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Title Page

Contents

ABSTRACT 13

CHAPTER 1. INTRODUCTION 14

1.1. Bioethanol from lignicellulosic biomass 14

1.2. Pretreatment 16

1.2.1. Pretreatment strategies 16

1.2.2. Liquid hot water pretreatment 17

1.3. Lignin chemistry 18

1.4. Lignin isolation 22

1.5. Lignin characterization 23

1.6. Inhibitory role of lignin on enzymatic hydrolysis 26

1.7. Enzyme adsorption onto lignins 27

1.8. Thesis objectives 30

CHAPTER 2. LITERATURE REVIEW 32

2.1. Lignin modification during pretreatment 32

2.2. Identifying the enzyme adsorption behaviors onto lignins 34

CHAPTER 3. MATERIALS AND METHODS 38

3.1. Materials 38

3.2. Liquid hot water (LHW) pretreatment 38

3.3. Enzymatic hydrolysis 39

3.4. Scanning electron microscopy (SEM) 39

3.5. Lignin isolation 39

3.6. Differential scanning calorimetry (DSC) 40

3.7. Fourier transform infrared spectroscopy (FTIR) 40

3.8. Lignin blocking effect by bovine serum albumin (BSA) on enzymatic hydrolysis 41

3.9. Brunauer, Emmett, and Teller (BET) surface area 41

3.10. Adsorption experiments 41

3.11. Enzyme activity assays 42

3.12. Protein analysis by electrophoresis 43

CHAPTER 4. RESULTS AND DISCUSSION 44

4.1. Changes of lignin in liquid hot water pretreated hardwood 44

4.1.1. Changes in lignin composition 44

4.1.2. Morphological changes of pretreated solids 48

4.1.3. Enzymatic hydrolysis of pretreated solids 50

4.2. Lignin isolation and characterization 52

4.2.1. Lignin isolation 52

4.2.2. Glass transition temperature of isolated lignins 53

4.2.3. Fourier transform infrared spectroscopy (FTIR) 56

4.2.4. Inhibitory effect of isolated lignin on Avicel hydrolysis 58

4.2.5. Lignin blocking effect by BSA on enzymatic hydrolysis 59

4.3. Enzyme adsorption onto lignins of LHW pretreated hardwoods 61

4.3.1. Profiles of isolated lignins 61

4.3.2. Time course of enzyme adsorption onto isolated lignins 62

4.3.3. Enzyme adsorption isotherms 64

4.3.4. Distribution of adsorbed enzyme components onto lignins 67

4.3.5. SDS-PAGE analysis of free T. reesei cellulase distribution 71

4.3.6. Effect of pH on enzyme adsorption onto lignin 74

4.3.7. Effect of NaCl on enzyme adsorption onto lignin 76

4.3.8. Enhancement in enzymatic hydrolysis of lignocellulose 78

CHAPTER 5. CONCLUSIONS AND FUTURE RESEARCH 80

LIST OF REFERENCES 83

APPENDIX 105

VITA 111

List of Tables

Table 1. The distribution of monolignols in lignin of plants 19

Table 2. Percentages of different linkages in hardwood and softwood lignins. 20

Table 3. Glass transition temperatures of lignins 25

Table 4. Langmuir adsorption constants of enzymes onto various lignins 36

Table 5. Effect of liquid hot water pretreatment on the composition of solid fraction. 45

Table 6. Lignin content changes during pretreatment 47

Table 7. Compositions of isolated lignins 52

Table 8. Glass transition point and ranges of isolated lignins 55

Table 9. Relative absorbance of functional groups of isolated lignins 57

Table 10. Measurement of specific surface area and nitrogen content of isolated lignins 61

Table 11. Maximum cellulase adsorption capacity (Emax), affinity (K) and partition...(이미지참조) 66

Table 12. Adsorption constants for isolated lignins from other sources 67

Table 13. Properties of β-glucosidases from T. reesei and A. niger 70

Table 14. Profile of putative enzymes produced from T. reesei 73

Table A1. Constants for SSF model 107

List of Figures

Figure 1. Precursors of lignins 18

Figure 2. Common phenylpropane linkages in lignin. 20

Figure 3. Proposed model structure of partial hardwood lignin 21

Figure 4. Lignin reactions occurring during steam explosion pretreatment 34

Figure 5. SEM micrographs of untreated (A and B, magnification 10k x and 20k x),... 49

Figure 6. Enzymatic hydrolysis of hardwood pretreated at different severities. Pretreated... 51

Figure 7. Glass transition temperature (Tg) of isolated lignins for severities over the..(이미지참조) 55

Figure 8. Fourier transform infrared spectra of isolated lignins 57

Figure 9. Effect of isolated lignins (of severity log Ro＝10.44, 11.39, 11.56 and 12.51 on...(이미지참조) 58

Figure 10. Enzymatic hydrolysis of liquid hot water pretreated solids with and without... 60

Figure 11. Time course of cellulase adsorption onto lignins of different severities of (A)... 63

Figure 12. Adsorption isotherms of cellulase (Cellic Ctec2) on lignins of different... 66

