PRODUCTION OF CELLULASE ENZYME FROM OIL PALM EMPTY FRUIT BUNCH (OPEFB) BY TRICHODERMA REESEI RUT C-30 USING SOLID
STATE FERMENTATION
BY
MANISYA ZAURI BINTI ABDUL WAHID
A thesis submitted in fulfilment of the requirement for the degree of Doctor of Philosophy in Engineering
(Biotechnology)
Kulliyah of Engineering International Islamic University
Malaysia
MARCH 2012
ii
ABSTRACT
Vast quantities of oil palm empty fruit bunches (OPEFB) are being produced during processing of oil palm in the oil palm industry. The OPEFB posses a pollution problem to the environment. The study focusses on the production of cellulases from oil palm empty fruit bunches (OPEFB) by solid state fermentation using commercial strain Trichoderma reesei RUT C-30 has been carried out using several approaches.
The ability of Trichoderma reesei RUT C-30 to produce cellulases on CMC agar and SSF of OPEFB particles in culture flask were compared to Penicillium (2 strains) and Trichoderma harzianum (2 strains) from the culture collection stock of Environmental Biotechnology Laboratory, IIUM. Trichoderma reesei RUT C-30 and strain Penicillium 1 gave high cellulolytic activity on day 2 and day 3 of growth culture, respectively, by forming clearing zones on the CMCase agar that has been stained with 1 mg/ml of congo red dye solution. However, Trichoderma reesei RUT C-30 produced the highest cellulases production from OPEFB under SSF culture flask compared to other tested strains with maximum CMCase, FPase and β- glucosidase production values of 1.831, 0.111 and 2.361 U/gds, respectively. The characterization of OPEFB fibres contained 49.2, 25.9 and 17.3 % of cellulose, lignin and hemicelluloses, respectively. On CMCase activity by Trichoderma reesei RUT C-30 using different OPEFB particle sizes of 0.105-0.225 mm, 0.225-425 mm, 0.425-0.6 mm and 0.6–0.816 mm showed that using OPEFB particle size of 0.225- 0.425 mm gave the highest CMCase activity with the value of 1.782 U/gds. The effect of pretreatment of the OPEFB particles (0.225-0.425 mm) with various concentrations of acid (1-3 N) or alkali (1-15 N) followed by autoclaving at different steaming time (10-30 min) on the cellulose, lignin and hemicelluloses content of the OPEFB particles was studied. The study found 58.4 % of cellulose was recovered from OPEFB soaked for 1h with 3N of HCl followed by autoclaving for 10 min.
Furthermore, 99.2 % of hemicelluloses were recovered on the OPEFB fibers that have been soaked in 3N of HCl for 1h followed by 20 min of autoclaving.
Meanwhile, the OPEFB particles treated with more concentrated alkali (15 N NaOH) followed by autoclaving for 20 min was able to remove 36.8 % of OPEFB lignin. All the pretreated OPEFB particles have not shown any apparent surface changes when examined under SEM micrograph. Result on untreated OPEFB particles were found to produce higher cellulases activities by Trichoderma reesei RUT C-30 compared to pretreated OPEFB particles. A total of 11 parameters comprising various concentration of peptone, urea, ammonium sulfate, calcium nitrate, yeast extract, tween 80, pH, incubation time, initial moisture content, inoculum size and substrate amount was screened for significant parameters affecting cellulase enzymes production. It was found that factors of initial moisture content (P>F=0.001), incubation time (P>F=0.001), inoculum size (P>F=0.023) and ammonia sulfate concentration (P>F=0.032) significantly affect cellulases production. The optimization of these parameters improved CMCase yield with maximum production of 5.341 U/ml with productivity of 1.22 U/mg/min glucosamine being obtained. It was found with ammonium sufate concentration, initial moisture content and inoculum density of 132 mg/L, 60 % and 1x107 spores/ml, respectively, gave high yield of celluase at day 11 of the grown culture in SSF. The cellulases of the
collected filtrate crude sample were characterized based on temperature, pH, substrate concentration and incubation time. Results indicate that endoglucanase (CMCase), exoglucanase (FPase) and β-glucosidase of the collected filtrate crude sample were stable at pH between 4 and 8. CMCase and FPase were found to be stable at temperature range of between 600C and 800C compared to β-glucosidase which has lower optimum stability temperature between 300C and 500C. The kinetic investigation on each of the cellulases of the collected filtrate crude sample revealed that the Vmax values for CMCase, FPase and β-glucosidase were 0.88, 0.144 and 0.5, respectively. Whereas, the KM for CMCase, FPase and β-glucosidase were 0.529, 0.286 and 0.448, respectively. Using 30L stirred-drum horizontal bioreactor fermentation conducted at 300C using 6% (v/w) of 1 x 107 spores/ml of Trichoderma reesei RUT C-30 as inoculum and 0.5 kg of OPEFB particles as fermentation substrate under various aeration (1-5 L/min) and intermittent agitation (6-90 rotation/day). Cellulase production of 6.317 U/gds CMCase with productivity of 2.01 U/mg/min of glutamine was obtained from the OPEFB with continuous supply of 4L/min of saturated air with intermittent agitation of 30 rotation/day. The findings from this research indicate OPEFB could be used as potential source for production of cellulase using Trichoderma reesei RUT C-30. This would contribute to the development of promising cellulase enzymes fermentation technology as an alternative for better management of OPEFB in the oil palm industries.
