SHORT-TERM LIME PRETREATMENT AND ENZYMATIC CONVERSION OF SAWDUST INTO ETHANOL
By
Augustine Omoniyi AYENI (CUGP050132)
Presented To
Department of Chemical Engineering
ABSTRACT
The utilization of lignocellulosic biomass as feedstock for the production of fuel ethanol has attracted considerable interests in the last few decades. The emergence of new technologies has provided hope for fuel ethanol potential uses. Lignocellulose is a valuable alternative energy source. The enzymatic hydrolysis of lignocellulosic biomass is constrained due to its complex structural features, so pretreatment is important to enhance its enzymatic digestibility. In this study, the influence of process parameters â€" temperature, air addition, hydrogen peroxide addition, and time â€" on the pretreatment of sawdust (a wood residue) was investigated. The optimization of the pretreatment step was performed by using the full factorial and central composite designs of experiments. The study assessed the compositional changes by applying short-term oxidative pretreatments such as alkaline wet air oxidation, alkaline peroxide oxidation, and alkaline peroxide assisted wet air oxidation methodologies, and their effects on the yields of reducing sugar. The best pretreatment condition based on the yield of the reducing sugar was the alkaline peroxide-assisted wet air oxidation at 150 oC, 1%H2O2, 10 bar air pressure, 45 min. The optimal 4-day reducing sugar yield was 335.35 mg equivalent glucose/g dry biomass at 40 g/L substrate concentration, 25 FPU/g dry substrate of cellulase enzyme,
and 5 IU/g dry substrate of β-glucosidase. Furthermore, when considering the fermentability of the treated solids, at 2% effective cellulose loading, 9.71 g/L ethanol (23.43% theoretical ethanol yield) was obtained for pretreatment at 150 oC, 1%H2O2, 10 bar air pressure, and 45 min. At the optimum pretreatment condition, 0.1 g Ca(OH)2/g dry biomass was enough to cause appreciable lignin removal. Lignin removal was largely dependent on temperature, and the prevailing oxidative conditions. Cellulose was highly preserved in the solid fraction, while more of the hemicellulose was solubilized/degraded. The high-lignin content of the raw material was a great obstacle to the digestibility of the treated material. The lignin remained largely undissolved in the solid fraction.
The utilization of lignocellulosic biomass as feedstock for the production of fuel ethanol has attracted considerable interests in the last few decades. The emergence of new technologies has provided hope for fuel ethanol potential uses. Lignocellulose is a valuable alternative energy source. The enzymatic hydrolysis of lignocellulosic biomass is constrained due to its complex structural features, so pretreatment is important to enhance its enzymatic digestibility. In this study, the influence of process parameters â€" temperature, air addition, hydrogen peroxide addition, and time â€" on the pretreatment of sawdust (a wood residue) was investigated. The optimization of the pretreatment step was performed by using the full factorial and central composite designs of experiments. The study assessed the compositional changes by applying short-term oxidative pretreatments such as alkaline wet air oxidation, alkaline peroxide oxidation, and alkaline peroxide assisted wet air oxidation methodologies, and their effects on the yields of reducing sugar. The best pretreatment condition based on the yield of the reducing sugar was the alkaline peroxide-assisted wet air oxidation at 150 oC, 1%H2O2, 10 bar air pressure, 45 min. The optimal 4-day reducing sugar yield was 335.35 mg equivalent glucose/g dry biomass at 40 g/L substrate concentration, 25 FPU/g dry substrate of cellulase enzyme,
and 5 IU/g dry substrate of β-glucosidase. Furthermore, when considering the fermentability of the treated solids, at 2% effective cellulose loading, 9.71 g/L ethanol (23.43% theoretical ethanol yield) was obtained for pretreatment at 150 oC, 1%H2O2, 10 bar air pressure, and 45 min. At the optimum pretreatment condition, 0.1 g Ca(OH)2/g dry biomass was enough to cause appreciable lignin removal. Lignin removal was largely dependent on temperature, and the prevailing oxidative conditions. Cellulose was highly preserved in the solid fraction, while more of the hemicellulose was solubilized/degraded. The high-lignin content of the raw material was a great obstacle to the digestibility of the treated material. The lignin remained largely undissolved in the solid fraction.
