CHAPTER NO. TITLE PAGES

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1 BRIEF CONTENTS CHAPTER NO. TITLE PAGES PREFACE 1 1 INTRODUCTION 3 2 REVIEW OF LITERATURE 6 3 MATERIALS & METHODS 70 4 OBSERVATIONS & RESULTS DISCUSSION SUMMARY & CONCLUSIONS 243 BIBLIOGRAPHY 248 i

2 DETAILED CONTENTS S. No. TITLE Page No. Brief Contents i Detailed Contents ii-xii List of Tables xiii-xv List of Figures xvi-xxiv List of Publication xxv-xxvii PREFACE 1 1. INTRODUCTION 3 2. REVIEW OF LITERATURE 6 History of ethanol as biofuel 6 Bioethanol: First and Second generations 6 First generation bioethanol 6 Second generation bioethanol 7 Current Status of Bioethanol 8 Current status of Bio-ethanol production worldwide 8 Status of bioethanol production in India 10 Lignocellulose 11 Cellulose 11 Hemicelluloses 13 Lignin 16 Bioconversion of lignocellulosic biomass into ethanol 18 Pretreatment of lignocellulosic biomass 18 Detoxification 30 Enzymatic hydrolysis 33 Fermentation 37 Ethanol Recovery: Distillation and Dehydration 45 Strain Improvement for ethanol fermentation 46 Mutation 46 ii

3 Protoplast Fusion 47 Adaptation 48 Genetic manipulation for improved ethanol fermentation 50 Economic evaluation of cellulosic ethanol production 59 Commercialization of bioethanol 61 Future Prospects MATERIALS & METHODS 70 I MATERIALS 70 II METHODS Pretreatment 73 Compositional analysis of P. juliflora 73 Selection of pretreatment strategies for the fractionation of P. 75 juliflora Chemical pretreatments 75 Acid pretreatment 75 Alkali pretreatment 75 Chlorite pretreatment 75 Biological pretreatments 76 Microorganism and inoculum preparation 76 Fungal treatment of P. juliflora under solid state fermentation (SSF) 76 conditions Evaluation of pretreatment strategies on the basis of their 77 enzymatic hydrolysis Optimization of acid hydrolysis of lignocellulosic substrates 77 One factor at a time (OFAT) approach 77 Effect of substrate consistencies on the hydrolysis of P. juliflora 78 Effect of pretreatment temperature on the hydrolysis of P. juliflora 78 Effect of acid concentration on the hydrolysis of P. juliflora 78 Effect of pretreatment time on the hydrolysis of P. juliflora 78 Response surface methodology (RSM) using Box Behnken design 79 (BBD) Scale up of acid hydrolysis 80 iii

4 Laboratory scale 80 Pilot scale 80 Optimization of Chlorite treatment of acid hydrolysed 80 lignocellulosic substrate One factor at a time (OFAT) approach 80 Effect of substrate consistencies on the delignification of P. juliflora 81 Effect of pretreatment temperature on the delignification of the acid 81 hydrolysed P. juliflora Effect of sodium chlorite concentration on the delignification of the acid 81 hydrolysed P. juliflora Effect of pretreatment time on the delignification of the acid hydrolysed P. 82 juliflora Response surface methodology (RSM) using Box Behnken design 82 (BBD) Scale up of chlorite treatment 83 Laboratory scale 83 Pilot scale 83 Fourier Transform Infrared Spectroscopy (FTIR) 84 characterization of biomass samples 3.2 Detoxification of acid hydrolysate 84 Detoxification of acid hydrolysate of P. juliflora by neutralization 84 Detoxification of acid hydrolysate by overliming 84 Detoxification of acid hydrolysate by activated charcoal treatment 84 Detoxification of acid by ion exchange resins 85 Detoxification of acid hydrolysate by enzymatic treatment 85 Characterization of detoxified hydrolysates by HPLC 85 Scale up of detoxification strategy 86 Laboratory scale 86 Pilot scale Enzymatic saccharification of delignified substrate 86 Batch enzymatic hydrolysis 86 Optimization of enzymatic hydrolysis using one factor at a time 86 (OFAT) approach Time course of enzymatic saccharification of delignified substrate 86 iv

5 Effect of FPase and β-glucosidase ratio on the enzymatic saccharification of 87 delignified substrate Effect of substrate consistencies on the enzymatic saccharification of 87 delignified substrate Effect of incubation temperature on the enzymatic saccharification of 87 delignified substrate Effect of initial ph of buffer on the enzymatic saccharification of delignified 88 substrate Effect of agitation rate on the enzymatic saccharification of delignified 88 substrate Effect of surfactants on the enzymatic saccharification of delignified 89 substrate Effect of various dosage of Tween 80 on the enzymatic saccharification of 89 delignified substrate Effect of divalent cations on the enzymatic saccharification of delignified 89 substrate Effect of various concentration (mm) of Cobalt ions (Co ++ ) on the enzymatic 90 saccharification of delignified substrate Effect of different enzyme dosage on the enzymatic saccharification of 90 delignified substrate Response surface methodology (RSM) using Box Behnken design 91 (BBD) Scale up of enzymatic hydrolysis 92 Laboratory scale 92 Bioreactor scale 92 Fed-batch enzymatic hydrolysis 93 Optimization of enzyme dosage on the fed-batch enzymatic hydrolysis of delignified substrate 93 Scale up of fed-batch enzymatic hydrolysis at pilot scale 93 Kinetic analysis and simulation of batch and fed-batch 94 enzymatic hydrolysis of delignified substrate 3.4 Fermentation of hydrolysates 96 Microorganisms and maintenance 96 Fermentation of pentose sugars (detoxified acid hydrolysate) 96 Screening of pentose fermenting microorganisms 96 Optimization of ethanol production from detoxified acid. Time course of ethanol production from detoxified acid hydrolysate using P. stipitis NCIM v