Figure 13. Distribution of Trichoderma reesei cellulase cocktail (Cellic Ctec2) remained... 68

Figure 14. Free protein content and activity of A. niger β-glucosidase (Novozyme 188) in... 69

Figure 15. SDS-PAGE analysis of free proteins (Cellic Ctec2) in the supernatant after the... 72

Figure 16. Effect of pH on enzyme adsorption onto the isolated lignins of severity log Ro...(이미지참조) 75

Figure 17. Effect of NaCl on enzyme adsorption onto the isolated lignins of severity log... 77

Figure 18. (A) Enzymatic hydrolysis of LHW pretreated hardwood (severity log Ro＝...(이미지참조) 79

Figure A1. Cellulose hydrolysis with 35 mg-cellulase/g-cellulose (left) and fermentation... 106

Figure A2. Simplified representation of the SSF process from cellulose to ethanol. 107

Figure A3. SSF with 35 mg-cellulase/g-cellulose 110

초록보기

 Lignin, one of the major component of lignocellulosic biomass, plays an inhibitory role on the enzymatic hydrolysis of cellulose. When hardwood was pretreated with liquid hot water at severities ranging from log Ro＝8.25 to 12.51, 80-90% lignin was recovered in the solid. The ratio of acid insoluble lignin (AIL) to acid soluble lignin (ASL) increased and the formation of spherical lignin droplets on the cell wall surface was observed as previously reported in the literature. When lignins were isolated from hardwoods pretreated at increasing severities and characterized based on glass transition temperature (Tg), the Tg of isolated lignins was found to increase from 171 to 180℃ as the severity increased from log Ro＝10.44 to 12.51. The increase in Tg suggested that the condensation reactions of lignin molecules occurred during pretreatment and altered the lignin structure. The more condensed lignin structure has a higher glass transition temperature and coincides with more extensive enzyme adsorption as severity increases. Since the enzyme components which are required to synergistically hydrolyze cellulose have different profiles (molecular weight, hydrophobicity, pI), they exhibit different adsorption behaviors with lignin, and thereby change the ratio of enzyme activities needed for synergism during cellulose hydrolysis. Among the enzyme components of Trichoderma reesei cellulase cocktail, β-glucosidase showed the strongest adsorption onto lignin. The adsorption of cellulase enzymes onto lignin is shown to be non-productive and in effect causes inhibition of enzymatic hydrolysis of cellulose in liquid hot water pretreated lignocellulose.

리그노셀룰로스 바이오매스의 주요 성분인 리그닌은 셀룰로스의 효소적 당화를 저해하는 역할을 한다. 목질계 바이오매스 (Mixed hardwoods)를 고온180-210℃에서 열수 전처리(Liquid hot water pretreatment)하였을 때, 헤미셀룰로오스(자일란)는 대부분 제거된 반면에80-90%의 리그닌이 바이오매스에 남아있음을 확인하였다. 전처리 온도가 높아짐에 따라 산에 녹는 리그닌 (Acid soluble lignin, ASL)에 대한 산에 녹지 않는 리그닌(Acidinsoluble lignin, AIL)의 비율(AIL/ASL)이 높아졌고, 전처리된 바이오매스 표면에 방울 (droplet) 형태의 리그닌이 형성됨을 확인하였다. 전처리에 따른 리그닌의 성질 변화를 확인하기 위해, 전처리된 바이오매스로부터 리그닌을 분리하였다. 리그닌의 성질 변화는 유리전이온도(glass transition temperature, Tg)를 측정하여 비교하였고, 전처리 조건이 심화됨에 따라 리그닌의 유리전이온도가 171℃에서 180℃로 증가함을 확인하였다. 이 리그닌의 유리전이온도(Tg) 값의 변화는 전처리하는 동안 리그닌의 응축 반응이 일어나고 그 구조가 변화되었음을 보여준다. 높은 전처리 조건에서 더 응축된 리그닌 구조는 리그닌의 유리전이온도(Tg)를 증가시켰고, 리그닌 표면으로의 셀룰레이즈 효소의 비효율적인 흡착(nonproductive adsorption) 현상 또한 증가시켰다. 셀룰로스를 효과적으로 분해하기 위해 필요한 셀룰로스 분해 효소의 구성 요소 (Cellobiohydrolase, endoglucanase, β-glucosidase, etc.)는 각각 다른 특성(molecular weight, hydrophobicity, pI)을 가지고 있으므로 리그닌 표면에의 흡착 작용이 다르고, 이는 셀룰로스 분해를 위해 최적화된 효소 구성 성분 비율을 변화시킨다. 본 연구에서는 곰팡이 균주Trichoderma reesei에서 생산된 효소의 구성 성분 중에서, β-glucosidase(셀로바이오스 분해 효소)가 리그닌에 대하여 가장 강한 흡착도를 가짐을 확인시켜주었다. 이러한 셀룰로스 분해 효소의 리그닌 표면에의 흡착은 전처리한 바이오매의 효소적 당화 효율성의 감소 현상에 기여함을 확인하였다.

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