iv
OPEFB OPEFB OPEFB
RUT C-30 RUT
C-30
IIUM RUT C-30
CMCase agar RUT C-30
CMCase FPase
CMcase
U/gds
RUT C-30
(P>F=0.001) (P>F=0.001)
،
( P>F=0.023)
>F=0.032)
CMCase
CMCase
FPase) CMCase
FPase
Vmax
CMCase
KM
(v/w RUT C-30
CMCaseU/gds
RUT C-30 .
ريوطت يف مهاسي دق اذه
ريمخت ايجولونكت
vii
APPROVAL PAGE
The thesis of Manisya Zauri Binti Abdul Wahid has been approved by the following:
___________________________
Mohamed Ismail Abdul Karim Supervisor
_________________
Faridah Yusof Co-supervisor
_________________
Md. Zahangir Alam Co-supervisor
_______________________
Suleyman Aremu Muyibi Internal Examiner
________________
Irwandi Jaswir Internal Examiner
__________________
Mohd. Ali Hassan External Examiner
___________________________
Nasr Eldin Ibrahim Ahmed Chairman
viii
DECLARATION
I hereby declare that this dissertation is the result of my own investigations, except where otherwise stated. I also declare that it has not been previously or concurrently submitted as a while for any other degrees at IIUM or other institutions.
Manisya Zauri Binti Abdul Wahid
Signiture ……… Date……….
ix
INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA
DECLARATION OF COPYRIGHT AND AFFIRMATION OF FAIR USE OF UNPUBLISHED RESEARCH
Copyright © 2011 by International Islamic University Malaysia. All rights reserved.
PRODUCTION OF CELLULASE ENZYME FROM OIL PALM EMPTY FRUIT BUNCH (OPEFB) BY TRICHODERMA REESEI RUT C-30 USING
SOLID STATE FERMENTATION
I hereby affirm that The International Islamic University Malaysia (IIUM) hold all rights in the copyright of this Work and henceforth any reproduction or use in any form or by means whatsoever is prohibited without the written consent of IIUM. No part of this unpublished research may be reproduced, stored in a retrieval system, or transmitted in any form or by means, electronic, mechanical, photocopying, recording or otherwise without prior written permission of the copyright holder.
Affirmed by MANISYA ZAURI BINTI ABDUL WAHID
Signiture ………. Date……….
x
TO MY PARENTS, BROTHERS, RELATIVES AND FRIENDS
xi
ACKNOWLEDGEMENTS
All praised and thanks to supreme Almighty Allah swt for His mercy, kindness and blessing that have enabled the author to accomplish her study. The author would like to express her gratitude to the supervisor, Prof. Dr. Mohammed Ismail Abdul Karim and not forgetting other members of the supervisory committee, Prof. Dr. Md.
Zahangir Alam and Assoc. Dr. Faridah Yusof for their ideas, critics, guidance and patience which the enable the author to complete her PhD research work. The author also would express her gratitude to Prof. Dr. Momoh-Jimoh E. Salami for his support and understanding throughout author’s study period. Not forgetting also sincere thanks to the staff of Deputy Dean, Postgraduate and Research, Kulliyah of Engineering, IIUM, Sr. Mariana and Sr. Roslina for their support. The author also would like to thank Dr. Raha Raus for her advice and help during author’s study.
Besides that, sincere thanks to Prof. Dr. Nassereldin Ahmed Kabashi for giving author a tuition session on statistical design.