TABLE OF CONTENTS PAGE
TITLE PAGE - - - - i
CERTIFICATION - - - - ii
DEDICATION - - - - iii
ACKNOWLEDGEMENTS - - - iv
TABLE OF CONTENTS - - - - vi
LIST OF FIGURES - - - - x
LIST OF TABLES - - - - xv
ABBREVIATIONS - - - - xvii
ABSTRACT - - - - xviii
CHAPTER ONE
1 INTRODUCTION - - - - 1
11 Background of the study - - - 1
12 Statement of research problem - - - 2
13 Objectives of the study - - - 3
14 Significance of the study - - - 3
15 Scope and limitation of the study - - - 4
CHAPTER TWO
2 LITERATURE REVIEW - - - 6
21 Sawdust as a renewable energy source - - - 6
22 Alcohol production from biomass - - - 8
23 Chemical structure of lignocellulosic biomass - - 12
231 The cell wall - - - - 13
232 Cellulose - - - - 16
233 Hemicellulose - - - - 18
234 Lignin - - - - - 18
24 Lignin-carbohydrate complexes - - - 20
241 Lignin-hemicellulose bonds - - - 22
25 Woody biomass and non-woody biomass - - - 22
2 6 Pretreatment methods - - - - 25
261 Alkaline hydrolysis - - - - 27
262 Oxygen delignification - - - - 28
2621 Hydrogen - - - - - 29
2622 Per-acetic acid - - - - 29
2623 Ozonolysis - - - - 30
2624 Wet air oxidation - - - - 30
2625 Alkaline peroxide assisted wet air oxidation - - 31
27 Chemical reactions during alkaline pretreatment - - 31
28 Oxygen species - - - - 31
29 Mechanism of carbohydrates degradation - - 35
210 Mechanism of lignin removal - - - 37
CHAPTER THREE
3 MATERIALS AND METHODS - - - 40
31 Statistical designs of experiments - - - 40
311 Choice of experimental design - - - 40
32 Preparation, storage and handling of raw material - - 43
33 Compositional analysis of raw and pretreated raw material - - 43
331 Extractives - - - - - 44
332 Hemicellulose - - - - 44
333 Ash - - - - - 45
334 Determination of lignin (acid insoluble and soluble) - - 45
335 Determination of cellulose content - - - 45
34 Reactor set up for pretreatments - - - 45
35 Pretreatment method - - - - 50
36 Lime as the alkaline pretreatment agent - - - 50
361 Specific lime consumption determination - - 51
37 Enzymatic conversion - - - - 53
371 Cellulase activity determination - - - 54
372 Convertibility of the sawdust material - - - 54
38 Fermentation of treated and untreated materials - - 55
CHAPTER FOUR
4 RESULTS - - - - 56
42 Composit ion of pretreated solid fraction - - - 60
421 Lignin removal (delignification) - - - - 59
43 Statistical optimization of the pretreatment process - - 60
431 Alkaline WAO pretreatments - - - 61
432 23 CCD alkaline peroxide oxidation pretreatment - - 64
433 22 CCD alkaline peroxide oxidation pretreatment - - 64
434 Alkaline peroxide assisted wet air oxidation pretreatment (APAWAO) 67
44 Enzymatic hydrolysis of raw and pretreated solids - - 67
441 Cellulase enzyme activity - - - 69
442 Hydrolysis of pretreated solids - - - 72
443 Effect of substrate concentration on enzymatic hydrolysis - 76
444 Effects of enzyme loading on enzymatic hydrolysis - - 76
445 Enzymatic hydrolysis of untreated and washed only biomass - 80
45 Simultaneous saccharification and fermentation - - 80
CHAPTER FIVE
5 DISCUSSIONS - - - - 84
51 Characterization of untreated (raw) biomass - - - 84
52 Changes of biomass during pretreatment - - - - 86
521 Pretreatment yields - - - - - - 86
522 Holocellulose removal in the pretreated solids - - 88
53 Alkaline WAO pretreatments optimization - - - 92
54 23 CCD alkaline peroxide oxidation pretreatments optimization - 107
55 22 CCD alkaline peroxide oxidation pretreatments optimization - 120
56 Alkaline peroxide assisted WAO pretreatments - - 125
57 Enzymatic hydrolysis of treated solids - - - 127
58 Substrate concentration on enzymatic hydrolysis - - 130
59 Enzyme loading studies - - - - 136
510 Hydrolysis studies of untreated and washed only biomass - - 142
511 Simultaneous saccharification and fermentation - - 143
CHAPTER SIX
6 CONCLUSIONS AND RECOMMENDATIONS - - 145
61 Conclusions - - - - - 145
62 Recommendations - - - - 146
REFERENCES - - - - - 147
APPENDIX A - - - - - 164
APPENDIX B - - - - - 166
