: Soils that have low concentrations
of pollutants can be cured using simple bioremediation alone and usually
it takes around 6 months to a year to decontaminate soil containing two
percent heavy oils, but at a concentration of 0.8 percent, the job can be
done in only about one to two months. This environmentally-friendly
method makes it possible to recycle and reuse soil without much effort.

1. How in situ bioremediation is different from ex situ bioremediation?
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2. Write a note on environmental factors that affect bioremediation.
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9.6 RISK ASSESSMENT (ADVANTAGES AND

LIMITATIONS OF BIOREMEDIATION)
The typical risk assessment is depends on the overall sum of the advantages
and limitations of applied bioremediation method for specific requests.

• It is natural, environmental friendly and sustainable approach for waste
cleanup.
• It is very cost effective as compared to other conventional methods of
waste removal.
• Pollutants are basically destroyed or degraded in to the less persistent
form or less toxic forms or permanently full degraded thus a better
option as compared to traditional dumping or waste transferring process.
• A conversion of waste in to organic matters directly reduces the amount
of waste and also offers a better environmental cycling of the matter and
nutrients.
208 • Comparatively very easy to apply, implement, operate and maintain
process compared with other conventional processes. Principles of
Bioremediation
• In or ex situ or their combination provides a flexible solution for
contaminates removal.
• The target pollutant classes are very broad thus a very huge potential is
present.

• Biodegradability is a critical criterion for bioremediation. The
compounds that are non-biodegradable are not the target for
bioremediation applications.
• Many times the byproducts or during bioremediation products become
more toxic than their untreated versions.
• The whole process control and maintenance of process is always a
critical factor.
• A bench or small scale successful process not always convertible for
large scale clean up.
• The type of pollutants are a great challenge.
• The required time for treatment is a critical factor. Often takes too much
time.
• Controllingindecision remains concerningsatisfactory performance
standards for bioremediation.
9.7 BIOREMEDIATION, SUSTAINABLE

DEVELOPMENT AND FUTURE PROSPECTS
Thus the environmental biotechnology or more specifically the
bioremediation is great technology waiting to be explored. It is a promise and
a strong hope for future environmental cleanup strategies along with the eye
catching attributes of environmental-friendly method, versatile technique and
cost-effective technology to reconstruct the planets clean resources, manage
the planet health dangers, save the biodiversity heritage and refurbish the
damaged ecosystems in a more natural way.
Bioremediation has the potential to reestablish and refurnish the

contaminated places/ecosystems/environments not just economically/
reasonablybut very effectively as well. This potation opens very huge
platforms for this promising technique towards the sustainable retreats and
sustainable development of the future of this planet.
It is also observed with this remarkable environmental cleanup technique

better understating, better instrumentation, correct control of operations,
better analysis, more sufficient training and necessary knowledge about the
bioremediation will surely facilitate this technique a became a more better
and useful near future and better outputs. Hence, future studies should invest
serious more and more efforts towards the adopting this scientifically and
rationale approaches and acquiring the highest quality standards as possible
to make this technique a gold standards. 209
Bioremediation Check Your Progress 3
1. Discuss the future scope of bioremediation
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REFERENCES
• Singh, B. D., & Singh, B. D. (2007). Biotechnology expanding horizons.
Kalyani publishers.
• Willey, J. M., Sherwood, L., &Woolverton, C. J. (2009). Prescott's
principles of microbiology.
• Fulekar, M. H., & Pathak, B. (Eds.). (2020). Bioremediation
Technology: Hazardous Waste Management. CRC Press.
• Fulekar, M. H. (2010). Environmental biotechnology. CRC Press.
• Sangeetha, J., Thangadurai, D., David, M., & Abdullah, M. A. (Eds.).
(2016). Environmental Biotechnology: Biodegradation, Bioremediation,
and Bioconversion of Xenobiotics for Sustainable Development. CRC
Press.
• Fulekar, M. H. (Ed.). (2012). Bioremediation technology: recent
advances. Springer Science & Business Media.

EXERCISE
Check Your Progress Exerscie 1

Refer Section Number 9.1,9.2
210
Bioremediation for
UNIT 10 BIOREMEDIATION FOR SOIL Soil Environment
ENVIRONMENT
Structure
10.1 Introduction
10.2 Objectives
10.3 Bioremediation
10.3.1 Bioremediation Approaches
10.3.2 Types of Bioremediation
10.3.3 Bioremediation Techniques
10.4 In situ Bioremediation

10.4.1 Biosparging
10.4.2 Bioventing
10.4.3 Biostimulation/Bioaugmentation
10.4.4 Phytoremediation
10.5 Ex situ Bioremediation

10.5.1 Landfarming
10.5.2 Composting
10.5.3 Biopile/Soil Mounds
10.5.4 Bioreactor
1.6 Bioremediation of Metals
10.6.1 Biotransformation
10.6.2 Biosorption
10.6.3 Bioaccumulation
10.7 Key Words

10.8 Suggested Further Readings/References
10.9 Answers to Check your Progress Exercise
10.1 INTRODUCTION
Heavy industrialization in developed countries led to industrial pollution of
land, air and water. The contamination of soil with inorganic and organic
pollutants is a major problem nowadays. These pollutants are heavy metals,
organic compounds, products derived from industrial processing, deliberate
and accidental release of harmful compounds. Table 1.1 shows the types of
pollutants released into the environment.
211
Bioremediation Table 10.1 Types of pollutants
S. No. Environmental pollutants

1. Organic
Petrochemicals: Diesel, oil, benzene, toluene, ethylbenzene, xylene
Synthetic: Pesticides, organohalogens
Biodegradable: Sewage, process waste and agriculture waste
2. Inorganic
Asbestos
Cyanides
Metals: Ag, Cd, Co, Cr, Cu, Fe, Hg, Pb
Radionucleotides
Nitrites, nitrates, phosphates
3. Biological
Pathogens: Viruses, bacteria
4. Gaseous
Gases: Methane, carbon dioxide, sulphur dioxide, nitrous oxide
Volatiles: Volatile organic compounds, chloroflourocarbons
Particulates
These contaminants can come from old industrial sites, unused mines,
industrial effluents, run off from landfills, excessive application of pesticides,
herbicides, weedicides and accidental spills.
The current methods for soil remediation are divided into five major
categories:
• Physical
• Chemical
• Biological
• Solidification/vitrification
• Thermal
Physical
This method includes physical removal of contaminated substance takes

place by excavation and concentration. This contaminated material has to be
further treated or disposed. Although this method is not a remediation
measure but it clean-up the affected site. This is a costly method due to
landfill tax and increasing cost of waste disposal. It is advantageous as it is
purely physical and no reagent is used. But a disadvantage is that further
treatment of contaminant is required.
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Bioremediation for
Chemical
Soil Environment
In this method chemical reactions destruct, fix or neutralize the toxic

chemicals. This approach is advantageous as biologically recalcitrant
chemical can also be destroyed. Moreover, the toxic substances are also
converted to biologically available form. The negative side of this treatment
is that chemical reagents are itself harmful for soil and a secondary treatment
might also be required.
Biological
Biological methods involves the modification of contaminants to less toxic

and more mobile or more toxic and less mobile which can be done by
accumulation of fixation in harvestable biomass crops. These methods are
beneficial as they decontaminate a large number of organic compounds and
do not harm soil structure and fertility. The only disadvantage is that this
process is slow and all pollutants can’t be treated by this method.
Solidification/Vitrification
In this process, toxic compounds which cannot be destroyed are made

unavailable to the environment by encapsulation inside a monolithic solid
having high structural integrity. Vitrification involves the use of high
temperature to fuse contaminated substance. The advantage of this method is
that future construction work might be done on these solidified stabilized
soils. On the other hand the chemical contaminants are not actually destroyed
and soil is permanently damaged.
Thermal
Heat treatments methods for example pyrolysis, gasification, incineration and

volatilization are used to eliminate the contaminants. The main advantage of
this method is that the contaminants are efficiently destroyed. This process
involves very high energy cost. Also, there is a strong possibility of
development of a new pollutant for more toxic contaminants. Soil structure
might also be disrupted in this process.
10.2 OBJECTIVES
After studying this unit you should be able to:
• define bioremediation and explain its significance;

• define and describe different types of in situ bioremediation techniques;
• define and describe different types of ex situ bioremediation techniques;
• explain bioremediation of metals;
• explain microbial transformation of metals;
• describe the process of biosorption and bioaccumulation.
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Bioremediation
10.3 BIOREMEDIATION
Bioremediation is a term composed of two words, bio from biological and
remediation means to remedy. The meaning of remediation is to resolve a
problem and bioremediation is the resolution of an environmental problem
such as contaminated soil and water using biological organisms such as
bacteria, fungi and other microorganisms. Overall, bioremediation is the
treatment and removal of pollutants from environment through biological
agents.
Bioremediation technology uses microorganisms to transform, reduce, and

destroy contaminants present in soil to harmless products. It depends on the
presence of suitable microorganisms present in appropriate amount in the soil
and proper environmental conditions. The indigenous microbes present in the
contaminated soil are well adapted to the contaminated conditions of the soil
(toxic components, temperature, pH etc.). Bioremediation process comprises
biotransformation and biodegradation by microbes. Microbes alter the
molecular or atomic structure of a compound in the process of
biotransformation. Biodegradation involves break down of organic or
bioaccumulation and biotransformation of inorganic compound into non-
hazardous products.
10.3.1 Bioremediation Approaches

Earlier physical and chemical methods are used to remediate contaminated
sites. However, the advancement in biotechnology leads to biological process
of contaminant degradation and removal called bioremediation. This method
is economical then previous methods but take longer duration of time. The
main approaches of bioremediation are as follows:
• Utilization of Indigenous microorganisms

• Amplification of indigenous microorganisms growth
• Bioaugmentation; the use of adapted microorganisms
• Inclusion of genetically modified organisms
• Phytoremediation
10.3.2 Types of Bioremediation

The major types of bioremediation are as below:
• Intrinsic Bioremediation
• Enhanced Bioremediation
• Bioaugmentation
10.3.3 Bioremediation Techniques

The bioremediation techniques depends on the contaminated area, the
properties of compounds, the concentration of contaminants and the time
214
Bioremediation for
required for completion of the process of bioremediation. There are various
Soil Environment
methods of bioremediation; the polluted/contaminated soil can be treated on
site called in-situ bioremediation or excavated and treated off site termed as
ex-situ bioremediation. The in-situ and ex-situ methods of bioremediation are
depicted in Fig. 1.
Fig. 10.1:Insitu and ex situ techniques for contaminated soil. Ex situ treatment can be
done off site or onsite
10.3 IN-SITU BIOREMEDIATION

10.4.1 Biosparging
Biosparging is a technique to enhance the bioactivity of soil by supplying
increased amount of oxygen through sparging air or oxygen into the soil.
Initially air was introduced but later replaced by oxygen to augment the
degradation rates. This process remediates contamination at or below water
table boundary. It superaerate the groundwater and stimulation increased
contaminant removal. Air/oxygen is injected through pipes into the
contaminated area (Fig. 10.2). The extra oxygen increased the aeration of the
contaminated soil and stimulates the activity of native microorganisms. This
accelerates the natural process of biodegradation of contaminants by these
microbes. The application of this method is limited due to its high expense in
extremely contaminated sites. However, onsite generation of oxygen lower
down the cost. Hydrogen peroxide is also used at some sites but it can be
toxic to microorganisms. The technique of air/oxygen delivery can be simple
or complex depending on the site specific requirements. The sparger control
system contains a pressure gauge and relief valve to release excess air
pressure. Flow meters and filter systems are also attached to clean the
contaminants from the site.
215
Bioremediation
Fig . 10.2: Biosparging
The structure of soil is a major factor for the success of this technique. The
most important step in in-situ bioremediation is the delivery and supply of
air/oxygen to the site of contaminants which in turn depends on the
conditions of the soil. The removal or degradation of the contaminants is
influenced by the channels and pores in soil organization, diffusion into
closed pores and presence of organic matter. Therefore, thorough site
investigation, site geology and hydrogeology are crucial before starting this
procedure.
This process is utilized for many contaminants such as benzene, toluene,

ethyl benzene, xylene and volatile organic compounds. Biosparging is
commonly recommended for removal of medium weight petroleum
hydrocarbon products such as diesel and jet fuels. Light pollutants like
gasoline can be easily mobilized into the unsaturated zones and removed by
physical means. Heavy contaminants such as oils take longer remedial time
period due to decreased microbial bioavailability with increasing carbon
chain length. It should be taken into account that the contaminants and vapors
released by this process require further treatment.
10.4.2 Bioventing
Bioventing is an in-situ technique used to remove volatile soil contaminants
present above the water table in unsaturated zone. This method also includes
superaeration to stimulate the enhanced degradation of contaminated
material. This process is not appropriate for sites having water table within
one meter or surface and waterlogged soil as air flow is restricted in these
circumstances.
This method combines an augmented oxygen supply along with the vapors
extraction. At a particular depth in contaminated soil a vacuum pump was
applied in a bore hole drilled into the contaminated soil, which draws out the
volatile compounds (Fig. 3). Volatile components already available in
216
Bioremediation for
contaminated soil or obtained as byproducts of biological degradation of
Soil Environment
pollutants are mobilized during the process and easily extracted. Bore holes
are also drilled around the polluted site to exchange the air extracted by the
vacuum. Supplementation of nutrients is also done by adding nutrients into
channels dug around the site. The supplementary provision of air will
increase the process of natural degradation by aerobic microorganisms. This
method is applicable only for volatile compounds and depends on the
permeability of the soil. The vapours obtained from contaminated soil may
need further treatment such as use of biofilters. Bioventing also requires
complete and extensive investigation of contaminated site before starting as
the accurate functioning of this method essentially requires the suitable
setting of the requisite pipework.
This technique is useful to treat aerobically biodegradable contaminants such

as non-chlorinated volatile organic compounds and petroleum hydrocarbons.
Bioventing is not suitable if soil oxygen levels are 2-5 %. It takes few years
to decontaminate a site depending upon pollutant concentration and removal
rates.
Fig. 10.3: Bioventing
10.4.3 Biostimulation/Augmentation
Biostimulation is the process to remediate deep contaminated soil by
provision of a combination of appropriate nutrients and specific
microorganisms. Bioaugmentation is addition of microbial cultures to fasten
the bioremediation process. This process is essentially required for those
contaminants which are not degraded by indigenous microbes. The
population of microbes incorporated from outside reduced quickly and
growth also become poor. This might be due to their inability to compete
with the indigenous population of microorganisms. However, supply of
specific substrate for augmented organism may reduce this problem.
Bioaugmentation is always applied in concurrence with biostimulation.
Microorganisms having the ability to degrade a specific contaminant are
217
Bioremediation added to the polluted site. Nutrients are injected into the contaminated soil
below the surface stimulates the indigenous/native microorganism through a
bore hole. Another bore hole is used to extract water to make space for
microorganisms and nutrients (Fig. 4).
Fig. 10.4: Biostimulation
Bioaugmentation has been used for crude oil removal. In laboratory studies
done by researchers augmented cultures degrades oil. However, studies for
removal of weathered diesel in arctic soils showed no degradation of oil.
Thus, bioaugmentation might found effective in laboratory but not in field
conditions. The possible causes of failure of bioaugmentation process are a
limiting availability of microbes; the concentration of pollutant was not
adequate to support growth; the presence of growth inhibiting substances in
soil environment; predation by protozoa; the augmented microbes may be
utilizing substrate other than the contaminant; the microbial culture was
unable to reach or penetrate the soil up to the site of contamination.
Moreover, active soil is found to be more suitable then pure cultures. The
activated soil contains indigenous microbes having recent exposure to the
contaminated soil. This technique introduces naturally occurring microbes
with mixed population of other microbes.
10.4.4 Bioslurping
Bioslurping is the technique of vacuum enhanced dewatering technology for
remediation of hydrocarbon-contaminated sites. It is the combination of two
bioremedial processes, bioventing and vacuum-enhanced free-product
recovery from groundwater and soil. The bioventing process stimulates the
aerobic bioremediation of hydrocarbon-contaminated soils in situ. Under
aerobic conditions, maximum aliphatic and aromatic components of
petroleum hydrocarbons are degradable. Vacuum-enhanced free-product
recovery extracts light non-aqueous phase liquids (LNAPLs) from the
capillary fringe and the water table.
To understand the bioslurping technique, comprehensive knowledge of both

218
Bioremediation for
these technologies is needed. Bioslurping can increase efficiency and less in
Soil Environment
cost and treatment times as compared to other hydrocarbon remediation
techniques as it removes free product and residual contamination in single
step.
The key components of a bioslurping system (Fig. 5) to extract groundwater,

free product, and soil gas contain:
• Recovery (slurper) well with slurp tube

• Vacuum pump to extract liquids and vapors (usually a liquid ring pump)
• Liquid/vapor and oil/water separation units
• Water and vapor treatment units
The bioslurping system has a “slurp” tube that extends into the free-product
layer. The pump extracts liquid (including free-product) and soil gas up the
tube in the same process stream. Light non-aqueous phase liquids (LNAPLs),
for example oil was pumped from the top of the water table and the capillary
fringe (an area just above the saturated zone, where water is held in place by
capillary forces). The LNAPLs are taken outward, where they are separated
from water and air. Bioventing pulls air into the soil while withdrawing soil-
gas through the recovery well and stimulates aerobic bioremediation of
hydrocarbon-contaminated soils (recovers product) in the vadose zone.
Vacuum-enhanced free-product recovery remediates vadose zone by
extracting LNAPLs from the capillary fringe. The system is designed
to minimize environmental discharge of ground water and soil gas. After
completion of free-product removal, the bioslurping system is converted to a
conventional bioventing system for completing the remediation process. It is
a medium to long term operation and takes few months to years for complete.
Fig. 10.5: Bioslurping
219
Bioremediation Bioslurping is less operational in low permeable soils. Extreme soil moisture
is the highest restraint to air permeability. Optimum soil moisture is very
soil-specific. Air permeability of the soil reduced in high soil moisture and
decreases its oxygen transfer capability. Low moisture will inhibit microbial
activity in the soil. Low temperature slows the process of remediation. In
deep and high permeability sites the system has difficulty in establishing a
vacuum.
10.4.5 Phytoremediation
Phytoremediation is the utilization of plants for degradation of contaminant
and metals from soil or water and make them harmless. The term
phytoremediation composed of two words- ‘phyto’ means plant and
‘remediation’ means to remedy. This method has many advantages such as
the top soil is not disturbed, applicable in case of low level of contamination,
metals can be recovered and is low cost. On the other hand, the process takes
more time than other methods, many growing seasons are required, plant
growth reduced and these plants with accumulated contaminant pose a hazard
to wildlife. Phytoremediation includes various mechanisms through which
living plants change the chemical composition of soil in which they grow. It
can be useful in remediation of metals, pesticides, crude oils, solvents,
explosives and various other contaminants. This process exploits plant’s
natural capacity to accumulate, absorb and break down pollutants from soil or
the medium of growth. Microorganisms living in soil also interact with plants
and promote phytoremediation.
The plants required for bioremediation should have certain characteristics

such as tolerance for specific contaminants, fast growth (high yield per
hectare), accumulation of metals in harvestable parts, highly developed root
system and high bioconversion factor. Bioconversion factor is the
concentration of contaminant in plant as compared with the environment.
Most of the plants have bioconversion factor less than 1.
Phytoremediation is divided into following processes (Fig. 10.6):
1. Phytoextraction (phytoaccumulation)
2. Phytodegradation
3. Phytovolatilization
4. Phytostabilization
Phytoextraction
Phytoextraction involves the removal of organic pollutants and metals by the

roots of the plants from the soil and their storage in plant parts such as roots,
stem and leaves. Plant roots have high surface area with high affinity
chemical receptors. Herbaceous plants are most appropriate for their fast
growth, high biomass and soil stabilization. Few examples of pollutants that
can be removed by this process are heavy metals, trinitrotoluene (TNT),
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Bioremediation for
trichloro ethylene (TCE), benzene, toluene, ethylbenzene, xylene (BTEX).
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Fern, Dicropteris dichotoma can accumulate rare earth elements like La, Ce,
Pr and Nd, vetiver grass (Vetiveria zizaniodes) and bana grass (Pennisetum
glaucum x P. purpureum) uptake cadmium and lead from mine areas,
johnson grass and canadian wild rye grass removed TNT from soil.
Phytomining utilize Alyssum spp. for removing nickel, manganese and
calcium from soils. Poplar trees can eliminate trichloethylene (TCE) from
polluted water applied to its roots. Trees are used for phytoremediation as
they are high biomass producer, low cost sustainable and ecologically
feasible solution. Examples of trees to be used for phytoremediation are
sycamore (Acer), willow (Salix), alder (Alnus), birch (Betula) and poplar
(Populus).
Fig. 10.6: Phytoremediation
Phytodegradation
Phytodegradation process is biological disintegration of pollutants internally

after extraction from plants or externally by enzymes secreted by them. The
complex organic pollutants break down into simpler compounds through the
process of biodegradation and integrated in the plant tissues. Organic
compounds can go through many changes: partial conversion into less toxic
substances, partial degradation, storage into plant and complete degradation.
In this process, plant species are used to degrade organic pollutants such as
chloroacetanilide, trinitrotoluene (TNT), trichloroethylene (TCE), RDX and
nitroglycerine to carbon dioxide, ammonium and nitrate as they have
enzymes such as nitroreductases, dehalogenases and laccases or they are
stored in vacuoles for degradation later on. Other important classes of
enzymes capable of degradation of organic pollutants are peroxidases,
peroxygenases, phosphatases and nitrilase. The degradation of pesticides and
herbicides by plants has been studied thoroughly. Oxygenation played a
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Bioremediation major role in the degradation of pesticides and herbicides as it makes them
water soluble and appropriate to disintegrate.
Phytovolatilization
The process of phytovolatilization is the uptake of pollutants by plants,

render them harmless by modify them in less toxic form and further release in
the atmosphere through stomata. It involves pollutant uptake, plant
metabolism and plant transpiration. This phytoremediation technique depends
on the transpiration pull by fast growing trees which enhance the uptake of
contaminants which then released in atmosphere through leaves.
Poplar tree volatilize trichloroethylene (TCE), eucalyptus extracts methyl t-

butyl ether (MTBE), Indian mustard change selenium to dimethyl selenide
and tobacco plant converts methyl mercury to mercury vapours. The genetic
engineering techniques have also been applied by developing plants with
better competence for phytoextraction and phytovolatilization processes.
Yellow poplar (Liriodendron tulipifera) was genetically modified by
introducing mercuric reductase gene (mer A). Thus, the plant was able to
tolerate high mercuric concentration and alter the ionic form of metals to
element form, allowing the plant to withstand polluted conditions, extract the
contaminant from the soil and volatilize it. This is advantageous as current
bioremediation technologies cause much disturbance to the contaminated site
due to extensive excavation.
Phytostabilization
Phytostabilization involves reducing the mobility of contaminant via

adsorption and precipitation by rhizosphere of green plants, preventing
dispersion of contaminated soil and thereby stabilizing the soil. This process
prevents contaminant movement by wind and air erosion, leaching and soil
dispersion. In this process, change in contaminant chemical form takes place
making them insoluble and immobilize on soil and subsequent accumulation
of contaminant in plant tissues and in the soil around the roots. This process
involves the formation of a vegetation cover on the surface of contaminated
soil or sediment, thereby reduced exposure of contaminated soil with wind,
water and direct contact with humans. Native or introduced plant varieties
which are able to tolerate high levels of contaminants are grown in such sites.
This grown vegetation becomes a barrier to erosion and human exposure to
contaminated sites. The roots of these plants exude proteins and enzymes into
the soil and precipitate and immobilize the contaminants in soil or root
surface. The pollutants are obtained by the plants and stored in the roots.
Heavy metals are unable to degrade but are made less toxic, more water
soluble or insoluble to precipitate. Some contaminants do not degrade and
concentrate into the root zone. For example, Lolium perenne stabilizes the
soil of an iron treatment plant and provide vegetative cover and no loss of
metal.
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Bioremediation for
Phytostabilization seems to be similar to phytoextraction as it uptakes,
Soil Environment
accumulate, adsorb and precipitate around the roots of the plants. Conversely,
this technique does not involve the harvesting of grown plants with
accumulated pollutants but fixed them in plant biomass or rhizosphere hence
reducing their bioavailabilty and migration from the contaminant site. Metals
do not degrade completely so fixing them in place is the best possible
bioremediation measure for low contamination sites and large polluted areas
where other measures are difficult to apply. Moreover, the polluted sites
where natural vegetation is repressed, the use of species with high tolerance
of contaminants establish a vegetation cover which further protect the
exposed site with the effects of soil leaching, wash off and wind erosion and
preventing the spread of pollutants to other areas.

1. Expalain Biosparging, Bioventing and Biostimulation/Bioaugmentation
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2. Explain various kinds of phytoremediation?
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10.5 EX-SITU BIOREMEDIATION

Ex-situ bioremediation technologies are generally used for bioremediation of
different solids, liquids and solid-liquid mixture. The various types of solid
bioremediation are landfarming, composting and bioreactors. The key
characteristic of ex-situ bioremediation is that the soil is removed from its
original site for treatment. The contaminated material can be taken to another
place for clean up or another part of same site. Contaminated soil is
excavated and treated externally in various treatment systems. It is most
suitable in small, severely contaminated site and when a hasty clean-up is
required. The major advantage is that the conditions are promptly optimized,
easy maintenance of process control, accurate and simpler monitoring of the
process. Moreover, the addition of specific microorganisms, when they are 223
Bioremediation required, is easier and safer and these approaches are faster than in-situ
processes. These techniques are important for a confined pollution of medium
to high concentration within a site. The main disadvantage of this technique
is increased transportation costs, surplus land requirements, and higher level
of engineering and increased risks of spillage leading to secondary pollution
due to these spillages.
10.5.1 Landfarming
Landfarming also called land treatment is enhanced aerobic biodegradation of
organic wastes in excavated soils or near surface by the amendment of
nutrients, lime (for pH) and moisture along with aeration improvement by
tilling and mechanical mixing. This type of biodegradation usually carried
out by the help of indigenous microorganisms however bioaugmentation
might also introduced to enhance biodegradation. In this process, the
contaminated soils are excavated from a site and transferred to a treatment
unit. Tilling of 8-12 inches of soil also takes place to increase aerobic
biodegradation of organic pollutants. This technique requires the addition of
nutrient amendments (N and P) to the soils to improve biodegradation of
pollutants. The mineral may be added into the soil as a solid or mixed with
water. Various methods such as ploughing, tilling and methods related to
mechanical mixing of the soil enhance the process of biodegradation.
The excavated soil from contaminated site initially screened for rubble,
rocks, oversize material prior to the start of remediation. Biodegradation
takes place in a lined earthwork separated from the surroundings by an
impermeable clay or high density polyethylene lining. Indigenous microbes
are being relied upon for the process of bioremediation. However, specific
bacteria and fungi might also be added if required. First layer in the
earthwork is of gravel followed by a sand layer. Drainage pipes are fitted into
the gravel layer. The whole system is isolated from the lower soil by an
impermeable clay or polymer layer (Fig. 7). Biological activity is stimulated
by addition of water and nutrients along with turning and ploughing which
aid in proper aeration. Since the process is simple, soil characteristics and
climatic conditions play a major role in efficiency of this technique. For
example in heavy clay soils, the oxygenation of soil is difficult to achieve and
uniform distribution of nutrients is quite cumbersome. In colder regions,
covering of soil is required to overcome the harsh effects of weather.
Assessment of progress of the process and compliance with standard
parameters is achieved by regular sampling and monitoring of the process.
After the remediation process, the soil can either return to original site or can
be utilized elsewhere.
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Bioremediation for
Soil Environment
Fig. 7: Landfarming
10.5.2 Composting
Composting is a controlled biological procedure that treats organic
contaminants with the help of microorganisms under thermophilic conditions
(temperature 40-50 oC). Ideal biological, physical and chemical conditions
are required for proper functioning of microorganisms through the process.
Temperature, moisture content and oxygen levels are observed and adjusted
to optimize the biodegradation. Frequent mixing maintains the oxygen
process and surface irrigation helps in maintaining proper moisture content.
When microorganisms begin to decompose the organic matter, carbon is
decomposed to it’s byproducts like carbon dioxide water and humic end
products. Microbes also need some carbon for producing new microbial cells
for increasing their population. Different kinds of microbes are successively
introduced until decomposable organic matter remains. Stabilization of
organic matter and mass reduction are the preliminary steps involved in this
process. The extent of this stage depends upon biodegradability of the
organic matter, moisture, aeration, and the preferred mass reduction. Larger
residence time leads to increased mass reduction. Mass reduction is brought
around 30-40 % and volume reduction about 50 %. Solid waste degradation
is achieved by a consortium of aerobic microbes, mainly bacteria, fungi and
actinomycetes. This consortium breaks down the complex organic matter to
simple and small molecules which might be used by microbes for its carbon
and energy needs. Compost the final product is dark brown or black in color,
water insoluble, earthy odour and loose in texture. The volume reduced to
half of its original.
10.5.3 Biopile/Soil Mounds

Biopiles are also called as soil mounds, biocells and bioheaps and utilize the
process of biodegradation to reduce concentrations of petroleum components
in excavated soils. In this process contaminated soils are heaped into piles in
a lined area to avoid leaching. Aeration, mineral, nutrients and moisture is
provided to the surface and piles are covered with polythene. This stimulates
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Bioremediation aerobic microbial activity in the soils. The increased microbial activity results
in degradation of adsorbed petroleum-product constituents through microbial
respiration. A suction system is applied to the base of the pile to improve
aeration. Leachate can be collected at the base through pipes and can be
recycled is required. This system is useful where space is limited. Bio filters
may also be added to restrict vaporemission if needed.
Both biopiles and landfarms are above-ground, engineered systems that use
oxygen, to stimulate the growth of aerobic bacteria thereby degrading the
petroleum substance adsorbed to soil. Though aeration in landfarms is done
by tilling or plowing, air is forced to move by injection or extraction through
perforated piping placed inside the pile to aerate biopiles.
10.5.4 Bioreactor
Soil removed from a polluted site can also be treated as a solid waste/slurry
in bioreactors. Excavated soil is transferred into a mixing tank and water is
added to make slurry. Nutrients are also added to augment microbial growth.
The slurry suspension is transferred to a series of aerated reactors with
efficient microorganisms to treat the contaminated soil. The treated soil slurry
is thickened in clarifiers and dewatered. The recovered liquid reintroduced
into the mixing tank to act as a wetting agent for next batch of contaminated
soil. Solid part is further dried and reused or disposed (Fig. 8).
Fig.10. 8: Bioreactor
Slurry bioreactors are used for soil, sediments, sludge and other solid and
semisolid wastes. Three phenomena are employed in slurry bioreactors
namely, suspension, aeration and mixing. Bioreactors are useful as various
parameters such as pH, temperature and oxygen level can be monitored and
controlled. Proper balance of these parameters is required for slurry reactor to
function properly. The solid part comprises of the absorbed contaminants, the
liquid part becomes the medium for microbial growth. Nutrients are added to
improve the process of biodegradation. Bioremediation of contaminants
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Bioremediation for
depends on the availability of constituents in water phase to the
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microorganisms. The design of bioreactors for solid state slurries are solid
bed, fluidized bed and stirred tank bioreactors (Fig. 9).
Xenobiotics such as 2,4 dichlorophenol, chloroanaline, chlorinated solvents,

aromatic hydrocarbons can be removed by this method, the activated sludge
is mixed with a special mixed culture.
Bioreactors are effective for soil, sediments, sludge etc as conditions for
enhancing bioremediation can be achieved easily. However, bioreactors still
needs improvement and further research is required to make them efficient
and economic.
Fig.10.9: Slurry Bioreactor

1. What are the different types of ex situ bioremediation?
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2. Describe the functioning of bioreactor?
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Bioremediation
10.6 BIOREMEDIATION OF METALS
Bioremediation of metals involves a complicated interaction of biological,
chemical and physical processes. Chemical and biological processes are
unable to degrade the metals therefore the process for metal treatment must
concentrate the metal for its recycling purpose. Bioremedial activities of
metal contaminated sites depend on actively metabolizing microbes and high
concentration of toxic heavy metals inhibits these microbes. Microbes are
present in biogeochemical matrices in soils and sediments and interact with
metals through many dynamic environmental factors. The microbial
processes for bioremediation of metal contaminants by influencing the
toxicity and transport of metals are biotransformation, biosorption,
bioaccumulation and degradation.
10.6.1 Biotransformation
Multivalent metals causing environmental pollution are reduced by metal
reducing microbes. These contaminants should be in oxidized form as
electron acceptors for enzymatic reduction to take place by these
microorganisms. The oxidized forms of heavy metals are soluble in aqueous
media while reduced form is much insoluble and precipitate from solution.
Geobacterium sulfurreducens reduces Fe (III) and U (VI) in subsurface
environment. Many bacteria reduce chromate ion to Cr (III) which under
suitable conditions precipitate as Cr (OH)3. Pseudomonasaeruginosa,
Arthrobacter, anaerobic surface reducing bacteria and algae have been
isolated from chromate contaminated sites and reduce Cr (IV). Similarly, Fe
(III) can be reduced by microorganisms to Fe (II), Mn (IV) to Mn (II) and S
(VI) (Sulphate) to S (II) (Hydrogen sulphide). The reduced forms of these
metals are insoluble and can precipitate as reduced oxide or hydroxide
minerals. Sulphate reducing bacteria produced hydrogen sulphide which
chemically reduces the contaminant in stable form for a long time.
10.6.2 Biosorption
The adsorption of variety of metals to biological material is called
biosorption. This process explains the involvement of soluble matter with cell
surface. This process does not require an active metabolism. The uptake of
metals by biological material is both active and passive. The adsorption to
cell surface or any extracellular polysacchcharides is called passive uptake.
Inactive uptake, which is a slow process, the metals are transported inside the
cells, react with metallothioneins and deposited there or stored in vacuole
(Fig. 10). Heavy metals are physically removed with microbes by their
association with biomass. Metals are bound up to 30 % of dry weight of
biological material. Metal bearing precipitates are deposited on charged
functional groups which act as nucleation sites. High concentration of metals
results in following response by microorganisms:
1. Chemical alteration of metals

228
Bioremediation for
2. Intracellular deposition of metals by specific proteins called as
Soil Environment
metallothioneins.
3. Extracellular deposition of metals on extracellular polysaccharides or
cell-wall.
4. Energy driven exit of metals from the cells.
In biosorption, low cost biomass is used, such as sewage sludge, marine

algae, and waste from fermentation industry. Marine macroalgae, both living
and dead adsorb metals and used in many cycles of adsorption and
desorption. Commercially available microalgae, AlgaSORB is immobilized,
non-living microalgae in a permeable matrix, comparable to ion exchange
resins and used to remove cadmium. Similarly, Ecklonia radiata, a marine
macroalga has been used to bind copper.
Fig. 10.10: Biosorption
10.6.3 Bioaccumulation
The retention and concentration of a metal within an organism is called
bioaccumulation. In this process, solvents are transported inside the cell
cytoplasm through cellular membrane and metals are deposited in cell
cytoplasm. The utilization of biological material to remove metals detoxify
contaminant site and help in recovery of valuable metals such as gold.