The author is also grateful to the Ministry of Science, Technology and Innovation (MOSTI), Malaysia and IIUM for providing three years scholarship through PASCA programme to author for her research work. Apart from that, the author would also like to express her gratitude to IIUM Research Management Centre for providing a number of endowment funds (EDW A08-294; EDW B0901-193;
EDW B0905-287 and EDW B09-455) and the Department of Biotechnology Engineering, IIUM for their support.
The author would also like to thank Seri Ulu Langat Palm Oil Mill in Dengkil, Selangor, Malaysia for their co-operation in providing OPEFB sample throughout the study. The author also would like to extend her gratitude to Sr. Azirah and Sr.
Rosminah from MARDI, Serdang, Malaysia for their technical assistance.
The author also would like to express thanks to all IIUM staff, Eng. Zahir Hossain, Eng. Raziff, Br. Aslan, Br. Hafizul, Sr. Suharti and Sr. Adila who author appreciate their friendship and help. Last but not least, thanks to Sr. Afifah Darani, Br. Fahrurazi, Sr. Ain, Br. Shah Simur Rashid for their co-operation and help throughout author’s study.
xii
TABLE OF CONTENTS
Abstract…...………...…ii
Abstract in Arabic………….………iv
Approval Page…….………...…………...vi
Declaration……...………...……….………....vii
Copyright…………..………..viii
Dedication……...………..ix
Acknowledgements……….………...……….…...x
List of Tables………..…………xvii
List of Figures……..………xx
List of Abbreviations…………..……….……….………..……..xxvii
List of Symbols…….………xxix
CHAPTER 1: INTRODUCTION………...1
1.1 Background………..1
1.2 Problem Statement and its Significance………...………....3
1.3 Research Philosophy...……….…………...……….………….…4
1.4 Research Objectives...…………...……….….…5
1.5 Research Methodology…………...………..….6
1.6 Research Scope……….………….…….……….….…8
1.7 Thesis Organization…….……….9
CHAPTER 2: LITERATURE REVIEW……….……...11
2.1 Introduction………..………..………11
2.2 Agro-industrial Residual…………..……….……….11
2.2.1 Composition of Agro-residue...………..………....12
2.2.1.1 Cellulose………..…13
2.2.1.2 Lignin………...15
2.2.1.3 Hemicelluloses……….16
2.2.2 Component of Agro-residue.………...18
2.3 Oil Palm Empty Fruit Bunches and Utilization………...…...18
2.3.1 Characteristics of OPEFB……….19
2.3.2 Recent Utilization of OPEFB………19
2.3.2.1 OPEFB as Substrate Fermentation for Cellulase Enzymes Production………..21
2.4 Lignocellulose Pretreatment ………...……….…..23
2.5 Cellulases Producing Microorganisms………..……….…24
2.5.1 Trichoderma reesei as Cellulases Producer…………..….….……..25
2.5.2 Cellulase Enzymes………27
2.5.2.1 Secretion System of Cellulases………...28
2.5.2.2 Classes of Cellulases……….28
2.5.2.3 Mechanism of Cellulose Hydrolysis……….………....30
2.5.3 Cellulases Production Technology………31
xiii
2.6 Solid State Fermentation (SSF)………..……….………...…33
2.7 Factors Affecting Cellulases Production………...……….…35
2.7.1 Nutrients……….………...35
2.7.2 pH……….……….37
2.7.3 Temperature………...38
2.7.4 Moisture Content……….………...….39
2.7.5 Incubation Time………....40
2.7.6 Inoculum Size……….……….………..40
2.7.7 Particle Size……….……….………….41
2.7.8 Aeration……….………....42
2.7.9 Agitation……….………..….43
2.8 Solid State Bioreactors……….………..……44
2.8.1 Tray Bioreactors……….………...44
2.8.2 Drum Bioreactors……….……….45
2.8.3 Packed-bed Bioreactors……….………....46
2.8.4 Different Bioreactors Used in Cellulase Production……….46
2.9 Statistical Optimization of Cellulase Production Through Respond Surface Method (RSM) and Fractional Factorial Design……....47
2.9.1 Plackett-Burman Design………48
2.9.2 Central Composite Design (CCD)……….50
2.9.3 Fractional Factorial Design………...52
2.10 Kinetics of Cellulases…………...………....53
2.10.1 Enzyme Kinetics………..53
2.10.2 Kinetic Analysis………..54
2.10.2.1 Effect of Substrate Concentration, Incubation Time, Temperature and pH……….………57
2.11 Summary…….………..………...60
CHAPTER 3: MATERIALS AND METHODS……….62
3.1 Introduction……...………..………62
3.2 Experimental Materials……….……….…………63
3.2.1 Oil Palm Empty Fruit Bunches……….63
3.2.2 Preparation of Sample………...63
3.2.3 Microorganisms……….………65