APPENDIX C - - - - - 168
APPENDIX D - - - - - 170
APPENDIX E - - - - - 172
APPENDIX F - - - - - 175
APPENDIX G - - - - - 178
APPENDIX H - - - - - 180
APPENDIX I - - - - - 182
APPENDIX J - - - - - 187
APPENDIX K - - - - - 189
APPENDIX L - - - - - 191
APPENDIX M - - - - - 193
APPENDIX N - - - - - 195
APPENDIX O - - - - - 196
LIST OF FIGURES
FIGURE PAGE
21 Schematic diagram of traditional biomass conversion to ethanol - 10
22 Mode of action of cellulolytic enzymes - - 11
23 Micro- and macro-fibrils (fibres) formation of cellulose and their
positions in the wood cell wall - - - 14
24 Schematic of goals of pretreatment on lignocellulosic materials - 15
25 The cellulose chain - - - - 17
26 Hemicellulose monomer units - - - 19
27 Distinguishing chemical structures of lignin building blocks - 21
28 Proposed model types of lignin carbohydrate linkages - - 23
29 Oxygen species derived from molecular oxygen in aqueous solution - 34
210 A proposed mechanism for carbohydrate degradation by hydroxyl
radical during oxidative delignification - - 36
211 Radical chain reactions during oxygen delignification in alkaline
condition - - - - 38
212 Proposed reaction of lignin via phenoxyradical - - 39
31 Schematic diagram of the pretreatment reactor set up - 47
32 Complete reactor set up with the process and power controllers - 48
33 Pictures of reactor system - - - - 49
34 Slurry and solid fraction of pretreated biomass - - 52
41 The average particle size distribution of dry raw sawdust - 57
42 Mass balance for the optimized alkaline peroxide
oxidat ion pretreatment - - - 65
43 Construction of glucose standard curve for cellulase activity
determination - - - - 70
44 Enzyme dilution vs glucose concentration for cellulase activity
determination - - - - 71
45 Pretreated samples preparation for enzymatic hydrolysis and
fermentation - - - - 74
51 Surface plots of lime consumed (g Ca(OH)2/g raw biomass): (a) vs
temperature and % H2O2, (b) vs temperature and time for 23 CCD
APO design - - - - 85
52 Surface plot of lime consumed (g Ca(OH)2/g dry biomass) vs time
and temperature for 22
CCD APO design - - 87
53 Surface plot of pretreatment yield of holocellulose (g recovered/100g
in raw biomass) vs lignin removal %(w/w) and temperature for 23
full factorial WAO pretreatments - - - 89
54 Surface plot of pretreatment yield of holocellulose (g recovered/100g
in raw biomass) vs lignin removal %(w/w) and temperature for 23
CCD APO pretreatments - - - 89
55 Surface plot of pretreatment yield of holocellulose (g recovered/100g
in raw biomass) vs lignin removal %(w/w) and temperature for 22
CCD APO pretreatments - - - 90
56 Surface plot of lignin removal %(w/w) vs lime consumed
(g Ca(OH)2/g raw biomass) and temperature - - 91
57 Surface plot of lignin removal %(w/w) vs lime consumed
(g Ca(OH)2/ g raw biomass) and time - - 91
58 (a) contour plot, (b) surface plot of cellulose content %(w/w) vs
time and temperature for 23
full factorial WAO pretreatment - 96
59 (a) contour plot, (b) surface plot of cellulose content %(w/w) vs
pressure and time - - - - 97
510 (a) contour plot, (b) surface plot of cellulose content %(w/w) vs
pressure and temperature - - - 98
511 (a) contour plot, (b) surface plot of hemicellulose solubilization %(w/w)
vs pressure and time - - - 99
512 (a) contour plot, (b) surface plot of hemicellulose solubilization %(w/w)
vs pressure and temperature - - - - - - 100
513 (a) contour plot, (b) surface plot of hemicellulose solubilization %(w/w)
vs time and temperature - - - 101
514 (a) contour plot, (b) surface plot of lignin removal %(w/w) vs
time and temperature - - - 102
515 (a) contour plot, (b) surface plot of lignin removal% (w/w) vs
pressure and time - - - - 103
516 (a) contour plot, (b) surface plot of lignin removal %(w/w) vs
pressure and temperature - - - 104
517 (a) contour plot, (b) surface plot of reducing sugars (g/L) vs
temperature and time - - - 105
518 (a) contour plot, (b) surface plot of pH vs pressure and