1. Explain how bioremediation of metals takes place?
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2. Explain the process of biosorption?
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Bioremediation
10.6 LET US SUM UP
Let us sum up the bioremediation process for soil environment. Industrial
pollution is contaminating soil, air and water and becoming a major problem
nowadays. Various methods such as physical, chemical, biological,
solidification and thermal are employed for soil bioremediation.
Bioremediation is a term composed of two words, bio from biological and
remediation means to remedy. It is the treatment and removal of pollutants
from environment through biological agents. The main approaches of
bioremediation are utilization of indigenous microorganisms, amplification of
indigenous microorganism’s growth, bioaugmentation, the use of adapted
microorganisms, Inclusion of genetically modified organisms and
phytoremediation. The major types of bioremediation are intrinsic
bioremediation, enhanced bioremediation and bioaugmentation. There are
various methods for bioremediation depending on the contaminated area, the
properties of compounds, the concentration of contaminants and the time
required for completion of the process of bioremediation.
The onsite treatment of contaminated soil is called in-situ bioremediation and
excavation of soil and off site treatment is known as ex-situ bioremediation.
Different in situ techniques are biosparging, bioventing, biostimulation and
phytoremediation. Biosparging is to enhance the bioactivity of soil by
supplying increased amount of oxygen through sparging air or oxygen into
the soil. Bioventing also involves the process of superaeration to stimulate
the enhanced degradation of contaminated material and is used to remove
volatile soil contaminants present above the water table in unsaturated zone.
In biostimulation a combination of appropriate nutrients and specific
microorganisms are added to remediate deep contaminated soil. This process
is useful for those contaminants not degraded by indigenous microbes.
Bioslurping process used for remediation of hydrocarbon-contaminated sites
is a combination of two processes, bioventing and vacuum-enhanced free-
product recovery from groundwater and soil. The bioventing process
stimulates the aerobic bioremediation and under aerobic conditions, most of
the aliphatic and aromatic components of petroleum hydrocarbons are
degradable.
The utilization of plants for degradation of contaminant and metals from soil
by exploiting plant’s natural capacity to accumulate, absorb and break down
pollutants making them harmless is called phytoremediation. This process is
low cost, top soil is not disturbed, applicable in case of low level of
contamination, recovery of metals takes place. But it takes more time than
other methods as many growing seasons are required. It can be useful in
remediation of metals, pesticides, crude oils, solvents, explosives and various
other contaminants. Phytoremediation is divided into phytoextraction
(phytoaccumulation), phytodegradation, phytovolatilization and
phytostabilization.
In ex-situ bioremediation the soil is removed from the site of contamiantion

for treatment. It includes landfarming, composting and bioreactors.
Landfarming is enhanced aerobic biodegradation of organic wastes in
230
excavated soils by the amendment of nutrients, lime (for pH) and moisture Bioremediation for
Soil Environment
along with aeration improvement by tilling and mechanical mixing.
Composting is a controlled biological procedure that treats organic
contaminants with the help of microorganisms under thermophilic conditions
(temperature 40-50 oC). Ideal biological, physical and chemical conditions
are required for proper functioning of microorganisms through the process.
Biopiles are the heaps of contaminated soils in a lined area to avoid leaching
to reduce petroleum components in excavated soils by the process of
biodegradation.
Contaminated soil can also be treated as a solid waste/slurry in bioreactors. It

contains a mixing tank with water to make slurry and nutrients are added to
increase microbial growth. The slurry suspension is transferred to a series of
aerated reactors with efficient microorganisms to treat the contaminated soil.
They are effective for soil, sediments, sludge etc as conditions for enhancing
bioremediation can be achieved easily.
Bioremediation of metal contaminated sites depend on actively metabolizing
microbes present in biogeochemical matrices in soils and sediments which
interact with metals through dynamic environmental factors. The various
microbial processes for bioremediation of metal contaminants are
biotransformation, biosorption, bioaccumulation and degradation.
The adsorption of variety of metals to biological material is called

biosorption. This process does not require an active metabolism. The uptake
of metals by biological material is both active and passive. Heavy metals are
physically removed with microbes by their association with biomass. Low
cost biomass such as sewage sludge, marine algae, and waste from
fermentation industry is used. The retention and concentration of a metal
within an organism is called bioaccumulation
10.7 KEY WORDS

In-situ bioremediation :The onsite treatment of contaminated soil is called in-
situ bioremediation Ex-situ bioremediation: Excavation of soil and off site
treatment is known as ex-situ bioremediation
10.8 SUGGESTED FURTHER READINGS/

REFERENCES
• Ali H, Khan E, Sajad MA. Phytoremediation of heavy metals—concepts
and applications. Chemosphere. 2013, 91: 869–881.
• Atlas, Ronald M., and Jim Philip, eds. Bioremediation. Washington, DC,
USA: ASM Press, 2005.
• Azubuike CC, Chikere CB, Okpokwasili GC. Bioremediation techniques
classification based on site of application: principles, advantages, limitations
and prospects. World J Microbiol Biotechnol. 2016, 32 (11): 180.
• Cummings, Stephen P., ed. Bioremediation. Totowa, NJ: Humana Press,
2010.
231
Bioremediation • Dick, Warren A. Bioremediation. Soil Science Society of America
Journal 70, 2006: 307.
• Emmi Lalaroo, H., Savita Devi, and Anania Arjuna. Bioremediation.
Biosciences, Biotechnology Research Asia 14, 2017: 367–72.
• H, Baker Katherine, and Herson Diane S, eds. Bioremediation. New
York: McGraw-Hill, 1994.
• J, Valdes James, ed. Bioremediation. Dordrecht: Kluwer Academic
Publishers, 2000.
• Mahesh A, Shreedharan PC, Ambika SR. Bioremediation for
Environmental Management. International Journal of Environmental
Science and Development, 2015, 555–558.
• Maulin P, Shah. Soil Bioremediation. International Journal of
Biotechnology and Bioengineering 3, 2017: 08–09.
• Prasad R, Aranda, E. Approaches in Bioremediation: The New Era of
Environmental Microbiology and Nanobiotechnology. Nanotechnology
in the Life Sciences. 2018. 403 p. Springer.
• Sharma, Sanjay K., ed. Bioremediation. Boca Raton, Florida:CRC Press,
2019.: CRC Press, 2019.
• Singh S N, Tripathi RD. Environmental Bioremediation Technologies.
Springer Science & Business Media, 2007, 520 p.
• Skipper, H. D., and R. F. Turco, eds. Bioremediation. Madison, WI,
USA: Soil Science Society of America, American Society of Agronomy,
and Crop Science Society of America, 1995.
• Wang LK, Ivanov V, Tay JH Environmental Biotechnology, Handbook
of Environmental Engineering, Humana Totowa, NJ 1, p 975.
10.9 ANSWERS TO CECK YOUR PROGRESS

EXERCISE
1. Refer Section Number 10.4.1, 10.4.1and 10.4.3
2. Refer section Number 10.4.5
1. Refer Section Number 10.5.
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UNIT 11 BIOREMEDIATION OF THE AIR the Air Environment
ENVIRONMENT
Structure
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.1 Introduction
11.2 Objectives
11.3 Bioremediation
11.3.1 Factors for remediation
11.3.2 Bioremediation strategies
11.4 Bioremediation for Air Pollutants

11.4.1 Bioreactors
11.4.2 Components of Bioreactor unit
11.5 Biofilters
11.5.1 Type of biofilter
11.5.2 Biofilter process description and mechanism
11.5.2.1 Process description
11.5.2.2 Mechanism Involved in Biofilter
11.5.3 Factors affecting Biofilter Operating Conditions
11.5.4 Advantages and Disadvantages
11.6 Biotrickling Filter

11.6.1 Operating Conditions
11.6.2 Principle Involved in Biotrickling Filter
11.7 Bioscrubber
11.7.1 Process Involved in Bioscrubber
11.7.2 Advantages and disadvantages of Bioscrubber
11.8 Let Us Sum Up

233
Bioremediation 11.9 Key Words
11.10 Suggested Further reading/References
11.11 Answer to Check Your Progress Exercise
11.1 INTRODUCTION
Off gases released from the industrial process have been identified as a
significant contributor of toxic pollutants in the air that causes the risk to life
forms and thus requires the implementation of management practices for
mitigating air pollution. Bioremediation has been recommended to be the best
practice for control of air pollution caused by pollutants released from
industrial units. The characteristic features of the bioremediation process
provide many positive attributes for the biological treatment of contaminants.
The biological removal of contaminated off-gas has been achieved by the use
of different devices like biofilters, biotrickling filters, bioscrubbers. In this
chapter, information has been provided on the process of bioremediation and
its types, general approaches to the bioreactors used in waste gas stream
treatment, and different biosystems such as biofilters, biotrickling filters, and
bioscrubbers. Their general presentation, operating conditions, and
advantages and disadvantages are discussed. The potential of bioreactors for
the treatment of air pollutants is further elaborated.
11.2 OBJECTIVES
After studying this chapter, you should be able to:
• Define the concept of bioremediation

• Define the types of bioremediation process for the decontamination of
contaminated sites
• Define the concept of bioreactors
• Identify different types of bioreactors used in the treatment of
contaminated off-gas
• Explain the concept of working of biofilter, biotrickling filter, and
bioscrubber
11.3 BIOREMEDIATION
The term bioremediation has been made up of two parts ‘bios’ which refers to
living organisms and ‘remediate’ means to solve a problem. So, the process
involving the use of living organisms particularly the microorganisms to
solve the problem of environmental dilapidation is known as bioremediation
and the microorganisms used for removing environmental contaminants are
called bioremediators (Sharma, 2012). It is a cleanup technology that
degrades or transforms various hazardous chemicals to less harmful or
nontoxic compounds using naturally occurring microorganisms and also
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Bioremediation of
helps to restore the contaminated sites and preventing further pollution. It is the Air Environment
considered to be the new eco-friendly and cost-effective technology for
decontamination of the polluted environment than other technologies used for
cleanup of hazardous wastes (Sharma, 2012).
Bioremediation technology exploits the abilities of microorganisms and

plants to metabolize and sequester contaminants from soil, groundwater, and
sediments. Bioremediation is based on the fact that organisms can use
organic pollutants as food. The enzymatic reactions yield both energy and
molecules useful for cell growth. Biological processes can be enhanced by
creating environmental conditions favorable for the growth of
microorganisms which in turn enhance the biodegradation process occurring
in nature. Several cleanup technologies have been in use in the past involving
dispersal, collection, removal, landfill, solidification, thermal desorption, and
incineration. But these techniques only dilute the contaminant or transfer
them into another environmental compartment. However, bioremediation is a
safer and cost-effective technique than other solutions. In the bioremediation
process, there is cradle to grave liabilities for the contaminants because the
contaminants have been turned into harmless by-products, carbon dioxide,
water, and biomass.
Bioremediation for the contaminated site works in one of two ways. In one
case, microorganisms eat and digest organic substances as the source of food
for nutrients and energy. When the microorganisms completely digest
chemicals, then by enzyme activity the digested chemicals are changed into
water and harmless gases, with end product like cell material, water, and
carbon dioxide. In other cases, the microorganisms degrade hazardous
substances into inert products. Sometimes, specialized microbes are added to
degrade the contaminants.
Bioremediation strategies are often more beneficial than traditional strategies

because it can be implemented in-situ (directly at the site of the contaminant
with no need to transport the contaminated material) or ex-situ (contaminant
removed from the original sites and then the degradation processes are
carried out).
11.3.1 Factors for Bioremediation

The bioremediation process is a complex system of many factors. These
factors include the presence of pollutants degrading microbial population;
availability of contaminants to the microbial population; environmental
factors (the type of soil, temperature, pH and the presence of oxygen or other
electron acceptors, and nutrients), and absence of bacterial growth-inhibiting
substances.
11.3.2 Bioremediation Strategies

Bioremediation treatment strategies have been broadly divided into two
categories based on the place where waste is to be treated. Various types of
235
Bioremediation bioremediation strategies are shown in Figure 1.
In-Situ Bioremediation
In-situ bioremediation is the application of microorganisms to the site of

contamination i.e. within the affected environment or action of
microorganisms that are naturally present at the contaminated sites to carry
out remediation. This microbial population consists of a variety of
microorganisms which invariably have minimum oxygen and nutrient
requirements. When the system is deficient in oxygen, the oxygen requiring
bacteria do not thrive and they give way to the anaerobic bacteria. It is the
superior method for cleaning up of contaminated environment as it saves
transportation costs, uses harmless microorganisms to eliminate the chemical
contamination, and also causes no disruption to the contaminated site.
In-situ bioremediation can be intrinsic or engineered bioremediation. An

intrinsic system only monitors remediation progress. It is applied when
bioremediation takes place naturally, and risk-based assessment indicates that
there is no urgent need for site cleanup. Engineered systems are designed to
enhance bioremediation. Some of these systems involve bioventing,
bioaugmentation, biosparging, biostimulation, natural attenuation.
Intrinsic Bioremediation/ Natural Attenuation
It is the simplest form of bioremediation where no intervention is needed at

all and simply allows the natural process to reduce the contaminant in an
environment and enhance the metabolic activity of indigenous or naturally
existing microfauna by improving nutritional and ventilation conditions.
Engineered In-Situ Bioremediation
This process is used to enhance the natural process of bioremediation by

alternating and manipulating physical/chemical factors of the environment.
This type of remediation is performed through the introduction of certain
microorganisms to a contaminated site. Some of the engineered systems
based on the in-situ concept are discussed below.
i) Biosparging and Bioventing
Biosparging involves air injection under pressure below the water table
to increase groundwater oxygen concentrations and enhance the rate of
biological degradation of contaminants by naturally occurring bacteria.
Biosparging increases the mixing in the saturated zone and thereby
increases the contact between soil and groundwater (Sharma, 2012).
Bioventing works on the principle of air and nutrient supply to
contaminated soil through constructed wells, to enhance the activity of
indigenous bacteria. Additional oxygen may also be supplied through
direct injection into residual contamination in soil. Bioventing uses
simple, inexpensive, low maintenance equipment that can be left
unattended for long periods (Lees and Senior, 1995).
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Bioremediation of
ii) Biostimulation the Air Environment
It involves the stimulation of degrading properties of indigenous

microorganisms by the addition of nutrients and electron acceptors like
phosphorous, nitrogen, oxygen, and carbon. This technology necessitates
the presence of well-distributed indigenous organisms that eliminates
complex contaminants from polluted sites by maintaining a proper
balance between desirable and undesirable addition of limiting nutrients
into the soil. In general, biostimulation depends primarily on the
modification of the environment to accelerate the decontamination rate
(Goswami et al., 2018).
iii) Bioaugmentation
In bioaugmentation, there is the addition of indigenous or exogenous,

autochthonous or allochthonous, or wild or genetically modified
microorganisms to the contaminated site to increase biodegradation.
Bioaugmentation ensures that the proper team of microorganisms is
present in the soil in sufficient type, and number to effectively and
efficiently attack the waste constituents and break them down into their
most basic compounds (Adams et al., 2015).
Ex-Situ Bioremediation
This process requires the excavation of contaminated soil or water to
facilitate microbial degradation. Ex-situ bioremediation is classified as:
i) Land farming
Contaminated soil is excavated, mixed with soil amendments (nutrients,
soil bulking agents), and spread over a prepared bed (that can be a
biotreatment cell or upper soil zone). Contaminated soils are then mixed
with uncontaminated soils to increase the volume of contaminated
material and dilute the contaminants (this is, however, a drawback and
must not be done). This contaminant media is then periodically tilled for
aeration until pollutants are degraded. Contaminants are degraded,
transformed, and immobilized by oxidation and microbial action.
Degradation rate can be optimized by controlling soil conditions (like
nutrients, pH, moisture, aeration) (Rubinos et al., 2007).
ii) Composting
Composting is an aerobic process by which organic wastes are degraded
by microorganisms, into humus-like material called compost, typically at
elevated temperatures and optimum moisture content, and periodic
turning to ensure good mixing and aerobic conditions. Remediation
through composting is done by arranging contaminated material in
windrows (long rows of compostable material), aerated static-piles or in-
vessel systems, mixing contaminated soils with high-energy feedstocks,
or finished/mature compost and then aerating/tilling timely. Mature
compost is added to accelerate the microbial degradation of organic 237
Bioremediation contaminants in soil and improve plant growth and establishment in toxic
soils. Typical compost temperatures are in the range of 55° to 65° C and
moisture level in the range of 40-60%. The increased temperatures result
from heat produced by microorganisms during the degradation of the
organic material in the waste (EPA, 1998; Ro et al., 1998).
iii) Biopiles
It is a hybrid of land farming and composting. Excavated soils from the
contaminated area are collected in the treatment area, mixed with soil
amendments, and further treated using forced aeration. It is typically
used for the treatment of surfaces contaminated with petroleum
hydrocarbons, with CO2 and H2O produced as end products. A biopile
consists of a treatment bed, aeration system, and irrigation/nutrient unit
(buried under soil, made available through the vacuum or positive
pressure), and a leachate collection unit. Essentially, engineered cells are
constructed as aerated compost piles (Das and Dash, 2014).
iv) Bioreactors
The bioreactor involves the processing of contaminated solid material
(soil, sediment, sludge) or water through an engineered containment
system.
11.4 BIOREMEDIATION FOR AIR POLLUTANTS

Proliferation in urban infrastructure and increase in population causes
immense pressure on the environment and leads to degrading the quality of
air. The air contaminated with vapors, fluid, or other pollutants present for an
adequate time under specific conditions interferes with human solace,
wellbeing, or welfare and causes harm to living beings of that area (Kolekar
et al., 2019). The flue gases emitted from different sources release various
volatile organic and inorganic compounds. So, there are strong arguments for
the development and use of new and original processes to control waste gas
emission from industrial, agricultural, and domestic sources while concerning
human health and welfare (the population in general and plant operators) and
environmental protection (domestic and wild animals, plants, paintwork and
damage to the building). Many international treaties for environmental
protection have been transcribed and applied in many countries. From these
treaties, local legislation has been written, particularly for solid waste
management, water, and wastewater treatment, and air quality improvement.
The air pollution control regulations reflect the concern of the government for
the protection of people and the environment (Cloirec et al., 2014).
Traditionally, pollutants were treated by non-biological methods such as
condensation, activated carbon adsorption, cooling, and compression of
contaminant from the air. The non-biological methods to remove pollutants
are feasible for compounds having a high boiling point and concentrated
vapors (Singh and Ward, 2004).
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Bioremediation of
In the adsorption process, pollutants are adsorbed onto the adsorbents such as the Air Environment
activated carbon, etc. (Shareefdeen et al., 2005). Once the adsorption bed is
saturated, the regeneration of the adsorbents can be done using steam or hot
air. Often, the recovery of compounds is costly and thus spent adsorbent
becomes solid waste, which needs to be landfilled or incinerated. Moreover,
the adsorption process is effective when the concentration in the air stream is
low.
In absorption/scrubbing processes, pollutants from the air are absorbed into

scrubbing solution such as water or solvents. By changing the pH of the
scrubbing solution, the mass transfer rate of contaminants from the air to the
water phase can be adjusted. Often chemical costs are high and the generated
liquid wastes need treatment. Frequent nozzle maintenance, complex feed
systems, and high operating costs are problems associated with the
absorption method (Shareefdeen et al., 2005).
Incineration technology although proved to be widely used due to its high

efficiency in the destruction of contaminant, but it is an expensive process
due to high energy and fuel requirements. The incineration process is not
economical if the contaminant concentration levels are low and the large
airflow volume needs treatment. This process also produces the highest
amount of CO2 and NOx, which contribute to greenhouse gases (Singh and
Ward, 2004: Shareefdeen et al., 2005).
Although these technologies aim at achieving effective control of gaseous

pollutants, in many cases, these control techniques yield by-products that
require further treatment before disposal. In the case of treatment of dilute
waste gas streams, however, these traditional methods are relatively less
effective and more expensive and necessitate the identification of alternative
control measures.
A suitable alternate air pollution control technology is the biological

treatment of waste gas. There is no end product release at end of remediation,
thus there is no need for further treatment and hence, appears to be a very
competitive and green technology to treat the waste gas stream before its
discharge into the atmosphere (Cloirec et al., 2014). They are believed to be
more valuable for the decontamination of released gas streams from
industrial processes and gaining more acceptance over other conventional
techniques (Kraakman et al., 2011). The biological gas treatment methods
include bioreactors which are discussed below.

1. Explain bioremediation of air pollutant takes place?
……………………………………………………………………………
…………………………………………………………………………… 239
Bioremediation ……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
11.4.1 Bioreactors
Biological waste gas purification technology currently includes bioreactors
known as biofilters, biotrickling filters, and bioscrubbers. The operation
modes of all these reactors are similar. Bioreactors have been used for
hundreds of years to treat sewage and other odoriferous water-borne waste.
About sixty years ago, Europeans began using bioreactors to treat
contaminated air (odors), particularly emissions from sewage treatment plants
using bioreactors to control air pollution. Air containing volatile compounds
is passed through the bioreactors, where these compounds are transferred
from the gas phase into the liquid phase. Microorganisms responsible for
biodegradation usually grow as a mixture of organisms. Such a mixture of
different bacteria, fungi, and protozoa depends on the number of interactions
and is referred to as microbial/biological community. The pollutants in the air
usually act as a source of carbon and energy for the growth and maintenance
of microorganisms. Microorganisms also require essential nutrients to
function and produce new cells. These nutrients include nitrogen,
phosphorous, sulfur, vitamin, and trace elements. Many times, these nutrients
are not present in the waste gases and have to be supplied externally. The
biomass mainly composed of aerobic species and microorganisms utilize
oxygen level provided by the air to be treated. Water is required to maintain
biological activity. The substrate equilibrium is necessary to stimulate the
activity of biomass and thus nitrogen compounds, phosphate, and trace
elements are often added through incorporation or solution into the
biosystem. The degradation of contaminants gives mostly CO2 and H2O
(Cloirec et al., 2014).
A general approach of biological treatment is to transform the contaminants

present in the gaseous phase, use them as a substrate for microorganisms
(bacteria, fungi, and yeast), and convert them into non-toxic compounds. A
very simplified reaction pathway given by Cloirec et al. (2014), expressing
the principle of degradation process in presence ofenzymes (E) of aerobic
bacteria (X) is given below.
Contaminant (substrate) E, X CO2 + H2O +X metabolites internal +

CO2,N, P, trace elements H2O and energy
As the degradation of compounds is carried out by microorganisms present in

the aqueous phase or saturated as a biofilm in water, the water solubility of
pollutants appears to be a major factor in choosing the technology. Biomass
is attached to the packing material of the bioreactor or in the form of
activated sludge. The material used for bed media includes peat, composted
yard waste, bark, coarse soil, gravel, or plastic shape. Water moistens the
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Bioremediation of
media in the biofilter and is recycled for two other processes (biotrickling the Air Environment
filter and bioscrubbers). Pollutants before degraded by the microorganisms
are required to be absorbed into the water or an organic solution (Cloirec et
al., 2014).
11.4.2 Components of Bioreactor Unit

There are various components of bioreactors like support rack, fan, etc. The
contaminated air is passed to a plenum as the emission flows through the bed
media, the pollutant absorbed in the biofilm and come in contact with the
microorganisms. The microorganisms then reduce the pollutant concentration
by consuming and metabolizing the pollutant. During the process of
digestion, the microbial enzymes convert the organic compounds into energy,
CO2, and water. Materials that remain undigested are leftover and become
residue. The main components of the bioreactor are as follows.
i) Fan: Fan is used to collect the contaminated air from buildings or

processes, and direct it to the plenum. As the off-gases pass through the
bed media, the pollutant is absorbed by the moisture on the bed media.
Microorganisms reduce pollutant concentration by consuming and
metabolizing pollutants. If the contaminated air is too hot, too cold, too
dry, or too dirty, then the contaminated air is pretreated to obtain
optimum conditions before introducing into the bioreactor (EPA, 2003).
ii) Support rack: The support rack used for the reactor is perforated to
allow the air from the plenum to move into bed media and make contact
with the microbes present in the bed media. Perforation also helps to
drain out excess, condensed mixture out of the bed to the plenum (EPA,
2003).
iii) Bed media: Media is mainly the microbial community, mainly

composed of aerobic species. Despite that, some anaerobic
microorganisms are also suspected to be present in potentially anoxic
zones caused by a non-ideal gas or liquid flow through the reactor
(Cloirec et al., 2014). The reactor used for the decontamination is not
under sterile conditions and can be naturally colonized by a large number
of bacteria. A consortium of microorganisms generally grows in the
biological reactor depending on the nature of the available biodegradable
substrate (Cloirec et al., 2014). These microorganisms form the biofilm
on the surface of the bioreactor. Figure 11.2shows the schematic diagram
of a bioreactor unit (EPA, 2003).
While studying the process of the bioreactor, an important question is how to

put microorganisms in contact with water and substrates present in the gas
phase. Taking into account the mobility of an aqueous phase and the
microorganisms, three processes generated are biofilter, biotrickling filter,
and bioscrubber as shown in Table 1. Bacteria are an integral part of the
bioreactor. Bacteria remove pollutants from the polluted gas stream. The
most common bacteria that are used to enhance the degradation efficiency of 241
Bioremediation bioreactors are shown in Table 11.2.
11.5 BIOFILTERS
Biofiltration is an air pollution control technology that uses microorganisms
to transform oxidizable vapors and gases into harmless end products (Janni et
al., 2001). Air purification by biofilter is an emerging new technology
applied for waste gas purification to control volatile organic compounds
(VOCs), inorganic volatile organic compounds (IVOCs) from odor
generating processes, wastewater treatment plant and it is particularly
suitable for the treatment of high air-flow rates at low concentration of a large
number of pollutants (Soccol et al., 2003; Cloirec et al., 2014;). It is a
cleanup technology as the byproducts of microbial oxidation are primarily
water, carbon dioxide, mineral salts, and microbial biomass (Janni et al.,
2001) and thereby, one of the leading technologies for controlling VOC
emissions (Zarook et al., 1997; Zarook et al., 1998; Spigno et al., 2003).
11.5.1 Types of Biofilter

Depending upon the layout, shape, and support media used, biofilters are
divided further into various types (Showqi et al., 2016).
A) Based on layout
Open bed: Uncovered and exposed to all weather conditions (such as
rain, snow, and temperature extremes).
Closed bed: Enclosed with a small exhaust port for venting of cleaned
air.
B) Based on shape
Horizontal: The horizontal biofilters have large footprints and they are
relatively less expensive.
Vertical: The vertical biofilters are designed to reduce the footprint as
required and also use less surface area compared to the horizontal
biofilter.
C) Based on support media
Compost biofilter: In compost biofilter, soil, peat, and compost
materials are used as support media.
Synthetic biofilter: In synthetic biofilter, ceramic and plastic are used.
11.5.2 Biofilter Process Description and Mechanism

This section covers the biofilter process description and Mechanism.
Process Description
The conventional biofilter is probably the oldest bioreactor used for waste gas
treatment. The conventional biofilter is a fixed-film or packed bed bioreactor
in which a natural filter bed is most often used.Biofilter uses microorganisms
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Bioremediation of
fixed on the filter media to break down pollutants present in the waste air the Air Environment
stream. The microorganisms grow in a biofilm on the surface of the medium
or are suspended in the water phase surrounding medium particles. The
medium used for biofilter consists of inert substances (like peat, compost)
which ensure a large surface attachment area and additional nutrient supply to
microorganisms. The contaminated gas is vented from the emitting source
into the filter, after getting sufficient residence time to the contaminated off-
gas, the gas will diffuse into the wet biological active layer (biofilm) which
surrounds the filter particles. In the biofilm layer, the target pollutants
undergo biological degradation. As the air passes through the bed media, the
contaminants in air streams are sorbed into the biofilm or onto the filter
medium, where pollutants undergo degradation and the end products of
biological treatment of flue gases (CO2, H2O, and microbial waste) are
released (Leson and Winer, 1991).
If the waste gas contains a high level of solid particles, a filter unit must be
installed before the humidification chamber so that the biofilters are
prevented from clogging by the particles. The contaminated air must be
saturated with water, before moving into the filter bed, otherwise, air without
moisture will result in the drying of filter material that can cause the death of
microorganisms and total loss of control efficiency (Leson and Winer, 1991).
Humidification has many benefits since it reduces the temperature by energy
consumption through water evaporation, helps in enhancing the development
of mesophilic microorganisms at the surface of packing material. It also helps
in removing the particle (dust, fat vesicles), reducing clogging of the reactor
and the packing material (Cloirec et al., 2014).
The biofilter is not a filter unit if strictly defined, the whole pollutant removal
process is completed by the combination of various processes of adsorption,
absorption, degradation, and desorption of gas-phase contaminant. The
overall efficiency of biofilter largely depends on the properties and
characteristics of the support medium, ability of microbial population for
remediation, water retention capabilities, gas residence time, medium pH (6-
8), temperature (15-30oC), and nutrient content (Devinny et al., 1998). The
filter medium must be such that it can hold dust-laden gas and resistant to
chemicals present in waste gas.
Biofilters are ideal for treating waste gas having a low concentration of
contaminants and high gas volume, a situation that vexes traditional treatment
methods (U. S. Environmental Protection Agency, 2003). Biofilters have a
large scale of application in industries like in oil and grease industry,
synthetic resins, paint and ink industries, in the pharmaceutical industry,
waste and wastewater treatment and also sewage industries (Kumar et al.,
2013; Kolekar et al., 2019).The use of biofilters to remove contaminants
from waste gas is advantageous because it does not require a large amount of
energy during operation and also produces a relatively low toxicity waste gas
stream after treatment (Soccol et al., 2003).
243
Bioremediation Mechanism Involved in Biofilter
The pollutant removal in a biofilter involves several successive steps

(Ardjmand et al., 2005; Cloirec et al., 2014) that are briefly schematized as
under and diagrammatic representation is shown in Figure 3.
Firstly, the pollutants transfer from the gaseous phase to the gas-liquid
interface
↓
Diffusion of pollutants occur within the liquid phase and the biofilm
↓
In the biofilm, pollutants undergo biodegradation by the microbial
population, and the pollutants are used as energy and carbon sources by the
microorganisms
↓
Diffusion of metabolites produced into the liquid phase and then to the gas
phase
Both diffusion of pollutants, as well as degradation of contaminants, is a

complex phenomenon. The sorption process of the contaminant at the
material surface is frequently observed at the startup of biofilter, namely
when biofilm formation is still weak. During this acclimation phase of the
microorganisms, the packing material act as a conventional adsorbent, and a
significant decrease in pollution load can be observed (Cloirec et al., 2014).
11.5.3 Factors Affecting Biofilter Operating Conditions and