3.2.4 Chemicals and Reagents………65
3.2.5 Mineral Solution………....65
3.2.6 Equipments and Instruments……….66
3.2.7 Consumable Items……….67
3.3 Experimental Methods…………..………..……...67
3.3.1 Characterization of Oil Palm Empty Fruit Bunches………...……….………67
3.3.1.1 Determination of Cellulose, Hemicellulose, Lignin and Ash Content of OPEFB…...67
3.3.1.1.1 Determination of Neutral Detergent Fiber (NDF) of OPEFB…………...……...……....68
3.3.1.1.2 Determination of Acid Detergent Fiber (ADF) of OPEFB………..………..…..68
xiv
3.3.1.1.3 Determination of Lignin Content of
OPEFB………...….………..…..69
3.3.1.1.4 Determination of Cellulose Content of OPEFB………..….70
3.3.1.1.5 Determination of Hemicellulose Content of OPEFB………..71
3.3.1.1.6 Determination of Ash Content of OPEFB…………...…………...………..…71
3.3.2 Determination of Cellulases Activities………..72
3.3.2.1 Determination of FPase Activity……….………...72
3.3.2.2 Determination of CMCase Activity…………...…73
3.3.2.3 Determination of β-glucosidase Activity……….….74
3.3.3 Determination of Lignin Peroxidase Activity………...75
3.3.4 Determination of Protein………...………..………..76
3.3.5 Determination of Glucosamine ……….……...77
3.3.6 Determination of Reducing Sugar of OPEFB………...…78
3.3.7 Determination of Reducing Sugar of Crude Sample…….……...….79
3.3.8 Extraction of Cellulase Enzymes………...……...79
3.3.8.1 Extraction Method for Culture Flask ……….………..79
3.3.8.2 Extraction Method for Bioreactor………..………...79
3.3.9 Cellulolytic Activity of Fungi Culture……….…….80
3.3.9.1 Cultural Cellulolytic Activity on Plates..…………...…….80
3.3.9.2 Cultural Cellulolytic Activity in Flask………...……...80
3.3.10 Pretreatment of OPEFB……...………...81
3.3.10.1 Effect of Particle Size on CMCase activity….……..…....82
3.3.10.2 Effect of the Autoclaving………...…….……..…....82
3.3.10.3 Effect of the Combination of Acid Pretreatment with Autoclaving………..…..………...83
3.3.10.4 Effect of the Combination of Alkali Pretreatment with Autoclaving..………..………...84
3.3.11 Optimization of Fermentation Condition for Cellulase Production………..………..…..85
3.3.11.1 Screening of Factors Effecting Cellulase Production………...…………...85
3.3.11.2 Optimisation of Significant Parameters Affecting Cellulase Production………..…87
3.3.11.2.1 Endoglucanase (CMCase) Production………...……..….…87
3.3.11.2.2 Exoglucanase (FPase) Production…..………..…..89
3.3.11.2.3 β-glucosidase Production……….…...…90
3.3.12 Characterization of Cellulases from Crude Fermentation Sample.………..…………...………...….90
3.3.12.1 Identification of Cellulases Dilution Factor……….………...……….…...90
3.3.12.2 Characterization of Endoglucanase (CMCase)………....91
3.3.12.3 Characterization of Exoglucanase (FPase)………..…….92
3.3.12.4 Characterization of β-glucosidase..…………..…………93
3.3.12.5 Kinetics of Cellulases………..……….94
xv
3.3.13 Optimisation of Operating Conditions in Stirred- horizontal Drum Bioreactor for Cellulase
Production………...………….…..95
3.3.13.1 Effect of Temperature………...………..….……..……..95
3.3.13.2 Effect of Inoculum Size…………...………….……...95
3.3.13.3 Effect of Amount of Substrate…………...……..……...95
3.3.13.4 Effect of Cellulases Production from the Optimization Process………...……….…...96
3.4 Summary…….………...97
CHAPTER 4: RESULTS AND DISCUSSION………98
4.1 Introduction………..……….…...98
4.2 Selection of Cellulase Producing Fungi………..…………...99
4.2.1 Cellulase Production on CMC Agar………..…………99
4.2.2 Cellulase Production From OPEFB in SSF flask culture….……...101
4.2.2.1 Endoglucanase Production (CMCase)………101
4.2.2.2 Exoglucanase Production (FPase)…...………..……..103
4.2.2.3 β-glucosidase Production……...………….………104
4.2.2.4 Protein Production……….……..106
4.2.2.5 pH Profile…...………..…...107
4.2.2.6 Reducing Sugar Profile…………...………108
4.2.3 Summary of Findings of the First Objective……….……..110
4.3 Characteristics of Native and Pretreated Oil Palm Empty Fruit Bunch (OPEFB)………...………110
4.3.1 Characteristics of OPEFB……….………..111
4.3.2 Endoglucanase (CMCase) Production on Different OPEFB Particle Sizes………....………...112
4.3.3 Effect of Autoclaving of Native OPEFB on Cellulose, Hemicelluloses and Lignin………....………..114