temperature - - - - - - - - - - - - - - - 106
519 (a) contour plot, (b) surface plot of cellulose content % (w/w) vs
time and temperature for 23
CCD APO pretreatment - - 111
520 (a) contour plot, (b) surface plot of cellulose content %(w/w) vs
temperature and %H2O2 - - - 112
521 (a) contour plot, (b) surface plot of cellulose content %(w/w) vs
time and %H2O2 - - - - 113
522 (a) contour plot, (b) surface plot of hemicellulose solubilization
%(w/w) vs time and temperature - - - - - - 114
523 (a) contour plot, (b) surface plot of hemicellulose solubilization
%(w/w) vs time and %H2O2 - - - 115
524 (a) contour plot, (b) surface plot of hemicellulose solubilization
%(w/w) vs temperature and %H2O2 - - 116
525 (a) contour plot, (b) surface plot lignin removal %(w/w) vs
time and temperature - - - 117
526 (a) contour plot, (b) surface plot lignin removal %(w/w) vs
%H2O2 and temperature - - - 118
527 (a) contour plot, (b) surface plot lignin removal %(w/w) vs
time and %H2O2 - - - - - - - 119
528 (a) contour plot, (b) surface plot of cellulose content %(w/w)
(22
CCD APO pretreatment) vs temperature and time - - 122
529 (a) contour plot, (b) surface plot of hemicellulose solubilization %(w/w)
(22
CCD APO pretreatments) vs temperature and time - - 123
530 (a) contour plot, (b) surface plot of lignin removal %(w/w)
(22
CCD APO pretreatment) vs temperature and time - 124
531 Correlations of pretreated samples with variations of
experimental conditions (WAO (A), APO (D), and APAWAO
(B, C, E, and F)) pretreatments compared - - - 126
532 3-d reducing sugar yields for the optimized pretreatment
conditions and their variations - - - 128
533 3-d reducing sugar yields for the 22
CCD APO pretreatments - 129
534 4-d Effect of time and substrate concentration on sugars yield
with supplemental β-glucosidase; Pretreatment condit ions:
150 o
C, 1% H2O2, 10 bar, 45 min - - - 132
535 4-d Effect of time and substrate concentration on sugars
yield with no supplemental β-glucosidase; Pretreatment condit ions: 150oC, 1% H2O2, 10 bar, 45 min - - - 133
536 4-d Effect of substrate concentration on biomass conversion (% Saccharification) with supplemental β-glucosidase; Pretreatment conditions: 150oC, 1% H2O2, 10 bar, 45 min - 134
537 4-d Effect of substrate concentration on biomass conversion
(% Saccharification) with no supplemental β-glucosidase;
Pretreatment conditions: 150oC, 1% H2O2, 10 bar, 45 min - 135
538 4-d Effect of time and substrate concentration on sugars yield
with supplemental β-glucosidase; Pretreatment condit ions:
120 oC, 1% H2O2, and 30 min - - - 137
539 4-d Effect of time and substrate concentration on sugars yield with no supplemental β-glucosidase; Pretreatment conditions: 120oC, 1% H2O2, and 30 min - - - 138
540 4-d Effect of enzyme loading on sugar yields; Pretreatment conditions:150 oC, 1% H2O2, 10 bar, 45 min - - 140
541 4-d Effect of enzyme loading on sugar yields Pretreatment conditions: 120 oC, 1% H2O2, and 30 min - 141
542 4-d Effect of time and substrate concentration on sugars yield for untreated and treated biomass - - - 144
LIST OF TABLES
TABLE PAGE
21 A 2007 Estimate of world oil statistics - - 7
22 Weight percent of cellulose, hemicellulose, and lignin in wood biomass - - - - 7
23 Composit ions of some woody and non-woody biomass %(w/w) - 24
24 Lignocellulosic biomass pretreatment methodologies - - 32
31 Statist ical 23- factorial design for WAO experiments - 41
32 Statist ical 23- central composite design for APO experiments - 41
33 Statist ical 22- central composite design for APO experiments - 41
41 Particle size distribution of the sawdust - - 57
42 Composition of raw sawdust - - - 58
43 Predicted and experimental (validated) responses for the WAO pretreatment at 170 oC, 10 bar, and 10 min optimized conditions - 63
44 Predicted and experimental (validated) responses for the 23 CCD APO pretreatment at 150 oC, 45 min, 1%H2O2 optimized conditions 63