Performance
The biofilter performance not only depends on its design but also the
operating conditions such as watering, pH, and especially waste gas velocity
which is low due to slow degradation kinetics (Cloirec et al., 2014).
i) Biofilter design: Depth of filter is in the range of 0.5 to 2.5 with a usual
value of 1m for organic packing materials, providing sufficient residence
time to treat pollutants while minimizing footprint and pressure drop and
energy consumption as well as favoring the formation of preferential
paths. These can induce a rise in local flow rate, leading to a lower
residence time and consequently lower treatment efficiency (Cloirec et
al., 2014).
ii) Packing material: The filter beds used in conventional biofilter were
originally composed of natural material i.e. soil, peat, or compost that
contain naturally occurring nutrients and microorganisms (Kannes et al.,
2009; Cloirec et al., 2014). The natural microbial community may not be
present in optimal concentrations, so the filter bed may get depleted of
essential nutrients due to gradual consumption by microorganisms
(Kannes et al., 2009). The success rate of the biofiltration process
depends on the media that provide all necessary environmental
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Bioremediation of
conditions to the microbial population of the media to maintain a high the Air Environment
biodegradation rate (Showqi et al, 2016). Several authors have
highlighted the following characteristics as suitable for biofilter material
(Elias et al., 2002; Shareefdeen et al., 2005).
- Packing material should have large porosity and high void for
helping the microbial growth
- A large surface area to improve the transfer of pollutants, oxygen,
and nutrients
- Low investment costs and a long life
iii) Biocatalyst/microorganisms: For the degradation of pollutants present

in the waste gas stream, several microorganisms including bacteria,
actinomycetes, and fungi are helpful. The filter units that are compost-
based typically show significantly higher population densities of these
microorganisms than soil and peat. The growth and metabolic activity of
microorganisms depend on the presence of dissolved oxygen (DO) in the
biofilm, nutrient availability, moisture presence, suitable range of pH and
temperature, and the absence of compounds that are toxic to the
microbial population (Leson and Winner, 2012).
Biofilter works primarily on heterotrophic microorganisms that use

organic off-gas constituents as carbon and energy sources. As a result,
microorganisms on the introduction of these pollutants shift the
distribution of the existing microbial population towards a strain that
metabolizes the target pollutant. Acclimatization will typically take about
ten days for easily biodegradable organic compounds. Inoculation with
an appropriate culture can reduce acclimation time for the compounds
that are less likely to be degraded by the microorganisms that are initially
present in the filter bed. If the compounds that are not likely to be
degraded by the initially present microorganisms are treated with
inoculation of appropriate culture (Hartenstein, 1987; Bardtke, 1990).
iv) Environmental conditions: To achieve and maintain a high degradation

rate, it is necessary to have the optimal environmental condition in the
filter bed, the environmental conditions that are necessary to be
maintained are discussed below.
a) pH: Most microorganisms prefer a specific pH range, changes in the pH

of filter material will strongly affect their activity. In some cases, the
biodegradation of air pollutants can generate acidic by-products like
oxidation of sulfur- or nitrogen-containing compounds and chlorinated
organics. The drop in pH can destroy the resident population and reduce
the filter’s degradation capacity. To reduce the effect of pH, sometimes
chemical buffers such as lime are added (Leson and Winer, 2012).
Optimal pH for biofilter operation is in the range of 7-8 to quicken the
absorption process, maximizes the microbial action, and hence enhances
the odor removal efficiency (Swanson and Loehr, 1997).
245
Bioremediation b) Nutrients and oxygen level: Microorganisms that are present on the
filter bed are aerobic in nature and hence require oxygen for their
metabolism. In the case of gases heavily loaded with organic
compounds, biofiltration efficiency can be negatively hampered by the
amount of oxygen in the gas to be treated (Cloirec et.al, 2014). There
must be a proper oxygen level for the efficient working of biofilter since
improper levels of oxygen affect the performance of biofilter (Showqi et
al., 2015). Besides oxygen, microorganisms also need nutrient supply for
better performance of the filter. Some researchers propose that the
metabolic activity of microorganisms is stimulated in the presence of key
elements in certain proportions, i.e. in a C: N:P ratio of 100:15:3 or
100:5:1. In nutrient deficiency, the metabolic activity of microorganisms
is reduced. While a high dose, from a continuous supply, induces
uncontrolled development of the biofilm and causes a clogging problem.
c) Temperature: Various species of microorganisms tolerate a wide range

of temperature, but efficient biofiltration processes have been achieved at
a temperature in the mesophilic range of 20-45oC and 35-37oC, often
noted as the optimum temperature (Swanson and Loehr, 1997). Lower
temperatures can result in freezing of the filter media and also reduce the
activity of microbial degradation (Showqi et al., 2015).
11.5.4 Advantages and Disadvantages of Biofilters

Advantages: Biofilters have several advantages associated with them like
they have lower capital costs, produce simpler end products like CO2, and
water, eco-friendly in nature, etc. (Soccol et al., 2003). Some of the
advantages are given below.
• Lower capital cost

• Lower operational cost
• Lower chemical usage
• No combustion source
• Biofiltration units can be designed to fit in shape and size to the
industrial unit set, optimizing space
• The system is versatile to treat odors, toxic compounds and VOCs
efficiency >90% for low contaminant concentration (<1000 ppm)
• Possibility of different media, microorganisms, and operational
conditions for many emission points
• Biofiltration produces simpler by-products such as waste biomass that
can be easily disposed off when compared to other methods such as
thermal processes that produce different types of oxides. Also, chemical
oxidation processes produce chlorine and chlorinated products which
have high negative impacts.
• Biofilter usually treat large volumes of low concentration VOCs and
other odorants (Showqi et al., 2016).
Disadvantages: Besides showing several advantages biofilters also have some

246
drawbacks such as large areas are required for large biofilters, sources of Bioremediation of
the Air Environment
emission show a detrimental effect on the biofilter performance and also to
the microbial population (Soccol et al., 2003: Kolekar et al., 2019). The main
disadvantages of biofilters are presented below.
• The system is not fitted for compounds, which have low adsorption and
degradation rates, mainly chlorinated VOCs.
• Large footprint requirement.
• The source of emission that varies severely or produces spikes, can be
detrimental to the biofilter performance and the microbial population.
• Biofilters require a long period of acclimation for microbial population,
weeks or even months, mainly for VOCs treatment.
• It is necessary to maintain proper humidity and temperature control in
biofilters. Also, the airstreams with large concentrations of VOCs
decrease the efficiency of biofilters (Showqi et al., 2016).
11.6 BIOTRICKLING FILTERS

Biofiltration employing biotrickling filters is currently considered to be the
most promising technology of biological air treatment (Zamir et al., 2011;
Kumar et al., 2017). This is because pH and other conditions (like
temperature, process stability, and continuous nutrient supply) can be
controlled very well, and also there is reduced space requirement comparing
to traditional biofilters which make them more popular (Rybarczyk et al.,
2019). The most important parameter for the better functioning of the
biotrickling filter is the pH of trickling liquid. It is because microorganisms
work properly with the optimal value of pH ensuring the efficient activity of
microorganisms. Usually, the optimal pH is close to neutral i.e. 7, which
depends on the pollutant to be treated (Rybarczyk et al., 2019). Another
important element of the biotrickling filter is the biofilm formed on the
packed bed. The biofilm layer contains various kinds of microbes including
bacteria, fungi, yeasts, and molds. The growth and formation of biofilm
depend on various factors including pH, temperature, oxygen concentration,
and the presence of nutrients. The thickness of biofilm varies from several
micrometers up to several millimeters depending on the packing material and
operating conditions (Rybarczyk et al., 2019).
In a biotrickling filter, the gas is passed through a packed bed irrigated with
the trickling liquid which contains essential nutrients allowing the growth of
microorganisms in the biofilm. The mechanism of the process consists in
simultaneously occurring processes of absorption of the target compounds in
the liquid surrounding the biofilm and their absorption onto the biofilm
covered particles followed by the subsequent biodegradation of the
compounds in the biofilm. The development of biofilm is possible after
inoculating the packed bed with microorganisms because inert packing
materials are used in the packed bed (Barbusinski et al., 2017).
247
Bioremediation These techniques treat pollutant effectively by the use of microbial
metabolism and proved to be superior to other types of bioreactors when both
efficiency and economy of the process is taken into account (Schmidt and
Anderson, 2017; Rybarczyk et al., 2019). The analysis and comparison with
traditional biofilter show good reliability, stable performance, and
applicability to a large volume of air containing a lower concentration of
VOCs (Schmidt and Anderson, 2017). Higher removal of H2S, organo-sulfur
compounds, and NH3, shows the high abatement performance of biotrickling
filters (Rybarczyk et al., 2019).
11.6.1 Operating Conditions

The performance of biotrickling filters not only depends on its design but
also the operating conditions i.e. removal efficiency, elimination capacity,
and inlet loading rate (Cloirec et al., 2014; Kennes et al., 2016; Rybarczyk et
al., 2019). The operating conditions of biotrickling factors are provided in
Table 3.
11.6.2 Process Description and Mechanism

A biotrickling filter consists of a column packed with solid materials
(inorganic grains, rashing rings, Berl saddles) which is covered by a biofilm
formed predominantly of aerobic microorganisms (Cloirec et al., 2014;
Rybarczyk et al., 2019). A packed bed is a crucial element of the biotrickling
filter because it provides the necessary support for the growth of
microorganisms and also helps to provide a high contact area between liquid
and gas phases (Wu et al., 2016; Barbusinski et al., 2017). The waste gas
stream is passed through the bottom of the packed column, irrigated with the
trickling nutrient-rich liquid that helps in the growth of microorganisms in
the biofilm, and the treated gas is vented to the atmosphere. The pH of water
is also controlled by the addition of base or acid to the fresh solution to
obtain appropriate bacterial growth (Cloirec et al., 2014; Rybarczyk et al.,
2019).
For the packing of the column, various materials are used, including ceramic
elements, polymeric materials, open-pore synthetic foams, glass beads, tire-
derived rubber particles, and organic materials like wood chips (Chen et al.,
2016). The packing material for better efficiency should have high porosity
for efficient distribution of gas phase, having high water retention capacity so
prevent the bed from drying. The material should be odorless and the material
should be cheap and durable (Rybarczyk et al., 2019).
The contaminants present in the gaseous phase are first transferred into the
aqueous phase and then degraded in the biofilm present at the surface of the
packing. Metabolites are removed by the solution flowing in the column.
Some bacteria are also detached and are carried away by water. Washing the
packing materials is sometimes necessary, due to clogging of the column by
uncontrolled biofilm growth (Cloirec et al., 2014).
248
Bioremediation of
Check Your Progress 2 the Air Environment
1. What is the proportion of nutrients used in the biofilters?
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
2. Which is the most important parameter for the better performance of the
biotrickling filter?
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
11.7 BIOSCRUBBER
As biotrickling filter is an enhancement of biofilter, bioscrubber is an
enhancement of biotrickling filter (U.N environmental protection agency,
2003). The biofilter and biotrickling filter involves immobilized organisms,
but the bioscrubber utilizes dispersed (suspended) culture. The bioscrubber
consists of two units: a usual scrubber in which VOCs and odorous
compounds are transported from the air to a water phase, and a classical
bioreactor where the water exiting the scrubber is subjected to biological
treatment in the liquid phase. To maintain the high level of degradation in the
bioreactor which contains suspended culture, sufficient oxygen through
aeration is required (Singh and Ward, 2004).
The bioscrubber tries to solve two problems associated with the biotrickling
filter. First, improvement of the absorption of pollutants into the liquid and
second, lengthening of the time the microorganisms have to consume the
pollutants (i.e. the microbes and pollutant interaction time). These processes
are accomplished in two ways: the bioreactor packing is flooded with a liquid
phase and the discharge effluent from the bioscrubber is collected in a storage
tank (sump) before being recycled back to the bioscrubber. The bioscrubber
unit is appropriate for the treatment of pollutants that are fairly well soluble
in water, as biodegradation is performed by suspended biomass in the
bioreactor (U. S. Environmental Protection Agency, 2003; Kannes et al.,
2009).
249
Bioremediation Flooding increases the bed's ability to absorb pollutants because as the gas
phase (emission) impacts the bed media, it forms tiny bubbles that greatly
increase the surface area of the interface between the gas phase and liquid
phase. As the interface area increases, it will enhance the liquid phase's
ability to absorb pollutants (U. S. Environmental Protection Agency, 2003).
The sump (storage tank) provides the additional reaction time for the
microbes to consume pollutants and act as a reservoir to carry out the
reaction. Reaction time depends on the recirculation rate of the liquid phase
and the size of the storage tank, and that can be increased to an hour or more,
which increases the time available for microbes to attach and destroy
pollutants (U. S. Environmental Protection Agency, 2003).
The first full-scale plant of bioscrubber was installed in German foundries in

the 1970s for the removal of waste gases containing amines, phenol,
formaldehyde, and odors. Bioscrubber can be operated at higher gas loads up
to 3000-4000 m3 m-2 h-1 (Kannes et al., 2009).
In bioscrubber, the gas contaminants are absorbed in a free liquid phase

before biodegradation by either suspended or immobilized microbes. Unlike
biofilter and biotrickling filter, the bioscrubber consists of two reactors. The
first part of the reactor is an absorption tower, where the pollutants are
absorbed by a liquid phase, then the liquid phase goes to a second reactor
which is a kind of active sludge unit, here the degradation process of the
contaminant is performed by a suspended consortium of microbes in a
separate vessel. The effluent of this unit is recirculated over the absorption
tower in a co- or counter-current direction to the flow of the waste gas. This
system is properly aerated to ensure maximum degradation. To date, the
majority of bioscrubbers in operation exist to eliminate odors from waste gas
streams (Sing and Ward, 2004).
11.7.1 The Process Involved in Bioscrubber

The process of bioscrubber is performed by an association of two steps and
the schematic presentation of bioscrubber is given in Figure 4 (Kannes et al.,
2009: Cloirec et al., 2014).
The polluted air is fed to an absorption tower first i.e. scrubber unit. By doing
so, target compounds are transferred from the gas phase (air) to the liquid
phase (water) and the decontaminated air i.e. clean air is released into the
atmosphere from the scrubber while the polluted water is fed to a bioreactor
where biological degradation of pollutants takes place.
Bioscrubber is suitable for well soluble pollutants as degradation is carried by

suspended biomass in the bioreactor. Poor solubility would negatively affect
the absorption rate and efficiency (Kannes et al., 2009). Bioscrubber has been
used for the removal of odour compounds such as H2S and NH3 from gas
originating from water purification plants, removal of odour arising from the
production of enzymes, aromas, and polymers, and also for the treatment of
250
Bioremediation of
flue gases coming from waste sites for dangerous substances, and ammonia the Air Environment
removal at cattle farms, etc. Bioscrubber can also treat waste gases containing
hydrophilic VOCs such as methanol.
11.7.2 Advantages and Disadvantages of Bioscrubber

The use of bioscrubber for the decontamination of waste gas has a lot of
advantages as these equipments are smaller in size, more predictable, and
reliable. This technology has some disadvantages also. Various advantages
and disadvantages of bioscrubbers are given below (U. S. Environmental
Protection Agency, 2003).
Advantages
• The volume of equipment is smaller

• In bioscrubber, pH can be better controlled
• More predictable and reliable
• No clogging problems of packing materials
• The chance of occurrence of toxic concentration is low in the water
phase
• The process is ideal for emissions that produce acids upon treatment
Disadvantages
• To attain an efficiency of higher than 98% is difficult

• Chancesof slowest growing microorganisms to wash out are high
• Disposal of sludge
• Operational cost is high
• The installation cost of the unit is very high
11.8 LET US SUM UP

Urbanization has resulted in a reduction in air quality. The release of
contaminated waste gas from various sources causes many environmental
problems. The traditional method for the treatment of the contaminated gas
release end products needs further treatment. To protect both human health
and the environment in the urbanizing world, we need to make drastic
changes in how we overcome the problem of environmental degradation by
the use of eco-friendly management practices. Green technologies including
bioreactors may be key factors for the treatment of contaminated gases to
reduce impact and also incomplete degradation of pollutants released from
industries. In the present unit, types, processes, and mechanisms, advantages
and disadvantages, operating conditions of biofilters, bioscrubbers, and
biotrickling filters are discussed. Biofilters, bioscrubbers, and biotrickling
251
Bioremediation filters can help in the complete degradation of pollutants by releasing end
products like CO2, water, and bacterial biomass which need no further
treatment. In general, the environmental benefits of these bioremediation
technologies are immense and also these technologies are more predictable
and reliable when compared with the traditional treatment technologies.
11.9 KEY WORDS

Biofilter: Biofilter uses microorganisms fixed on the filter media to break
down pollutants present in the waste air stream. The microorganisms grow in
a biofilm on the surface of the medium or are suspended in the water phase
surrounding medium particles.
Bioscruber: The biofilter and biotrickling filter involves immobilized

organisms, but the bioscrubber utilizes dispersed (suspended) culture.

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Table 11.1. Classification of Bioreactors used for Waste Gas Purification the Air Environment
Reactor type Bioreactor Water phase
Biofilter Fixed Stationary
Biotrickling filter Fixed Flowing
Bioreactor Suspended Flowing
Table 11.2: Examples of particular microorganisms degrading specific

pollutants present in the air (Clolrec et al., 2014)
Contaminants Microorganism
Ethanol Pseudomonas sp., Pseudomonas putida

Phenol, benzene, toluene, Psudomonas putida
xylene, ethylbenzene
Toluene Pseudomonas putida ToIIA
Styrene and Xylene Narcodia
Butanol Pseudomonasfluorescens
Hydrogen sulfide Pseudomonas putida CH11
Methyl Hydromicrobium sp.
Dimethylsulfur Pseudomonas
Dichloromethane Hudromicrobium sp.
Dichloroethane Xanthobacter
Trichloroethylene Methylosinus
Ammonia Actinomadura nitrigens sp.
Table11.3: General operating conditions for waste gas stream treatment

in biofilters (Clolrec et.al, 2014).
Parameter Value Comments

Gas-phase velocity 50-500 Low value due to low kinetics of
degradation
Air residence time/s 15-90 Depending on the molecule degradation
kinetic.
Alcohols > ketones > n-alkanes >
aromatics
Bed porosity 0.4-0.95 High values avoid clogging
Filter depth (H)/m 0.5-2.5 Compromise between the residence 255
Bioremediation time and pressure drop
Pressure drop/m H2O 0.1-0.5 Depending on packing material
clogging and compaction
Air humidity 60-100 High values are advised to maintain
humidity in the biofilter
Water pH 5-9 Depending on pollutant solubility
Temperature (T)/oC 10-40 Mesophilic microorganism at a
temperature ranging from 50-70oC
Efficiency/% 90-99 Depending on molecules
Lifetime/yr 2-5 Structured and inorganic material
z increase lifetime
Bioremediation
In-situ Ex-situ
Intrinsic Engineered in-situ Land Bioreactors Composting Biopiles

bioremediation bioremediation farming
Biosparging and Biostimulation Bioaugmentation

Bioventing
Fig. 111: Schematic representation of various types of bioremediation
256
Bioremediation of
the Air Environment
Fig. 11.2: Schematic diagram of a bioreactor unit.
Fig. 11.3: Mechanism involved in biofilter: The contaminated air enters in the
biofilter where the biofilm layer (over the filter bed) is present and in this
layer, the present microorganism converts the contaminant into water and
carbon dioxide (Ardjmand et al., 2005).
257
Bioremediation Fig. 11.4: Bioscrubber having two separate units in a combination of a suspended
growth bioreactor and a scrubber (Kannes et al., 2009; U.S. Environmental Protection
Agency, 2003).
11.13 ANSWERS TO CHAECK YOUR PROGRESS

EXERSCISE

1. Some authors propose that the metabolic activity of microorganisms is
stimulated in the presence of key elements in certain proportions, i.e. in a
C:N:P ratio of 100:15:3 or 100:5:1 (carbon: nitrogen: phosphorus).
2. The most important parameter for the better functioning of the
biotrickling filter is the pH of trickling liquid.
TERMINAL QUESTIONS
1. Describe the term bioremediation.
2. List the main biofilter operating conditions for better efficiency.
3. Describe the difference between In-situ and Ex-situ bioremediation.
4. How does the continuous supply of nutrients in the filter bed affect its
performance?
Answers to terminal questions
1. Bioremediation is a process in which microorganisms are used to solve

the problem of environmental degradation.
2. The main biofilter operating conditions include the optimum pH of the

filter bed, temperature, oxygen, and nutrient availability.
3. In-situ bioremediation is the application of microorganisms to the site of

contamination i.e. within the effected environment but in the case of ex-
situ process requires excavation of contaminated soil or water to
facilitate microbial degradation.
4. The continuous supply of nutrients into the biofilm, will induce the
uncontrolled development of biofilm and cause a clogging problem in
the packing material and leads to a decrease in the overall efficiency of
the biofilter.
258
Phytoremediation
UNIT 12 PHYTOREMEDIATION
Structure
12.1 Introduction
12.2 Objectives
12.3 Definition, Scope and Types
12.3.1 What is phytoremediation?
12.3.2 Scope
12.3.3 Types of phytoremediation
12.4 Process and Mechanism
12.4.1 Inorganics and Organics
12.4.2 Heavy Metals
12.4.3 Process and mechanism
12.5 Environmental Factors
12.6 Advantages, Disadvantages and Limitations
12.7 Phytoremediation in Wetland Ecosystems
12.7.1 Natural Wetlands and Constructed/ Engineered Wetlands
12.8 Role of Genetically Engineered Plants
12.9 Let us Sum Up
12.10 Key Words
12.12 Answers to Check your Progress Exercise
12.1 INTRODUCTION
Several methods employed for the treatment of a variety of wastewater are
available viz., activated sludge, aerated lagoon, up-flow anaerobic sludge
blanket digestion, reverse osmosis, ion-exchange, membrane bioreactor,
trickling filter etc. However, these methods are quite expensive, which divert
the industrialist’s attention from treating the effluents and have become one
of the most important detriments of any industry. Hence, there is an urgent
need to develop simple and cost-effective technologies for treatment of
effluents to reduce their pollution loads. Phytoremediation offers such a
technology. Phytoremediation is in situ use of plants to stabilize, reduce,
remediate, or restore contaminated ground water, sediment, soil or surface. It
relies on plant’s ability to act as a solar driven pumping and filtering system
and enhances the natural tendency of ecosystem to restore itself.
Phytoremediation is a technology that uses plants to treat soil and water
contamination thereby reducing environmental pollution. Plants are used
either to remove or to stabilize (hold in place) pollution in the soil. The
ultimate goal is to use plants to reduce the risk of human exposure to various
environmental hazards. Although, phytoremediation may take a long time,
but it is often less-expensive compared to other methods used to remove toxic 259
Bioremediation substances from the soil/ water. Another benefit is that it covers the site with
an attractive layer of plants that prevents wind and water from carrying the
pollution to other places.
The paradigm that all such plants can be used for environmental remediation
is definitely very old and cannot be traced to one particular reference.
Constructed wetlands, reed beds, floating plant systems, and aquatic weeds
have been common for the treatment of waste waters for many years.
In this unit, we shall focus our attention, primarily on phytoremediation

method to improve the environmental sustainability. We shall discuss the
basic process involved in phytoremediation. Then we shall take up types of
phtoremediation, process and mechanism, and its advantages, disadvantages
and limitations. Subsequently we would study, phytoremediation in wetland
ecosystem. We shall also talk about the role of genetically engineered plants.
12.2 OBJECTIVES
After reading this unit, you will be able to:
• define phytoremediation and its types;

• describe the various processes involved and mechanism of
phytoremediation;
• understand the advantages, disadvantages and limitation of
phytoremediation; and
• apply the phytoremediation in wetland ecosystems
12.3 DEFINITION, SCOPE AND TYPES

Phytoremediation comes from the Greek word phyto meaning "plant" and the
Latin word remedium meaning "restoring balance”- the process of correcting
a problem or to put back in proper condition.
12.3.1 What is Phytoremediation?

Phytoremediation is a bioremediation process that uses various types of
plants to remove, transfer, stabilize, and/or destroy contaminants in the soil
and groundwater and can be simply defined as the use of green plant to
remove pollutants from the environment or to render them harmless.
Phytoremediation is a green technology for clean environment and is a form
of bioremediation and applies to all chemical or physical processes that
involve plants for degrading or immobilizing contaminants in soil and
groundwater. While the technology is not new, current trends suggest its
popularity is growing. Several comprehensive reviews have been written on
this subject, summarizing many important aspects of this low-cost plant-
based technology by Cunningham et al. (1996); Cunningham and Ow (1996);
Raskin et al, (1997); Salt et al. (1995); Salt et al. (1998) and Srivastava and
260
Phytoremediation
Purnima, (1998). The basic idea that plants can be used for environmental
remediation is very old and cannot be traced to any particular source.
However, a series of fascinating scientific discoveries combined with an
interdisciplinary research approach have allowed the development of this idea
into a promising, cost-effective and environment friendly technology.
Phytoremediation can be applied to both organic and inorganic pollutants,
present in solid substrates (eg., soil), liquid substrates (e.g., water) and the air.
12.3.2 Scope
Cunningham et al. (1997) defined phytoremediation as the use of green plant-
based systems to remediate contaminated soils, sediments and water. Relative
to many traditional techniques, phytoremediation is a fledgling technology
intended to address a wide variety of surficial contaminants.
Phytoremediation targets currently include contaminating metals, metalloids,
petroleum hydrocarbons, pesticides, explosives, chlorinated solvents and
industrial by-products. Watanabe (1997) defined phytoremediation on the
brink of commercialization. Although phytoremediation has been tested on
sites contaminated with petroleum products, heavy metals, munitions and
radio nuclides and at abandoned mines, wood treatment sites, and sewage
treatment sites, it remains unclear how large the phytoremediation market
will be. Phytoromediation experts opine that the growth of interest in the
field is driven by its relative cost-efficiency compared to standard
remediation methods for government-mandated site clean-up.
Several workers have employed plants for the removal of organic and
inorganic matters from the wastewater and soil. It is not exaggerated to
compare green plants with that of lungs of nature with ability to uptake,
tolerate and even hyper-accumulate heavy metals and toxic substances from
soil and water through the roots and concentrate them in their various parts.
Different phytoremediation applications to polluted/ contaminated soil and
water are shown in Figure 5.1. Some of the aquatic weeds used for this
purpose are in floating macrophytes based system viz., water hyacinth, water
lettuce, salvinia and some species of duck weeds (Hammer, 1990; Mandi et
al., 1993), in submerged macrophytes based systems such as hydrilla, elodea,
egeria and hornwort (Ceratophyllum demersum) (Brix and Schierup, 1989),
in energent macrophytes based systems and wetlands viz., common reed
(Phragmities australis) (Hammer, 1990). In this context, Eichhornia
crassipes particularly has received much attention, because of its high
efficiency of removing pollutants from industrial wastewater (Jebanesan,
1997; Jain, 2000 and Narayana and Parvez, 2000).
12.3.3 Types of Phytoremediation

The following is a list of six different types of phytoremediation with
explanations describing how they work:
261
Bioremediation a) Phytosequestration
Phytosequestration can also be termed as phytostabilization. It comprises
of many different processes involving absorption by roots, adsorption to
the surface of roots or the production of biochemicals by the plant that
are released into the soil or groundwater in the immediate vicinity of the
roots, and can sequester, precipitate, or otherwise immobilize nearby
contaminants.
b) Rhizodegradation
Rhizodegradation takes place in the soil or ground water
immediate surrounding of the plant roots. Under this process exudates
from plants stimulate rhizospheric bacteria to enhance biodegradation of
soil/ water contaminants.
c) Phytohydraulics
Phytohydraulics is the process to use deep-rooted plants like trees to
contain, sequester or degrade ground water contaminants those come into
contact with their roots. Poplar trees are such examples, which were used
to contain a ground water plume of methyl-tert-butyl-ether (MTBE)
(Hong et al. 2001).Poplar trees act as natural pumps to keep toxic
herbicides, pesticides, and fertilizers out of the streams and groundwater
as demonstrated by Environmental Protection Agency (EPA).
d) Phytoextraction
Phytoextraction is also known as phytoaccumulation. Plants take up or
hyperaccumulate contaminants through their roots and store them in the
stem or leaves tissues rather than degrade them. The contaminants are
not necessarily degraded but are removed from the environment after
harvesting the plants. This is useful especially in removing metals from
soil and, in some cases, the metals can be recovered for reuse, by
incinerating the plants, in a process called phytomining.
e) Phytovolatilization
Phytovolatalization is a process in which plants take up volatile
compounds through their roots, and transpire the same compounds, or
their metabolites, through the leaves, thereby releasing them into the
atmosphere.
f) Phytodegradation
Phytodegradation is a process under which contaminants are taken up
into the plant tissues where they are metabolized/destroyed, or bio-
transformed. The transformation depends on the type of plants used, and
can occur in roots, stem or leaves.
Check Your Progress Exercise 1
Put a tick (√) mark against the correct answer:
1) Phytoremediation is:
262
Phytoremediation
a) treatment of soil pollution through plants
b) treatment of air pollutants through plant
c) treatment of wastewater through plants
d) all of the above
2) Phytoremediation technology involves:
a) low-cost
b) high cost
c) no related to cost
d) none of the above
3) Rhizodegradation process includes:
a) stimulation of rhizospheric bacteria to enhance biodegradation of
contaminants
b) hyperaccumulate contaminants through their roots
c) take up volatile compounds through roots and their transpiration
d) adsorption of contaminants to the surface of roots
Phytoremediation
Applications
SOIL WATER
• Phytostabilisation • Rhizofiltration
• Phytoimmobilisation • Hydraulic barriers
• Vegetative caps
• Phytoextraction • Constructed wetlands
• Phytovolatalisation
Fig. 12.1: Different phytoremediation applications to polluted/ contaminated soil and

water
263
Bioremediation
The complex nature of most synthetic For chemicals that are easily degraded, the
chemicals encountered by presence of 100-fold more microorganisms
microorganisms can require interaction in the rhizosphere compared with non-
of microbial communities to achieve vegetated soil leads to increased rates of
transformation. The plant root zone chemical transformation.
fosters these types of interactions.
Synergistic Biomass
activity
 
MECHANISMS
 
Diversity Exudates
By providing a niche suitable to a diverse Root exudates may serve as structural

population of microorganisms, vegetation analogs to contaminants as well as enhance
may enhance microbial degradation co-metabolism of contaminants.
because of the presence of a key group of
microorganisms involved in the
metabolism of the contaminant.
Fig. 12.2 Mechanism showing microbial degradation in the rhizosphere and its
implications for bioremediation (Anderson and Coats, 1995)
Rhizosphereaccumu
Rhizofiltration
lation
Inorganics
Phytostabilisation Complexation
Phytoextraction Hyper-accumulation
Organics
Phytovolatilization Volatilisation by
leaves
Phytotransformation Degradation in plant
Fig. 12.3Phytoremediation includes the following processes and mechanisms of

contaminant removal
12.4 PROCESS AND MECHANISM

Phytoremediation technique can be applied for inorganic/ organic compounds
as well as for heavy metal removal.
264
12.4.1 Inorganics and Organics Phytoremediation
Unlike the case with organic compounds that can be mineralized, the
remediation of contamination with an inorganic contaminant must either
physically remove the contaminant from the system or convert it into a
biologically inert form. Removal can be accomplished by removing the
biomass or, with certain inorganic contaminants, by contaminant'
volatilization.
12.4.2 Heavy Metals

The plants with their sophisticated metabolic and for sequestration
detoxification mechanisms, have the ability to accumulate essential heavy
metals from soil and water which help in the plant growth like Fe, Mn, Zn,
Cu, Mg, Mo, and Ni and non-essential heavy metals with unknown biological
function like Cd, Cr, Pb, Co, Ag, Se, Hg. A lot of species of plants have
potential in absorbing contaminants such as arsenic, cadmium, chromium,
lead, and radionuclides from soils.
12.4.3 Process and Mechanism

Anderson and Coats (1995) presented an overview of microbial degradation
in the rhizosphere and its implications for bioremediation (Fig. 5.2). This
figure shows that the complex nature of most synthetic chemicals
encountered by microorganisms can require interaction of microbial
communities to achieve transformation. The plant root zone fosters these
types of interactions. For chemicals that are easily degraded, the presence of
100 fold more microorganisms in the rhizosphere compared with non-
vegetated soil leads to increased rates of chemical transformation. By
providing a niche suitable to a diverse population of microorganisms,
vegetation may enhance microbial degradation because of the presence of a
key group of organisms involved in the metabolism of the contaminant. Root
exudates may serve as structural analogs to contaminants as well as enhance
cometabolium of contaminants.
Fig. 12.3 shows the variousprocesses and mechanisms of contaminant

removal included in Phytoremediation, which are already discussed under
section 12.4.3.
12.5 ENVIRONMENTAL FACTORS

The various factors involved for phytoremediation of contaminants were
described in details by Anderson (1996). These factors are shown in Figure
5.4.
The following criteria were suggested by Abbasi and Ramasami (1999) over
the years for selecting a plant species or combination of species as the main
bioagent(s) in water treatment systems:
a) adaptability to local climate, 265

Bioremediation b) high photosynthetic rates-in other word, high growth rate,
c) high oxygen transport capability,
d) tolerance to adverse concentration of pollutants
e) high pollutant-uptake efficiency,
f) tolerance to adverse climate conditions,
g) resistance to pests and diseases, and
h) ease of management
The effectiveness of mechanical aeration in floating aquatic macrophyte

based wastewater treatment system was studied by Debusk et al. (1989).
Light aeration (0.003 and 0.021 Lm-2 min-1) had no effect on the treatment of
primary domestic effluent in the batch-fed water hyacinth tanks. Heavy
aeration (1.03 and 3.53 Lm-2 min-1) raised waste water dissolved oxygen
(D.O.) concentrations but did not improve Biochemical Oxygen Demand
(BOD5) removal efficiency or increase plant growth rates during 21-days
experiments. Debusk et al. (1991) reviewed wastewater treatment methods
based on floating aquatic macrophytes in pond systems. Pennywort was the
most efficient plant to provide secondary wastewater treatment, followed by
water hyacinth and common duckweed. Similar study has been reported by
Walsh et al. (1991) with Echinochloa crusgalli and Sesbania macrocarpa.
The phytoremediation experiments have been popular in several academic

laboratories and industries (Brown, 1995). The plants called hyper-
accumulators are categorized as the best for phytoremediation. They can be
herbs, shrubs, and even trees. Hyper-accumulators concentrate trace
elements, heavy metals or radio nuclides at level 100-fold or greater than
normal.
Comis (1996) also described eco-friendly approach for remediation of soil

i.e., used plants to clean the soil. Cunningham and Ow (1996) described in
details the promises and prospects of phytoremediation. Cunningham et al.
(1999) described phytoremediation as an affordable technology that is most
useful when contaminants are within the root zone of plants (top 3-6 ft [1-2
m]). He also reported various plants for the treatment of wastewater
viz.,Lemna, Pistia, alfa alfa water hyacinth etc. Further popularization of
phytoremediation technology from laboratory to the market place has been
attempted by Dushenkov et al. (1997), Nyer and Gatliff (1996), Boyajian and
Carreira (1997), Bishop (1997).
Plant’s ability to absorb, translocate and concentrate metals has been studied
by Chandra et al. (1997). The potential to accumulate chromium by Scirpus
lacustris, Phragmites karka and Bacopa monnicri was assessed by
subjecting them to different chromium concentrations under laboratory
condition. The plants caused significant reduction in chromium
concentrations. While there was an increase in biomass, no visible
phytotoxic symptoms were shown by treated plants. Similar reports were
266
Phytoremediation
made by Satyakala and Jamil (1997) with Pistia stratiotes.
Rice et al. (1997) conducted an investigation to test the hypothesis that

herbicide-tolerant aquatic plants could remediate herbicide-contaminated
water. The addition of Ceratophyllum demersum (Coontail, hornwort),
Elodea Canadensis (American elodea, Canadian pondweed) or Lemna minor
(common duckweed) significantly (p ≤ 0.01) reduced the concentration of
[14C] metolachlor (MET) remaining in the treated water. Sarkar (1997)
studied seasonal influence of the plant Lemnamajor in the treatment of
eutrophicated ponds. The concentrations of total ammonical nitrogen (TAN),
nitrate-nitrogen (NN) and phosphate of water in different seasons decreased
in ponds exposed to aquatic plants and the mean percentages of nutrients in
ponds with plants exhibited different trends and in the following orders:
monsoon > winter > summer for TAN, monsoon > summer > winter for NN
and summer > winter > monsoon for phosphate.
Using water lettuce plants, heavy metals can be effectively removed when
they are present at a concentration of 10 mg/l or less (Selvapathy et al.,
1998). Siciliano and Germida (1998) described the mechanisms of
phytoremediation. They further specified the biochemical and ecological
interactions between plants and bacteria. Zayed et al. (1998) reported
phytoaccumulation of trace elements by wetland plants viz., duckweed.
Phytoremediation generally reduced B.O.D., NH4+-N, P, turbidity and

volatile suspended solids. Populations of faecal coliforms were consistently
reduced by 90 to 99 per cent by 2-days detention in microcosms with and
without plants. Trivedy and Nakate (1999) studied aquatic weeds based waste
water treatment plants in India and reported a very high degree of reduction
in suspended solids, BOD and COD, nitrogen, phosphorous and oil and
grease. Rose (2000) inferred that Lemna minor is efficient in removing
BOD, solids and nutrients from the wastewater and has high potential for
treating organically rich wastewater and reuse possibilities. Trivedy and
Nakate (2000) studied treatment of diluted distillery waste by using
constructed wetland on laboratory scale using Typha latifolia. The reduction
in BOD and COD was 47.59 per cent and 78.77 per cent in 10 days. On the
basis of above reports Central Pollution Control Board (CPCB, 2001)
emphasized that phytoremediation is a natural biological treatment of
wastewater.