4.3.4 Acid Pretreatment of OPEFB………...115
4.3.4.1 Effect of Acid Pretreatment on Cellulose Content of OPEFB………116
4.3.4.2 Effect of Acid Pretreatment on Hemicellulose content of OPEFB………...118
4.3.4.3 Effect of Acid Pretreatment on Lignin Content of OPEFB………...….119
4.3.4.4 Surface Examination of Acid Pretreated OPEFB by Scanning Electron Microscope (SEM)……..………..120
4.3.5 Alkali Pretreatment of OPEFB……….………...…122
4.3.5.1 Effect of Alkali Pretreatment on Cellulose Content of OPEFB……….………...122
4.3.5.2 Effect of Alkali Pretreatment on Hemicellulose Content of OPEFB……….……….124
4.3.5.3 Effect of Alkali Pretreatment on Lignin Content of OPEFB………125
4.3.5.4 Surface Examination of Alkali Pretreated OPEFB by Scanning Electron Microscopy (SEM)………….…..…126 4.3.6 Comparison Between Acid Dilute and Alkali
xvi
Dilute Pretreatment……….…….127
4.3.7 Cellulase Production from Pretreated OPEFB………128
4.3.7.1 Effect of Endoglucanase (CMCase) Production………….128
4.3.7.2 Effect of Exoglucanase (FPase) Production……..…….….130
4.3.7.3 Effect of β-glucosidase Production………….…...……….131
4.3.8 Summary of Findings of the Second Objective………...……135
4.4 Optimization of Fermentation Condition for Cellulase Production...136
4.4.1 Screening of Significant Parameters Affecting Cellulase Production by Plackett-Burman Design………..136
4.4.2 The Optimization of Cellulases Production through Solid State Fermentation of Oil Palm Empty Fruit Bunches by Response Surface Method (RSM)…….………..141
4.4.2.1 Optimization of Parameters for Enhancing Endoglucanase (CMCase) Production……..………...142
4.4.2.2 Effect Optimization Process on Exoglucanase (FPase) Production……..……….152
4.4.2.3 Effect of Optimization Process on β-glucosidase Production………..………..157
4.4.2.4 Validation of optimization process of CMCase Production………172
4.4.3 Summary of Findings of the Third Objective………….…..….….176
4.5 Characterization of Cellulases Activities Derived from Solid State Fermentation (SSF) Oil Palm Empty Fruit Bunches (OPEFB) by Trichoderma reesei RUTC-30 Through Respond Surface Methodology (RSM)……….177
4.5.1 Identification of Cellulases Dilution Factor………...….177
4.5.1.1 Dilution Factor for CMCase………178
4.5.1.2 Dilution Factor for FPase……….………179
4.5.1.3 Dilution Factor for β-glucosidase………181
4.5.2 Characterization of Endoglucanase (CMCase) activity………...182
4.5.3 Characterization of Exoglucanase (FPase) Activity…………...194
4.5.4 Characterization of β-glucosidase Activity………...207
4.5.5 Kinetic of Cellulases………...220
4.5.5.1 Kinetic of CMCase……….….…………220
4.5.5.2 Kinetic of FPase...……….…………..223
4.5.5.3 Kinetic of β-glucosidase……….………….225
4.5.6 Summary of Findings of the Fourth Objective………..…………..227
4.6 Optimization of Operating Condition of the Stirred-drum Bioreactor for Cellulase Production…….……….228
4.6.1 Effect of Temperature………..………..….228
4.6.2 Effect of Inoculum Size………....……...229
4.6.3 Effect of Amount of Substrate………..…………...230
4.6.4 Effect of Cellulases Production from the Optimization of Process Parameters…….………...………..231
4.6.4.1 Effect of Exoglucanase and β-glucosidase Production from the Optimization Process……….……….235
4.6.4.2 Prediction of Cellulase Production from a Range of Optimum Aeration and Intermittent Agitation Rates………...….…236
xvii
4.6.4.3 Validation of Cellulase Production in SSB….………..…..240
4.6.5 Summary of Findings of the Fifth Objective………..241
4.7 Summary of Results……….241
CHAPTER 5: CONCLUSION AND RECOMMENDATION.…...……...……..244
5.1 Conclusion………..………..244
5.2 Recommendation………...………...…..248
BIBLIOGRAPHY………..……..………250
APPENDICES...……….……….………277
APPENDIX A: Culture Collection ……….…..…….………....277
APPENDIX B: List of Chemicals..………...…..……….…….…………..….278
APPENDIX C: Equipment ……….………279
APPENDIX D: Glassware and Consumable Items ……….………...…..281
APPENDIX E: Reagents Preparation ………..……….282
APPENDIX F: Standard Curves………283
APPENDIX G: The Student Achievement…...……….……….286
xviii
LIST OF TABLES
Table No. Page No.