45 Cellulose content, hemicellulose solubilization, lignin removal %(w/w) after raw biomass pretreatments for 22 CCD Alkaline peroxide oxidation - - - - 66
46 Optimized conditions and alkaline peroxide/air pressure variations for WAO and 23
CCD APO pretreatments - - 68
47 Glucose concentrations of samples as determined from standard curve for cellulose enzyme activity determination - - 71
48 Summary of enzymatic hydrolysis conditions for pretreated biomass as specified in Table 46 - - - 73
49 3-d RS yields for the optimized conditions of WAO, 2 CCD APO Pretreatments and variations - - - 73
410 3-d Reducing sugar (RS) yields for the 22
CCD APO pretreatments 75
411 4-d Effect of substrate concentration with corresponding increase in enzyme concentration and incubation period on
the enzymatic saccharification of pretreated sawdust conditions
of 150 oC, 1% H2O2, 10 bar, and 45 min - - 77
412 4-d Effect of substrate concentration with corresponding increase in enzyme concentration and incubation period on the enzymatic saccharification of pretreated sawdust conditions of 120 o
C, 1% H2O2, and 30 min - - - 78
413 Effect of enzyme loading on the reducing sugar yield of treated sawdust - - - - 79
414 4-d Effect of substrate concentration with corresponding increase
in enzyme loading and incubation period on the enzymatic saccharification of pretreated and untreated sawdust - - 79
415 Ethanol concentration and yields dur ing SSF of pretreated sawdust 83
ABBREVIATIONS
ANOVA Analysis of variance
APAWAO Alkaline peroxide assisted wet air oxidation
APO Alkaline peroxide oxidation
BSS British standards specification
CCD Central composite design
DNS Dinitrosalicylic acid
DOE Design of experiment
FPU Filter paper unit
HMF Hydroxymethylfurfural
IUPAC International union of pure and applied chemistry
LCW Lignocellulosic waste
NREL National renewable energy laboratory
OVAT One value at a time
PID Proportional-intergral-derivative
RSM Response surface methodology
SSF Simultaneous saccharification and fermentation
WAO Wet air oxidat ion
TITLE PAGE - - - - i
CERTIFICATION - - - - ii
DEDICATION - - - - iii
ACKNOWLEDGEMENTS - - - iv
TABLE OF CONTENTS - - - - vi
LIST OF FIGURES - - - - x
LIST OF TABLES - - - - xv
ABBREVIATIONS - - - - xvii
ABSTRACT - - - - xviii
CHAPTER ONE
1 INTRODUCTION - - - - 1
11 Background of the study - - - 1
12 Statement of research problem - - - 2
13 Objectives of the study - - - 3
14 Significance of the study - - - 3
15 Scope and limitation of the study - - - 4
CHAPTER TWO
2 LITERATURE REVIEW - - - 6
21 Sawdust as a renewable energy source - - - 6
22 Alcohol production from biomass - - - 8
23 Chemical structure of lignocellulosic biomass - - 12
231 The cell wall - - - - 13
232 Cellulose - - - - 16
233 Hemicellulose - - - - 18
234 Lignin - - - - - 18
24 Lignin-carbohydrate complexes - - - 20
241 Lignin-hemicellulose bonds - - - 22
25 Woody biomass and non-woody biomass - - - 22
2 6 Pretreatment methods - - - - 25
261 Alkaline hydrolysis - - - - 27
262 Oxygen delignification - - - - 28
2621 Hydrogen - - - - - 29
2622 Per-acetic acid - - - - 29
2623 Ozonolysis - - - - 30
2624 Wet air oxidation - - - - 30
2625 Alkaline peroxide assisted wet air oxidation - - 31
27 Chemical reactions during alkaline pretreatment - - 31
28 Oxygen species - - - - 31
29 Mechanism of carbohydrates degradation - - 35
210 Mechanism of lignin removal - - - 37
CHAPTER THREE
3 MATERIALS AND METHODS - - - 40
31 Statistical designs of experiments - - - 40
311 Choice of experimental design - - - 40
32 Preparation, storage and handling of raw material - - 43
33 Compositional analysis of raw and pretreated raw material - - 43
331 Extractives - - - - - 44
332 Hemicellulose - - - - 44
333 Ash - - - - - 45
334 Determination of lignin (acid insoluble and soluble) - - 45
335 Determination of cellulose content - - - 45
34 Reactor set up for pretreatments - - - 45
35 Pretreatment method - - - - 50
36 Lime as the alkaline pretreatment agent - - - 50
361 Specific lime consumption determination - - 51
37 Enzymatic conversion - - - - 53