1. Describe the various criteriafor selecting a plant species or combination

of species as the main bioagent(s) in water treatment systems.
……………………………………………………………………………
…………………………………………………………………………… 267
Bioremediation ……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
12.6 ADVANTAGES, DISADVANTAGES &

LIMITATIONS
Some of the advantages of phytoremediation technology are mentioned
below:
• phytoremediation both in situ and ex situ is low cost treatment

technology as compared to the traditional processes
• the plants used in the phytoremediation can be easily monitored
• the accumulated valuable metals in the harvested biomass can be
recovered for reuse through "phyto mining" technique
• phytoremediation is eco-friendly and the least harmful method as it uses
plants/ organisms occur in the nature, which also help to preserve the
environment and ecological sustenance.
• phytoextraction can clean up the soil without causing any kind of harm
to soil quality and its productivity.
Disadvantages
Contamination concentration, toxicity and bioavailability and Plant choice

and stress tolerance are the main disadvantages of phytoremediation. Some of
the reported disadvantages (Farraji et al., 2016) are as below:
• Accumulation of pollutant in fruit and other edible parts of crop and

vegetables.
• Growing of phytoremediator plants (hyperaccumulators)
• Low biomass production in phytoremediators, so several planting and
harvesting required for decontamination
• Generally, specific selective unique accumulation of one metallic
element in hyperaccumulator
• Environmental pollution caused by chelate–enhanced phytoremediation
• Very slow and seasonally effective treatment method
• Handling and disposing contaminated plants through the
phytoremediation is the major foot print of this green technology
• Mobilization of radionuclides through the translocation in plants
• Not applicable for all compounds
• Dissolved contaminant in groundwater are not suitable case for aquatic
phytoremediation
268
Phytoremediation
LIMITATIONS
There are always certain limitations while we use any technology. As such,
phytoremediation technology has also its certain limitations, which are
mentioned below:
• phytoremediation is limited to the surface area and depth occupied by the

roots.
• slow growth and low biomass require a long-duration
• with plant-based systems of remediation, it is not possible to completely
prevent the leaching of contaminants into the groundwater without the
complete removal of the contaminated ground
• the survival of the plants is affected by the toxicity of the contaminated
land and the general condition of the soil.
• The product produced during degradation may be accumulated in the
animals via food materials obtained by plants, bio-accumulation of
contaminants, especially metals, into the plants pass into the food chain,
from primary level consumers to top level consumers
• the affected plant material requires the safe disposal
• as atmospheric layer stored the degraded materials for short period and
via rain water it may again stored in ground water, the concerns are
shown in the remediation of toxic contaminants through the
phytoremediation process.
• Further research need to provide evidence that leaves fall down during
autumn do not produce any contamination in the soil or fire wood also
not release any gas which causes harmful effect to the environment.
• clearance of collecting plants can be difficult, if the amount of heavy
metals is high.
• effectiveness mainly limits to surface or shallow soils and stream water.
• In-case of ground water treatment, pumping method is needed to collect
the water, which is also added cost.
• effectiveness of phytoremediation is seasonal; due to plant development
is mainly season based. A large area is required for cultivation of plants
or tree used in phytoremediation.
• if contaminant concentrations are too high, plants may die.
12.7 PHYTOREMEDIATION IN WETLAND

ECOSYSTEMS
Phytoremediation of metals is a cost-effective green technology based on the
use of selected metal-accumulating plants to remove toxic metals from soils
and water. Wetland plants are important medium in heavy metal removal.
The Ramsar convention, one of the earlier modern global conservation
269
Bioremediation treaties, was adopted at Ramsar, Iran, in 1971 and became effective in 1975.
This convention emphasized the wise use of wetlands and their resources. In
the convention, salient features of wetland ecosystems, their vegetation
component, and the pros and cons involved in heavy metal removal were
discussed extensively. Wetland plants are preferred over other bio-agents due
to their low cost, frequent abundance in aquatic ecosystems, and easy
handling. The extensive rhizosphere of wetland plants provides an enriched
culture zone for the microbes involved in degradation. The wetland sediment
zone provides reducing conditions that are conducive to the metal removal
pathway. Constructed wetlands proved to be effective for the abatement of
heavy metal pollution from acid mine drainage; landfill leachate; thermal
power; and municipal, agricultural, refinery, and chlor-alkali effluent. The
physicochemical properties of wetlands provide many positive attributes for
remediating heavy metals. Typha, Phragmites, Eichhornia, Azolla, Lemna,
and other aquatic macrophytes are some of the potent wetland plants for
heavy metal removal. Biomass disposal problem and seasonal growth of
aquatic macrophytes are some limitations in the transfer of phytoremediation
technology from the laboratory to the field. However, the disposed biomass
of macrophytes may be used for various fruitful applications like biogas
production (Singhal and Rai, 2003) using anaerobic digestion, manure
production and valuable metal recovery. Disposed biomass is stored solar
energy in plant mass and materials having combustible organic matter. The
main constituents of disposed biomass material are lignin, hemicellulose,
cellulose, mineral matter and ash. Biogas production also ensures reducing
odor, energy production and improve the storage and handling characteristics
of manure. Genetic engineering and biodiversity prospecting of endangered
wetland plants are important future prospects in this regard.
Relationship between phytoremediation and on-site treatment of septic

effluents in sub surface flow constructed wetlands was studied by Neralla et
al. (1999).
12.7.1 Natural Wetlands and Constructed/ Engineered

Wetlands
Wetlands are inundated land areas with water depths typically less than 2 ft
that can support the growth of emergent plants. Floating aquatic plants
system contains the floating species such as water hyacinth and duck weed,
where an average depth of water ranges from 1.6 - 6.0 ft. Supplementary
aeration has been used with floating plant system for improved treatment
efficiency and to maintain an aerobic condition for the biological control of
mosquitoes. Plants in a natural wetland provide a substrate (roots, stems, and
leaves) upon which microorganisms can grow as they break down organic
materials and uptake heavy metals. However, as a result of the exponentially
increasing demands of human expansion and resource exploitation, it has
been recognized that natural wetland ecosystems cannot always function
efficiently for desired objectives and stringent water quality standards. These
270
Phytoremediation
and many other factors have led to the rapid development of "constructed
wetlands" for waste (especially wastewater) treatment. A constructed wetland
(CW) is an artificial marsh or swamp, which have been designed and
constructed to utilize the natural processes involving wetland vegetation,
soils, and their associated microbial assemblages to assist in waste treatment.
It usually consists of a number of individual rectangular and/or irregularly-
shaped basins (cells) connected in series and surrounded by clay, rock,
concrete or other materials. Three types of cells may be used in a constructed
wetland system (CWS): free water surface (FWS) cells, sub-surface flow
(SSF) cells, and hybrid cells that incorporate surface and subsurface flows
(Zhang et al, 2001). Phytoremediation in engineered wetland is an
aesthetically pleasing, solar-driven, passive technique useful for cleaning up
wastes including metals, pesticides, crude oil, poly-aromatic hydrocarbons,
and landfill leachates and has become an increasingly recognized pathway to
advance the treatment capacity of wetland systems.
12.8 ROLE OF GENETICALLY ENGINEERED

PLANTS
Breeding programs and genetic engineering are powerful methods for
enhancing natural phytoremediation capabilities, or for introducing new
capabilities into plants. Genes for phytoremediation may originate from
a micro-organism or may be transferred from one plant to another variety
better adapted to the environmental conditions at the cleanup site. For
example, genes encoding a nitroreductase from a bacterium were inserted
into tobacco and showed faster removal of TNT (Trinitrotoluene-
C6H2(NO2)3CH3 ) and enhanced resistance to the toxic effects of
TNT.Mechanism in plants has also been discovered that allows them to grow
even when the pollution concentration in the soil is lethal for non-treated
plants. Some natural, biodegradable compounds, such as
exogenous polyamines, allow the plants to tolerate concentrations of
pollutants 500 times higher than untreated plants, and to absorb more
pollutants.
A plant is said to be a hyper accumulator if it can concentrate the pollutants

in a minimum percentage which varies according to the pollutant involved
(for example: more than 1000 mg/kg of dry weight for nickel, copper, cobalt,
chromium or lead; or more than 10,000 mg/kg for zinc or manganese). This
capacity for accumulation is due to hypertolerance, or phytotolerance: the
result of adaptative evolution from the plants to hostile environments through
many generations. A number of interactions may be affected by metal
hyperaccumulation, including protection, interferences with neighbour plants
of different species, mutualism (including mycorrhizae, pollen and seed
dispersal), commensalism, and biofilm.
The understanding of phytoremediation reactions has recently progressed

from basic uptake studies to quantifying the importance of chemical 271
Bioremediation speciation for bioavailability and the role of genetic engineering for
hyperaccumulating species. Genetic engineering of wetland plants may aid
phytoremediation and more focused work is needed in this regard.

1. Write down the three major advantages and disadvantages of
phytoremediation technology.
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
2. What are wetlands?
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
12.9 LET US SUM UP

The attention on vascular aquatic plants has been mainly directed towards
their elimination from water bodies, since dense stands of these plants harm
water resources in terms of quality as well as quantity. These plants
adversely affect fisheries, impede navigation, hasten water loss, encourage
insects, pests and disturb the fragile oxygen balance of water bodies through
decay. Volumes of literature have been generated on methods to control the
growth of aquatic plants. The negative characteristics of these weeds-
hardiness, ability to survive under adverse environmental conditions and high
productivity can be harnessed to make them efficient bioagents for treating
wastewaters. Till recently, the successful exploitation of aquatic plants in this
respect had been constrained by the lack of economically viable methods of
post-harvest utilization of the ‘spent’ plants, but now it has been
demonstrated that it is feasible to use such plants as energy feed stock, animal
fodder, feed, biogas production and fertilizers. This has triggered
considerable interest in aquatic macrophytes based wastewater systems
(AMS). The economic success of energy production and water treatment
using an AMS for water treatment/biomass production depends to a large
extent on the photosynthetic activity and growth rate of plants.
272
Phytoremediation
12.10 KEY WORDS
Phytoremediation: Phytoremediation is a bioremediation process that uses
various types of plants to remove, transfer, stabilize, and/or destroy
contaminants in the soil and groundwater and can be simply defined as the
use of green plant to remove pollutants from the environment or to render
them harmless.
Wetlands: Wetlands are inundated land areas with water depths typically less
than 2 ft that can support the growth of emergent plants
12.11 TERMINAL QUESTIONS

1) What is contamination? Write down its different types.
2) Write down the types of phytoremediation with explanations in your own
words.
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
3) Explain the mechanism involved in microbial degradation in the
rhizosphere and its implications for bioremediation with the help of a
suitable diagram.
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
4) How genetically engineered plants are useful for phytoremediation?
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
273
Bioremediation
12.12 ANSWERS TO CHECK YOUR PROGREE
EXERCISE
Check your Progress Exercise 1
1) 1) d 2) a 3) a
1) There are the following criteria for selecting a plant species or

combination of species as the main bioagent(s) in water treatment
systems:
a) adaptability to local climate,

b) high photosynthetic rates-in other word, high growth rate,
c) high oxygen transport capability,
d) tolerance to adverse concentration of pollutants
e) high pollutant-uptake efficiency,
f) tolerance to adverse climate conditions,
g) resistance to pests and diseases, and
h) ease of management
2) Hyper-accumulator plants concentrate trace elements, heavy metals or

radio nuclides at level 100-fold or greater than normal.
1) Adantages:
a) phytoremediation both in situ and ex situ is low cost treatment
technology as compared to the traditional processes
b) the plants used in the phytoremediation can be easily monitored
c) phytoremediation is eco-friendly and the least harmful method as it uses
plants/ organisms occur in the nature, which also help to preserve the
environment and ecological sustenance.
Disadvantages:
a) Accumulation of pollutant in fruit and other edible parts of crop and
vegetables.
b) Very slow and seasonally effective treatment method
c) Handling and disposing contaminated plants through the
phytoremediation is the major foot print of this green technology
2) Wetlands: Wetlands typically occur in topographic settings where

surface water collects and ground water level remains near the surface,
making the area wet through most period of the year.
274
Phytoremediation
Terminal Questions
1) Write from your own experience/ knowledge.
2) Refer section 5.2.3 for answer.
3) Anderson and Coats (1995) presented an overview of microbial

degradation in the rhizosphere and its implications for bioremediation.
The complex nature of most synthetic chemicals encountered by
microorganisms can require interaction of microbial communities to
achieve transformation. The plant root zone fosters these types of
interactions. For chemicals that are easily degraded, the presence of 100
fold more microorganisms in the rhizosphere compared with non-
vegetated soil leads to increased rates of chemical transformation. By
providing a niche suitable to a diverse population of microorganisms,
vegetation may enhance microbial degradation because of the presence
of a key group of organisms involved in the metabolism of the
contaminant. Root exudates may serve as structural analogs to
contaminants as well as enhance cometabolium of contaminants. Refer
Fig. 5.2.
4) Breeding programs and genetic engineering are powerful methods for

enhancing natural phytoremediation capabilities, or for introducing new
capabilities into plants. Genes for phytoremediation may originate from
a micro-organism or may be transferred from one plant to another variety
better adapted to the environmental conditions at the cleanup site.
12.13 SUGGESTED FURTHER READINGS/

REFERENCES
• Abbasi, S. A. and Ramasami, E. 1999. Aquatic macrophytes in
wastewater treatment systems: suitability, mechanism of action, design
considerations, economics and environmental impact In:
Biotechnological Methods of Pollution Control. Hyderguda, Hyderabad,
India, University Press Ltd. pp. 8-51.
• Anderson, T. A. 1996. Rhizosphere technology for phytoremediation. In:
International Phytoremediation Conference, Arlington, VA, May 8-10,
1996. International Business Communications, Southborough, MA.
• Anderson, T. A. and Coats, J. R. 1995. An overview of microbial
degradation in the rhizosphere and its implications for bioremediation.
In: Skipper, H. D. and Turco, R. F. eds. Bioremediation: Science and
Applications, SSSA Special Publication, 43, Madison, WI, Soil Science
Society of America. pp. 135-143.
• Bishop, J. 1997. Phytoremediation: A new technology gets ready to
bloom. Environ. Solu. 10(4): 29.
• Boyajian, G. E. and Carreira, L. H. 1997. Phytoremediation: A clean
275
Bioremediation transition from laboratory to marketplace. Nat. Biotechnol. 15(2): 127-
128.
• Brix, H. and Schierup, H. H. 1989. The use of aquatic macrophytes in
water pollution control. Ambio. 18: 100-107.
• Brown, K. S. 1995. The green clean: The emerging field of
phytoremediation takes root. Bioscience. 45: 579-582.
• Chandra, P.; Sinha, S. and Rai, U. N. 1997. Bioremediation of chromium
from water and soil by vascular aquatic plants. In: Kruger, E. L.;
Anderson, T. A. and Coats, J. R. eds. Phytoremediation of Soil and
Water Contaminants. Washington, D.C., American Chemical Society.
pp. 274-282.
• Comis, D. 1996. Green remediation: using plants to clean the soil. J. Soil
Water Conserv. 51(3): 184-187.
• CPCB, 2001. Biotechnologies for Treatment of Wastes. Information
manual on pollution abatement and cleaner technologies series:
Impacts/6/2000-01. Delhi, India, Central Pollution Control Board,
Ministry of Environment and Forests. pp. 23-24.
• Cunningham, S. D., Shann, J. R.; Crowley, D. E. and Anderson, T. A.
1997. Phytoremediation of contaminated water and soil. In: Kruger, E.
L.; Anderson, T. A. and Coats, J. R. eds. Phytoremediation of Soil and
Water Contaminants. Washington, D.C., American Chemical Society.
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BLOCK 4
ECOFRIENDLY BIOPROCESSES
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BLOCK 4 ECOFRIENDLY BIOPROCESSES
This block covers role of environmental biotechnology in ecofriendly process
like biofuels, bioplastics, biofertilizers, mining and biobleaching and
Biomarkers.
Unit 13 “Biofuel” gives the concept, characteristics, current status of

biofuels.
Unit 14 “Bioplastics” elaborate the production, characterization and

application of Bioplastics.
Unit 15 “Biofertilizers” gives an idea about biofertilizers, Biological nitrogen

fixation, phosphate solubilisation, PGPR bacteria, general mode of action of
plant growth promoting microorganism.
Unit 16 “Mining and Bioleaching” describes microbial transformation,

accumulation and concentration of metals, metal leaching, extraction and
exploitation of microbes in metal extraction.
Unit 17 “Biomarkers” Definition, application and scope in environmental

Monitoring
the ecofriendly bioprocess.
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Biofuels
UNIT 13 BIOFUELS
Structure
13.1 Introduction
13.2 Objectives
13.3 Biofuels
13.4 Categories of Biofuels
13.4.1 First Generation (1G) Biofuels
13.4.1.1 Limitation of First Generation (1G) Biofuels
13.4.2 Second Generation (2G) Biofuels
13.4.2.1 Biochemical Pathway of Biomass to Biofuel
13.4.2.2 Thermo-Chemical Pathway of Biomass to Biofuel
13.4.2.3 Limitation of Second Generation (2G) Biofuels
13.4.3 Third Generation (3G) Biofuels
13.4.3.1 Limitation of Third Generation (3G) Biofuels
13.4.4 Fourth Generation (4G) Biofuels
13.5 Potential for Biofuels
13.5.1 Ethanol Production Potential of Biomass
13.5.2 Biodiesel Production Potential of Biomass
13.5.3 Other Renewable Fuel Production Potential of Biomass
13.6 Let Us Sum Up
13.7 Keywords
13.8 Suggested Further Reading / References
13.1 INTRODUCTION
Biofuel is fuel primarily derived or produced from living matter called
biomass. It is known as a non-conventional liquid or gaseous fuels, can be
used to replace or as an additive to diesel, petrol or other fossil fuels for
transport, stationary, portable and other purposes.The concept of biofuels is
surprisingly old. RudolfDiesel, whose invention now bears his name, had
envisioned vegetable oil as a fuel source for the engine. Similarly, Henry
Ford expected his Model T to run on ethanol, a corn product. However,
discoveries of enormous petroleum deposits kept gasoline and diesel cheap
for decades, and biofuels were mostly forgotten. However, with the
concurrent increase in oil prices, along with rising concern of energy
security and global warming induced by carbon dioxide emissions, biofuels
have been regaining popularity.In this unit, we would be discussing the
various generations of biofuels, along with its potential and limitations.
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Ecofriendly
Bioprocesses 13.2 OBJECTIVES
After studying this unit, you will be able to:
• Explain the different generations of biofuels;

• Discuss the potential and limitations of various categories of biofuels.
13.3 BIOFUELS
The fossil fuels burning is a significant contributor to raising the level of CO2
in the atmosphere which is instantly linked with global warming observed in
recent decades (Leonardo et al., 2008). The adverse effects of GHG
emissions on the environment have been realized. Therefore, the quest for
sustainable sources of energy for the Indian economy and consumer societies
has become urgent in recent years (Prasad et al., 2014). Energy consumption
in India is increasing at 6.5% per year, while global petroleum reserves are
decreasing continually.
India’s share of crude oil production is about 1% of global crude oil

production, whereas consumption amounts to 3.1% of global consumption.
India imports 82% of its crude oil (fossil fuels) demand and aims to bring it
down to 67% by 2022 by replacing it with renewable energy and domestic
biofuels. India was the 3rd top net crude oil importer of 189 Mt in 2017 (BP
Statistical Review, 2018). Currently, the fossil resources are regarded as non-
sustainable and questionable from the economic, ecology and environmental
point of views.
The biofuels are found to be appropriate for such a transition for the
following reasons: “(i) Simplicity; (ii) production via well-known agricultural
technologies; (iii) potential for mitigation of global warming without
complete restructuring of the current working energy system; (iv) the use of
existing engines for their transportation; (v) potential to facilitate worldwide
mobilization around a standard set of regulations; (vi) potential as a directly
available energy source with real public acceptance; (vii) more uniform
distribution than the distributions of fossil fuel and nuclear resources; and
(viii) potential to create benefits for rural areas, including employment”
(Attal, et al., 2014). Biofuels have the potential to fulfill the energy needs and
contribution in energy security, mitigation of GHGs from the transport sector
and also in delivering rural economic development benefits (Winrock, 2009).
13.4 CATEGORIES OF BIOFUELS

The technologies to treat biomass for conversion into fuel (or energy) can be
logically placed into four general categories as described below:
13.4.1 First generation (1G) Biofuels

First generation biofuels are the biofuels made from sugar, starch, vegetable
oil, or animal fats using conventional or established technology. The primary
282 feedstock for the production of first generation biofuels are often sugarcane
Biofuels
juice or molasses, grains starch is fermented into bioethanol, or sunflower
seeds, which are pressed to produce an oil that can be converted to biodiesel
through a chemical process. Biodiesel can be produced from oil extracted
from seeds, animal fats, and used cooking oil. Biodiesel is commonly
produced through a process called transesterification which enable
transformation of triglycerides (fats and oils) into biodiesel and glycerin (Fig.
11.1). In other words, chemical reaction involving about 100 pounds of oil or
fatwith 10 pounds of alcohol (methanol or ethanol) in the presence of sodium
hydroxide or potassium hydroxide result in the production of about 100
pounds of biodiesel and 10 pounds of glycerin. The by-product of the
transesterification process viz. Glycerin is used for the synthesis of cosmetics
and pharmaceuticals(AFDC, 2019).
Fig. 11.1: First generation (1G) biofuels
Cane juice and molasses usually contain the appropriate amount of soluble
sugars, which can be directly fermented to produce ethanol (Fig. 13.1). At the
industrial scale, Saccharomyces cerevisiae has been used for fermentation of
hexoses from juice and molasses or other sugar-containing materials (Prasad
et al., 2007). It is the most efficient yeast strain, often used in the ethanol
production industry. The typical feature of this yeast is that it carries out
fermentation by Embden Meyerhof Parnas (EMP) pathway and can utilize
glucose. The end products of anaerobic fermentation of hexoses (glucose) are
two moles of ethanol (C2H5OH) and carbon dioxide (CO2) by glycolysis
(Ingram et al., 1998).
Grain starch is a viable raw material for ethanol production (Fig. 13.1).
Chemically, grain starch is a polymer of glucose, and yeast cannot use it
directly to ferment ethanol. Hence, grain starch must be hydrolyzed
(saccharification) by a combination of enzymes (amylase and
amyloglucosidase) into glucose, before its fermentation to produce ethanol
(Prasad et al., 2007). The process includes milling of grain first, then
enzymatic hydrolysis or saccharification of starch to liberate fermentable
sugar (glucose), and finally fermentation of sugar-containing stock by yeast
(Almodares et al., 2007).
283
Ecofriendly 13.4.1.1 Limitation of First Generation (1G) Biofuels
Bioprocesses
Feedstocks such as sugar, starch, vegetable oil are essential sources of human
foods in India. These feedstocks cannot be diverted entirely for the
generation of biofuels, because the demand for oils and sugars increased to
the point where these fuels started to compete with food feedstock, raising the
prices of arable crops (Seljak and Katrašnik, 2019). Foodstuff sources are not
sustainable for biofuel production in India (Prasad et al., 2012; Dutta et al.,
2014). To offset this adverse effect, further stimulation of 1st generation
biofuels is limited, and efforts are made to displace them by subsequent
generations of biofuels (Eggert and Greaker, 2014). However, significant
production capacities, particularly of biodiesel are expected to grow more,
because most of the non-edible seed-bearing trees seeds are not utilized
adequately and are going as wastes.
13.4.2 Second Generation (2G) Biofuels

The second generation biofuels are produced from the non-food crops,
including lignocellulosic biomass, especially crop-residue or waste biomass
in the form of stalks and straw of wheat, rice, corn, and other biomass
residues. Biomass feedstock can be converted into different fuels using a
range of processes to generate cost-effective biofuels production such as
biomethanol, Bio-DME, Fischer-tropsch diesel, biodiesel, mixed alcohols,
bio-oil and biohydrogen, syngas, biogas, etc.
Lignocellulosic biomass represents a significant fraction of the most

abundant plant material on the Earth. Typical examples of lignocellulosic
biomass are farm residues, remaining organic fraction of municipal solid
waste (MSW), industrial and urban residues, wastes, and dedicated non-food
energy crops grown on marginal land unsuitable for crop production as
feedstocks for the production of fuels. As shown in Fig. 13.2, the chief
fraction of such biomass, typically of the order of 35-50%, is a glucose
polymer known as cellulose. The next sizable fraction, of the order of 20-
35%, is hemicellulose.
Hemicellulose is also a polymer of sugars, but the types and distributions of

these sugars vary depending upon the particular biomass source. For many
types of lignocellulosic biomass, the five-carbon sugar xylose represents the
dominant fraction of the hemicellulose part. The third most significant
fraction, of the order of 15 to around 25%, is typically lignin, a phenyl-
propene polymer of complex components that cannot be broken down to
form sugar molecules. Some other compounds such as plant oils, proteins,
and ash make up the remaining fraction of the lignocellulosic biomass
structure (Charles E. Wyman, 1994).
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Biofuels
Fig. 13.2: Major fractions of lignocellulosic biomass materials
Source: Charles E. Wyman, 1994, Bioresource Technology 50 (1994) 3-16
Second-generation biofuels are produced from a range of feedstocks and

therefore may require various technologies to derive valuable energy/fuel
from them. The conversion process for the production of second-generation
biofuels is done according to two different approaches, generally referred to
as biochemical or thermochemical pathways (Lee and Lavoie, 2013).
Fig. 13.3. Schematic processes of the process of second-generation biofuels
13.4.2.1 Biochemical Pathway of Biomass to Biofuel
The biochemical pathway as shown in Fig. 13.3 involves four necessary steps
that convert lignocellulosic biomass to ethanol: (i) pretreatment of raw
biomass, (ii) enzymatic hydrolysis for fermentable sugar production (iii)
ethanol fermentation and (iv) distillation. The cellulosic biomass is first
pretreated to break down the material and generate more surface area for the
second step, hydrolysis. Hydrolysis is accomplished either by the use of
enzymes or chemicals. In this step, the complex carbohydrate chains in the
biomass are broken down into simple sugars.
285
Ecofriendly Finally, these sugars are fermented by microorganisms (yeast, or bacteria),
Bioprocesses
which produce ethanol in a dilute form. In order to concentrate the fuel-
grade anhydrous ethanol, distillation techniques are used. There are some
limitations to the processes, mostly from an economic point of view as the
price of the enzymes is forecasted to reach US$0.12 to US$0.20/L of ethanol
produced in 2015 (Mielenz, 2001). On the other hand, chemical processes
rely on rather inexpensive chemicals (e.g., sulfuric acid), although they have
to be recuperated at a low cost to keep the process economically viable.
Anaerobic digestion is a versatile, efficient and well-established method that

is being used worldwide to produce energy in the form of biogas. Organic
waste materials of agricultural, industrial and municipal origin can be
anaerobically digested or transformed into biogas by the action of rumen
microorganisms. This technology has the potential for the energy transition of
biomass to 'modern' fuels. Biogas technology is also helpful to improve the
air quality, as it is a cleaner fuel compared to the traditional fuels like wood
and fossil used for cooking, lighting, and heating in rural households.
13.4.2.2 Thermo-chemical Pathway of Biomass to Biofuel
The thermochemical approach covers specific procedures where biomass is

heated with a minimal amount of oxidizing agent. All methods in this
category lead to the conversion of biomass into three fractions: one solid
known as biochar, one liquid currently referred to as pyrolytic oil or bio-oil,
and one gas known as syngas, which is usually composed of carbon
monoxide, hydrogen, short-chain alkanes, and carbon dioxide. When
processed at low temperatures (250 to 350°C) without oxygen, biomass
undergoes a torrefaction process, and the primary conversion product is
biochar.
At higher temperatures (550 to 750°C), also without oxygen, the process is

known as pyrolysis (either fast or slow depending on the heat exchange rate
with the biomass) and the primary product is bio-oil. At higher temperatures
(750 to 1,200°C) and with limited inputs of oxygen, gasification occurs
producing mostly syngas with biochar and bio-oils as by-products. Thermal
processes are to a certain extent self-sufficient in terms of energy because the
energy required to heat the biomass up to the requested temperatures can be
supplied by the partial or total oxidation of carbon from the biomass,
reactions that are usually very exothermic (Lee and Lavoie, 2013).
Biochar, considered as a solid biofuel, is gaining much attention in the

pelletizing business, especially in parts of the world where lignocellulosic
biomass is rather inexpensive (Clarke and Preto, 2011). Nevertheless, for
transportation fuel, production of pyrolytic oil or syngas is usually considered
as more promising intermediaries. Pyrolytic oil is a liquid intermediary
which, to a certain extent, looks similar to petroleum but is very different
chemically. Therefore, to produce transportation fuel from this intermediary,
a second transformation must be made, which is a rather difficult task
286
Biofuels
because of the high water content as well as the corrosive nature of bio-oil.
Gasification, in contrast to pyrolysis, produces syngas, mostly composed of
single carbon compounds and hydrogen. Nevertheless, although the
production of transportation fuels is possible out of syngas, it relies on the
use of complex catalysts to induce the production of carbon-carbon bonds.
An example of such a process is the Fisher-Tropsh process (Jun et al., 2004;
Lee and Lavoie, 2013).
There are certain advantages of 2G biofuels. They can be produced on

degraded agricultural lands and processes are highly sustainable and ensure
stable fuel supply. The 2G biofuels do not contaminate the groundwater and
help maintain cleaner rivers. Economically, they provide more energy per
acre than food-based biofuels in India. India has a tremendous amount of
agriculture land area, so massive residues are produced here. This residue
contains the potential of biomass feedstock for the use of energy generation
(Prasad et al., 2007).
13.4.2.3 Limitation of Second Generation (2G) Biofuels
The capital costs of second-generation ethanol plants are much higher than
they are for the first generation plants. Again, detailed information is not
available, but the best estimates for the capital costs are three to five times
higher than for first generation technology plants. Some process
configurations may be even higher. The high capital costs combined with the
about the same operating expenses currently make these plants a less
attractive investment than the 1st generation plants. Thus, for the same return
on investment, these second-generation biofuels are still more expensive than
the existing first-generation biofuels production pathway.
13.4.3 Third Generation (3G) Biofuels

The third generation biofuels are biofuels produced from algae. Algal
biomass is a feedstock to reckon with, as they have potential to produce
biodiesel, bioethanol, biogas, biohydrogen, etc. Additionally, algal feedstock
find no competition from the food and feed production (Demirbas, 2007).
Production of biofuel processes or transformation ofalgal biomass into
biodiesel through transesterification (as shown Fig. 13.1) or conversion of
algal biomass into bioethanol and biohydrogen (fermentation), biogas
(anaerobic digestion), bio-oil (pyrolysis) and syngas (gasification)are shown
in Fig. 13.3.The interest in algae as a potential biofuel feedstock stems from
two main features. Many algae species are remarkably efficient at
photosynthesis, converting up to 6% of incident solar energy into biomass.
The production of algae for processing into biofuels has greater potentials in
India because of the availability of abundant solar energy. Further, high
growth rates and near continuous harvesting which is possible with algae
makes them an essential alternative energy resource for biofuel.
287
Ecofriendly 13.4.3.1 Limitation of Third Generation (3G) Biofuels
Bioprocesses
Biofuels from third-generation sources have limitations in terms of economic
performance, ecological-footprint, reliance on sunlight, geographic allocation
and so are inadequate to substitute for fossil fuels. They are cultivated in high
yields using bioreactors. It is estimated that microalgae could produce about
10 to 300 times more oil (for biodiesel) than traditional or dedicated energy
crops in the future (Dutta et al., 2014). However, the algal-based oil
production platform is technologically immature.
13.4.4 Fourth Generation (4G) Biofuels

Fourth generation biofuels are merely a step further from the third generation
biofuels. It is mostly based on metabolicengineering, a powerful tool to
improve biofuel production, either through genetic modification to increase
CO2 capture and lipid production as well as to develop low-input, fast-
growing energy crops with reduced fertilizers, insecticides, and water
requirements ortargeted alteration of metabolic pathways into a model
organism toward better biofuel yields. Many efforts have been made to
develop fast-growing, high yielding trees/crop and are also being designed
that sequester more CO2 and make the promise of carbon-negative biofuels.
The thermo-chemical conversion of biomass to biofuel coupled with carbon
capture and storage technologies are seen as sustainable and more eco-
friendly. The carbon-rich crops biomass can be converted into fuel and gases
using second generation techniques. In this way, fourth generation biofuels
are thought to contribute better to reducing greenhouse gas emissions, by
being more carbon neutral or even carbon negative compared to the other
generation biofuels.