2.1 Type of crops based on different group of dry residue. 12
2.2 Main components of agro-residues 18
2.3 Cellulose, hemicellulose and lignin composition of different lignocellulosic wastes 19
2.4 Nutrients content of OPEFB 19
2.5 List of common cellulase producing microorganisms 25
2.6 Cellulase production from various mutant Trichoderma reseei 26
2.7 Commercial cellulase from major USA cellulase manufacturers 33
2.8 Agro-industrial residues used for value added products 34
2.9 Agro-residues used in SSF for enzyme production 34
2.10 Study on effect of carbon and nitrogen in cellulase production 36
2.11 Various types of solid state bioreactors that have been used for fermentation production 45
2.12 Cellulases production studied in SSB 47
2.13 Various studies on optimization process using statistical approach in cellulase production 51
2.14 Characteristics of cellulases from different types of fungi 60
3.1 Composition of the mineral solution used in SSF 66
3.2 Variables of the moisture and autoclaving time applied on native OPEFB 83
3.3 Variables of treatments on OPEFB with different HCl concentrations and autolaving time 83
3.4 Variables of treatments on OPEFB with different NaOH concentrations and autoclaving time. 84
xix
3.5 Variables tested at different levels for cellulase production 86 3.6 Experimental design of central composite design
(CCD) for flask scale cellulase production 88 3.7 Serial dilution for crude sample 90 3.8 Experimental design CCD for characterization of
CMCase 92 3.9 Experimental design CCD for characterization of
FPase 93 3.10 Experimental design CCD for characterization of
β-glucosidase 94 3.11 Experimental design for bioreactor process using
Factorial design of DOE 96 4.1 Degree of clearing zone produced by strains on
CMC agar 101 4.2 Comparison of OPEFB composition 112 4.3 Effect of autoclaving of OPEFB fibre on hemicellulose,
cellulose and lignin composition 115 4.4 Plackett Burman experimental design matrix with observed and
predicted responses of different trials 137 4.5 Levels of variables tested and their effect on cellulase
production 138 4.6 Central composite design matrix for optimization of parameters
For CMCase production 143 4.7 Analysis of variance of parameters for CMCase production 144 4.8 Central composite design matrix for optimization of parameters
for FPase production 153 4.9 Analysis of variance of parameters for FPase production 154 4.10 Central composite design matrix for optimization of
parameters for β-glucosidase production 158 4.11 Analysis of variance of parameters for β-glucosidase production 159 4.12 Model validation experiments for CMCase production 172
xx
4.13 Central composite design matrix for characterization of
CMCase activity 183 4.14 Analysis of variance of parameters for CMCase activity 184 4.15 Central composite design matrix for characterization of
FPase activity 195 4.16 Analysis of variance of parameters for FPase activity 197 4.17 Central composite design matrix for characterization
of β-glucosidase activity 208 4.18 Analysis of variance of parameters for
β-glucosidase activity 209 4.19 Data of CMCase activity at different substrate concentrations
with calculated data for kinetic study 221 4.20 Data of FPase activity at different substrate concentrations
with calculated data for kinetic study 223 4.21 Data of β-glucosidase activity at different substrate
concentrations with calculated data for kinetic study 225 4.22 Experimental design matrix with observed and predicted
responses of different trials of endoglucanase (CMCase)
production. 232 4.23 Analysis of variance (ANOVA) for respond surface
model of SSB operational conditions 233 4.24 Experimental and predicted results of FPase and
β-glucosidase from the optimization of parameters
condition in SSB 236 4.25 Predicted production of CMCase at different values of
test variables 237
4.26 Comparison of endoglucanase production in different SSB 239
xxi
LIST OF FIGURES
Figure No. Page No.