371 Cellulase activity determination - - - 54
372 Convertibility of the sawdust material - - - 54
38 Fermentation of treated and untreated materials - - 55
CHAPTER FOUR
4 RESULTS - - - - 56
42 Composit ion of pretreated solid fraction - - - 60
421 Lignin removal (delignification) - - - - 59
43 Statistical optimization of the pretreatment process - - 60
431 Alkaline WAO pretreatments - - - 61
432 23 CCD alkaline peroxide oxidation pretreatment - - 64
433 22 CCD alkaline peroxide oxidation pretreatment - - 64
434 Alkaline peroxide assisted wet air oxidation pretreatment (APAWAO) 67
44 Enzymatic hydrolysis of raw and pretreated solids - - 67
441 Cellulase enzyme activity - - - 69
442 Hydrolysis of pretreated solids - - - 72
443 Effect of substrate concentration on enzymatic hydrolysis - 76
444 Effects of enzyme loading on enzymatic hydrolysis - - 76
445 Enzymatic hydrolysis of untreated and washed only biomass - 80
45 Simultaneous saccharification and fermentation - - 80
CHAPTER FIVE
5 DISCUSSIONS - - - - 84
51 Characterization of untreated (raw) biomass - - - 84
52 Changes of biomass during pretreatment - - - - 86
521 Pretreatment yields - - - - - - 86
522 Holocellulose removal in the pretreated solids - - 88
53 Alkaline WAO pretreatments optimization - - - 92
54 23 CCD alkaline peroxide oxidation pretreatments optimization - 107
55 22 CCD alkaline peroxide oxidation pretreatments optimization - 120
56 Alkaline peroxide assisted WAO pretreatments - - 125
57 Enzymatic hydrolysis of treated solids - - - 127
58 Substrate concentration on enzymatic hydrolysis - - 130
59 Enzyme loading studies - - - - 136
510 Hydrolysis studies of untreated and washed only biomass - - 142
511 Simultaneous saccharification and fermentation - - 143
CHAPTER SIX
6 CONCLUSIONS AND RECOMMENDATIONS - - 145
61 Conclusions - - - - - 145
62 Recommendations - - - - 146
REFERENCES - - - - - 147
APPENDIX A - - - - - 164
APPENDIX B - - - - - 166
APPENDIX C - - - - - 168
APPENDIX D - - - - - 170
APPENDIX E - - - - - 172
APPENDIX F - - - - - 175
APPENDIX G - - - - - 178
APPENDIX H - - - - - 180
APPENDIX I - - - - - 182
APPENDIX J - - - - - 187
APPENDIX K - - - - - 189
APPENDIX L - - - - - 191
APPENDIX M - - - - - 193
APPENDIX N - - - - - 195
APPENDIX O - - - - - 196
LIST OF FIGURES
FIGURE PAGE
21 Schematic diagram of traditional biomass conversion to ethanol - 10
22 Mode of action of cellulolytic enzymes - - 11
23 Micro- and macro-fibrils (fibres) formation of cellulose and their
positions in the wood cell wall - - - 14
24 Schematic of goals of pretreatment on lignocellulosic materials - 15
25 The cellulose chain - - - - 17
26 Hemicellulose monomer units - - - 19
27 Distinguishing chemical structures of lignin building blocks - 21
28 Proposed model types of lignin carbohydrate linkages - - 23
29 Oxygen species derived from molecular oxygen in aqueous solution - 34
210 A proposed mechanism for carbohydrate degradation by hydroxyl
radical during oxidative delignification - - 36
211 Radical chain reactions during oxygen delignification in alkaline
condition - - - - 38
212 Proposed reaction of lignin via phenoxyradical - - 39
31 Schematic diagram of the pretreatment reactor set up - 47
32 Complete reactor set up with the process and power controllers - 48
33 Pictures of reactor system - - - - 49
34 Slurry and solid fraction of pretreated biomass - - 52
41 The average particle size distribution of dry raw sawdust - 57
42 Mass balance for the optimized alkaline peroxide
oxidat ion pretreatment - - - 65
43 Construction of glucose standard curve for cellulase activity
determination - - - - 70
44 Enzyme dilution vs glucose concentration for cellulase activity
determination - - - - 71
45 Pretreated samples preparation for enzymatic hydrolysis and
fermentation - - - - 74
51 Surface plots of lime consumed (g Ca(OH)2/g raw biomass): (a) vs
temperature and % H2O2, (b) vs temperature and time for 23 CCD
APO design - - - - 85