6 Effect of initial ph of the medium on the ethanol production from detoxified 97 acid Effect of incubation temperature on the ethanol production from detoxified 97 acid Effect of agitation (rpm) on the ethanol production from detoxified acid 98 Effect of inoculum size on the ethanol production from detoxified acid 98 Effect of inoculum age on the ethanol production from detoxified acid 98 Effect of soybean meal on the ethanol production from detoxified acid 98 Effect of casamino acid on the ethanol production from detoxified acid 99 Effect of yeast nitrogen base on the ethanol production from detoxified acid 99 Effect of vitamins on the ethanol production from detoxified acid 99 Effect of divalent cations on the ethanol production from detoxified acid 100 Scale up of ethanol production from detoxified acid hydrolysate 100 using P. stipitis NCIM 3499 Laboratory scale 100 Bioreactor scale 101 Fermentation of hexose sugars (enzymatic hydrolysate) 101 Optimization of ethanol production from enzymatic 101 at laboratory scale Time course of ethanol production 101 Effect of initial ph of the medium on the ethanol production from enzymatic Effect of incubation temperature on the ethanol production from enzymatic Effect of agitation (rpm) on the ethanol production from enzymatic Effect of inoculum size on the ethanol production from enzymatic Effect of inoculum age on the ethanol production from enzymatic Effect of soybean meal on the ethanol production from enzymatic vi

7 Effect of casamino acid on the ethanol production from enzymatic 104 Effect of yeast nitrogen base on the ethanol production from enzymatic 104 Effect of vitamins on the ethanol production from enzymatic hydrolysate 104 using S. cerevisiae HAU Effect of metal ions on the ethanol production from enzymatic 105 Scale up of ethanol production from enzymatic hydrolysate 105 using S. cerevisiae HAU Laboratory scale 105 Bioreactor scale Distillation of ethanol Mass balance of ethanol production from P. juliflora Analytical methods 107 HPLC characterization of sugars 107 Determination of ethanol 107 Determination of total reducing sugars 108 Determination of phenolics 108 Determination of pentosans 108 Determination of furans 108 Determination of enzymatic saccharification efficiency 108 Enzyme assays OBSERVATIONS & RESULTS Pretreatment 110 Compositional analysis of lignocellulosic substrates 110 Pretreatment strategies 110 Chemical pretreatments 110 Acid pretreatment 110 Alkali treatment 111 Chlorite treatment 112 Biological pretreatment 114 Evaluation of pretreatment strategies for their enzymatic 115 saccharification of pretreated substrates vii

8 Optimization of acid hydrolysis of lignocellulosic substrates 117 One factor at a time (OFAT) approach 117 Effect of substrate consistencies on the acid hydrolysis of P. juliflora 117 Effect of pretreatment temperature on the hydrolysis of P. juliflora 117 Effect of acid concentration on the hydrolysis of P. juliflora 118 Effect of pretreatment time on the hydrolysis of P. juliflora 118 Response surface methodology (RSM) using Box Behnken design 119 (BBD) Scale up of acid hydrolysis of P. juliflora 124 Optimization of Chlorite treatment of acid hydrolysed 124 substrates One factor at a time (OFAT) approach 124 Effect of substrate consistencies on the delignification of acid hydrolysed P. 124 juliflora Effect of pretreatment temperature on the delignification of acid hydrolysed 125 P. juliflora Effect of sodium chlorite concentration on the delignification of acid 125 hydrolysed P. juliflora Effect of pretreatment time on the delignification of acid hydrolysed P. 126 juliflora Response surface methodology (RSM) using Box Behnken design 127 (BBD) Scale up of delignification of acid hydrolysed P. juliflora 131 Fourier Transform Infrared Spectroscopy (FTIR) 131 characterization of biomass samples 4.2 Detoxification 133 Acid hydrolysis of lignocellulosic substrate 133 Detoxification of acid hydrolysate 133 Neutralization 133 Overliming 134 Activated charcoal treatment 134 Ion exchange resins 135 Enzymatic treatment 136 Comparison among different treatments 137 HPLC characterization of acid hydrolysates 137 Scale up of detoxification strategy 142 Recycling of activated charcoal Enzymatic saccharification of delignified lignocellulosic biomass 144 viii