1.1 Flowchart of research methodology 7 2.1 Chemical structure of cellulose 14 2.2 Hydrophorbic and hydrophilic nature in cellulose
structure 14 2.3 Chemical structure of lignin 16
2.4 Chemical structure of hemicelluloses 17 2.5 General schematic sketch of a fungal cellulase 29 2.6 Cellulose hydrolysis by cellulases 31 2.7 Reaction velocity (v) as a function of the substrate
Concentration [S] for an enzyme-catalyzed reaction.
At high substrate concentrations the reaction velocity reaches a limiting value, Vmax. Km is the substrate
concentration at which the rate is half maximal. 56
2.8 Plot of the rate (1/v0 as function of the substrate concentration (1/[S]) fits a straight line. Extrapolating the line to its intercept on the infinite substrate
concentration gives 1/Vmax. Extrapolating to the
intercept on the abscissa gives -1/Km. 57
3.1 Experimental procedure 64
3.2 Schematic diagram of a 30 L stirred-horizontal drum
bioreactor 66 4.1a-e Ability of strains to hydrolyse cellulose on
CMC agar 100 4.2 Endoglucanase production in SSF by different
fungal strains 103
4.3 Exoglucanase production in SSF by different fungal
strains 104
4.4 β-glucosidase production in SSF by different fungal strains 105
xxii
4.5 Protein profile of different fungal strains 106
4.6 pH profile in cellulase production in SSF by different
fungal strains 107
4.7 Reducing sugar profile in SSF of OPEFB by different
fungal strains 109 4.8 CMCase production on different
particle sizes 113
4.9 Effect of OPEFB pretreatment with hydrochloric acid on hemicelluloses, cellulose and
lignin composition 117
4.10 SEM of (a) untreated OPEFB, (b) OPEFB autoclaved for
15 minutes 120
4.11a-i OPEFB treated with 1N HCl followed by autoclaving for (a) 10 minutes, (b) 20 minutes and (c) 30 minutes; OPEFB treated with 2N HCl followed by autoclaving for
(d) 10 minutes, (e) 20 minutes and (f) 30 minutes;
OPEFB treated with 3N HCl followed by autoclaving for (g) 10 minutes, (h) 20 minutes and
(i) 30 minutes 121 4.12 Effect of OPEFB pretreatment with sodium hydroxide on
hemicellulose, cellulose and lignin
composition 123
4.13a-i OPEFB treated with 1N NaOH followed by autoclaving for (a) 10 minutes, (b) 20 minutes and (c) 30 minutes;
OPEFB treated with 2N HCl followed by autoclaving for (d) 10 minutes, (e) 20 minutes and (f) 30 minutes; OPEFB treated with 3N HCl followed by autoclaving for (g) 10 minutes,
(h) 20 minutes and (k) 30 minutes 126
4.14 Production of CMCase on pretreated OPEFB 129
4.15 Production of FPase on pretreated OPEFB 131
4.16 Production of β-glucosidase on pretreated OPEFB 133 4.17 Sugar content of OPEFB due to HCl/NaOH treatment 135
4.18 Effect of independent variables on cellulase production 140
4.19 Three-dimensional response surface and corresponding contour plot for CMCase production (U/gds) in relation
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to initial moisture (%) and ammonium sulfate
concentration (mg/L) 146
4.20 Three-dimensional response surface and corresponding contour plot for CMCase production (U/gds) in relation
to initial moisture (%) and incubation time (day) 146
4.21 Three-dimensional response surface and corresponding contour plot for CMCase production (U/gds) in relation to spore density (1 x 10x spores/ml) and ammonium
sulfate concentration (mg/L) 147
4.22 Three-dimensional response surface and corresponding contour plot for FPase production (U/gds) in relation
to initial moisture (%) and incubation time (day) 155 4.23 Three-dimensional response surface and corresponding
contour plot for β-glucosidase production (U/gds) in relation to incubation time (day) and initial
moisture (%) at ammonium sulfate concentration 118 mg/L 160
4.24 Three-dimensional response surface and corresponding contour plot for β-glucosidase production (U/gds) in relation to spore density (1 x 10x spores/ml) and initial moisture (%) at ammonium sulfate concentration
118 mg/L 161 4.25 Three-dimensional response surface and corresponding
contour plot for β-glucosidase production (U/gds) in relation to spore density (1 x 10x spores/ml) and incubation time (day) at ammonium sulfate
concentration 118 mg/L 161 4.26 Three-dimensional response surface and corresponding
contour plot for β-glucosidase production (U/gds) in relation to spore density (1 x 10x spores/ml) and
