臺灣博碩士論文加值系統

此研究致力於探討最具經濟效益之稻稈醣化及Acetone-Butanol-Ethanol (ABE) 發酵產醇之操作流程，並以中央合成設計及反應曲面法 (CCD-RSM) 找出於滅菌及無滅菌狀態下發酵分別之最佳化細胞植種濃度及培養溫度兩操作條件，以獲得最大之丁醇產率、丁醇產值、與Gompertz 模擬之丁醇生產速率。未前處理之稻稈 (NPRS)、前處理之稻稈 (PRS) 及前處理稻稈與酸水解液之混合物 (MPRSH) 分別於一系列之批次反應瓶中進行酵素醣化實驗，結果顯示葡萄糖為主要之醣化產物，NPRS的葡萄糖產值為每克之NPRS稻稈產出0.52 g之葡萄糖，與每克PRS及MPRSH的葡萄糖產量不相上下，PRS及MPRSH的葡萄糖產值分別為0.50及0.58 g/g。然而以操作成本及時間為考量，只經研磨之無化學性前處理稻稈NPRS為最具效益之醣化及發酵原料。模擬醣化結果所合成之NPRS水解液中含有2.73 g/L 阿拉伯糖、28.10 g/L葡萄糖、10.00 g/L半乳糖與5.00 g/L之乙酸則用於ABE 發酵批次實驗中。傳統ABE 發酵皆於滅菌的環境下進行實驗，而滅菌過程中所損耗的能量及時間為成本來源之一，有鑑於此，本研究之發酵實驗分別於滅菌與無滅菌環境下進行，以探討ABE發酵於無滅菌條件下之可行性。各個批次反應皆於pH 5.42±0.03及100 rpm震盪之條件下進行。批次發酵結果用以計算丁醇產率、丁醇產值、與Gompertz方程式推估之丁醇生產速率。發酵反應期間，葡萄糖最容易被Clostridium saccharoperbutylacetonicum N1-4所利用，半乳糖次之，而阿拉伯糖則幾乎沒有被利用，乙酸則被微生物再利用轉換為丁醇、丙酮或乙醇。高濃度之初始細胞植種濃度，可抑制無滅菌操作實驗中污染之落菌或其他微生物，使丁醇生產之效率不受影響。低濃度之初始細胞濃度 (< 800 mg/L) 及過高之溫度 (> 42℃) 則使產醇量下降，甚至造成細胞無活性或死亡。經表面曲面分析，滅菌組ABE發酵實驗之最大的丁醇產率 (1.45 g/L/d)、丁醇產值 (0.22 g/g)、及丁醇生產速率 (4.05 g/L/d) 可分別於初始細胞濃度1.96 g/L、2.01 g/L及2.33 g/L結合相對應之培養溫度32.3℃、26.3℃及30.5℃之操作條件下獲得; 而無滅菌組發酵實驗之最大丁醇產率 (1.45 g/L/d)、丁醇產值 (0.32 g/g) 及丁醇生產速率 (3.74 g/L/d) 則是分別於26.4℃、25.0℃及25.0℃之培養溫度結合2.33 g/L之初始細胞濃度的操作條件下獲得。於分別所適當的條件下進行ABE發酵，滅菌與無滅菌環境下之反應可達到相近的丁醇產率、產值與生產速率。總括本實驗的結果，可知以無滅菌方式進行ABE發酵未化學性前處理稻稈進行醣化後之水解液為一經濟且可行的生物產生質能源之方法。