52 Surface plot of lime consumed (g Ca(OH)2/g dry biomass) vs time
and temperature for 22
CCD APO design - - 87
53 Surface plot of pretreatment yield of holocellulose (g recovered/100g
in raw biomass) vs lignin removal %(w/w) and temperature for 23
full factorial WAO pretreatments - - - 89
54 Surface plot of pretreatment yield of holocellulose (g recovered/100g
in raw biomass) vs lignin removal %(w/w) and temperature for 23
CCD APO pretreatments - - - 89
55 Surface plot of pretreatment yield of holocellulose (g recovered/100g
in raw biomass) vs lignin removal %(w/w) and temperature for 22
CCD APO pretreatments - - - 90
56 Surface plot of lignin removal %(w/w) vs lime consumed
(g Ca(OH)2/g raw biomass) and temperature - - 91
57 Surface plot of lignin removal %(w/w) vs lime consumed
(g Ca(OH)2/ g raw biomass) and time - - 91
58 (a) contour plot, (b) surface plot of cellulose content %(w/w) vs
time and temperature for 23
full factorial WAO pretreatment - 96
59 (a) contour plot, (b) surface plot of cellulose content %(w/w) vs
pressure and time - - - - 97
510 (a) contour plot, (b) surface plot of cellulose content %(w/w) vs
pressure and temperature - - - 98
511 (a) contour plot, (b) surface plot of hemicellulose solubilization %(w/w)
vs pressure and time - - - 99
512 (a) contour plot, (b) surface plot of hemicellulose solubilization %(w/w)
vs pressure and temperature - - - - - - 100
513 (a) contour plot, (b) surface plot of hemicellulose solubilization %(w/w)
vs time and temperature - - - 101
514 (a) contour plot, (b) surface plot of lignin removal %(w/w) vs
time and temperature - - - 102
515 (a) contour plot, (b) surface plot of lignin removal% (w/w) vs
pressure and time - - - - 103
516 (a) contour plot, (b) surface plot of lignin removal %(w/w) vs
pressure and temperature - - - 104
517 (a) contour plot, (b) surface plot of reducing sugars (g/L) vs
temperature and time - - - 105
518 (a) contour plot, (b) surface plot of pH vs pressure and
temperature - - - - - - - - - - - - - - - 106
519 (a) contour plot, (b) surface plot of cellulose content % (w/w) vs
time and temperature for 23
CCD APO pretreatment - - 111
520 (a) contour plot, (b) surface plot of cellulose content %(w/w) vs
temperature and %H2O2 - - - 112
521 (a) contour plot, (b) surface plot of cellulose content %(w/w) vs
time and %H2O2 - - - - 113
522 (a) contour plot, (b) surface plot of hemicellulose solubilization
%(w/w) vs time and temperature - - - - - - 114
523 (a) contour plot, (b) surface plot of hemicellulose solubilization
%(w/w) vs time and %H2O2 - - - 115
524 (a) contour plot, (b) surface plot of hemicellulose solubilization
%(w/w) vs temperature and %H2O2 - - 116
525 (a) contour plot, (b) surface plot lignin removal %(w/w) vs
time and temperature - - - 117
526 (a) contour plot, (b) surface plot lignin removal %(w/w) vs
%H2O2 and temperature - - - 118
527 (a) contour plot, (b) surface plot lignin removal %(w/w) vs
time and %H2O2 - - - - - - - 119
528 (a) contour plot, (b) surface plot of cellulose content %(w/w)
(22
CCD APO pretreatment) vs temperature and time - - 122
529 (a) contour plot, (b) surface plot of hemicellulose solubilization %(w/w)
(22
CCD APO pretreatments) vs temperature and time - - 123
530 (a) contour plot, (b) surface plot of lignin removal %(w/w)
(22
CCD APO pretreatment) vs temperature and time - 124
531 Correlations of pretreated samples with variations of
experimental conditions (WAO (A), APO (D), and APAWAO
(B, C, E, and F)) pretreatments compared - - - 126
532 3-d reducing sugar yields for the optimized pretreatment
conditions and their variations - - - 128
533 3-d reducing sugar yields for the 22
CCD APO pretreatments - 129
534 4-d Effect of time and substrate concentration on sugars yield
with supplemental β-glucosidase; Pretreatment condit ions:
150 o
C, 1% H2O2, 10 bar, 45 min - - - 132
535 4-d Effect of time and substrate concentration on sugars
yield with no supplemental β-glucosidase; Pretreatment condit ions: 150oC, 1% H2O2, 10 bar, 45 min - - - 133