9 Optimization of enzymatic saccharification 144 One factor at a time (OFAT) approach 144 Time course of enzymatic saccharification of delignified lignocellulosic 144 biomass Effect of β-glucosidase dosage in the enzymatic saccharification of 145 delignified lignocellulosic biomass Effect of substrate consistency on the enzymatic saccharification of 146 delignified lignocellulosic biomass Effect of temperature on the enzymatic saccharification of delignified 146 lignocellulosic biomass Effect of agitation (revolution per minute) on the enzymatic saccharification 147 of delignified lignocellulosic biomass Effect of initial ph of the buffer on the enzymatic saccharification of 148 delignified lignocellulosic biomass Effect of various surfactants on the enzymatic saccharification of delignified 149 lignocellulosic biomass Effect of divalent cations on the enzymatic saccharification of delignified 151 lignocellulosic biomass Effect of different enzyme dosages on the enzymatic saccharification of 152 delignified lignocellulosic biomass Optimization of enzymatic hydrolysis of delignified lignocellulosic 153 biomass using Box Behnken design (BBD) of Response surface methodology (RSM) Scale up of enzymatic hydrolysis of delignified lignocellulosic 158 biomass Scale up of enzymatic saccharification at laboratory scale 158 Scale up of enzymatic saccharification at bioreactor level 158 Fed-batch enzymatic hydrolysis of delignified lignocellulosic 160 biomass Optimization of enzyme dosage on the fed-batch enzymatic 160 hydrolysis of delignified substrate Scale up of fed-batch enzymatic hydrolysis at bioreactor scale 161 Kinetic analysis and simulation of batch and fed-batch 163 enzymatic hydrolysis of delignified substrate Batch enzymatic hydrolysis 163 Kinetics of fed-batch enzymatic hydrolysis 166 Comparison between batch and fed-batch enzymatic hydrolysis 166 Model prediction for feeding policy Fermentation of detoxified acid hydrolysate 172 Screening of yeast strains for xylose utilization and xylose to ethanol conversion ability 172 ix

10 Optimization of ethanol production from detoxified acid 174 at laboratory scale. Time course of ethanol production from detoxified acid hydrolysate 174 using P. stipitis NCIM 3499 Effect of initial ph of the medium on the ethanol production using 175 P. stipitis NCIM 3499 Effect of incubation temperature on the ethanol production using 177 P. stipitis NCIM 3499 Effect of agitation (rpm) on the ethanol production using P. stipitis 179 NCIM 3499 Effect of inoculum size on the ethanol production using P. stipitis 181 NCIM 3499 Effect of inoculum age on the ethanol production from detoxified 183 acid Effect of soybean meal on the ethanol production from detoxified 185 acid Effect of casamino acid on the ethanol production from detoxified 187 acid Effect of yeast nitrogen base on the ethanol production from 189 detoxified acid Effect of different vitamins on the ethanol production from 189 detoxified acid Effect of divalent cations on the ethanol production from detoxified 190 acid Scale up of ethanol production from detoxified acid 191 hydrolysate Scale up of ethanol production from detoxified acid hydrolysate 191 using P. stipitis NCIM 3499 at laboratory level. Scale up of ethanol production from detoxified acid hydrolysate 193 using P. stipitis NCIM 3499 at bioreactor level. Optimization of dissolved oxygen for the ethanol production from detoxified 193 acid at 3.0 L bioreactor Scale up of ethanol production from detoxified acid hydrolysate using P. 195 stipitis NCIM 3499 at 13L and 30L bioreactor scale. 4.5 Fermentation of enzymatic hydrolysate 197 Optimization of ethanol production from enzymatic at laboratory scale Time course of ethanol production from the enzymatic hydrolysate using S. cerevisiae HAU x

11 Effect of initial ph of the medium on the ethanol production from 198 enzymatic Effect of incubation temperature on the ethanol production from 200 enzymatic Effect of agitation (rpm) on the ethanol production from enzymatic 202 Effect of inoculum size on the ethanol production from enzymatic 204 Effect of inoculum age on the ethanol production from enzymatic 206 Effect of soybean meal on the ethanol production from enzymatic 208 Effect of casamino acid on the ethanol production from enzymatic 210 Effect of yeast nitrogen base on the ethanol production from 210 enzymatic Effect of vitamins on the ethanol production from enzymatic 211 Effect of divalent cations on the ethanol production from 212 enzymatic Scale up of ethanol production from enzymatic hydrolysate 213 Scale up of ethanol production from enzymatic hydrolysate using 213 S. cerevisiae HAU at laboratory level Scale up of ethanol production at pilot scale 215 Optimization of dissolved oxygen for the production of ethanol from 215 enzymatic at bioreactor level. Scale up of ethanol production from enzymatic hydrolysate using S. 217 cerevisiae HAU at bioreactor scale. 4.6 Distillation of ethanol Mass balance evaluation for the ethanol production 220 process 5 DISCUSSION SUMMARY & CONCLUSION 243 BIBLIOGRAPHY 248 xi