This study aimed to integrate a cost-effective approach on the conversion of rice straw into fermentable sugars and biobutanol production through Acetone-Butanol-Ethanol (ABE) fermentation. The optimal initial cell concentration and incubation temperature for ABE fermentation under both sterile and non-sterile conditions were resolved by central composite design and response surface methodology (CCD-RSM). Saccharification experiments of non-pretreated rice straw (NPRS), pretreated rice straw (PRS), and mixture of pretreated rice straw and acid hydrolysate (MPRSH) were conducted in a series of batch reactors. Glucose was the major product. The results show that the glucose yield of 0.52 g glucose/g rice straw for NPRS was compatible to those of 0.50 and 0.58 g glucose/g rice straw for PRS and MPRSH, respectively. Thus, the saccharification of the rice straw grinded only without other pretreatment is more cost-effective if concerning to save operating time, energy and chemical cost. Simulated NPRS hydrolysate contained 2.73 g/L arabinose, 28.10 g/L glucose, 10.00 g/L galactose, and 5.00 g/L acetic acid was then used as the medium for ABE fermentation batch experiments with pH 5.42±0.03 and 100 rpm agitation. Conventional ABE fermentations are conducted under sterile condition to avoid contaminations from other microbes. However, sterilization is one of the costly steps in conventional ABE fermentation. To evaluate the feasibility of non-sterile ABE fermentation, the fermentation experiments in this study were performed under sterile and non-sterile environmental conditions. The results from the batch experiments were used for determine the maximum butanol productivity, butanol yield, and butanol production rate estimated by the modified Gompertz equation. During the fermentation, glucose was easily and sharply utilized by Clostridium saccharoperbutylacetonicum N1-4 while arabinose was hardly utilized. Acetic acid was reutilized by cell to form butanol, acetone or ethanol. When batch experiments conducted under non-sterile condition, high initial cell concentration of C. saccharoperbutylacetonicum N1-4 can constrain the contaminations from other microbes and ensure the biobutanol production compatible with those under sterile condition. Low initial cell concentration (&lt; 800 mg/L) or high incubation temperature (> 42 ℃) cause low biobutanol production. As results from the statistical approach by RSM, the maximum butanol productivity (1.45 g/L/d), butanol yield (0.22 g/g), and butanol production rate (4.05 g/L/d) were obtained at the initial cell concentrations and incubation temperatures of 1.96 g/L and 32.3℃, 2.01 g/L and 26.3℃, and 2.33 g/L and 30.5℃, respectively, under sterile condition. Meanwhile, under non-sterile condition, similar butanol productivity (1.45 g/L/d), butanol yield (0.32 g/g), and butanol production rate (3.74 g/L/d) could be achieved when the initial cell concentrations and incubation temperatures were controlled at 2.33 g/L and 26.4℃, 2.33 g/L and 25.0℃, and 2.33 g/L and 25.0℃, respectively. To overlook this study, the biobutanol production from non-pretreated rice straw powder can be achieved feasibly and economically under non-sterile environmental condition.

摘要 i
Abstract iii
謝誌 v
Table of Contents vi
List of Tables ix
List of Figures xii
Chapter 1 Introduction 1
1.1 Background 1
1.2 Objectives 2
Chapter 2 Literature Review 3
2.1 History of Acetone-Butanol-Ethanol fermentation 3
2.2 Current developments of biofuel technology 3
2.2 Biofuels and fossil fuel 5
2.3 Acetone-Butanol-Ethanol fermentation 6
2.3.1 Fermentation microorganisms and metabolic pathway 8
2.3.2 Factors affecting the solvent production 13
2.3.2.1 Intracellular status of Clostridia 13
2.3.2.2 Medium conditions 13
2.3.2.3 Solvent toxicity 15
2.4 Substrates 16
2.4.1 Monosaccharide 16
2.4.2 Starch/sugar-based crop 17
2.4.3 Lignocellulosic biomass 21
2.4.2.1 Pretreatments 23
2.4.2.2 Enzymatic saccharification 29
2.5 Alternative operation strategies of ABE fermentation to reduce the effect of solvent toxicity 31
Chapter 3 Materials and Methods 35
3.1 Instruments and chemicals 36
3.2 Rice straw 41
3.3 Dilute acid/base pretreatment 41
3.4 Enzymatic saccharification 42
3.5 Medium preparation 46
3.6 Culture development 48
3.6.1 Laboratory stock Clostridium saccharoperbutylacetonicum 48
3.6.2 Preparation of inoculums for ABE fermentation 48
3.7 Experimental design (central composite design) 48
3.8 Batch experiments 50
3.9 Analytical methods 51
3.9.1 Composition analysis of rice straw 51
3.9.2 Carbohydrate analysis 52
3.9.3 Fermentation products analysis 52
3.9.4 Cell concentration analysis 53
3.10 Data analysis 53
Chapter 4 Results and Discussion 55
4.1 Rice straw composition 55
4.2 Different pretreatment methods 56
4.3 Enzymatic saccharification 59
4.3.1 Enzyme loading 59
4.3.2 Saccharification of NPRS, PRS, and MPRSH 60
4.3.2.1 Saccharification profiles and performances of NPRS, PRS, and MPRSH 60
4.3.2.3 Saccharification kinetics of NPRS, PRS, and MPRSH 64
4.4 Profiles of ABE fermentation 66
4.4.1 Solventogenesis dominant reaction (maximum solvents/acids ratio > 1) 67
4.4.2 Acidogenesis dominant reaction (maximum solvents/acids ratio &lt; 1) 69
4.5 Performances of ABE fermentation 72
4.5.1 ABE fermentation under sterile condition 72
4.5.2 ABE fermentation under non-sterile condition 75
4.6 Kinetics of butanol production 78
4.7 Response surface analysis 80
4.7.1 The second-order model and analysis of variance (ANOVA) 81
4.7.2 Responses optimization 85
Chapter 5 Conclusions and Future Prospects 91
References 96
Appendix A Metabolic product (maximum solvents/acids > 1) 106
Appendix B Metabolic product (maximum solvents/acids ratio &lt; 1) 109
Appendix C Inactive runs with no metabolic products 110
Appendix D Total sugar, pH, and cell concentration, and solvents/acids ratio 112
Appendix E Modified Gomperz model for butanol 115
Appendix F Modified Gomperz model for sugar 116

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