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研究生:費恩祥
研究生(外文):FerdianWirawan
論文名稱:開發以木質纖維素料源進行連續式酒精生產之共醱酵策略
論文名稱(外文):Developing co-Fermentation Strategies for Continuous Ethanol Production from Lignocellulosic Feedstock
指導教授:張嘉修張嘉修引用關係
指導教授(外文):Jo-Shu Chang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:化學工程學系碩博士班
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:英文
論文頁數:124
中文關鍵詞:纖維酒精固定化 Z. mobilisP. stipitisCSTRSHFSSF共醱酵
外文關鍵詞:Cellulosic ethanolimmobilized Z. mobilissuspended P. stipitisCSTRSHFSSFco-fermentation
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從木質纖維材料生產酒精一直被視為取代化石燃料最有重要的生質燃料選項之一。基於在連續流過程中能被滯留且重複使用的特性,固定化酵母或細菌經常被使用在連續式操作的生物程序中。本研究由木質纖維素材料中獲得葡萄糖和木糖,並利用於培養聚乙烯醇(PVA)-固定化 Zymomonas mobilis細胞及懸浮的Pichia stipitis細胞進行醱酵以生產酒精。在固定化Z. mobilis的葡萄糖批次醱酵實驗中,發現最適化酒精生產條件為20%w/v 固定化細胞負載量和50 g/L的初始糖濃度。在固定化Z. mobilis和P. stipitis的同步發酵系統中,發現若要達到酒精生產之最佳狀態,必須先以Z. mobilis消耗葡萄糖,再使用P. stipitis消耗木糖。

在以固定化Z. mobilis進行葡萄糖發酵之饋料批次操作中,發現當酒精濃度高於70 g/L時會有產物抑制的現象。為避免酒精之累積,本研究採用連續發酵(CSTR)進行酒精之生產。以聚乙烯醇固定化Z. mobilis進行CSTR操作的最佳酒精生產速率(4.78 g/L/h)發生在固定化細胞負載量為35% w/v和葡萄糖進料量為10.64 g/L/h時。而最佳的葡萄糖轉化率(96.87%),則發生在相同的固定化細胞負載量(35% w/v),但較低的葡萄糖進料量(8.64 g/L/h)。

我們亦使用聚乙烯醇固定化P. stipitis進行CSTR連續式戊糖發酵以生產酒精,結果發現其酒精生產速度非常緩慢(小於0.2 g/L/h),顯示固定化P. stipitis細胞可能不適合木糖發酵,其原因可能是質傳阻礙之影響。當我們改以懸浮P. stipitis細胞進行饋料批次醱酵時,發現其酒精生產之表現遠優於使用固定化細胞之連續式發酵。因此,最佳的五-六碳糖共醱酵策略,乃以固定化Z. mobilis進行連續式六碳糖醱酵,而利用懸浮P. stipitis進行五碳糖饋料批次醱酵。此外,P. stipitis 五碳糖醱酵連續與膜分離技術相結合能維持高細胞濃度和高酒精生產力,但長期操作仍會發生細胞活性衰減之現象,造成酒精生產效率之降低。

本研究亦進行以木質纖維素為料原之纖維酒精生產。在這項研究中我們分別以酸前處理蔗渣為碳源,進行批次 SHF (separate hydrolysis and fermentation)及SSF (simultaneous saccharification and fermentation)程序之六碳糖醱酵生產酒精;以及以鹼處理蔗渣進行連續式 SHcF (separate hydrolysis and co-fermentation)及 SScF (simultaneous saccharification and co-fermentation)程序之五-六碳糖共醱酵生產酒精。在批次SHF的結果中,PVA固定化細胞生產酒精之最大濃度為6.24 g/L (理論產量的79.09%),而藻酸鈣(CA)固定化細胞生產酒精之最大濃度為5.52 g/L (理論產量的69.96%)。而PVA固定化細胞之的酒精生產速率為1.52 g/L/h,而CA固定化細胞則是0.92 g/L/h。SSF的結果中,PVA固定化細胞生產的酒精濃度5.53 g/L (理論產量的70.09%),而CA固定化細胞則是5.44 g/L (理論產量的68.95%),PVA和CA固定化細胞之酒精生產速率分別為0.679與0.691 g/L/h。至於連續式纖維酒精共醱酵生產系統中,SHcF系統之整體酒精生產速率達1.868 g/L/h,為平均酒精生產理論產量的70.65%;而SScF系統的整體酒精生產速率為0.705 g/L/h,為平均酒精生產理論產量的81.18%。與其他相關研究相較之下,我們提出的方法有良好之纖維酒精生產效率,應能適用於生質酒精生產,並具有未來工業應用之潛力。

