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Characterization and optimization of cellulase enzyme from

bacteria isolated from fruit industrial waste


A
DISSERTATION SUBMITTED TO
MITS SCHOOL OF BIOTECHNOLOGY
AFFILIATED TO

UTKAL UNIVERSITY,
BHUBANESWAR
IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE
AWARD OF DEGREE OF MASTER OF SCIENCE IN
BIOTECHNOLOGY
BY
MRUTYUNJAY DAS

Roll No:15254U204031
Under the guidance of
Dr. RASHMIRANJAN MISHRA

Associate Professor
Department of Biotechnology

MITS SCHOOL OF BIOTECHNOLOGY


2 (P), Infocity, Patia, Chandaka Industrial Estate, Bhubaneswar, Odisha,
Pin - 751024
2022
DEPARTMENT OF BIOTECHNOLOGY
COLLEGE OF ENGINEERING AND TECHNOLOGY
(A Constituent College of BijuPatnaik University of Technology, Odisha)
TECHNO CAMPUS, P.O:- GHATIKIA, KALINGA NAGAR,
BHUBANESWAR-751 003, INDIA

Dr. S. Mohapatra
Assistant Professor
Certificate
This is to certify that the dissertation entitled “CHARACTERIZATION AND OPTIMIZATION
OF CELLULASE ENZYME FROM BACTERIA ISOLATED FROM FRUIT INDUSTRY
WASTE” submitted by Mrutyunjay Das bearing University Roll no:15254U204031 in
partial fulfillment of the requirement for the degree of Msc in Biotechnology from Utkal
university ,Bhubaneswar odisha is a faithful record of bonafide research work carried out
under the supervision of Mrs. Sonali Mohapatra, Assistant Professor, Department of
Biotechnology, CET. The results of the investigation reported in the thesis have not been
submitted for the award of any other degree.

Place: Bhubaneswar (Sonali Mohapatra)


DECLARATION

I, Mr. Mrutyunjay Das do hereby declare that the dissertation thesis entitled “Characterization
and optimization of cellulose enzyme from bacteria isolated from fruit industry waste”
which is being submitted to Department of Biotechnology, MITS School of Biotechnology, 2(P),
Infocity, Patia, Chandaka Industrial Estate, Bhubaneswar751024, Odisha, India affiliated to
Utkal University, Bhubaneswar, Odisha, India for the Degree of Master of Science in
Biotechnology is done by me at MITS SCHOOL OF BIOTECHNOLOGY, Bhubaneswar-
751024, Odisha, India under the guidance of Dr. Sonali Mohapatra,Assistant professor, Dept.
of Biotechnology ,college of engineering and technology , Bhubaneswar, Odisha. It has not
formed in the basis for the award of any Degree/Diploma/Fellowship or other similar degree to
any candidate of any University. To reiterate, the help and support received has been duly
acknowledged.

Date:
Place:Patia, Bhubaneswar (Mrutyunjay Das)
ACKNOWLEDGEMENT

I express my sincere reverence to my respected Supervisor, Dr. Sonali


Mohapatra ,Assistant professor CET Bhubaneswar-751016, Odisha, India
for his sincere supervision, kindness and support during entire endeavor of this
dissertation work despite his bustling schedule.

I convey my heartfelt respect and thanks to Administrative Officer, all


teaching and non-teaching staff and to all my friends who have given me
mental strength, support, encouragement, valuable time and suggestions in
completing my dissertation work.
I would like to thank Rasmi ranjan Mishra Assist. professor, MITS School of
Biotechnology, 2 (P), Infocity, Patia, Chandaka Industrial Estate, Bhubaneswar-
751024, Odisha, India for his help.
Last but not the least; I humbly convey my immense reverence, devotion
and appreciation to my loving parents, family members and GOD for the
heavenly blessings and constant support

(Mrutyunjay Das)
MITS SCHOOL OF BIOTECHNOLOGY
Ref. No./

FROM Date:

PROF.(DR.) G.K. PANDA

DIRECTOR

MITS SCHOOL OF BIOTECHNOLOGY


BHUBANESWAR-751024, ODISHA

TO WHOM SOEVER IT MAY CONCERN

This is to certify that Mrutyunjay Das is a bonafide student of M.Sc. in Biotechnology (Fourth
Semester), MITS School of Biotechnology, Bhubaneswar-24, Odisha with University Roll
No._15254U204031. he has carried out her dissertation work under the direct Supervision of
Mrs Sonali Mohapatra, Associate Professor, Dept. Biotechnology, CET, Odisha, India within
duration of six (06) months i.e. from January, 2022 to June 2022. As per my knowledge is
concerned, no part of this work has been submitted for any other degree or diploma..