1. State the reasons for adoption of biofuels as a sustainable strategy to
replace the fossil fuels?
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
2. What are the first generation and second generation biofuel feed stocks?
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
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Biofuels
13.5 POTENTIAL FOR BIOFUELS
Biofuels made from biomass are attracting increasing interest worldwide.
Biofuels have the potential to fulfill the energy needs and contribution in
energy security, diversifying energy sources, and mitigation of GHG
emissions from the transport sector and also in delivering rural economic
development benefits (Winrock, 2009). The first generation (1G) has specific
limitation and feedstocks used for biofuels especially food grains, sugars,
edible oil cannot be diverted entirely for biofuels production. However,
second (2G) and third-generation (3G) biofuel production technologies have
enormous potential, because they are produced from biomass feedstock
(residues and waste materials) and can be converted into varieties of biofuels
such as bioethanol, Bio-DME, Fischer-tropsch diesel, biodiesel, mixed
alcohols, bio-oil and biohydrogen, syngas and biogas, etc.
13.5.1 Ethanol Production Potential of Biomass

Bioethanol is considered a prospective substitute for fossil energy because of
its clean-burning characteristics and high octane value (Prasad et al., 2007).
Currently, ethanol has been promoted as an alternative automotive fuel,
because of its anti-knocking properties which help to increase octane ratings
and improve fuel efficiency (Prasad et al., 2014). Also, the use of biomass-
based transportation biofuels can help in reducing CO2 build up by recycling
CO2 that is liberated when ethanol is combusted as fuel (Hasunuma and
Kondo, 2012). The ethanol yields per acre from various feedstocks at several
locations is presented in Table 13.1.
Table 13.1:Ethanol yields by feedstocks gallons per acre
Feedstocks Ethanol yields (gallons per acre)

Maize (US) 401
Maize (China) 213
Sugar Cane (Brazil) 585
Sugar Cane (India) 483
Cassava (Nigeria) 158
Cellulosic Switchgrass 1150
Source: CSS, 2018
Global production peaked in 2017 after a dip in 2011 and 2012. The United
States (US) is the world's largest producer of ethanol, has produced nearly 16
billion gallons in 2017 alone (Table 13.2). Together, the U.S. and
Brazil produce 85% of the world's ethanol in 2016 (CSS, 2018). The reason
for the large volume of US production is, among others, the Renewable Fuel
Standard (RFS). In force since 2005 and enhanced and extended in 2007, the
standard requires transport fuels that are sold in the US to contain a specific,
annually increasing percentage of renewable transport fuels. The second
largest bioethanol-producing country is Brazil with an output of 7.06 billion 289
Ecofriendly gallons (Table 11.2). Brazil adopted its National Alcohol Programme in
Bioprocesses
response to the oil crisis in the seventies to decrease the country‘s
dependence on fossil-energy imports. In the EU-28, the directive on the
promotion of the use of biofuels or other renewable fuels for transport
(28/30/EG) and the Energy Tax Directive (30/96/EG) created the framework
conditions for European biodiesel and bioethanol production in 2003 (AFDC,
2019).
Table 13.2:Countries shares in Global Ethanol Production
Country/Region Billion gallons Countries shares

United States 15.80 58%
Brazil 7.06 26%
Europe 1.42 5%
China 0.88 3%
Rest of World 1.90 8%
Source: CSS, 2018
13.5.2 Biodiesel Production Potential of Biomass

There is strong energy demandwhich is bound to happen because of
differences in the economic sectors. The biodiesel is being looked at as the
best substitute for diesel fuel. It can be produced from new and used
vegetable oils, animal fats, and recycled grease. Its physical properties are
comparable to those of petroleum diesel, but it is a cleaner-burning renewable
alternative. Using biodiesel in place of petrol diesel significantly reduces
carbon emissions and toxic air pollutants from many on-road and off-road
applications. Biodiesel yieldsper acre from various feedstocks are presented
in Table 11.3. Microalgae have been recognized as potential sources
for biodiesel production because of its relatively high oil content in the form
of triglycerides, and its rapid biomass production. Kim et al. (2014) used
microalgae spp. Scenedesmus for biodiesel production through
transesterification and reported 55.07±2.18%, based on lipid by weight of
biodiesel conversion using NaOH as an alkaline catalyst than using H2SO4 as
48.41±0.21% of biodiesel production.
Algae are organisms that grow in aquatic environments and use light and
carbon dioxide (CO2) to create biomass. Many species of green algae like
Spirogyra and Chlorococcum contain a high level of polysaccharides.
Microalgae species C. vulgaris contain about 37% of starch on a dry weight
basis and found the best source for bioethanol production with 65%
conversion efficiency (Eshaq et al., 2010; Lam and Lee, 2012).Microalgae
are capable of all year round production using the non-agricultural land.
Therefore, oil productivity of microalgae cultures exceeds the yield of the
best oilseed crops, e.g., biodiesel yield of 5020 gallons per acre for
microalgae compared with 506 gallons per acre for oil palm and 110 gallons
290
Biofuels
per acre for rapeseed (Table 13.3). Oil content of microalgae is usually
between 20 to 50% (dry weight), while some strains can reach as high as 80%
(Spolaore et al., 2006). Algae also grow in aqueous media but need less water
than terrestrial crops, therefore, reducing the load on freshwater sources.
Table 13.3:Biodiesel yields by feedstock (gallons per acre)
Biodiesel Biodiesel yields (gallons per acre)

Soybean 59
Oil Palm 506
Rapeseed 110
Castor 140
Jatropha 170
Microalgae 5020
Source: CSS, 2018
In 2016, world biodiesel production amounted to just over 34 million tonnes.

By far the most crucial biodiesel producer is the European Union, which
accounts for almost 34 percent of global output. The EU‘s key feedstock is
rapeseed. On the American continent, biodiesel production is based on
soybeans. The most important American biodiesel producers are the US,
Brazil, and Argentina. Southeast Asia is gaining more and more critical in the
biodiesel market. In the vital palm oil producing countries - Indonesia and
Malaysia, biodiesel production is on a steady increase, driven by structural
glut and the associated pressure on prices in the vegetable oil markets.
Regional distributions of world biodiesel production and use in 2024 is
presented in Fig. 13.4.
Fig. 13.4. Regional distributions of world biodiesel production and use in 2024
Source: OECD/FAO (2015), “OECD-FAO Agricultural Outlook,” OECD Agriculture
statistics (database), http://dx.doi.org/10.1787/888933229645
13.5.3 Other Renewable Fuel Production Potential of Biomass

Most of the seaweeds are considered as an excellent feedstock for anaerobic
digestion and biogas production. Various algae species like Scenedesmus, 291
Ecofriendly Spirulina, Euglena, and Ulva has been reported as a good source and suitable
Bioprocesses
for biogas production. Sangeetha et al. (2011) reported the biogas production
from anaerobic digestion of green algal species Chaetomorpha litorea with
the generation of 80.5 liters of biogas per kg of dry biomass under 299 psi
pressure. Many species of algae can be used to produce biohydrogen through
different processes like biophotolysis and photofermentation. Park et al.
(2011) found red alga species Gelidium amansii produced 53.5 mL of H2
from 1 g of dry algae with a hydrogen production rate of 0.518 L H2 per g
VSS per day. Microalgal biomass is also suitable for bio-oil and syngas
production even better than the wood. Porphy and Farid (2012) successfully
produced bio-oil which composed of 50 wt% acetone, 30 wt% methyl ethyl
ketone from pyrolysis of algae (Nannochloropsis sp.) at 300°C after lipid
extraction. Similarly, Choi et al. (2014) reported 47% bio-oil yield from
brown algal species Saccharina japonica at a temperature of 450°C. Mass-
energy balance (inputs/outputs) of thermochemical energy production from
wood chips forest residues are presented in Table 11.4.
Table 13.4: Mass-energy balance (inputs/outputs) of wood chips forest

residues
Inputs (forest residue)/ output Amount (Kg/ha) Yield (%)

Inputs (wood chips) 81,000 ----
Outputs
Bio-oil 48,345 59.7
Syngas 10,496 13
Char 12,377 15.3
Ash 745 0.9
Water vapor loss during the 9.037 11.2
process
Total 81,000 100
Source:Steele et al. (2013). Life-Cycle Assessment of Pyrolysis Bio-Oil
13.6 LET US SUM UP

Biofuels hold the great potential to provide a more sustainable energy source
for the transport and other sectors if produced sensibly. It is a well-
established fact that biofuels offer several environmental benefits over fossil
fuels; howevercrop-based, 1G-biofuels have increasing concerns over
potential conflicts with food supplies and land protection.Biofuels produced
from non-food biomass using 2G-and-4G-biofuel technology has been
reviewed to contribute to the future energy supply mix, the cost is a
significant barrier to increasing commercial production in the near to medium
term. 4G-biofuel technology to produce these replacement fuels is still being
developed. There are many successful developments in the areas of enzyme
production and cellbiology related to generating more biofuels, and a range
292 of high-value co-products, that will further reduce the cost of biofuel
Biofuels
production and make it more competive to fossil fuels in the near future.
13.7 KEYWORDS
Biomass: The total mass of living organisms in a given area or volume; dead
plant material can be included as dead biomass.
First-generation manufactured biofuel: First-generation manufactured

biofuel is derived from grains, oilseeds, animal fats, and waste vegetable oils
with mature conversion technologies.
Second-generation biofuel: Second-generation biofuel uses non-traditional

biochemical and thermochemical conversion processes and feedstock mostly
derived from the lignocellulosic fractions of, for example, agricultural and
forestry residues, municipal solid waste, etc.
Third-generation biofuel: Third-generation biofuel would be derived from

feedstocks such as algae and energy crops by advanced processes still under
development.
13.8 SUGGESTED FURTHER READING /

REFERENCES
• AFDC (2019). Alternative Fuels Data Center, Biodiesel Production and
Distributionhttps://afdc.energy.gov/fuels/biodiesel_production.html
• Almodares, A., M.R. Hadi, M. Ranjbar, R. Taheri, 2007. The effects of
nitrogen treatment, cultivars and harvest stages on stalk yield and sugar
content in sweet sorghum, As. J. Plant Sci., 6, 423-426.
• Biopact
(2007)https://global.mongabay.com/news/bioenergy/2007/10/quick-
look-at-fourth-generation.html
• BP Statistical Review of world energy June 2018" (PDF). Retrieved 6
January 2019.
• Choi, J.H., Woo, H.C., and Suh, D.J. (2014). Pyrolysis of seaweeds for
bio-oil and biochar production. Chem. Eng. Trans. 37, 121–126.
• Clarke, S., & Preto, F. (2011). Biomass densification for energy
production. Guelph, Ontario, Canada: Ontario Ministry of Agriculture,
Food and Rural Affairs.
• CSS (2018). Center for Sustainable Systems, University of Michigan.
"Biofuels Factsheet." Pub. No. CSS08-09.http://css.umich.edu/sites/
default/files/Biofuels_Factsheet_CSS08-09_e2018.pdf
• Demirbas A. (2007). Progress and recent trends in biofuels. Prog. Energy
Combust. Sci. 33, 1–1810.1016/j.pecs.2006.06.001
• Dutta K, Daverey A, Lin JG. Evolution retrospective for alternative
fuels: First to the fourth generation. Renew Energy 2014; 69:114–22.
• Eggert, H., & Greaker, M. (2014). Promoting second generation biofuels:
does the first generation pave the road?. Energies, 7(7), 4430-4445.
293
Ecofriendly • Energy Statistics Report (2003) New Delhi: Central Statistical
Bioprocesses
Organization. Min. of Statistics and Programme Implementation
(MoSPI), GoI, New Delhi, India
• Eshaq, F.S., Ali, M.N., and Mohd, M.K. (2010). Spirogyra biomass a
renewable source for biofuel (bioethanol) production. Int. J. Eng. Sci.
Technol. 2, 7045–7054.
• Hasunuma, T., & Kondo, A. (2012). Development of yeast cell factories
for consolidated bioprocessing of lignocellulose to bioethanol through
cell surface engineering. Biotechnology advances, 30(6), 1207-1218.
• Ingram, L. O., Gomez, P. F., Lai, X., Moniruzzaman, M., Wood, B. E.,
Yomano, L. P., & York, S. W. (1998). Metabolic engineering of bacteria
for ethanol production. Biotechnology and bioengineering, 58(2‐3), 204-
214.
• Jun, K.W., Roh, H.S., Kim, K.S., Ryu, J.S., and Lee, K.W., 2004.
“Catalytic investigation of FTS from biomass derived syngas”, App. Cat.
A, 259, pp. 221-226.
• Kim, G.V., Choi, W.Y., Kang, D.H., Lee, S.Y., and Lee, H.Y. (2014).
Enhancement of biodiesel production from a marine alga, Scenedesmus
sp. Through in situ transesterification process associated with acidic
catalyst. Bio Med. Res. Int. 2014:391542
• Lam, M.K., and Lee, K.T. (2012). Microalgae biofuels: a critical review
of issues, problems, and the way forward. Biotechnol. Adv. 30, 630–690.
• Lee RA and Lavoie Jean-Michel (2013).From first- to third-generation
biofuels: Challenges of producing a commodity from biomass of
increasing complexity, Animal Frontiers, 3 (2):6-11.
• Leonardo D. Gomez Clare G. Steele‐King Simon J. McQueen‐Mason
(2008). Sustainable liquid biofuels from biomass: the writing's on the
walls New Phytologist 178, 3: 473-485.
• Mielenz J. 2001. Ethanol production from biomass: Technology and
commercialization status. Curr. Opin. Microbiol. 4: 324–329
• OECD/FAO (2015), “OECD-FAO Agricultural Outlook” OECD
Agriculture statistics (database), http://dx.doi.org/10.1787/ 88893322
9645
• Park, J.H., Yoon, J.J., Park, H.D., Kim, Y.J., Lim, D.J., and Kim,
S.H.(2011). Feasibility of biohydrogen production from
Gelidiumamansii. Int. J. Hydrogen Energy 36, 13997–14003
• Porphy, S.J., and Farid, M.M. (2012). Feasibility study for producing
biofuel and chemicals from marine microalgae Nannochloropsis
sp.basedonbasicmass and energy analysis. ISRN Renew. Energy.
2012:156824.
• Prasad, S., Amit, K., & Muralikrishna, K. S. (2014). Biofuels production:
a sustainable solution to combat climate change. Indian Journal of
Agricultural Sciences, 84(12), 1443-1452.
• Prasad, S., Dhanya, M. S., Gupta, N., & Kumar, A. (2012). Biofuels
from biomass: a sustainable alternative to energy and environment.
Biochem Cell Arch, 12(2), 255-260.
• Prasad, S., Singh, A., & Joshi, H. C. (2007). Ethanol as an alternative
294
fuel from agricultural, industrial and urban residues. Resources, Biofuels
Conservation and Recycling, 50(1), 1-39.
• Sangeetha, P., Babu, S., and Rangasamy, R. (2011).Potential of green
alga Chaeto- morphalitorea (Harvey) for biogas production. Int. J. Curr.
Sci. 1, 24–29.
• Seljak T, Katrašnik T: Emission reduction through highly oxygenated
viscous biofuels: Use of glycerol in a micro gas turbine, Energy 2019,
doi: 10.1016/j.energy.2018.12.095.
• Spolaore, P., C. Joannis-Cassan, E. Duran, and A. Isambert. 2006.
Commercial application of microalgae. Journal of Bioscience and
Bioengineering 101: 87–96.
• Steele, Philip, Maureen E. Puettmann, Venkata Kanthi Penmetsa, and
Jerome E. Cooper. L of Pyrolysis Bio-Oil Production
(2013).https://www.semanticscholar.org/paper/Life-Cycle-Assessment-
of-Pyrolysis-Bio-Oil
• Winrock (2009). The Impact of Expanding Biofuel Production on GHG
emissions, White paper #1: Accessing and interpreting existing data.
http://www.globalbioenergy.org/
uploads/media/0904_Winrock_International__White_paper__1_GHG_i
mplications_biofuel.pdf
• Wyman, C. E. (1994). Ethanol from lignocellulosic biomass: technology,
economics, and opportunities. Bioresource Technology, 50(1), 3-15.

1. The biofuels are considered as a sustainable strategy for the reasons that
include but not limited to production via well-known agricultural
technologies; huge potential for climate change mitigation; the use of
existing engines for their transportation; potential as a directly available
energy source with real public acceptance; potential to create benefits for
rural areas, including employment.
2. The primary feedstock for the production of first generation biofuels are
often sugarcane juice or molasses, grains starch is fermented into
bioethanol, or sunflower seeds, which are pressed to produce an oil that
can be converted to biodiesel through a chemical process. Biodiesel can
be produced from oil extracted from seeds, animal fats, and used cooking
oil.The second generation biofuels are produced from the non-food
crops, including lignocellulosic biomass, especially crop-residue or
waste biomass in the form of stalks and straw of wheat, rice, corn, and
other biomass residues.
295
Ecofriendly
Bioprocesses UNIT 14 BIOPLASTICS
Structure
14.1 Introduction
14.2 Objectives
14.3 What is Plastic?
14.4 Present Scenario of Plastics Production
14.5 Concerns over the increased use of plastics
14.6 Harmful Effect of Plastic
14.7 Bioplastic- A Sustainable Alternative to Plastic
14.8 Applications of Bioplastics
14.9 Main Group of Bioplastic
14.10 Advantages of Bioplastics
14.11 Challenges for Bioplastics
14.12 Keywords
14.13 Let Us Sum Up
14.14 References and Further Suggested Readings
14.1 INTRODUCTION
The need and demand for the development of bioplastics has been increased
immensely in the backdrop of environmental degradation caused by
petroleum-based plastics and the fast depletion of fossil resources.
Bioplastics are environmentally friendly in nature. They present a sustainable
alternative for waste management. Bioplastics not only provide an alternative
for discarded plastic waste and waste management but also provide a
potential substitute for the traditional “petro-based” polymers. Bioplastics
obtained from renewable resources represent a new generation of sustainable
plastics that reduces the impact on the environment both in terms of energy
consumption and the amount of greenhouse gas emissions. Dear learners in
this unit we have given an introduction of bioplastic, what are the advantages
and challenges of bioplastic.
14.2 OBJECTIVES
• explain harmful effect of plastic;

• define bioplastic;
• describe types of bioplastics;
296
Bioplastics
• discuss advantages and challenges of bioplastics.
14.3 WHAT IS PLASTIC?

Today, we are living in a “plastic age” to the extent that life without plastic
seems unimaginable. Plastics are an integral part of modern life. It is one of
the most versatile and relatively cheap materials invented by mankind. It is
are almost omnipresent, in our homes, offices, hospitals, in our modes of
transport, in our clothes, in our gadgets, from pharmaceutical to automobiles
as well as packaging for what we eat. The biggest increase in the use of
plastic has come in the packaging industry, from water bottles to plastic
layers in tea bags to plastic straws, glasses, plates and just about all that we
package for our consumption. Single-use plastics, often also referred to as
disposable plastics, are commonly used for plastic packaging and include
items intended to be used only once before they are thrown away or recycled.
These include, among other items, grocery bags, food packaging, bottles,
straws, containers, cups and cutlery. But we also wear it; we sleep on it; we
build our homes with it and we pipe our water and oil in it. Growing
mountains of non-biodegradable garbage mainly plastic is our nightmare. It is
choking our drains, our rivers and our streets.
What is Plastic:
Plastics belong to a group of molecules called polymers, which are large

molecules made of repeating units called monomers. Most plastics contain
between 500 and 20,000 or more repeating units. Plastics can be produced by
bonding together monomers in a reaction called polymerization. For example,
a plastic called polyethylene, which is commonly used in grocery bags and
packaging, is formed by adding together molecules of ethylene (C2H4).
Plastics have chemical and physical properties that allow them to be shaped
for multiple uses. Plastic is versatile, lightweight, flexible, moisture resistant,
strong, and relatively inexpensive. These beneficial qualities lead to an over-
consumption of plastic good. Plastic can be moulded in a variety of ways and
utilized in a wide range of applications. Unlike metals, plastics do not rust or
corrode.
There are basically two categories of plastics.
• First one is thermoplastics are a family of plastics that can be melted

when heated and hardened when cooled. These characteristics, which
lend the material its name, are reversible. That is, it can be reheated,
reshaped and frozen repeatedly. The most common Thermoplastics are:
PolyethyleneTerephtalate (PET), Polypropylene (PE), Low Density
Polyethylene (LDPE), High Density Polyethylene (HDPE), Polystyrene
(PS), Expanded polystyrene (EPS), Polyvinyl-chloride (PVC),
Polycarbonate, Polypropylene (PP); Polylactic acid (PLA) and
Polyhydroxyalkanoates (PHA).’’
297
Ecofriendly • Second type of plastics isthermosets. Thermosets are a family of plastics
Bioprocesses
that undergo a chemical change when heated, creating a three
dimensional network. After they are heated and formed, these plastics
cannot be re-melted and reformed. The most common Thermosets
are:Polyurethane (PUR), Phenolic resins, Silicone, Vinyl ester, Acrylic
resins, Urea-formaldehyde (UF) resins’.plastics do not biodegrade, but
instead they photodegrade, meaning that they slowly break down into
small fragments known as microplastics.
14.4 PRESENT SCENARIO OF PLASTICS

PRODUCTION
Plastics production increased from 15 million tonnes in the 1960s to more
than 350 million tonnes in 2018 and it is expected to triple by 2050, when it
would account for 20 percent of global annual oil consumption. Plastics use
has increased twenty fold in the past half-century and is expected to double
again in just the next 20 years. A primary reason the figures seem so alarming
is plastics packaging. Plastic packaging is and will remain the largest
application. currently, packaging represents 26 percent of the total volume of
plastics used. Besides this urbanisation and population growth had given rise
to new kind of Disposable products, designed to be thrown away after brief
use, constituted another new kind of trash.
Average per capita consumption of plastic in India is about 11kgs. An

estimate by the Ministry of Petroleum and Natural gas suggests that the
annual per capita consumption in India would be 20 kgs by 2022. As per
CPCB reports (2017), plastic contributes to 8% of the total solid waste, with
Delhi producing the maximum quantity followed by Kolkata and
Ahmedabad.Only 60% of the total plastic waste is being recycled.
Households generate maximum plastic waste, of which water and soft drink
bottles form a large number. In India, around 43% of manufactured plastics
are used for packaging purpose and most are of single use. Multi Layered
Plastics are categorized under either recyclable, energy recoverable or with
some other alternate use, but their recycling is an expensive process.
Focus has to be on mobilising the world to tackle plastic pollution, including
creating support for a global effort to eliminate single-use plastics along with
global regulation for the disposal of plastics. Government should make
sincere efforts to educate millions of people about the health and other risks
associated with the use and disposal of plastics, including pollution of our
oceans, water, and wildlife, and about the growing body of evidence that
decomposing plastics are creating serious global problems.
14.5 CONCERNS OVER THE INCREASED USE OF

PLASTICS
Plastic waste is ubiquitous and is reported from the Arctic to the Antarctic,
from the surface to sediment. It is estimated that nearly half of all plastic ever
298 manufactured has been manufactured since 2000. The world production of
Bioplastics
plastics has increased exponentially Many plastic products are designed for
single use without planning for the potential after-use pathways. Plastic waste
represents a single-use throw away culture. It is estimated that 40 percent of
plastics produced in the world is for packaging, used just once and then
discarded. In good old days, we used banana leaves to pack our food. Now
we use plastic sheets which are non bio-degradable. The throw-away culture
is suffocating Mother Earth. If we don’t change the way we produce and use
plastics, there will be more plastic than fish in our oceans by 2050. It is a
proven fact that plastic waste is adversely impacting ecosystems and human
beings. International beach clean-up data from the Ocean Conservancy shows
that plastic drinks bottles, food wrappers, plastic bottle caps, straws and
stirrers, plastic bags and plastic lids are amongst the top ten most common
items collected. All of these are single-use plastics.The crisis turns acute for
single-use plastics, largely used for packaging, almost 26 percent of plastics
produced. 95 percent of packaging plastic, after a ridiculously short use, gets
lost to the economy, It gets worse. 32 percent of single-use plastics escapes
the collection system, clogs urban infrastructure and ends up in the oceans,
disrupting a valuable natural production system. At the end of its lifetime, a
product or packaging is recycled, incinerated, landfilled, dumped in
uncontrolled sites, or littered in the environment. According to recent
estimates, 79% of the plastic waste ever produced now sits in landfills,
dumps or in the environment, while about 12% has been incinerated and only
9% has been recycled. If current consumption patterns and waste
management practices do not improve, by 2050 there will be about 12 billion
tons of plastic litter in landfills and the natural environment. Single-use
plastics end up littering the environment in part because of irresponsible
individual behavior. But poor waste management systems also play an
enormous role. Due to plastic’s versatility, light weight and relatively
inexpensive production, the use of single use plastic items has become
widespread. Unmanaged plastic waste has become a challenge for all
countries as it is contaminating our soil, air and water. Plastics are non-
biodegradable, they do not vanish from the face of the Earth. For example, it
may take more than 500 years for a plastic bag in a landfill to completely
break down.
14.6 HARMFUL EFFECT OF PLASTIC

Plastics in the environment pose significant hazards to wildlife both on land
and in the ocean. High concentrations of plastic materials, particularly plastic
bags, have been found blocking the breathing passages and stomachs of
hundreds of different species. Plastic bags in the ocean resemble jellyfish and
are often ingested by turtles and dolphins who mistake them for food. There
is emerging evidence that the toxic chemicals added during the
manufacturing process transfer from the ingested plastic into the animals’
tissues, eventually entering the food chain for humans as well. When plastic
breaks down into microplastic particles, it becomes even more difficult to
299
Ecofriendly detect and remove from the open oceans. Plastic bags can choke waterways
Bioprocesses
and exacerbate natural disasters. In 1988, poor drainage resulting from plastic
bag litter clogging drains contributed to devastating floods in Bangladesh,
causing several deaths as two-thirds of the country was submerged.
Styrofoam items contain toxic chemicals such as styrene and benzene. Both
are considered carcinogenic and can lead to additional health complications,
including adverse effects on the nervous, respiratory and reproductive
systems, and possibly on the kidneys and liver. Several studies have shown
that the toxins in Styrofoam containers can transfer to food and drinks, and
this risk seems to be accentuated when people reheat the food while still in
the container. In low-income regions, domestic waste - including plastics - is
often burnt for heating and/or cooking purposes, exposing largely women and
children to prolonged toxic emissions. Illegal disposal practices of plastics
often take the form of open burning, accentuating the release of toxic gases
that include furans and dioxins.
PET bottles after plastic bags, the most omnipresent single use plastics item
are not safe. PET bottles leach phthalates. They also leach antimony.
Phthalates disrupt the endocrine system. Antimony is a suspected carcinogen.
The longer a liquid, water or fruit juice, is left in a PET container the greater
the potential for release of toxins. Warm temperatures inside cars, garages
and enclosed storage areas increase the release of antimony into the liquid.
Significant amount of toxic heavy metals like copper, zinc, lead and cadmium
recovered from plastic wastes from sea shores have an adverse effect on the
coastal ecosystems. Lead and Cadmium pigments, commonly used in most
of the plastics as additives are hazardous in nature and are known to leach
out. Several GHG gases are emitted from the landfills. Among them, carbon
dioxide and methane constitute 90 to 98%.
Many are concerned about the impact that microplastics may have on bodies
of water due to their ubiquitous nature. Microplastics are the tiny plastic
fragments (less than five millimeters in diameter) that fall off decomposing
plastic bottles and bags. Manufacturers often produce consumer products,
like cosmetics, that include microplastics. Scientists have discovered
microplastics in most marine habitats around the world, particularly in lakes
and rivers. These microplastics interact with various marine species,
including birds, fish, and turtles, and they are readily ingested and
subsequently passed through food chains. The spread of microplastics will
inevitably continue to rise as society in-creases plastic production,
consumption, and disposal.
As previously mentioned, plastic waste and microplastics, if ingested by fish

or other marine life, can enter our food chain Microplastics have already been
found in common table salt and in both tap and bottled water. Although in
recent years research on the effects of microplastics has been growing, still
little is known about the exact impacts on human health. Stranded single-use
300 plastics create visual pollution and are increasingly becoming a priority
Bioplastics
especially in countries that rely heavily on tourism as a major source of GDP,
such as Small Island Developing States. For instance, Asia-Pacific Economic
Cooperation (APEC) estimated a $1.3 billion economic impact of marine
plastics to the tourism, fishing and shipping industries. Species living there
are affected Seabirds and fish mistake these toxic globules for food and
gobble them up. As do sea turtles.Over 90 percent of seabirds’ stomachs
contain plastics. Even plankton, one of the tiniest creatures in our oceans, are
eating microplastics and absorbing their toxins. The substance displaces
nutritive algae that creatures up the food chain require.
Plastics are usually made from petroleum, with the associated impacts in
terms of fossil fuel depletion but also climate change. The carbon embodied
in fossil resources is suddenly released to the atmosphere by degradation or
burning, hence contributing to global warming. This corresponds to about
400 million metric tonnes of CO2 per year worldwide, almost half of the total
greenhouse gases that Germany emitted to the atmosphere in 2017. It is
estimated that by 2050, plastics could already be responsible for 15% of the
global CO2 emissions.