initial moisture (%) at day 10 162 4.27 Three-dimensional response surface and corresponding
contour plot for β-glucosidase production (U/gds) in relation to spore density (1 x 10x spores/ml) and
ammonium sulfate concentration (mg/L) at day 10 163
4.28 Three-dimensional response surface and corresponding contour plot for β-glucosidase production (U/gds) in relation to initial moisture (%) and ammonium
sulfate concentration (mg/L) at day 10 164 4.29 Three-dimensional response surface and corresponding
xxiv
contour plot for β-glucosidase production (U/gds) in relation to ammonium sulfate (mg/L) and incubation
time (day) at spore density of 1 x 107 spores/ml 165
4.30 Three-dimensional response surface and corresponding contour plot for β-glucosidase production (U/gds) in relation to initial moisture (%) and ammonium sulfate concentration (mg/L) at spore density
of 1 x 107 spores/ml 166 4.31 Three-dimensional response surface and corresponding
contour plot for β-glucosidase production (U/gds) in relation to initial moisture (%) and incubation
time (day) at spore density of 1 x 107 spores/ml 167
4.32 Three-dimensional response surface and corresponding contour plot for β-glucosidase production (U/gds) in relation to incubation time (day) and ammonium sulfate concentration (mg/L) at initial
moisture of 58% 168
4.33 Three-dimensional response surface and corresponding
contour plot for β-glucosidase production (U/gds) in relation to spore density (1 x 10x spores/ml) and
ammonium sulfate concentration (mg/L) at initial moisture
of 58% 169 4.34 Three-dimensional response surface and corresponding
contour plot for β-glucosidase production (U/gds) in relation to spore density (1 x 10x spores/ml) and
incubation time (day) at initial moisture of 58% 170
4.35 Profile of cellulase enzymes production in flask culture SSF 173
4.36 Logarithmic fungal growth during SSF 176 4.37 CMCase activity profile from a range of diluted sample 179
4.38 FPase activity profile from a range of
diluted sample 180 4.39 β-glucosidase activity profile from a range of
diluted sample 181 4.40 Three-dimensional response surface and corresponding
contour plot for CMCase activity (U/ml) in
relation to pH and temperature (%) at substrate concentration
of 2.25 % and incubation time of 16 min 185
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4.41 Three-dimensional response surface and corresponding contour plot for CMCase activity (U/ml) in relation to substrate concentration (%) and temperature (00C)
when pH and incubation time were at 6 and 16 min. 187 4.42 Three-dimensional response surface and corresponding
contour plot for CMCase activity (U/ml) in relation to incubation time (min)
and temperature (0C) at pH and substrate concentration
of 6 and 2.25 %, respectively. 188 4.43 Three-dimensional response surface and corresponding
contour plot for CMCase activity (U/ml) in relation to substrate concentration (%)and pH at temperature 500C
and incubation time of 16 min. 189 4.44 Three-dimensional response surface and corresponding
contour plot for CMCase activity (U/ml)
in relation to incubation time (min) and pH when temperature and substrate concentration were at
500C and 2.25 %, respectively. 190
4.45 Three-dimensional response surface and corresponding contour plot for CMCase activity (U/ml)
in relation to incubation time (min) and substrate concentration (%) when temperature and pH were at
500C and 6, respectively. 192
4.46 Three-dimensional response surface and corresponding contour plot for FPase activity (U/ml) in relation to pH and temperature (0C) at substrate concentration
of 67 mg/ml and incubation time of 70 min 198
4.47 Three-dimensional response surface and corresponding contour plot for FPase activity (U/ml)
in relation to substrate concentration (mg/ml) and
temperature (0C) at pH 6 and incubation time of 70 min. 200
4.48 Three-dimensional response surface and corresponding contour plot for FPase activity (U/ml)
in relation to temperature (0C) and incubation time (min) at pH 6 and substrate concentration of
67 mg/ml. 201 4.49 Three-dimensional response surface and corresponding
contour plot for FPase activity (U/ml) in relation to pH and substrate concentration (mg/ml) and pH when temperature and incubation time of 60 0C
and 70 min, respectively. 202