Mrutyunjay Das is a fit and suitable candidate for the award of Degree of Master of Science
in Biotechnology conferred by Utkal University. She has duly acknowledged the assistance
and help received during the course of this investingation.

Dr. Mousumi Mishra prof.(Dr.) G.K. Panda


Course coordinator Director
CONTENTS

TITLE Page No.

List of tables (a)


List of figures (b)
List of abbreviation (c)
Abstract 2
1.Introduction 3
2.Review of Literature 7
2.1 Lignocellulosic biomass 7
2.2 Cellulose 8
2.3 Cellulose source material 9
2.3.1 wood 9
2.3.2 Plant 9
2.3.3 Algae 9
2.3.4 Tunicate 9
2.4 Structural organisation of cellulose 10
2.5 Isolation of cellulose 11
2.6 Mechanical treatment 11
2.7 Acid hydrolysis 12
2.8 Hemicelluloses 12
2.9 Lignin 13
2.10 Lignocellulosic microorganism 14
2.11Cellulase enzyme 15
2.12Hemicelluloses enzyme 18
2.13 Ligninolytic enzyme 19
3.Bacterial and methods 25
3.1 collection of soil sample 26
3.2 isolation of cellulolytic bacteria 26
3.2.1 Serial dilution method 26
3.2.2 pour plate method 26
3.3 preparation of pure bacterial isolates 27
3.3.1 streak plate method 27
3.4 preservation of bacteria isolates 27
3.4.1 Bacteria culture in nutrient agar slant 28
3.4.2 bacteria culture in nutrient broth 28
3.5 screening of cellulolytic bacteria 28
3.5.1 CMCase assay 28
3.6 Quantitative assay of bacterial cellulase activity 28
3.6.1 DNS (3,5-dinitro salicylic acid) method 28
3.7 phenotypic characterisation of cellulase degrading bacteria 30
3.7.1 morphological character 30
4. results 31
4.1 isolation of cellulose degrading bacteria 31
5.1 pure culture prepairation 32
6.1 screening of cellulase activities 33
6.1.1 CMSase plate assey 33
7.1 quantative assey of bacterial cellulase activities 36
8.1 characterisation and identification of bacteria 38
.REFERENCE- (41-45)

LIST OF TABLES
SL.NO TABLE NO PAGE.NO TITLE
1 Table 1 20 Cellulose degrading bacteria isolated from different
natural reserves
2 Table 2 35 Cellulase activities of different bacterial isolase based
on the ration between hydrolyzing zone diameter and
colony diameter
3 Table 3 37 Absorbance values of cellulase activity of 24 bacterial
isolates from fruit industry soil by DNS method after
the incubation of 24 hours
4 Table 4 40 Phenotypical characteristics of isolated cellulolytic
bacterial strains
(a)

LIST OF ABBREVATION
µl Microliter
pH Hydrogen ion concentration
ml Mililiter
M Mole
DNS Di-nitro salicylic acid
MR Methyl red
MRVP Methyl red voges-proskauer
CMC Carboxy-methyl cellulose
SCB Sub cuticular bacteria
ddH2o Double distilled water
(c)

LIST OF FIGURES
SL NO FIG NO PAGE NO TITLE
1 Fig.1 9 Single cellulose chain repeat unit showing
directionality and hydrogen bond.
2 Fig.2 13 Structure of hemicellulose
3 Fig.3 14 Chemical structure of lignin
4 Fig.4 25 Flow diagram of study design
5 Fig.5 26 Serial dilution method of soil samples
6 Fig.6 29 Reaction mixture in cellulase assay
7 Fig.7 29 Reaction mixture mixed with DNS
8 Fig.8 31 Growth of Bacterial colony on CMC agar after
incubation
9 Fig.9 32 Growth of Bacteria isolates by striking method on
agar plate.
10 Fig.10 32 Preservation of bacterial slant from SCB1-SCB34

11 Fig.11 33 Preservation of bacterial slant (SCB1-SCB34) in


nutrient broth.
12 Fig.12 34 Growth of bacteria isolates on CMC agar before
congo red staining.
13 Fig.13 34 Clearing zone generated by cellulose degrading
bacteria in CMC plate
14 Fig.14 36 Cellulase activity of different bacteria on the basis
of H:C ratio.
15 Fig.15 38 Qantitative analysis of cellular activity of different
bacterial isolates as per DNS method
16 Fig.16 39 Microscopic view(100X) for different bacterial
isolates after gram staining.
(b)

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