Note: a) Write your answer in about 50 words.
b) Check your progress with the answers given at the end of this
unit.
1. Discuss the harmful effect of plastic?
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
14.7 BIOPLASTIC- A SUSTAINABLE

ALTERNATIVE TO PLASTIC
Bioplastics has emerged as a replacement for fossil fuel based plastics in way
back in 1980s. European Bioplastics, an organization for bioplastics, coined
the term “bioplastics”.The bioplastics gained currency on account of growing
plastic disposal crisis, environmental effects of plastics. Bioplastics are
biobased plastic and are broadly described as the plastic derived from the
plant material or the material which have ability to biodegrade into natural
compounds. These polymers can be completely converted by living
organisms, usually microorganisms, to carbon dioxide, water, and humic
material. Use of renewable resources like agricultural wastes and municipal
solid waste in place of fossil fuels, increasing costs of petroleum products and
environmental crisis due to petroleum based plastics place utmost need for
bioplastics. However, globally bioplastics account for only 1% of the total 301
Ecofriendly plastics production. There is indeed huge potential for upscaling bioplastics
Bioprocesses
production as there is growing awareness about the recycling of plastics,
technological breakthrough in bioplastics production and diversification of
feedstocks. Further, it is reported that global bioplastics production require a
meagre amount of arable land. Further, it is projected that the bioplastics
demand at the global level by 2025 may cross 0.4 million per year.
Bioplastics are plastics which are bio-based, biodegradable or both. Bio-

based plastics are produced from biomass, which helps to decrease our
dependence on fossil carbon resources (the feedstock for conventional
plastics). They can offer significant CO2 emissions savings compared to
conventional plastics. Biodegradable and compostable plastics, complying
with standards such as EN 134321 or NF T 51-8002 also contribute to the
circular economy, for example through improved end of life management
(organic recycling) and applications which can help facilitate efforts to
separately collect bio-waste and turning it into valuable resources
Bioplastics are biobased polymers with two sustainability concepts:

biodegradability and renewability. On the one hand, bioplastics that
biodegrade to CO2 and H2O in the environment can be produced, eg,
avoiding litter and damage to marine organisms. On the other hand,
renewable feedstocks instead of petroleum can be used, for instance, corn,
sugarcane, and algae, reducing dependence on crude oil and reducing the
impact on the climate. Currently, bioplastics have a market share of ∼1%, yet
they experience annual growth rates in excess of 20–30%. Important
bioplastics are TPS (thermoplastic starch), PLA (polylactic acid), PHAs
(polyhydroxyalkanoates), and bio‐PE, bio‐PP, and bio‐PET, which contain at
least some renewable carbon. Common bioplastics are starch, thermoplastic
starch (TPS), polylactic acid (PLA), polybutylene adipate terephthalate
(PBAT) and polyhydroxy butyrate (PHB). Common products are cutlery,
organic waste bags, shopping bags, flexible and rigid packaging, and
consumer goods. There also exist partly bio based “conventional” plastics
such as sugar-cane-derived polyethylene (PE), which are considered
bioplastics despite their non-biodegradability
Currently, conventional petroleum-based plastics such as polyethylene,

polypropylene (PP), and polyethylene terephthalate (PET) constitute more
than 95% of the plastics market. Because of the environmental degradation
caused by petroleum-based plastics and the depletion of fossil fuel resources,
there is, however, an increasing interest in bioplastics. Bioplastics are a
family of plastic materials that are either bio-based or biodegradable or
possess both properties. The term “bio-based” means the material is derived
from biomass such as corn, sugar cane, and wheat straw, whereas
biodegradation is a chemical process in which microorganisms present in
nature degrade the materials into natural substances such as water, carbon
dioxide, and compost without the addition of artificial additives.
Starting in the 1980s, bioplastics were introduced to reduce the use of

petroleum based plastics, because they possess a number of advantages
302
Bioplastics
including high resource efficiency, reduction of carbon footprint and
greenhouse gas emissions, and saving fossil resources. Nowadays, there are
numerous manufacturers producing bioplastics for a wide range of
applications including biomedical, packaging, consumer electronics,
automotive, textiles, and agricultural fields. Because of the growing demand
for a sustainable bio-economy, according to European Bioplastics the
production capacity for bioplastics has been increased from 1.5 million tons
in 2012 to 2.11 million tonnes in 2018, But as demand is rising, and with
more sophisticated biopolymers, applications, and products emerging, the
market is continuously growing. According to the market data compiled by
European Bioplastics in cooperation with the research institute nova Institute,
global bioplastics production capacity is set to increase from around 2.11
million tonnes in 2018 to approximately 2.62 million tonnes in 2023.
However, compared to conventional plastics, bioplastics accounted for only
less than 5% of the current market share because of their limited mechanical
properties and relatively high production costs. Therefore, the recent research
focuses on the improvement of bioplastics’.
Bioplastics are often promoted as a climate-friendly alternative to petroleum-

based plastics. Bioplastics are in principle climate-neutral since they are
based on renewable raw materials such as maize, wheat or sugar cane. These
plants get the CO2 that they need from the air through their leaves. Producing
bioplastics therefore consumes CO2, which compensates for the amount that
is later released at end-of-life. Overall, their net greenhouse gas balance is
assumed to be zero. Bioplastics are thus often consumed as an
environmentally friendly alternate
14.8 TYPOLOGY OF BIOPLASTICS

Though the concept of bioplastics is archaic, the disruptive technologies
paved way for novelties in production, diverse feedstocks, and applications in
wide array of sectors like agri-horticulture, medical sector, automobile,
electronics, etc. According to the Society of the Plastics Industry (SPI)
Bioplastics Council, bioplastics are defined as a plastic that is biodegradable,
has biobased content, or both. Bioplastics which are also known as
biopolymers, are classified based on the raw material used for the
production. Plastics can be grouped into four groups based on characteristic
features like biodegradability, etc. (European Bioplastics 2011).
• The first group comprises of plastics that are non-biodegradable and

produced from fossil fuels. This category includes polystyrene,
polyethylene, polyvinyl chloride, etc., which can be construed as
classical plastics.
• The second group includes biodegradable plastics, which are produced

from renewable resources. Since they are produced from carbon rich
biomass, these plastics are biodegradable. Common bioplastics formed
from renewable resources are starch blends, polyhydroxyalkanoate, etc.
303
Ecofriendly • Another group of plastics is biodegradable plastics, that are produced
Bioprocesses
from non-renewable resources. Such plastics are used in conjunction
with starch and other bioplastics. Such blending on account of
biodegradable properties aid in upscaling the “application-specific
performance” of starch. Plastics like polycaprolactone, polybuthylene
adipate terephthalate, and polybutylene succinate are grouped in this
category.
• Yet another group of plastics include non-biodegradable plastics that are

produced from renewable resources. Such plastics are produced from
biofuels and other renewable resources. First generation biofuels like
bioethanol that are produced from crops like sugarcane in Brazil, are
used to produce biopolyethylene. Plastics like polyvinyl chloride,
polypropylene, polyethylene terephthalate, etc., can also be produced
from renewable resources.
14.9 APPLICATIONS OF BIOPLASTICS

Bioplastics are used in an increasing number of markets, from packaging,
catering products, consumer electronics, automotive, agriculture/horticulture
and toys to textiles and a number of other segments. Packaging remains the
largest field of application for bioplastics with almost 65 percent (1.2 million
tonnes) of the total bioplastics. Bioplastics are used for disposable items like
packaging and catering items (crockery, pots, cutlery, straws, bowls) and also
often used for bags, trays, containers for fruit, vegetables, eggs and meat,
bottles for soft drinks and dairy products and blister foils for fruits and
vegetables. Nondisposable applications include mobile phone casings, carpet
fibres, and car interiors, fuel line and plastic pipe applications, and new
electroactive bioplastics are being developed that can be used to carry
electrical current. Medical implants made of PLA, which dissolve in the
body, save patients a second operation. Compostable mulch fi lms for
agriculture, already often produced from starch polymers, do not have to be
collected after use and can be left on the fields.
Biodegradable plastics, like conventional plastics, offer a large range of

packaging applications. Some of the packaging options include bags for
compost, agricultural foils, horticultures, nursery products, toys, and textiles.
Other areas include packaging for contact articles including disposable
cutlery, drinking cups, salad cups, plates, overwrap and lamination film,
straws, stirrers, lids and cups, plates and containers. Disposable housewares
made from biodegradable plastics are now seen as marketable options,
replacing traditional plastics such as polystyrene and polyolefin. Housewares
such as kitchen tools and utensils, washable storage containers and cups,
bathroom accessories, toys, hangers, and hooks are now being produced
using biodegradable plastics. RecentlyThe biodegradable plastic has been
developed by IIT-G’s Centre of Excellence-Sustainable Polymers.
304
Bioplastics
14.10 MAIN GROUP OF BIOPLASTIC
Some of the main groups of bioplastics are Cellulose-based plastics,
Thermoplastics, Polylactic Acid (PLA), Polyhydroxybutyrate (PHB), and
Polyamide. Cellulose-based plastics are usually produced from wood pulp
and used to make film-based products such as wrappers. Starch based plastics
are known as Thermoplastic. These account for about 50% of the bio-plastics
market. Pure starch’s ability to absorb humidity has led to it being widely
used for the production of drug capsules in the pharmaceutical sector.
Plasticizers, such as sorbitol and glycerine, are added to make it more flexible
and produce a range of characteristics. Polylactic acid (PLA) is produced
from the fermentation of starch from crops (commonly corn starch or
sugarcane), into lactic acid, that is then polymerised. Its blends are used for
wide range of applications including computer and mobile phone casings,
foil, biodegradable medical implants, moulds, tins, cups, bottles and other
packaging. Polyhydroxybutyrate (PHB) is used for packaging, ropes, bank
notes and car parts. It is a transparent film and biodegradable. PHB is
produced by enzymes in bacteria and genetically modified plants.Innovative
biopolymers such as PLA (polylactic acid) and PHAs (polyhydroxyal-
kanoates) are the main drivers of this growth in the field of bio-based,
biodegradable plastics. PHAs are an important polymer family that has been
in development for a while and that now finally enters the market at
commercial scale. These polyesters are 100 percent bio-based and
biodegradable, and feature a wide array of physical and mechanical
properties depending on their chemical composition. Production capacities of
PLA are also predicted to double by 2023 compared to 2018. PLA is a very
versatile material that features excellent barrier properties and is available in
high-performance PLA grades that are an excellent replacement for PS
(polystyrene) and PP (polypropylene).
14.11 ADVANTAGES OF BIOPLASTICS

The future of biodegradable plastics shows great potential. It is a sustainable
alternative to petroplastics.The helps in waste reduction Plastic makes up
around 13 percent of the waste stream, representing millions tons of waste.
While around 9 percent of that plastic goes into recycling programs, the
remainder enters landfills, where it takes up space for hundreds of years or
more. Biodegradable plastics, on the other hand, may break down over the
course of several months, depending on the materials involved and the
conditions of their disposal. While not every form of landfill-friendly
biodegradable plastic will completely break down, any reduction in the space
required to dispose of this material will ease pressures on the waste stream.
Bioplastics can be produced from plant starch, cellulose, lignin (wood), oils
and proteins. Like petroplastics, bioplastics are compounds constructed of
linked molecules that form long polymer chains (biopolymers). Most
bioplastics can be broken down in the environment by micro-organisms in a
process called biodegradation
305
Ecofriendly Biodegradable plastics also help conserve petroleum supplies. Traditional
Bioprocesses
plastic comes from heating and treating oil molecules until they turn into
polymers. Bioplastics come from natural sources, including crops like corn
etc. Production of bioplastics uses less energy than conventional plastics.
What makes bioplastic especially important is that petroleum oil plastics

increases tremendously the concentration of GHG in the atmosphere and its
stock will end in the near future. It is important for the global community to
have an alternative for the product derived from petroleum oil such as
plastics. Bioplastic also generates fewer greenhouse gasses and contains no
toxins. Bioplastics contribute clearly to the goal of mitigating GHG
emissions.
It has Potentially, a much lower carbon footprint. A plastic made from a

biological source sequesters the CO2 captured by the plant in the
photosynthesis process. If the resulting bioplastic degrades back into CO2
and water, this sequestration is reversed. But a permanent bioplastic, made to
be similar to polyethylene or other conventional plastics, stores the
CO2forever.
14.12 CHALLENGES FOR BIOPLASTICS

At the same time, bioplastics face critical challenges before they achieve
large scale market penetration.
• They are much more costly than conventional plastics. It is acclaimed

that bioplastics costs two times more than conventional plastics.
However, the amount of large-scale industrial production of bioplastics
which are more common in the future with the implementation of cost
reduction is expected.
• Often, bioplastics can only be composted in industrial, high temperature
composting facilities. Bioplastics are generally not accepted for recycling
with standard plastics, so mixed collections pose a problem. For instance,
a small fraction of PHB in PET can render the recycled material useless
for high value applications. Dedicated infrastructure for bioplastics
collection will be necessary, which is not in place yet. Bioplastic material
might actually contaminate the recycling process if not separated from
conventional plastics.
• The density of bioplastics is generally higher than that of polyolefins

(1.2–1.3 g/cm3 vs 0.9 g/cm3). Used in cars, bioplastics mean more
weight, resulting in higher fuel consumption,.
• Upstream technology is still evolving, and hence there are uncertainties

in technologiesand processes. Uncertainties are also present in post-use
processing and end of life options for bioplastics.
• Starch based bio plastics are manufactured generally from plants like
corn, potatoes and so on. This sets huge pressure on the agricultural
306 crops as they have to gratify the needs of the ever growing populace. To
craft out bio-plastics, crops have to be grown and this could lead to Bioplastics
deforestation. Bio plastics are commonly produced from crops like corn,
potatoes, and soybeans. These crops are often genetically modified to
advance their resistance to diseases, pests, insects etc. and increase their
yield. This exercise however holds a high risk to the environment as such
crops can be toxic for humans as well as for animals.
• Production of bioplastics is projected to increase to over But still, many
countries have not used any law or legislation about their production,
usage or waste management.

Note: a) Write your answer in about 50 words.
b) Check your progress with the answers given at the end of this unit.
1. Describe advantage and challenges of bioplastic?
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
14.13 KEYWORDS
Biodegradable: The term “biodegradable” is used to describe plastics and
materials able to degrade and break down into their individual molecules and
biomass through the use of living organisms (mainly microorganisms like
bacteria, fungi, algae or their enzymes).
Synthetic Polymers : Synthetic polymers simply mean “made by chemical

synthesis” or “man-made”, and can be derived from petroleum/fossil fuels or
bio-based materials (like corn), and once processed, can have the same
properties, including lack of ability to degrade, regardless of raw material.
Synthetic plastics frequently contain toxic additives to make them have
preferred qualities like being malleable, water resistant, heat resistant, and
more.
Bioplastic: The term “bioplastics” is used to describe both fossil fuel-derived

plastics that are biodegradable (they break down to some level at some point
in time- could even be thousands of years out), and biomass or renewable
resource-derived plastics (termed bio-based plastics).
Bio-derived Plastics: Bio-derived (or bio-based) plastics are plastics derived

from biomass or renewable sources, instead of fossil fuels. It’s preferred that
bio-based products are made from waste materials, as opposed to raw
materials, to prevent additional environmental stressors and land use change.
One of the most popular bio-based plastics is polylactide (PLA), which is
generally certified compostable 307
Ecofriendly
Bioprocesses 14.14 LET US SUM UP
Numerous types of plastics have been made. It is estimated that the plastic
industry size may double up to 800 million tons per year predicted by
2040.With landfills being no longer a viable option for their end-of-life
treatment in a circular economy, plastic recycling aiming at material recovery
is a necessity. It is of high importance for resource saving, thereby reducing
CO2 emissions. Bioplastics has emerged as a replacement for fossil fuel
based plastics in way back in 1980s. European Bioplastics, an organization
for bioplastics, coined the term “bioplastics”.The bioplastics gained currency
on account of growing plastic disposal crisis, environmental effects of
plastics. Bioplastics are biobased plastic and are broadly described as the
plastic derived from the plant material or the material which have ability to
biodegrade into natural compounds. Bioplastics are renewable and
sustainable alternatives to oil-based plastics. Currently，the main
opportunities for bioplastics are in packaging materials，but in future
bioplastics maybeusedmore in higher value applications（electronics and
vehicle parts. Implementation of the correct disposal methods and
corresponding infrastructure are vital if the bioplasticsindustry is to flourish
and deliver environmental benefits.
14.15 REFERENCES AND SUGGESTED FUTHER

READINGS
• OECD. The Application of Biotechnology to Industrial Sustainability - A
Primer. Paris: OECDPublishing. 2001. http://en.european-
bioplastics.org/about-us/.
• Srikanth Pilla，Handbook of Bioplastics and Biocomposites
Engineering Applications. Massachusetts: Wiley-Scrivener Publishing
LLC. 2011.
• Degnan T (2015) Bioplastics – Plenty of room to grow. Focus on
Catalysts 2015:1-2. doi:
• 10.1016/j.focat.2015.07.001.
• European Bioplastics (2011) Fact sheet. European Bioplastics. Accessed
at http://en.european-bioplastics.org/wp-content/uploads/2011/04/fs/
Bioplastics_eng.pdf.
• European Bioplastics (2016) Accessed at https://www.european-
bioplastics.org/
• Lackner M (2015) Bioplastics. Kirk-Othmer Encyclopedia of Chemical
Technology 1-41. doi:10.1002/0471238961.koe00006
• Rivero C, Hu Y, Kwan T, Webb C, Theodoropoulos C, Daoud W, Lin C
(2017) BioplasticsFrom Solid Waste. Current Developments in
Biotechnology and Bioengineering 1-26. doi:10.1016/b978-0-444-
308 63664-5.00001-0.
Bioplastics
• Ross G, Ross S, Tighe B (2017) Bioplastics. Brydson's Plastics
Materials 631-652. doi:10.1016/b978-0-323-35824-8.00023-2.
• Rujnić-Sokele M, Pilipović A (2017) Challenges and opportunities of
biodegradable plastics: A mini review. Waste Management &
Research 35:132-140. doi: 10.1177/0734242x16683272.
• Soroudi A, Jakubowicz I (2013) Recycling of bioplastics, their blends
and biocomposites: A review. European Polymer Journal 49:2839-2858.
doi: 10.1016/j.eurpolymj.2013.07.025.
• Gerngross, T.; Slater, S. How Green Are Green Plastics? Scientific
American, Aug 2000.
• https://docs.european-
bioplastics.org/publications/fs/EuBP_FS_What_are_bioplastics.pdf

Refer Section Number 14.11,14.12
309
Ecofriendly
Bioprocesses UNIT 15 BIOFERTILIZERS
Structure
15.1 Introduction
15.2 Objectives
15.3 What are biofertilizers?
15.4 Classification of Bioftilizers
15.4.1 Nitrogen fixing biofertilizers
15.4.1.1 Free living biofertilizers
15.4.1.2 Symbiotic biofertilizers
15.4.1.3 Associative symbiont
15.4.2 Phosphorus contributing biofertilizers
15.4.2.1 Phosphate solubilizers
15.4.2.2 Phosphate absorbers
15.4.3 Organix matter decomposers
15.5 Let Us Sum Up
15.6 Key Words
15.8 Answers to Check your Progress
15.1 INTRODUCTION
During the past four decades we have seen doubling of human population and
a concurrent doubling of food production. Increases in crop production have
been made possible through the use of commercial man-made fertilizers. The
increasing use of fertilizers and highly productive systems have also created
environmental problems such as deterioration of soil quality, surface water,
and groundwater, as well as air pollution, reduced biodiversity, and
suppressed ecosystem function. The fertility of soils from the chemical point
of view depends mainly on the presence in sufficient quantities of four
essential constituents of plant-food, viz., nitrogen, phosphoric acid, potash
and lime. If a soil contains 0.1 to 0.5% of nitrogen and 0.08 to -5% of
phosphoric acid, it may be classified as a good soil. Soil containing 1% of
potash or lime (the latter not as insoluble silicate but as carbonate) are to be
considered quite rich in these substances. The object of manuring is to give a
larger quantity of really available plant-food to growing crops and to help in
dissolving the plant-food of the soil, and thus augmenting its quantity. A
judiciously manured soil, also forest and pasture land, may go on getting
more and more fertile. So, few pounds of the chemical constituents of
manures are taken up ordinarily by crops, that it is easy to more than recoup
these by the use of proper manures. To ascertain, however, whether a
particular soil needs the addition of any one of these constituents, whether
nitrogen, phosphoric acid, potash or lime in the form of manure, or if it is
310
Biofertilizers
already sufficiently rich in this or that constituent, and it will be superfluous
to use one or another of the manures, it is not absolutely necessary to have
recourse to chemical analysis.
Use of chemical fertilizers is limited to rich farmers with certain drawbacks

like it depends on energy derived from fossil fuels, which are limited to use.
Many crops suffer potassium deficiency because of excessive use of nitrogen
based fertilizers, similarly excessive potassium treatment decrease available
nutritive food such as Vitamin C (ascorbic acid) etc. Use of ammonium based
fertilizers increase soil acidity, with decreased effect on succeeding crops. On
the other hand, biofertilizers are advantageous over chemical fertilizers due to
low cost, simple methodology of production, eco-friendly to agroecosystem
and considerable residual effect on succeeding crop.
15.2 OBJECTIVES
After studying this unit, you will be able to:
• describe the basics of biofertilizers

• explain Types of biofertilizers
• differenciate between biofertilizers and manures.
15.3 WHAT ARE BIOFERTILIZERS?

Microbiome mediated management of soil nutrients uptake is a very low cost
and eco-friendly approach. Moreover, the approach is reducing
environmental risks of getting contaminated with chemical based fertilizers.
There is an urgent need to address this issue of serious concern and which
can be effectively tackled by the application of biofertilizers.
Biofertilizers are especially defined fertilizers containing variety of microbes
to enhance plant nutrient uptake, which become easily accessible to plant root
hairs. Useful soil microbes can help plants to absorb nutrients efficiently.
Thus, the critical input in biofertilisers is the micro organisms. Microbes used
in the formulation of biofertilizers can be bacteria, fungus or algae. Microbes
typically used as biofertilizers are: nitrogen-fixing cyanobacteria (Anabaena),
nitrogen-fixing soil bacteria (Rhizobium, Azotobacter), AM (Arbuscular
mycorrhiza) fungi, and phosphate solubilizing bacteria (Pseudomonas sp.).
15.4 CLASSIFICATION OF BIOFERTILIZERS

To understand the classification of biofertilizers, we first need to understand
the type of microbe used in the formulation of biofertilizer. Based on the
nature of microbe used in making biofertilizer, they are classified as given
below:
311
Ecofriendly 15.4.1 Nitrogen fixing biofertilizers
Bioprocesses
Atmospheric air contains approximately 80 percent Nitrogen of total air we
breathe. Nitrogen being most abundant and ubiquitous in air, still it is a
limiting nutrient to plant growth because the atmospheric N can’t be directly
utilized by the plants. To cover up this, some bacteria are capable of fixing
atmospheric N2. Biofertilizers contain bacteria that can fix atmospheric N2.
Nitrogen fixation is defined as a process of converting di-nitrogen molecules
into ammonia (NH3). Such bacteria would associate themselves to root
nodules of leguminous plants, so as to easily carry out N2 fixation in the soil.
These bacteria associate with plants in various forms such as: free living,
symbiosis, close association. Nitrogen fixing biofertilizers are basically
classified on the type of bacteria used in the formulation of biofertilizer.
15.5.1.1 Free living biofertilizers
Many free living bacteria such as Azotobacter, Derxia, Rhodospirillum,

Rhodopseudomonas, Chromatium, Beijerinckia, Acetobacter, etc. are used in
the preparation of free living biofertilizer. They are saprotrophic anaerobes,
free-living soil bacteria, perform nitrogen fixation. Another example is
Frankia, which fixes atmospheric Nitrogen in non-host plants. So. Frankia
may be utilized for application in biofertilizers. Frankia is observed to find
around the rhizosphere of Betula pendula. It has been studied that free living
microbes such as Clostridium spp., Beijerinckia mobilis stimulated plant
growth in barley and cucumber, when used as biofertilizers. However, the
amount of N2 fixed by the free-living bacteria is difficult to estimate.
15.5.1.2 Symbiotic biofertilizers
The best known and most exploited symbiotic N2-fixing bacteria are those
belonging to the family Rhizobiaceae. To be used as biofertilizers, the
bacterial strain should have high N2-fixation rate and be able to compete with
the indigenous rhizobia to maximize infection of the target crops. Microbes
such as Bradyrhizobium, Mesorhizobium, Azorhizobium, Sinorhizobium,
Allorhizobium, Rhizobium, etc. are used to prepare symbiotic biofertilizers.
Rhizobium is one of the vital symbiotic nitrogen-fixing bacteria. Here
bacteria seek shelter and obtain food from plants. In return, they help by
providing fixed nitrogen to the plants. Blue-Green algae or Cyanobacteria
from the symbiotic association with several plants. Liverworts, cycad roots,
fern, and lichens are some of the nitrogen-fixing cyanobacteria. Anabaena is
found in leaf cavities of fern plants, responsible for nitrogen fixation. The
fern plants decay and release the same to be utilized by rice plants. From a
practical perspective, the inoculum must be easily produced and have a high
survivorship in field applications once inoculated on target seeds.
Inoculation of legumes to the field is a very old practice that is being carried
out more than 10 decades in agricultural sector in different parts of the world.
Also different soil types have varying densities of rhizobial symbiont, e.g.,
312 acidic soil have very low or no rhizobial symbiont while basic soil have low
Biofertilizers
density of rhizobial symbiont. Therefore, it is essential to know the kind of
soil and type of rhizobia for effective for inoculation. Prior to start a
commercial inoculation program, it is mandate to initially perform a low-cost
based pilot analysis. The rhizobial inoculum can per prepared as granular,
powdered or liquid form. Generally, granular formulations are preferred to
over others as they are easy to use and apply. The inoculums is used along
with a carrier, i.e., a carrier may be perlite, peat, charcoal, mineral soil etc. to
maximize the effect of inoculum in term of survival and infection rate.
15.5.1.3 Associative Symbiont
The associative symbiont bacteria mainly comprises from family Spirillaceae.

Azospirillum is a nitrogen-fixing bacteria that lives around the roots of higher
plants but do not develop direct association with plants. It is often termed as
rhizosphere association as these bacteria collect plant exudate and the same is
used as food by them. This process is termed associative mutualism.
Azospirillum improves plant growth by increasing mineral uptake, water
uptake ultimately increasing shoot and root growth. Species such as A.
lipoferum, A. brasilense, A. amazonense are commercially sold as nitrogen-
supplying biofertilizers. Other example of associative symbiont is
Herbaspirillum.

1. Classify biofertilizers.
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
15.5.2 Phosphorus Contributing Biofertilizers

Phosphorus contributing biofertilizers are formulation in which bacteria
solubilise the P present in soil to be readily available to plants.
15.5.2.1 Phosphate Solubilizers
Phosphate solubilizers or phosphorus-solubilizing bacteria (PSB) belong the

genus Bacillus and Pseudomonas. After N, P is the second most important
nutrients for plants. The P content of soil is generally high (0.05) but same
can not be utilized by the plants directly. The important role of these bacteria
is that, they have the capacity to mobilize phosphorus from soil to plants
through mineralization or solubilization. PSBs were introduced to agricultural
sector as phosphorus biofertilizer. Organic acids such as formic acid, acetic
acid, glycolic acid, lactic acid, fumaric acid, succinic acids are released by
313
Ecofriendly PSBs in soil to be taken up by plants. Sugarcane yield was reported to
Bioprocesses
increase by 12% on applying inoculums consist of Bacillus megaterium with
rock-phosphate. Agriculturists are preferring PSBs as inoculum in soil for
better plant growth and yield. Table 15.1 shows PSBs based biofertilizer
production unit in India and abroad.
Use of PSBs
• On an average use of PSBs in soil reduces phosphorus fertilizer

requirement by 25-30%.
• Use of PSB biofertilizer encourages more nutrients, water uptake and
faster root growth.
• Micronutrients such as Fe, Mg, Mn, B, Mo, Zn, Cu, become readily
available to plants.
• Provides better plant cell development so the plants become more
resilient to different environmental abiotic stresses.
• They are more compatible to other microbiomes associated with the
plants.
• It is an eco-friendly approach for better plant growth and yield for
sustainable food production in near future.
• PSBs can be applied to all crop plants such as oilseeds, paddy, millets,
vegetables and pulses.
Table 1: PSBs based biofertilizers manufacturer’s

Unit name Product Constituen Formulatio Reference
name ts n
Monarch Bio- Phosphobacte Bacteria Powder http://www.monarchbio.co.i

Fertilizers ria n/
and Research
Centre/India
TNAU Phosphobacte Two Powder/liqu https://agritech.tnau.ac.in/
Agritech ria and bacteria and id
Portal/ India Phosphatika two fungal
species
Agro Bio- Phosphobacte Bacillus Powder/liqu www.abtechbiofert.com
Tech ria megaterium id
Research var.
Centre phosphaticu
Ltd/India m
International Phosphofix Bacteria Liquid https://www.iplbiologicals.c
Panaacea om/
Limited/India
Novozymes Jumpstart Penicillium Powder/ https://www.novozymes.co
Biologicals LCO bilaii granular m/en
Limited/Cana
da
Ajay Bio- Biophos Bacteria Powder https://www.linkedin.com/
Tech (India) and fungi company/ajay-biotech-
Ltd/ India indialtd/?originalSubdomain
314
=fr Biofertilizers
AgriLife/Indi P Sol B-BM Bacillus Powder http://www.agrilife.in/

a megaterium
IARI, New IARI P. striata, Packed in
Delhi, India microphos B. polymyxa wood
and A. charcoal
awamori, A. and soil mix
niger, and
P. digitatum
15.5.2.2 Phosphate Absorbers
This type of biofertilizer consists of mycorrhizal fungi, these fungi are also
called as phosphate adsorbers. These fungi develop symbiotic association
with roots of crop plants such as wheat, rice, maize, potato. They form bridge
like structure between roots and the plants, extracting nutrients from plants
and supplying to plant roots. Generally, the mycorrhizae are of 2 types
depending upon the place they invade: if occurring on the surface of the host
plant they are called as ectomycorrhizae; if occurring inside the host plant
cell they are endomycorrhizae. For e.g., fungal hyphae of Glomeromycota sp.
penetrate upto root cortical cells and make highly branched structures called
arbuscules. The host plant is able to obtain essential nutrients especially P,
through fungal hyphae. P is readily absorbed by the soil which makes it
inaccessible to plants. In this case, the hyphae of ectomycorrhizae helps in
extending the root surface area and small diameter (20-50 micron) of hypahe
gets easy access to soil particles, which the roots can’t access. Therefore, a
plant roots with mycorrhizae have higher surface area and can absorb more
nutrients from soil as compared to non-mycorrhizal roots.
Now a days, there is an increase in the use of mycorrhizae based biofertilizers

for sustainable agriculture. For e.g., VAMRI (Vescicular Arbuscular
Mycorrhiza Root Inoculant) is a widely used biofertilizer prepared from
mixing Glomusmosseae/fasciculatum infecting dried corn roots. Different
crop plants such as tomato, onion, papaya, sugarcane, peanut, banana, egg
plant, watermelon, fruit crops etc.
15.5.3 Organic Matter Decomposers

Process of break down complex organic matter into its simplest form is called
as decomposition while the microbes/organisms involved in the process of
decomposition are called as decomposers. The sole purpose of decomposition
is recycling of nutrients back into the environment. Decomposition is carried
out for dead animal as well as plant matter in the ecosystem. Decomposers
are heterotrophic saprophytes and derive energy from dead organic matter
and occur at lower most position in the eco-pyramid.
Importance of decomposers
Disintegration of dead or decay matter
Provide balancing to nature through ecological cleansing.
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Ecofriendly Nutrients recycling into the environment
Bioprocesses
Types of decomposers
• Fungi- For e.g., Mushroom

• Insects- For e.g., dung beetles, maggots, ants, insects
• Invertebrates- For e.g., Earthworms and millipedes
• Bacteria-
Stages of decomposition
• Fragmentation- In this process large piece of mass are converted into

smaller pieces, so that decomposers mat act on it faster. This step is
basically carried out by detritivores.
• Leaching- Water soluble nutrients are dissolved by the water that
percolates through soil. The soil becomes enriched with these nutrients,
all this processes are termed as leaching.
• Catabolism- Once the water soluble nutrients are leached out, bacteria
and fungi present in the environment act on detritus by releasisng
enzymes and break them down further to more simpler molecules.
• Humification-It is defined as the process by which simple molecules
resultant of dead organic matter are further converted to dark coloured
colloidal, amorphous substance called as humus.
• Mineralization- It the process of further break down of humus by
microbes such as bacteria and fungi present in the soil, resulting into
release of inorganic substances in the soil.
Table 2: Differences between Manure and fertilizers are given below.
Manure Fertilizer
Manure is an organic substance that is