536 4-d Effect of substrate concentration on biomass conversion (% Saccharification) with supplemental β-glucosidase; Pretreatment conditions: 150oC, 1% H2O2, 10 bar, 45 min - 134
537 4-d Effect of substrate concentration on biomass conversion
(% Saccharification) with no supplemental β-glucosidase;
Pretreatment conditions: 150oC, 1% H2O2, 10 bar, 45 min - 135
538 4-d Effect of time and substrate concentration on sugars yield
with supplemental β-glucosidase; Pretreatment condit ions:
120 oC, 1% H2O2, and 30 min - - - 137
539 4-d Effect of time and substrate concentration on sugars yield with no supplemental β-glucosidase; Pretreatment conditions: 120oC, 1% H2O2, and 30 min - - - 138
540 4-d Effect of enzyme loading on sugar yields; Pretreatment conditions:150 oC, 1% H2O2, 10 bar, 45 min - - 140
541 4-d Effect of enzyme loading on sugar yields Pretreatment conditions: 120 oC, 1% H2O2, and 30 min - 141
542 4-d Effect of time and substrate concentration on sugars yield for untreated and treated biomass - - - 144
LIST OF TABLES
TABLE PAGE
21 A 2007 Estimate of world oil statistics - - 7
22 Weight percent of cellulose, hemicellulose, and lignin in wood biomass - - - - 7
23 Composit ions of some woody and non-woody biomass %(w/w) - 24
24 Lignocellulosic biomass pretreatment methodologies - - 32
31 Statist ical 23- factorial design for WAO experiments - 41
32 Statist ical 23- central composite design for APO experiments - 41
33 Statist ical 22- central composite design for APO experiments - 41
41 Particle size distribution of the sawdust - - 57
42 Composition of raw sawdust - - - 58
43 Predicted and experimental (validated) responses for the WAO pretreatment at 170 oC, 10 bar, and 10 min optimized conditions - 63
44 Predicted and experimental (validated) responses for the 23 CCD APO pretreatment at 150 oC, 45 min, 1%H2O2 optimized conditions 63
45 Cellulose content, hemicellulose solubilization, lignin removal %(w/w) after raw biomass pretreatments for 22 CCD Alkaline peroxide oxidation - - - - 66
46 Optimized conditions and alkaline peroxide/air pressure variations for WAO and 23
CCD APO pretreatments - - 68
47 Glucose concentrations of samples as determined from standard curve for cellulose enzyme activity determination - - 71
48 Summary of enzymatic hydrolysis conditions for pretreated biomass as specified in Table 46 - - - 73
49 3-d RS yields for the optimized conditions of WAO, 2 CCD APO Pretreatments and variations - - - 73
410 3-d Reducing sugar (RS) yields for the 22
CCD APO pretreatments 75
411 4-d Effect of substrate concentration with corresponding increase in enzyme concentration and incubation period on
the enzymatic saccharification of pretreated sawdust conditions
of 150 oC, 1% H2O2, 10 bar, and 45 min - - 77
412 4-d Effect of substrate concentration with corresponding increase in enzyme concentration and incubation period on the enzymatic saccharification of pretreated sawdust conditions of 120 o
C, 1% H2O2, and 30 min - - - 78
413 Effect of enzyme loading on the reducing sugar yield of treated sawdust - - - - 79
414 4-d Effect of substrate concentration with corresponding increase
in enzyme loading and incubation period on the enzymatic saccharification of pretreated and untreated sawdust - - 79
415 Ethanol concentration and yields dur ing SSF of pretreated sawdust 83
ABBREVIATIONS
ANOVA Analysis of variance
APAWAO Alkaline peroxide assisted wet air oxidation
APO Alkaline peroxide oxidation
BSS British standards specification
CCD Central composite design
DNS Dinitrosalicylic acid
DOE Design of experiment
FPU Filter paper unit
HMF Hydroxymethylfurfural
IUPAC International union of pure and applied chemistry
LCW Lignocellulosic waste
NREL National renewable energy laboratory
OVAT One value at a time
PID Proportional-intergral-derivative
RSM Response surface methodology
SSF Simultaneous saccharification and fermentation
WAO Wet air oxidat ion
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