Fertilizers are inorganic substances
obtained from decomposition of
manufactured in factories.
vegetables and animal waste.
Manures are relatively less rich in plant Fertilizers are very rich in plant
nutrients, they only remove general nutrients like Nitrogen (N),
deficiency of soil. Phosphorus (P), Potassium (K)
They ensure healthy growth and

They add humus to soil by providing development of plants by providing
organic substances and nutrients. nutrients however do not add
humus to soil.
The addition of fertilizer to soil

The addition of manure to soil does not requires special guidelines like
require any special guideline. dose time and post addition
precautions should be followed to
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Biofertilizers
avoid mineral toxicity.
Fertilizers are readily soluble in

Manure is not readily soluble in water
water thus are rapidly absorbed by
thus it is absorbed by plants slowly.
plants.
Its excessive use causes water

It protects the environment and helps in
pollution and can not replenish
recycling farm waste.
organic matter of soil.
Note: Fertilizers provide faster and more growth of plants on the cost of its
quality and productivity while manures ensure slow and healthier growth of
plants along with maintaining humus of soil.
15.6 LET US SUM UP

This unit discusses about biofertilizers, types and their potential uses.
Biofertilizers are low cost agricultural input playing a significant role in
improving nutrient availability to the crop plants by colonizing the
rhizosphere or entering into the plant root cell, thereby increasing the nutrient
availability to host plant. Biofertilizers add nutrients to plant basically by the
process of nitrogen fixation, solubilization of Phosphorus, stimulating plant
growth related substances. Biofertilizers are classified, depending of the type
of microbes used in the formulation at the time preparation. For e.g., free
living biofertilizers, associative and symbiotic biofertilizers. Others are:
phosphate solubilizing biofertilizers, phosphate adsorbers etc. Different
industries have come which are producing biofertilizers by using one or more
microbes in their formulation. If used within proper limits, use of green
manure or biofertilizers would result into another green revolution.
15.7 KEY WORDS

Manure:Manure is an organic substance product derived mainly from animal
and plant waste products, generally classified into 2 main categories i.e.,
Bulky organic manure and concentrated organic manure. The former is
generally derived from plant and animal wastes such as cow dung etc., while
the latter is derived from blood meal, meat meal, oil cakes etc. The main use
of manure is to provide nutrients to plants when they are mixed with soil. To
rejuvenate the soil and a good crop, use of green leaf manures is mandatory.
While using manures, different parameters should be kept in mind such as
soil type, crop to be sown, time of sowing etc to get a good crop.
PSB: Phosphate solubilizers or phosphorus-solubilizing bacteria (PSB)

belong the genus Bacillus and Pseudomonas.
317
Ecofriendly
Bioprocesses 15.8 SUGGESTED FURTHER READING/
REFERENCES
Khan MS, Rizvi A, Ahmed B, Lee Jintae (2022) Phosphate biofertilizers:
Recent trends and new perspectives. Doi: 10.1016/B978-0-323-91595-
3.00002-1

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Mining and
UNIT 16 MINING AND BIOLEACHING Bioleaching
Structure
16.1 Introduction
16.2 Beginning of bioleaching process
16.3 Microorganisms in bioleaching
16.4 Operating factors that affect the process of bioleaching
16.5 Recovery of metals
16.6 Methods in mineral recovery
16.7 Commercial processes of Bioleaching
16.8 Recovery of Copper by dump leaching:
16.9 Uranium bioleaching
16.10 Microbial sorption in metal recovery
16.11 Oil recovery
16.12 Petroleum prospecting
16.13 Let us Sum Up
16.14 Suggested further reading/ References
16.15 Answers to check your Progress Exercise
16.1 INTRODUCTION
With the advent of industrialization as well as urbanization, there is an
exponential increase in the demand of industrially important minerals along
the exponential increase in world population. As the technology advances so
does the need for minerals of industrial importance. The high-grade deposits
of ores were easily available earlier as there were huge reserves however;
many of them are either depleted or soon be depleted with their present rate
of extraction and consumption. Hence, it becomes increasingly important to
find innovative and economical methods to recover such metals from lower-
grade deposits, which for technical or economic reasons have not been
extracted for usage. Various physicochemical and biological methods are
available and can play an important role in recovering of valuable minerals
from low-grade ores. Among biological methods, microbes are used in
recovering low-grade ores acting as biocatalyst in bio mining processes
meeting some of the needs of industrialized society. Such recovery processes
employ microbial metabolic activities to gain access to, rather than to
produce, desired products; in the form of soluble minerals or metals, this
process is called bioleaching. During bioleaching for the recovery of metals,
methods modify the physicochemical properties of metallic ore, so that
metals can be extracted. Bioleaching is unaffected by low concentrations of
the metals in the solution. Currently, biomining is at the forefront of the
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Ecofriendly accessible applied mining sciences. The techniques of biomining are
Bioprocesses
inexpensive, nontoxic and efficient. Moreover, the techniques are
environment friendly as bioleaching results in less air pollution and little
disruption to geological formations, and the microbes used are naturally
present. In short mining with microbe is both ecofriendly and economical.
Bioleaching (or biomining) is a biohydrometallurgy process that extracts

valuable metals from a low-grade ore with the help of microorganisms. In
other words, it is also defined as metal dissolution from their mineral
resources by certain naturally occurring microorganisms or their utilization to
change elements, so that when water is sifted through it, elemental extraction
from a material is possible. Bioleaching generally refers to the transformation
of metals via microorganisms into their water-soluble form. For instance,
copper sulfide (CuS2) is oxidized microbially to copper sulphate (CuSO4) in
case of copper (Cu), and metals are available in the fluid stage and remaining
solids are disposed off. Conversely, bio-oxidation” (a type of bioleaching),
defines the microbial oxidation of minerals, containing metal compounds of
interest. Subsequently, metals stay in solid deposits in concentrated form.
Additional terms such as bio-extraction, biomining, and bio-recovery are also
used to describe metal mobilization from solid materials interceded by
microorganisms or parasites or planktonic build-ups. Biomining, primarily
concerns the wide-ranging implementation of microbial courses for economic
metal regeneration in the mining sector.
Fig.16.1: Electron microscopic view of bacteria embedded with mineral ore during
bioleaching
16.2 BEGINNING OF BIOLEACHING PROCESS

Evidences shows that ancient people figured out, how to naturally recover the
bioleached Copper. For example, one model was found at the hour of the
Roman Empire, Spain, harking back to the eighteenth century, where
recouping of copper from acidic water has been proved.However, this
biotechnology development began in around 1950, after the isolation and
characterization of bacteria capable of leaching Copper. This fundamental
knowledge enabled the inception of understanding relationship between this
unique microbial action and Cu disintegration, and its capability as an
elective innovation for Copper recovery. Nowadays, this procedure is very
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Mining and
popular in various countries with several thousand tons of extraction in case Bioleaching
of Copper and other commercial metals. The extraction of metal sulfides was
first demonstrated with the mobilization of zinc from zinc sulfide (ZnS). It
was found that the transformation of ZnS to ZnSO4 was microbially
mediated. Later in 1947, Thiobacillus ferrooxidans, one the significant
organism in bioleaching methods was found in an acid mine drainage. A first
patent in this field of metal extraction was granted in 1958. Further
exploration in this field continues which led to the popularization of this
technique. Now biomining is used to recover lead (Pb), arsenic (As),
antimony (Sb), nickel (Ni), molybdenum (Mo), gold (Au), silver (Ag) and
cobalt (Co) in various countries esp. in India, China, Chile, South Africa,
Australia, Iran, Mexico, and the United States.
16.3 MICROORGANISMS IN BIOLEACHING

Bioleaching includes various ferrous iron and sulfur oxidizing bacteria,
including one of the most commonly applied Acidithiobacillus ferrooxidans
and Acidithiobacillus (formerly called Thiobacillus). It is a bacterium that is a
non-spore forming, motile, rod-shaped and gram-negative. It is a chemo-
lithotrophic bacterium which derives growth energy from sulfur or iron
oxidation. It oxidizes ferrous iron (Fe+2) into ferric form (Fe+3), and convert
soluble or insoluble sulfides, thiosulfate to sulfate (SO2-4).
T.ferrooxidans and T. thiooxidans, are synergistic bacteria and improves the

efficiency of metal extraction from the ores when put together. Mixture of
Leptospirillum ferrooxidans and Thiobacillus organoparpus can efficiently
degrade pyrite (FeS2) and chalcopyrite (CuFeS2) to extract minerals of
interest. Besides this, the other microbes used in bioleaching process are
Sulfolobus acidocaldarius and S. brierlevi. These are thermophilic and
acidophilic bacteria which grow in acidic hot springs and often used to
extract Mo and Cu respectively from molybdenite (MoS2) and CuFeS2.
Pseudomonas aeruginosais also used in mining low grade Uranium (0.02%)
ores. Among fungal strains Aspergillus niger can extract Cu and Ni while
Aspergillus oryzae is employed for the extraction of Au. Rhizopus arrhizus is
known to extract Uranium (U) from waste water. Bioleaching, in nutshell is
the oxidative sulfide mineral solubilization interceded by microbial activity.
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Ecofriendly Chemolithotrophs, a group of microbes specifically use inorganic reduced
Bioprocesses
compounds as a source of energy. These organisms are regularly utilized as
acidophilic bioleaching microorganisms. They utilize Fe+2 or reduced sulfur
composites as vitality source and commonly found chemical elements in ores,
especially in Cu ores. The generation of ferric ion by the activity of
microbes, is a strong Cu2S oxidant, leading to the deliverance of metal into
the solution. Reduced sulfur compounds are oxidized to sulfuric acid (H2SO4)
via microbes during ferric attacks, upholding low pH, which is vital for
acidophiles and the solubility of ferric iron. Microorganisms re-oxidize
reduced iron, produced during mineral attacks at the end of the cycle.
16.4 OPERATING FACTORS THAT AFFECT

THE PROCESS OF BIOLEACHING
Microorganisms are undoubtedly, the primary performers in bioleaching.
Since pile bioleaching is not conducted under aseptic settings, the process is
taken care of by the mine site's in-situ mixed microbial consortia. A
microbial consortium is a group of two or more organisms living together. A
wide variety of microbes generally bacteria and archaea are available during
heap bioleaching process. .Acidithiobacillus ferrooxidans, Leptospirillum
ferrooxidans, and Acidithiobacillus thiooxidans are some of the most
important and first recognized bioleaching bacteria. These microorganisms
need nutrients for growth and to bioleach, which they must discover in their
environment. A significant number of nutrients become accessible from
similar ores and leaching solution, however, some of them like phosphorus
and nitrogen may turn out to be rare and influence the bioleaching procedure.
If economically practicable, these nutrients need to be supplemented to the
leaching solution.
Bioleaching is profoundly influenced by biological, physicochemical, and

environmental parameters, which in turn influence the yield of metal
extraction. Two gaseous components are needed by the bioleaching
microorganisms: carbon dioxide as an energy/carbon source, and oxygen as
an electron acceptor. They must be shifted from gaseous phase to leaching
solution, to become accessible to microorganisms. This implies that their
accessibility will rely on the bioleaching framework's mass exchange
characteristics.
Environmental factors like oxidation-reduction potential (ORP), pH, and

temperature are significant operating conditions for microorganisms and
sulfide oxidation done by ferric iron. Low pH (1-2) favors both the processes.
While as, microbial tolerance fixes an upper limit in the event of temperature
which obviously doesn't favor the sulfide ferric oxidation. High temperature
loving organisms have appeared to get greater percentage of extraction than
medium and low temperature organisms. While in ORP case, microbial
action tend to increase the leaching solution as bioleaching continues. The
322 speed of bioleaching process is more dependent on the kind of metal sulfide.
Mining and
Generally primary Cu minerals (CuFeS2, Enargite) are considerably harder to Bioleaching
solubilize than the secondary ones (Chalcocite, Covellite, Bornite). Particle
size influences the efficiency and degree of leaching in any extraction
operation. The rate and stretch out of sulfide oxidation will rely upon the
surface region presented to the leaching fluid. In turn, due to intensive
smashing of the ore, elevated interface area is acquired. Diffusion phenomena
control the solubilization with an enormous particle size. Initial bioleaching
efforts were carried out in dump bioleaching mode, described by an
exceptionally dissimilar solid bed, established by rocks as large as 1 m in
distance across. In this context, the principle working factors are not
controlled, resulting in low productivity. Heap is an improved and better
framework, where homogeneous arrangement significantly increases the
chances of bioleaching and the efficiency of the whole process. These are the
contentions, why on industrial scale, it is utmost important. An outrageous
circumstance is the utilization of unsettled reactors, where it is conceivable to
apply tight compliance over temperature, accessibility of gaseous component,
pH, and ORP. The effectiveness of bioleaching relies mainly on the expertise
of microorganisms and the structure of mineralogical and chemical ores.
Maximum metal extraction can be acquired only when leaching conditions
are an optimal range of bacterial development conditions. Bioleaching factors
and their related impacted have been listed in Table. 1
Table 16.1. Bioleaching factors and their impacts (adapted from Wasim and
group, 2019 )
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Ecofriendly
Bioprocesses 16.5 RECOVERY OF METALS
Bioleaching process is used to recover the metals from the ore’s that are
unsuitable for direct smelting because of their low-grade content. Under ideal
laboratory conditions, nearly 97% recovery of Cu from the ores takes place
by bioleaching, which is seldom attained in actual mining methods. Even 50
to 70% recovery of copper by bioleaching from an ore that would otherwise
be completely unproductive would be an important achievement.
If the ore establishment is enough porous and over layers a water-

impermeable stratum, the mineral can be leached in situ without first mining
it. An appropriate boreholes pattern is created, with some holes used for
leaching liquor injection and others used for leachate recovery. More often,
though, this bioleaching method is accomplished after mining, splitting, and
piling of the material in heaps on a water-resistant surface or on a specially
constructed apron. Water is then pumped to the top of the heap and runs
down through the ore of the apron. The leaching water and ore usually
supplies enough dissolved mineral nutrients to satisfy the needs of T.
ferroxidans, but in some cases, minerals such as ammonia and phosphate
must be added. In most of these bioleaching operations, the leached metal is
then extracted with an organic solvent and subsequently removed by
stripping from the solvent. The leaching liquor and the solvent are recycled.
The characteristics of the ore have an important effect on its susceptibility to

bioleaching. The rate of leaching is determined in large part by the size of the
mineral particles. Increasing the surface area, accomplished by crushing
and/or grinding, generally increases bioleaching however, must also be
conductive for efficient leaching to occur. Optimal conditions for bioleaching
use T. ferroxidans area temperature of 30 to 50 °C, a pH of 2.3 to 2.4, and an
iron concentration of 2 to 4 g/L of leach liquor. Available oxygen and
nutrients such as ammonium, nitrogen, phosphorus, sulfate and magnesium
are essential for the growth of T. ferroxidans.
The oxidative activities of Thiobacilli can produce high temperature in some

mineral deposits and may increase the tolerance limits of the species being
used. Obviously, this would lead to decreased bioleaching activity and
mineral production. Because of these high temperatures, thermophilic sulfur-
oxidizing microorganisms may be useful for some bioleaching processes.
Members of the genus Sulfolobus are obligate thermophiles that oxidizes Fe+2
and sulfur in a way, like that of the members of the genus Thiobacillus. These
acid-tolerant thermophilic bacteria can oxidize inorganic substrates and are
used in the bioleaching of metallic sulfides. Sulfolobus has been used for
MoS2 (Molybdenum sulfide) bioleaching, whereas Thiobacillus is not
tolerant to high Mo, mercury, and Ag concentrations. Two methods of sulfide
mineral oxidation have been proposed and relay on its composition. The
suggested pathway for thiosulfate oxidizes species like FeS2, MoS2, and
tungstenite (WS2), while sphalerite ((Zn, Fe) S), CuFeS2, arsenopyrite
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Mining and
(FeAsS), and galena (PbS) are oxidized by means of polysulfide pathway. Bioleaching
16.6 METHODS IN MINERAL RECOVERY

Methods play a key role in the leaching of metals from mineral bearing rocks
but their activities were discovered only recently. Thiobacillus ferroxidans
was isolated from coalmine drainage in 1947 and was subsequently
associated with most of the natural and artificial leaching sites. Methods are
used for recovery of minerals from low-grade ores and they do metal
leaching without polluting the atmosphere. Besides T. ferroxidans, other
acidophilic chemolithotrophic bacteria that are believed to be important to the
leaching processes are T. thioxidans, Leptospirillum, ferroxidans and species
belonging to genus sulphobus.T. ferroxidans is a small gram-positive straight
rod-shaped bacterium. It grows best in acidic solution at pH range 1.5-2.5
with an optimal temperature range of 10-30 °C and upper limit of 37 °C. It
obtains energy by the oxidation of ferrous to ferric form and the reduced form
of sulfur to H2SO4 using oxygen as a terminal electron acceptor. It uses CO2
as a carbon source. Other thiobacillus sp. that also plays a role in leaching
include T. thioxidans, T. acidophilus and T. organoborous. Many commercial
minerals of utilities are MS (metal sulphite) that are extremely insoluble.
Microbial activity causes solubilization of metals from their ores either by
direct leaching or by indirect leaching. These methods can be employed
together for efficient recovery of the metals (Fig.2).
Direct leaching:
In this process, the microbe act directly on the ore to extract metal. T.
ferroxidans become attached to mineral particles. Enzymes associated with
the cell walls catalyze oxidative attack on crystal lattice of the MS and
oxidation of mineral occurs in two steps.
Cu S + 0.5 O2 + 2H+ → Cu+2 + So + H2O
So + 1.5 O2 + H2O → H2SO4
T. thioxidans and T. ferroxidans cooperate in leaching of sulphite mineral.

When sulfide minerals are oxidized to Cu+2, the So (elemental sulfur) is
formed as a byproduct and coats the (covering Cu) remaining mineral
particles and limit the further access of Cupric to mineral sulfide.T.
thioxidans attacks this “passivating” sulfur layer and enhances the leaching
process by exposing the mineral surface and by generating H2SO4.
Leptospirillum ferroxidans is somewhat more acidophilic than T. ferroxidans
and grows at pH 1.2 on FeS2 and at temperature up to 40 °C. Archaebacteria
of genus sulphobolus may also contribute to the leaching process; the
organisms grow autotrophically at pH 1-3 and at temperature ranging 50-90
°C.
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Ecofriendly Indirect leaching:
Bioprocesses
In this process the microbe produce certain substances or oxidizing agents
such as ferric iron or sulphuric acid which solubilize the metal for extraction.
For indirect leaching acidic environment is important to extract metals. It
depends on the ability of various species of acidophilic sp. like Thiobacillus
ferroxidans to generate metabolic energy by oxidizing ferrous or sulfide
leading to ferric sulphate production according to following equation:
FeS2 + 3.5 O2 + H2O → FeSO4 + H2SO4

2FeSO4 + 0.5 O2 + H2SO4 → Fe2 (SO4)3 + H2O
Ferric sulphate is a potent oxidizing agent capable of dissolving many

essential minerals of copper sulfide.
CuFeS2 + 2Fe2 (SO4)3 → CuSO4 + 5FeSO4 + 2So
(Chalcopyrite)
Cu2S + 2Fe2 (SO4)3 → 2CuSO4 + 4 FeSO4 + So
(Chalcocite)
Cu5FeS4 + 6Fe2 (SO4)3 → 5CuSO4 + 13FeSO4 + 4So
(Bornite)
Leaching by ferric sulphate is called as indirect because it is independent of

presence of oxygen or microbial activity; an acidic pH is required for dump
leaching. In its absence, the oxidation of ferrous is very slow and hence
mineral leaching would be very slow. Thus, the indirect leaching means that
the microbial activity supply necessary conditions and efficiency to the
process. T. ferroxidans also derive energy by oxidizing sulfur generated in
the process and give H2SO4.
2So + 3O2 + 2H2O → 2H2SO4
H2SO4 maintains low pH that is optimal for acidophilic T. ferroxidans and
suppresses the loss of ferric sulphate by hydrolysis.
Fe2 (SO4)3 + 2H2O → 2Fe (OH)SO4 + H2SO4
H2SO4also leaches several copper oxide minerals for eg:
Cu3 (OH)2(CO3)2 + 3H2SO4 → 2CuSO4 + 2CO2 + 4H2O
(Azurite)
Fig. 16.2: The Indirect and direct mechanism of bioleaching
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Mining and
16.7 COMMERCIAL PROCESSES OF Bioleaching
BIOLEACHING
The natural process of bioleaching is very slow. In order to increase the
efficiency of the process, various methods are used commercially for
maximum extraction of the minerals. The type of resource is the main factor
that decides about the process involved in bioleaching. Generally three main
methods are used according to the ore to be processed (Fig. 3).
a) Slope/ Dump Leaching:
It is one of the most commonest and cheap method in which the finely
powdered ore are made into large piles along the slopes of a range, and
water containing microbes (Thiobacillus) is continuously sprinkled over
the slope. The collected water at the bottom is used to extract the metals.
After extraction, the microbial population is regenerated in an oxidation
pond.
b) Heap Leaching:
This method is used to extract low grade minerals from ore. The
powdered ore is arranged in a big heap on an impervious natural surface,
and then the same process of metal leaching is followed as in case of
slope leaching.
c) In-Situ Leaching:
This process takes place at the point of generation of ore, hence called as
in-situ process. The ore is exposed through sub surface blasting and
passages for acidic water are drilled through this ore. The acidic water is
pumped along with the microbe (Thiobacillus sp.) through these
passages. A pit is made at the bottom of the ore surface to collect this
percolating water. This water rich in minerals is pumped out for
extraction of desired minerals. After extraction the water is reused for
generation of microbial species.
Fig. 16.3: Commercial processes of bioleaching (a) Slope (b) Heap (c) In-situ
Case studies:
Copper bioleaching
Copper, an element with high thermal conductivity and ductility has always
been in elevated demand for electricity, construction, transportation and other
industries. With its increased demand there has always been a felt a limited
327
Ecofriendly supply of this resource hence, bioleaching has been commonly applied to
Bioprocesses
extract this mineral from low grade ores in many countries like the United
States, Australia, Canada, Mexico, South Africa and Japan. The United States
alone produces 10% of total copper through bioleaching. Types of copper
ores used in bioleaching processes are Covellite, chalcocite and Chalcopyrite.
These ores also contain fractions of other elements such as Chalcopyrite
contains 26% copper, 25.9% iron, 20.5% zinc and 33% sulphur. Thiobacillus
ferrooxidans oxidizes insoluble CuFeS2 and transforms it into soluble copper
sulfate (CuSO4). Sulphuric acid, a byproduct produced in this reaction,
maintains an acidic environment (low pH) necessary for microbial growth.
During the oxidation of Chalcopyrite the following reaction occurs:
2CuFeS2 + 8 ½ O2 + H2SO4 → 2 CuSO4 + Fe2 (SO4)3 + H2O
Similarly covellite is oxidized to copper sulphate:
CuS + 2O2 → CuSO4
In copper leaching processes, the action of Thiobacillus includes both direct

and indirect oxidation of CuS via generation of ferric ions from ferrous
sulfide (FeS). FeS is present in most of the important copper ores, like
CuFeS2. Copper is recovered by solvent extraction or by using scrap iron. In
the latter case, Cu replaces Fe according to the equation:
CuSO4 + Fe0 → Cu0 + FeSO4
16.8 RECOVERY OF COPPER BY DUMP

LEACHING:
Copper ore containing more than 0.5 % of Cu is prone to smelting whereas
Cu in lower grade ore is recovered by dump or heap leaching. A method in
which broken rocks are piled 100 or more feet high on an impermeable
surface and water. The same water is repeatedly circulated and recirculated
through pile of the rock. Same time FeS2 oxidize causes ore to become
strongly acidic and rich in ferric sulphate. This water slowly percolates down
through the pile and creates the applicable conditions for the growth of T.
ferroxidans within the pile.
The effluent becomes progressively enriched in metals such as Cu. Finally

the metal rich effluent is pumped in to a basin called as launder and iron
scraps are added to precipitate the copper by following eq.:
Cu+2 + Fe0 → Cu0 + Fe+2

This Fe+2 rich solution is transferred to shallow oxidation ponds when T.
ferroxidans rapidly oxidizes ferrous to ferric and forms some additional
H2SO4 by oxidation of sulfur compounds. Much of Fe+3 formed in oxidation
ponds precipitate as Fe(OH)3. The acidic supernatant Fe2(SO4)3 solution is
then pumped back to top of dump. A dump can be view as a continuous flow
reactor in which solubilization of metal is performed by bacteria attached to
ore particles.
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Mining and
The bacteria thus play roles at two places: Bioleaching
(i) in the leached dump

(ii) in oxidation pond
At present, large-scale Cu bioleaching is mostly conducted by heaps

percolation. Minerals are squashed to a particle size of about 1 cm or more in
few smashing stages, healed with diluted H2SO4 and agglomerated before
stacking into tiny mechanically resistant spheres. Using drip irrigation or
sprinkling, an acid solution is applied over heaps and rehashed many times as
needed to get the ideal extraction and concentration of Cu. To facilitate
percolation of leaching solution, and flow of upcoming gas inoculated at
bottom, it is critical to construct a homogenous heap with a high void portion
to provide necessary oxygen and CO2. Loaded liquor at that point enters the
recovery segment, comprising of solvent extraction unit, which purifies and
concentrates fluid in Cu and Cu recuperation by means of electrowinning.
The duration of leaching may last two months or more. The schematic
diagram of a typical Cu heap bioleaching operation is shown in Fig.16. 4.
Fig. 16.4: The schematic diagram of a copper heap bioleaching operation (adapted
from Gentina, 2013)
16.9 URANIUM BIOLEACHING

Uranium (U) bioleaching is commonly used in Canada, the United States,
India and many other nations. This process helps to recover U from low
grade ores (0.01 to 0.5% U) and low-grade nuclear wastes. The recovery of
329
Ecofriendly Uranium, a fuel required by the nuclear power generation industry, can easily
Bioprocesses
be enhanced by microbial activities. The microbial recovery of Uranium from
otherwise useless low-grade ores is helpful in overcoming the international
energy shortage. Nuclear safety and waste disposal problems, as well as the
limited supply of Uranium, render current nuclear fission generators
controversial; for all of these reasons, it may very well be only a stopgap
solution to the international energy problem. Although bioleaching cannot
influence safety considerations, this process can have an immediate and
direct bearing on the economics of nuclear power production by providing a
mechanism for commercial use of low-grade nuclear waste. Recovery of
Uranium from radioactive wastes is extremely important because it
overcomes the problem of waste disposal, a major shortcoming of using
nuclear power generators.
Bacterial leaching of Uranium is most feasible in geological strata where the

ore is in the tetravalent state. Insoluble tetravalent Uranium oxide (UO2)
occurs in low-grade ores. Although there is no evidence for the direct
oxidation of UO2 by T. ferroxidans, UO2 can be converted to the leachable
hexavalent form (UO2SO4) indirectly by the action of this microorganism.
Uranium ore occurs not as sulfide but as oxide UO2 and is frequently
associated with FeS2 minerals. The Uranium is leached from ore by indirect
mechanism. T. ferroxidans oxidizes Fe+2 in FeS2 (which often accompanies
the U ores) to ferric iron. The oxidized iron acts as an oxidant, converting
UO2 chemically to UO2SO4, which is then recovered through leaching. The
optimal conditions for extraction of Uranium are: 45-50 °C temperature, 1.5-
3.5 pH, and around 0.2% of incoming CO2 air. The soluble form of U from
the leach liquor is extracted into organic solvents (tributyl phosphate) which
is then precipitated and recovered through ion exchange chromatography.
Uranium recovery through this leaching process ranges from 30-90%.
UO2 + Fe2(SO4)3 + 2H2SO4 → [UO2 (SO4)3]-4 + 2FeSO4 + 4H+
The technical and economic feasibility of employing Thiobacillus for the

recovery of Uranium and copper minerals depends on various factors. The
geological formation in which the minerals occur is also important in
determining the suitability of the bioleaching process. In situ bioleaching is
ideal when there is a natural drainage system, as through a fault with an
impermeable basin, that will permit economic recovery of the minerals.
However, recovery of Uranium is much higher in heap leaching method.

1. Explain methods of mineral recovery?
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Mining and
…………………………………………………………………………… Bioleaching
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1. Discribe Uranium biobleaching.
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16.10 BIOLEACHING OF OTHER METALS

Bioleaching technique is also used for extraction of other metals such as gold,
silver, nickel, molybdenum, cobalt and antimony. This method has been
considered as most promising in case of precious metal ores like Au and Ag.
Removal of iron is again significant here, prior to the actual process of
leaching. This is done by using the organism Thiobacillus ferrooxidans
which precipitate iron under aerobic conditions. Au is acquired through
bioleaching of arsenopyrite/FeS2 ore and its cyanidation process. Ag is more
easily solubilized than Au during microbial leaching of iron sulfide.
Bioleaching is also helpful in removing certain impurities from the metal rich
ores. The microorganisms such as Rhizobium sp. and Brady rhizobium sp.
can remove silica from bauxite (aluminium ore) in metal purification process
Advantages of Bioleaching process

• Bioleaching is simple and effective technique in recovering metals from
low grade ores.
• It is an ecofriendly and cost effective technique as compared to smelting
process.
• It can used to concentrate metals from dilute mixtures and wastes.
• Compared to other processes this process does not produce any toxic
emissions and other health risk to miners
• Bioleaching also offer different ways to extract valuable metals from
low-grade ores that have already been processed.
However, the process has a major limitation or disadvantage due to its very
slow speed in recovering metals through biological processes. Though
researches are going on to make the process faster and efficient.
16.11 MICROBIAL SORPTION IN METAL

RECOVERY
In the field of biotransformation and biogeochemical cycling, metal and
mineral transformations by microbes are significant. Different microbial
properties can cause changes in metal speciation, toxicity and mobility, along 331
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properties, biosorption is most important in case of microbial transformation
of metals. Biosorption is commonly referred to as the mechanisms involved
in extracting metals from solution through microorganisms and related
materials. A wide range of microorganisms (bacteria, algae, yeasts, molds)
are used for biosorption of metals.
The cell membranes of microorganisms possess negatively charged ions due

to presence of hydroxyl (OH–), phosphoryl (PO3-4), carboxyl (COO–) and
sulfhydryl (HS–) groups. Metals being positively charged ions are easily
adsorbed on microbial cell surfaces. Several potential microbial metal
biosorbents are members from Bacillus, Pseudomonas, Streptomyces,
Aspergillus, Trichoderma, Yarrowialipolytica, Rhizopus, and Penicillium sp.
Rhodospirullum sp. can bioaccumulate Cd, Hg and Pb. Bacillus circulansis
used to bioadsorb metals such as Cd, Co, Cu and Zn. Among fungal species,
immobilized fungal biomass has been widely used in metal biosorption due
to mechanical strength, increased density, and resistance to chemical
environment. Aspergillus niger, A. oryzae, Mucor haemalis, Penicillium
chrysogenum helps in selective adsorption of several metal ions like
Uranium, Thorium. Pencillium lapidorum, P. spimuiosum are useful for the
bio sorption of metals such as Hg, Zn, Pb, Cu. Several species of fresh water
or marine algae are also known to bio accumulate metals. For example,
Chlorella vulgaris and C. regularis can accumulate metals like Pb, Hg, Cu,
Mo and U. All these processes are confirmed by use of electron microscopy,
which record the deposition of metals on the microbial cell walls which
proves the composition of cell wall plays a key role in the metal adsorption.
16.12 OIL RECOVERY

Bioleaching of oil shales (oil containing rocks) likewise can possibly enhance
hydrocarbons recovery. Many oil shales contains huge amount of carbonates
and FeS2, and evacuation of these minerals builds the porosity of the shale,
indirectly enhancing oil recovery. Acid dissolves the carbonates and can be
produced by Thiobacillus species growing on the sulfur and iron in FeS2.
Such microbial leaching appears to have the potential for making recovery of
hydrocarbons from oil shales economically feasible.
Recovering oil involves two to three stages. Primary recovery is a stage

where 12% to 15% of the oil in the well is recovered without need of any
external agent. Secondary recovery involves the use of water and other
substances to extract 15-20 % of more oil from well. The tertiary oil recovery
is the utilization of biological and chemical agents to improve oil recovery
from oil shales. Tertiary recovery of oil employs solvents, surfactant, and
polymers to dislodge oil from the geological formations (Fig.5). The tertiary
recovery has the potential for recovering 60 to 120 billion barrels of oil in the
United States reserves alone that otherwise could not be recovered. Xanthan
gum produced by bacteria such as Xanthomonas campestries, is a promising
332
Mining and
compound for the tertiary recovery of oil. Such polymers have higher Bioleaching
viscosity and flow characteristics that enables them to move in the rock
layers containing oil deposits through small pores. When introduced during
operations of aqueous flooding, that is, injected into petroleum reservoirs to
force out the oil, Xanthan gums help push the oil toward the production
wells. These polymers are formed through conventional fermentation
processes in which X. campestries is grown and the xanthan gums are
retrieved.
Fig. 16.5. Tertiary oil recovery process using surfactant injection method
16.13 PETROLEUM PROSPECTING

Petroleum prospecting is one of the most interesting way in which these
methods aid to the petroleum industry. Associated with liquids and solid part
of petroleum, a gaseous fraction also occurs. It consists of methane, ethane
and propane. In petroleum producing region, these gases may seep to the
surface and provide nutrient for the growth of specific hydrocarbons utilizing
bacteria. When one find bacteria capable of oxidizing these gases there is a
strong suggestion that a petroleum deposit is nearby. However, CH4 utilizing
bacteria are not the indicator of petroleum because CH4 is produced
biologically in many systems that are not related to petroleum (by cattle and
rice field). However, C2H6 is not produced biologically in significant
amounts and it is almost always associated only in with petroleum. Detection
of C2H6 utilizing methods can be used as an indication of petroleum
resources. Since geological methods of locating petroleum deposit are
adequate, microbiological prospecting at present is not in much use.
However, it may become more important in future as petroleum is getting
depleted.
16.14 LET US SUM UP

In this unit we have discussed about bioleaching process, microorganisms in
bioleachingand various operating factors that affect the process of
bioleaching. The unit also covers recovery of metal and prospects of
commercial processes of bioleaching.
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Bioprocesses 16.15 KEY WORDS
Biobleaching: Bioleaching process is used to recover the metals from the
ore’s that are unsuitable for direct smelting because of their low-grade
content.

REFERENCES
• Douglas E. Rawlings (Ed.) Biomining Theory, Microbes and Industrial
Processes. Springer Science & Business Media. 2013
• Edgardo R. Donati, Wolfgang Sand. Microbial Processing of Metal
Sulfides. Springer Science & Business Media. 2007
• Pradipta Kumar Mohapatra. Textbook of environmental biotechnology, I
K International. 2006
• K A Natarajan. Biotechnology of Metals Principles, Recovery methods
and Environmental Concerns. Elsevier. 2018

EXERCISE

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Biomarkers
UNIT 17 BIOMARKERS
Structure
17.1 Introduction
17.2 Objectives
17.3 Definition of biomarkers
17.4 Classification of biomarkers
17.5 Application of biomarkers
17.5.1 Disease diagnostic
17.5.2 Clinical monitoring of the patient
17.5.3 Disease prognostic
17.5.4 Therapeutic development
17.5.5 Anti-doping test of athletes
17.5.6 Environmental monitoring
17.6 Biomarkers in environmental monitoring
17.7 Future of biomarkers
17.8 Importance of biomarkers
17.9 Keywords
17.10 References/suggested further readings
17.11 Answers to check your progress
17.1 INTRODUCTION
The extensive exploitation of natural resources and disposal of all types of
wastes including organic waste, chemicals, industrial waste, and electronic
wastes into water causes water pollution. The government from time to time
has started some environmental monitoring programs in the coastal areas and
most of these programs consisted only chemical and physical monitoring of
the water. These measurements include the oxygen concentration, salinity,
temperature, nutrients, and the presence of toxic heavy metals in the water. In
some programs, the water transparency measurement using secchi disc
method gave the information about the algae bloom. However, these
measurements do not give information regarding the effect of contaminants
on the flora and fauna in the water. One of the first development occur in
1960s when scientists find very difficult to monitor the organochlorine
contaminants in water such as DDT and PCBs. These organochlorines were
present in very low concentrations and concentrations were lower than
detection limits or required expensive techniques and skilled analysts. This
was the time when scientists felt that focus should be move to effects
monitoring rather than contaminants monitoring. The researchers focused on
development of novel methods that could provide the effects and warning
indicators of different contaminants on flora and fauna in the marine
environment. These indicators were called biomarkers. 335
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in the area of science and technology. Researchers have made great
developments in the area of biomarkers in the last decade due to the increase
in demand to develop new drugs in the pharmaceutical industry and
challenges in environmental monitoring. A deep understanding of various
concepts of biomarkers is required to understand the scopes and challenges of
biomarkers in the area of environmental monitoring.
17.2 OBJECTIVES
• define biomarkers and distinguish it from related fields such as

bioindicators, COA (clinical outcome assessment);
• explain the classification of biomarkers based on several parameters;
• explain the various applications of biomarkers in science and technology;
• explain the scope of biomarkers in environmental monitoring in detail;
• explain the limitations and future prospects of biomarkers;
17.3 DEFINITION OF BIOMARKERS

Pollutants like herbicides, insecticides, and other chemicals started affecting
the marine environment, this causes to focus on environmental monitoring of
water rather than contaminant monitoring. Research scientists started to
design new methods that can provide early warning signs for the effect of
contaminants on the aquatic environment. These indicators are called
biomarkers. The biomarkers can be defined in many ways and some
definitions of biomarkers are discussed in this section.
Any measurable early warning indicator of any biological process,

pathogenic process, or xenobiotic exposure can be termed a biomarker.
Broadly, the biomarkers encompass therapeutic interventions, which can be
derived from molecular, radiographic, or physiological characteristics. Some
simplest examples of biomarkers used in disease diagnostics are listed in
table 1.
Table 1: Some simple biomarkers used in disease diagnostics.
Biomarker Biological state/ disease

Temperature Fever
c-reactive protein (CRP) Inflammation
Antibody Infection
Blood pressure Hypertension/ heart stroke
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The National Academy of Sciences in the USA defined biomarkers as Biomarkers
follows: ‘‘A biomarker is a xenobiotically induced variation in cellular or
biochemical components or processes, structures, or functions that is
measurable in a biological system or sample’’. In other words, the
biomarkers are the endpoint results of an ecotoxicological test performed on
living organisms. However, the term biomarker is surrounded by some
confusion as different scientists view it differently. Some scientists define
biomarkers just as responses at molecular, physiological, or biochemical
levels occurring within the organism’s tissues, cells, or cellular fluids. While
some scientists view the wider perspectives of biomarkers and consider the
accumulation of toxic chemicals within the organism and responses occurring
at the population level, community, or ecological level of organisms. And it
is always assumed that sub-organismic responses occur first and are followed
by the responses at the organismic level, population, and ecological level.
These sub-organismic responses can be useful in the detection of early
warning signals for various monitoring.
Depledge defined the biomarkers more comprehensively “A biomarker is

defined as a change at a biochemical, cellular, physiological or behavioural
level; it can be measured in tissues and/or cellular fluids and/or in the whole
organism and shows the exposure and/or the effects of one or more chemical
contaminants (and/or radiations)”. The above definition clearly states that the
key principle of biomarkers is that the early warning signals occur long
before the effects at the ecological/population level.
Difference between biomarker and bioindicator

The terms biomarker and bioindicator are a little confusing as both look like
synonyms to each other. But there is a clear difference exist between the two
terms. The use of the term biomarker is restricted to the response or warning
signal for the biological, physiological, or molecular change in the organism
(tissues, body fluids, organs) level. Thus, the biomarker is a warning sign of
the toxic effects of any exposure. The term bioindicator is used for changes
occurring at the population/ecological level. In short, we can say that the
biomarker term is used for warning signs, and the bioindicator term is used
for the endpoint effects of the exposure.
Difference between biomarker and Clinical outcome assessment (COA)
When therapy for any disease is developed the outcome assessments are used
to define the endpoint efficacy of the therapy. The efficacy of therapy is
defined by the clinical assessment of the patients. Thus, the clinical outcome
assessment (COA) can be defined as the clinical assessment used in the
clinical trial outcomes. COAs are the direct measure of a person’s feelings,
behavior, or function. The biomarkers and COA are different from each other
because COAs are directly important measures for the patients and COAs can
be used for approval of any novel medicine or therapeutic. On other hand, a
biomarker is used for different purposes and one of the uses of biomarkers is
that their measurement gave a prediction of COA.
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1. Define biomarkers. How biomarkers are different from bioindicators?
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17.4 CLASSIFICATION OF BIOMARKERS

Biomarkers can be classified based on different parameters such as
characteristics and applications. Based on characteristics, biomarkers can be
classified into imaging biomarkers (Magnetic resonance imaging, computed
tomography) and molecular biomarkers. A biomarker or biological state
detectable in an image is known as an imaging biomarker. For example, the
warning signs of lung cancer can be determined by a large number of
biomarkers. An x-ray, CT scan, or MRI of the lung can detect the presence of
any lesion within the lung. Imaging biomarkers are cost-effective, easily
available, and non-invasive diagnostic tools for screening any patient. These
are readily used in cancer diagnostic, development of cancer therapeutics,
chest infections, diagnosis of bone fracture etc.
Molecular biomarkers can be defined as a set of biomarkers that are

detectable or measurable using genomics or proteomics technologies. A
broader definition of molecular biomarkers is “All set of biomarkers existent
or to be discovered, that is detectable/ measurable using the characteristics of
molecules and any redesign version of these analytes. This definition of
molecular biomarkers recognizes the importance of molecular design and its
characteristics. Examples of molecular biomarkers are glucose, cholesterol,
insulin, m-RNA genes, DNA adducts, cytochrome P4501A, etc.
On the basis of applications, biomarkers can be classified as diagnostic

markers, staging of disease biomarkers, disease prognosis biomarkers,
clinical monitoring biomarkers, pharmacodynamic biomarkers, and
environmental monitoring biomarkers. The schematic classification of
biomarkers based on different parameters is represented in figure 1.
Diagnostic markers detect and confirm the presence of type or subtype of
disease in people. Diagnostic markers not only confirm the disease
338
Biomarkers
Biomarkers
Based on Based on
application characteristics
Diagnostic imaging
biomarkers biomarkers
staging of disease molecular

biomarker biomarkers
disease prognosis
biomarkers
clinical monitoring
biomarkers
pharmacodynamic
biomarkers
environmental
monitoring biomarkers
Figure 17.1: Classification of biomarkers
but also help in the classification of biomarkers. The class of diagnostic

biomarkers will evolve in the future as we are moving in the era of medicinal
precision. For example, earlier the cancer diagnosis depended on organ-based
biomarkers but rapidly moved to molecular and imaging-based biomarkers. A
biomarker that can detect the stage of a disease can be classified as a staging
of disease biomarker. A clinical monitoring biomarker can be classified
as a biomarker that is measured periodically to check the status of the disease
or medical condition after exposure to a medical product or biological agent.
For example: if a patient with high blood pressure is treated with medicines,
the progress of that patient can be monitored using low-density lipoprotein
(LDL) cholesterol biomarker. Likewise, a person undergoing HIV treatment
can be monitored using clusters of differentiation (CD4) counts biomarker.
339
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progression in a patient suffering from any disease or a state of medical
condition of interest are called prognostic biomarkers. The prognostic
biomarkers are used in clinical trials to set the trial entry and identify the
higher risk population. The risk or susceptibility biomarkers deals with
change from healthy state to disease and hence are different from the
prognostic biomarkers. The prognostic biomarkers are also different from the
predictive biomarkers as latter deals with effect of exposure to xenobiotic. In
response to exposure to any medicine or environmental agent when the level
of a biomarker changes, it can be known as a pharmacodynamic
biomarker. The pharmacodynamic biomarkers are also called response
biomarkers and are used in both early therapeutic development and clinical
practice. For example, if a person with hypertension disease takes therapy
and there is no reduction in blood pressure then it is better to give up that
invention and pursue another development. Similarly, pursuing a candidate
drug for any condition that does not change the key parameter of that
biomarker in phase 1 trial is not worth it.
Biomarkers used in environmental monitoring and assessment to identify the

exposure to xenobiotics and their effects are called environmental
monitoring biomarkers. There are various environmental monitoring
biomarkers that have been used or are under development and all of them
have their strengths and weaknesses. The selection of a biomarker for a
particular environmental monitoring program is done on the basis of five
properties including ecological relevance, chemical specificity, ability to
detect different types of chemicals and mixtures, ability to give early warning
signs, and current status in environmental monitoring. Various countries
perform environmental monitoring using biomarkers to detect early warning
signs and to protect the ecosystem from the harmful effects of chemicals. The
importance, future prospects and other important parameters of
environmental monitoring biomarkers will be studied in detail in the later
sections of this unit.
17.5 APPLICATIONS OF BIOMARKERS

Biomarkers have vast application areas including disease diagnosis, clinical
monitoring of the patient (disease management), disease prognostic (clinical
trials), therapeutic development, anti-doping test of athletes, environmental
monitoring etc.
17.5.1 Disease Diagnosis

Disease diagnosis is the first challenge faced by doctors in the treatment of a
disease. An accurate, fast and low-cost diagnosis of disease can help the
medical industry to better the medical facilities. An early diagnosis of some
of the deadly diseases can save the life of patients. A disease can be
diagnosed either using a single biomarker or a set of biomarkers. For
340
example, diagnosis of typhoid can be done by performing a Widal test, which Biomarkers
depends on O and H antigens. Similarly, Malaria disease can be diagnosed
using various biomarkers including histidine-rich protein II (HRP II) and
plasmodial lactate dehydrogenase. The early biomarkers for COVID-19
disease should include high CRP, high LDH, and high D-dimer. Diagnosis of
chronic kidney disease can be done using a set of metabolites biomarkers
including amino acids (valine alanine, glycine, etc.), glycolysis metabolites
(glucose, lactate), tricarboxylic acid (succinate, fumarate). It was found that
the development of chronic kidney disease was closely related to the
dysfunction of the lipids, carbohydrates, and amino acids metabolism. This
relation between metabolomics and the development of chronic kidney
disease is used to diagnose the disease and in therapeutic development. Thus,
we can conclude that a disease can be diagnosed by using various biomarkers
or a set of them and the selection of biomarkers for disease diagnosis should
be based on accuracy, fast, cost, and availability. The research to find novel
biomarkers will go on to match the characteristics of an ideal biomarker.
Table 17.2: Diseases and biomarkers to diagnose these diseases.
S.No. Disease Biomarkers for disease diagnosis

1 Typhoid O and H antigens
2 Malaria histidine-rich protein II (HRP II) and
plasmodial lactate dehydrogenase
3 COVID-19 high CRP, high LDH, and high D-dimer
4 Chronic kidney Metabolites (amino acids, lipids,
disease carbohydrates)
5 Alzheimer’s nerve growth factor precursor protein, MRI
Disease scanning
17.5.2 Clinical Monitoring of Patient

Clinical monitoring of patients or disease management is very crucial during
the treatment of any disease. This helps doctors to identify the severity of the
disease and to change the medicines or therapy accordingly. For example,
when dengue is treated platelets count is monitored at a regular interval.
Similarly, low-density lipoprotein (LDL) cholesterol and CD4 counts are
monitored during the treatment of high blood pressure and cancer treatment
respectively. Monitoring of these biomarkers helps doctors in the decision-
making and efficiency of drugs or therapy. The safety of human research
participants can also be ensured using these monitoring biomarkers. For
example, the safety of a drug with possible liver toxicity can be monitored by
performing a liver function test at regular intervals.
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S.No. Disease Monitoring biomarkers

1 High blood pressure LDL-cholesterol
2 Dengue Platelets count
3 Cancer CD4 counts
17.5.3 Disease Prognosis

One of the important applications of biomarkers is in clinical trials to filter
the trial entry and to point out the high-risk populations. Biomarkers are
important to identify the risk of an event in a person so that they can be
excluded from the clinical trials. Trials performed in this way are more
effective and safer for humans under trials. Biomarkers are also used to
identify the chances of recurrence of a particular disease and to check on the
progression of a disease. This information can be very useful in determining
how many days the patient will stay in the hospital or intensive care unit and
thus helps in hospital patient management.
17.5.4 Therapeutic Development

Biomarkers particularly pharmacodynamic/response biomarkers help in early
therapeutic development. For example, if we are developing a drug for
dengue disease and we find out that after consumption of the drug there is no
improvement in the number of platelets count then it is better to give up that
development. So, in this case, platelets biomarker helped in developing the
therapy or drug for dengue disease. Likewise, various biomarkers are used in
the medical industry for the development of drugs or therapy of diseases.
Table 3: Biomarkers used in the therapeutic development of a disease
S. No. Disease Biomarkers for therapeutic

development
1 Migraine CGRP (calcitonin gene-related peptide)
concentration
2 Chronic back pain Nerve growth factor
3 Dengue Platelets count
4 Inflammatory pain TRPV (transient receptor potential cation
channel subfamily V) expression
17.5.5 Anti-doping Tests of Athletes

The key challenge to sports authorities is to identify and detect the use of
prohibited substances by athletes with a degree of certainty and accuracy.
This detection can be done either by direct detection of prohibited substances
or detection of their metabolites. Biomarkers are used in doping tests of
athletes to detect prohibited substances with good accuracy. The biomarker of
342 doping can be defined as the biological variable that indicates the effect of a
prohibited substance with a high degree of certainty. For example, primary Biomarkers
biomarkers used in anti-doping tests may include hemoglobin concentration
and the ratio of urinary testosterone to epitestosterone (T/E). the secondary
doping biomarkers can not confirm the doping alone because of the low
specificity of these biomarkers but can be supportive evidence. An example
of secondary doping biomarker would be the immature reticulocyte fraction,
which can provide evidence of altered erythropoiesis doping.
17.5.6 Environmental Monitoring

Biomarkers are also used in biomonitoring of environmental change due to
xenobiotics. These uses and scope of biomarkers in environmental
monitoring are discussed in section 6 of this unit in detail.

1. Discuss the classification of biomarkers in detail.
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2. Explain with examples the role of biomarkers in clinical monitoring of
patients.
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17.6 BIOMARKERS IN ENVIRONMENTAL

MONITORING
The environmental monitoring programs in the early phase consisted of only
the chemical and physical monitoring of aquatic life in the coastal areas. With
developments in the area of biomarkers, these programs were incorporated
with biological monitoring to obtain the warning signals of ecological
toxicity. The chemical analysis of aquatic water included oxygen
concentration, heavy metals, and sediment concentrations. This analysis gave
useful information regarding the extent of pollutants in water but did not tell
about the effect of these pollutants on aquatic life. Early biomonitoring of the
effect of insecticides, pesticides, and antifouling agents on biota was started
in the 1960s when it was the need of the hour to focus on effects monitoring
rather than contaminant monitoring. Till the 1980s there were a large number
343
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was disagreement between the scientists regarding the reliability and method.
A conference was held as the Oslo conference to validate the methods and
biomarkers and it was not surprising that some methods detected the
contamination with high reliability and some were unable to detect the
contamination. In the last decade, various biomarkers have been developed
by researchers with the aim to achieve more sensitivity, reliability, and the
ability to detect new effects on biota.
DNA/RNA
alterations
Physiological Protein
biomarkers responses
Biomarkers in
environmental
monitoring
Immunological Metabolism
biomarkers products
Hysto-
cytopathological
biomarkers
Figure 17.2: Biomarkers used in environmental monitoring
Some of the major biomarkers used in various environmental monitoring

programs are discussed below:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
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11. Biomarkers
12.
13.
14.
15.
16.
17.
17.6.1 DNA-RNA Alterations

Some techniques, like mRNA fingerprinting, are used to identify the
variability in gene expression for the entire genome and these techniques can
also be applied to validate the presence of toxins in the field of environmental
toxicology. The mRNA fingerprinting technology is based on the
amplification and resolution of the 3’ terminal part of the mRNA on a DNA
sequencing gel where the change in the function of mRNA transcription or
degradation rate is observed. The limitations of this process include more
labour requirements and time-consuming.
Various techniques are employed to verify the induction of a particular gene
caused by a class of contaminants or toxins. For example, the induction of
gene expression in Cyprinodon variegatus (sheepshead minnows) exposed in
vivo to xenoestrogens (diethylstilbestrol, estradiol, ethinylestradiol ) can be
measured using a designed molecular-based assay. A characteristic
expression pattern of gene alterations caused by these contaminants is
observed as a result of this study.
Several contaminants such as dioxins, PAH, etc. are also capable to change
the genetic materials. Molecules like benzopyrene bind gradually to the
double helix and formed the adducts, these adducts are reactive chemical
intermediates that formed the covalent bond with the DNA bases. These
adducts are used to detect warning signals for polluting events. The alteration
in genes caused by genotoxicants can be very harmful because these
contaminants can deform future generations. These alterations can be
identified using the enzyme-linked immunosorbent assay (ELISA) method
and the radiochemical technique (32P-post labelling method).
The secondary alterations are easy to measure because these are caused by
adducts by fractures of the double helix of DNA. These alterations can be
detected as a warning signal of environmental contamination. Generally, the
secondary alterations of DNA are reversible in nature but sometimes the
alterations are so significant that they can not be reversed back and this
situation is called chromosomal aberrations. the tests including Sister
Chromatid Exchange, chromosomal analysis, and micronucleus test can be
used to evaluate biomarkers for these DNA alterations. The verification of
alteration up to mutation level can be done using the oncogene activation 345
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17.6.2 Protein Responses
Protein responses are the responses of an organism when the activity of the
protein is induced or inhibited by several classes of contaminants. This group
of biomarkers contains the protective or adaptive detoxification mechanism
of the chemical compounds and heavy metals. These biomarkers are very
specific and identified the presence of a particular type of contaminant.
Protein response biomarkers associated with an incident of inhibition of the
enzymatic activities for example organophosphorus insecticide inhibit the
blood or brain esterases. The induction of plasma protein and stress proteins
are the other general biomarkers.
Metallothionein protein is another example of protein responses that are

identified in approximately 50 species of crustaceans and molluscs. The
function of Metallothionein is the process of metal detoxification and
preservation of homoeostasis. Metallothionein is a low molecular weight but
high cysteine content protein. Metallothionein has a high attraction for some
metals like Cu, Ag, Cd, Zn, and Hg. Metallothionein can be detected using
spectrophotometry, polarography, and radioimmunological analysis
techniques. The concentrations of metallothionein in molluscs can correlate
with the environmental contamination of Cu and Cd metals.
17.6.3 Metabolism Products

The metabolism of porphyrin is affected by some contaminants such as PCB,
lead, and HCB in the liver and other parts of animals. As a result, there is an
increase in the concentrations of intermediate products of the metabolism of
porphyrins and this can act as a biomarker for some classes of contaminants.
In such cases, biomarkers can be represented by the high concentration of
metabolics such as uroporphyrins, protoporphyrins, and coproporphyrins in
the liver. Earlier, the measurement of these porphyrins is carried out in a
destructive way (in the liver) but the current research has helped us in
establishing the non-destructive methods of measurement (in the excreta and
biological fluids such as the blood of animals. Some scientists have
established non-destructive methods for determining porphyrins in the birds
and sea lions contaminated with PCBs.
17.6.4 Hysto-cytopathological Biomarkers

In particular organs, histopathological alterations can be caused by various
polluting compounds. These alterations are easy to measure because they
affect the structure of tissues, cells, and organs, or individual organisms. for
example, the placenta of human is suggested as a good dual biomarker for
monitoring contamination caused by trace elements. The maternal health of a
pregnant lady can be assessed by monitoring the concentration of Pb in the
placenta as a useful indicator of Pb exposure. Some scientists related the Hg
concentration in the blood of fetal/maternal with eating fish in large
346
quantities. Thus, measuring the Hg in the blood of maternal can be a Biomarkers
biomarker for Hg contamination in aquatic ecology. The development of
male sexual characteristics in females is called Imposex. The biomarker for
imposex is attributed to exposure to tributyl tin (TBT). The van deferens
sequence index (VDSI) is one of the most important indices for monitoring
environmental contamination caused by TBT. The degree of imposex in
animals can be quantified by the sequence of formation of VDSI and the
penis in TBT-exposed animals. The imposex in the population can be
quantified using an index namely the relative penis length index (RPLI). This
index is best suitable for the areas of low TBT contamination. Lysosomes
present in the digestive system are the main sites of toxic metals and organic
pollutants. These pollutants can damage and destabilize the membranes of the
lysosomes as a response to stress.
17.6.5 Immunological Biomarkers

Xenobiotics act as an immune moderator and give multidimensional
responses to the immune system of aquatic animals. These biomarkers can
detect the effects at very low concentrations of chemical concentrations.
Other advantages of immunological biomarkers are that these can provide the
link between the toxic contaminants and diseases spread in fishes and aquatic
animals. However, there are also some limitations like these biomarkers have
low specificity and it is difficult to obtain reliable results; xenobiotics
contaminating ecology must be already known, etc. some scientists have used
cellular and humoral immunological responses of earthworms to test for the
contamination caused by carbaryl, PCBs, and 2.4-dichloro phenoxyacetic
acid. The non-specific immunity was also studied in invertebrates like
earthworms and phagocytosis decreased dramatically in earthworms exposed
to soil contaminants. The immunological biomarkers can be classified into
three categories based on the immune system like tissues and cells involved,
based on the mechanism involved, and based on the efficacy of the response
in terms of disease susceptibility.
17.6.6 Physiological Biomarkers

Physiological biomarkers have not been studied on a large scale because
these biomarkers require continuous measurements of the speed of the
various processes involved. The activities studied in the physiological
biomarker’s field include the measurements of breathing, cardiac activity,
growth and energy metabolism of species. The measurement of the behaviour
of species like fish after exposure to contaminants is also studied in
physiological biomarkers. The blood parameters including protein
concentration and coagulation time are also valid to monitor toxic
environments in aquatic life. The scope for growth is also one of the indexes
of physiological integrity and its measurement indicates the energetic state of
the organism which is highly employed in toxicological studies. Monitoring
growth in organisms is one of the best ways of measurement of the
physiological responses to man-induced environmental contaminations. 347
Ecofriendly These physiological biomarkers can be converted into measurements of
Bioprocesses
energy flow. The scope for growth depends on the various biochemical or
physiological processes such as speed of food digestion, the efficiency of
food consumption and nutrient absorption, and decrease or increase in the
breathing speed of organisms. A high value of scope for growth value for
animals is indicative of environmental contamination.
17.6.7 Behavioural Biomarkers

Environmental contamination can also be detected using the changes in the
behaviour of organisms under different classes of contaminants. These types
of biomarkers can not be used as an early warning signal but can be used to
observe responses at a high level of contamination. Different types of
measurements such as sensory measurements (i.e. chemotaxis, phototaxis,
temperature preferences, larval settlement, tactile inhibition), motor activities,
rhythmic activities, and interindividual responses (i.e migration, predation
vulnerability, aggression, etc.). The parameters like distance covered by
animals, movement frequency, and mean speed measurement can be done
with the help of cameras and video recording.

1. DNA/RNA alterations can be used as a potential biomarker for
environmental contamination. Comment.
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
2. Write a short note on behavioural biomarkers in environmental
monitoring.
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
……………………………………………………………………………
17.7 FUTURE OF BIOMARKERS

The induction of DNA/RNA alterations and protein response biomarkers in
aquatic animals as a biomarker for xenobiotic contaminations have been
extensively and with reliability in many studies. The developments in the
area of capacity to store, organize and compute a large amount of information
will improve the biomarker’s prospects. The applications of these widespread
biomarkers will increase tremendously in the future because of enhanced
commercial availability of biotechnological products particularly gene probes
and antibodies and specific adducts. In the future, these biomarkers will be
348
rediscovered with improved reliability, specificity and prognostic Biomarkers
capabilities.
17.8 IMPORTANCE OF BIOMARKERS

Biomarkers play important role in detecting early warning signals for
xenobiotics stress and these signals are useful to save ourselves from
hazardous effects. Another importance of biomarkers is that biomarkers can
indicate biological effects while chemical-based detectors can not do so.
Different classes of biomarkers fulfill different purposes in the fields of
environmental monitoring and medicinal chemistry. Biomarkers can also
detect the overall toxicities caused by the mixture of contaminants. Also, a
set of biomarkers can be used to verify or confirm the state of stress caused
by a particular contaminant. Another advantage of biomarkers is that they are
more economical than many chemical analysis of chemical contaminants. For
example, chemical analysis of contaminant dioxin is very expensive and the
solution to this problem is to analyze it through biomarkers.
17.9 KEYWORDS
Biomarkers, environmental monitoring, contaminants, DNA/RNA alterations,
protein responses, xenobiotics.
17.10 REFERENCES/SUGGESTED FURTHER

READINGS
• Lam, Paul KS. "Use of biomarkers in environmental monitoring." Ocean
& Coastal Management 52.7 (2009): 348-354.
• Huss, Ralf. "Biomarkers." Translational regenerative medicine.
Academic Press, 2015. 235-241.Lam,
• Paul KS, and John S. Gray. "The use of biomarkers in environmental
monitoring programmes." Marine Pollution Bulletin 46.2 (2003): 182-
186.
• Lam, Paul KS. "Use of biomarkers in environmental monitoring." Ocean
& Coastal Management 52.7 (2009): 348-354.
• Califf, Robert M. "Biomarker definitions and their applications."
Experimental Biology and Medicine 243.3 (2018): 213-221.
• Grice, Kliti, and Christiane Eiserbeck. "The analysis and application of
biomarkers." Treatise on geochemistry. Elsevier Science, 2013. 47-78.
• Hanson, N., M. Halling, and H. Norin. "Biomarkers for Environmental
Monitoring: Suggestions for Norwegian monitoring programs."
Norwegian Environment Agency, Norway (2013): 70.
• Livingstone, David R. "Biotechnology and pollution monitoring: use of
molecular biomarkers in the aquatic environment." Journal of Chemical
349
Ecofriendly Technology & Biotechnology 57.3 (1993): 195-211.
Bioprocesses

Answers to Check Your Progress 1
1. Any measurable early warning indicator of any biological process,
pathogenic process, or xenobiotic exposure can be termed a biomarker.
The use of the term biomarker is restricted to the response or warning
signal for the biological, physiological, or molecular change in the
organism (tissues, body fluids, organs) level. The term bioindicator is
used for changes occurring at the population/ecological level.
Answer to Check Your Progress 2
1. See section 4 for detailed classification of biomarkers.
2. See section 5.2 for application of biomarkers in clinical monitoring.
Answers to Check Your Progress 3
1. See section 6.1
2. See section 6.7
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MEVE-013

Dr Sandeep Kumar. Center for Environmnetal

Welcome to the course MEVE-013 Environmental Biotechnology!

Environmental pollution and waste generation has become a major global

Environmental biotechnology has very wide scope in environmental safety.

Unit 1 “Introduction of Environmental Biotechnology” gives a brief

Unit 2 “Environmental Biotechnology in waste water treatment” covers the

• Define environmental Biotechnology and its scope;

1.3 WHAT IS ENVIRONMENTAL

Figure 1.1: Different contaminants in the environment.

• To adopt biotechnology for controlling contaminants at the source level

Overall, the major aim of environmental biotechnology is to imply

1.4 SCOPE OF ENVIRONMENTAL

Fig1.2 : Major Scope of environment biotechnology. 13

Different sets of genetic manipulations are conducted to develop strains that

Table 1.1 List of microbes employed for abatement of environmental

S.No Environmental Micro-organism References

1.6.2 Waste Treatment

Microorganisms are used to create pesticides that are extremely effective,

1.8 POLLUTION CONTROL

1.9 WASTE WATER TREATMENT

1.9.1 Activated Sludge Process

1.9.2 Tricking Filters

1.9.3 Membrane Bioreactors

1.9.5 Oxidation Pond

Check Your Progress 1

1.10 BIODIVERSITY CONSERVATION

1.12 LETS US SUM UP

Biotechnological techniques like metagenomics (functional), proteomics,

• Alternative energy resources

1.14 SUGGESTED FURTHER READING/

1.15 ANSWERS TO CHECK YOUR PROGRESS

1. Refer Section Number 1.6

“Cleaning of environment through nature’s scavengers”

Environmental biotechnology employs a diverse set of methodological

• Define biotechnological processes used for wastewater treatment;

2.3 PRINCIPLES OF BIOTECHNOLOGY FOR

Technological advancements in the environmental biotechnology sectors are

2.4 PRACTICES OF BIOTECHNOLOGY FOR

According to an OECD report, biotechnology for the treatment of waste

2.5 USE OF BIOTECHNOLOGY IN

2.5.1 Activated Sludge

Activated sludge reactors are aerobic suspended-growth type processes.

The flocs (agglomerations of sludge particles), which form in the aerated

Activated sludge processes are one part of a complex wastewater treatment

The design must be based on an accurate estimation of the wastewater

Although aerobic bacteria are the most dominant microorganisms in the

Detailed Treatment Process

When both nitrogen and phosphorus are to be removed, the combination

Nowadays, activated sludge systems, where valuable nutrients (phosphorus

To achieve specific effluent goals for BOD, nitrogen and phosphorus,

i) Sequential Batch Reactors (SBRs)

ii) Oxidation Ditches

Fig. 2.6 Oxidation ditch activated sludge system.

iii) Deep Shafts

Where land is in short supply, sewage may be treated by injection of

Excess sludge contains even higher amounts of microorganisms, as well as

Operation & Maintenance

Highly trained staff is required for maintenance and trouble-shooting. The

1) A phenomenon known as bulking, in which the sludge from the aeration