Bioethanol produced from lignocellulosic materials has been considered as one of the most promising fuels to replace the fossil fuel. Immobilized yeasts or bacteria have been frequently used in continuous system due to its feasibility for repeated uses with high biomass retention during the continuous process. In this work, glucose and xylose liberated from lignocellulosic materials were used to produce ethanol by PVA-immobilized Zymomonas mobilis and suspended Pichia stipitis. The optimal condition for batch glucose fermentation by immobilized Zymomonas mobilis was a particle loading of 20%-w/v and an initial sugar concentration of 50 g/L. The batch co-fermentation system between PVA-immobilized Z. mobilis and P. stipitis reached its best condition when glucose was first consumed by Z. mobilis, followed by xylose consumption by P. stipitis.

The fed-batch strategy was employed for glucose fermentation by immobilized Z. mobilis. It was found that product inhibition occurred when the ethanol concentration was higher than 70 g/L. Continuous fermentation could prevent the ethanol accumulation, so in this study, continuous fermentation using continuous stirred tank reactor (CSTR) was employed for bioethanol production. Optimization of the CSTR system for PVA-immobilized Z. mobilis shows that the optimum ethanol productivity (4.78 g/L/h) can be achieved at a high particle loading (35%-w/v) and a high glucose loading (10.64 g/L/h), while optimum glucose conversion (96.87%) was achieved at high particle loading (35%-w/v) but a lower glucose loading (8.64 g/L/h).

For pentose fermentation, continuous fermentation using CSTR by PVA-immobilized P. stipitis was implemented. It is found that even with the full nutrient support, the ethanol production rate was very slow (less than 0.2 g/L/h), indicating that immobilized cells might be not suitable for xylose fermentation. The reason for this may be related to mass transfer (especially oxygen transfer) limitations. Alternatively, fed-batch study using suspended P. stipitis exhibited better bioethanol production performance than the continuous fermentation using immobilized cells. In addition, the combination of continuous pentose fermentation by P. stipitis with membrane separation can maintain high cell concentration and ethanol productivity, whereas the decrease in performance still occurred during long-term operations due the cell activity decay.

The cellulosic ethanol fermentation was also investigated. The experiments were divided into two groups: batch SHF & SSF ethanol production from acid pretreated sugarcane bagasse and continuous SHcF & SScF ethanol production from alkaline pretreated sugarcane bagasse. The batch SHF process gave maximum ethanol concentration of 6.24 g/L (79.09% of theoretical yield) for PVA-immobilized Z.mobilis cells and 5.52 g/L (69.96% of theoretical yield) for CA-immobilized Z. mobilis cells, with ethanol productivity of 1.52 g/L/h and 0.92 g/L/h for PCA and CA cells, respectively. The SSF study gave a maximum ethanol concentration of 5.53 g/L (70.09% of theoretical yield) for PVA cells and 5.44 g/L (68.95% of theoretical yield) for CA cells, with ethanol productivity of 0.691 g/L/h and 0.679 g/L/h for PVA and CA cells, respectively. For continuous cellulosic ethanol production system, the SHcF system process displayed an overall process efficiency of 70.65% of theoretical yield with an average ethanol productivity of 1.868 g/L/h, while the SScF process gave an overall efficiency of 81.18% of the theoretical yield with an average ethanol productivity of 0.705 g/L/h. Comparing with the performance obtained from the related studies, the result from the proposed methods appears to be highly competitive and are thus suitable for cellulosic bioethanol production and have a good potential for industrial applications.

中文摘要 i
ABSTRACT iii
ACKNOWLEDGEMENT vi
CONTENTS viii
LIST OF TABLES xiii
LIST OF FIGURES xiv
Chapter 1 INTRODUCTION 1
1.1 Motivation and Purpose 1
1.2 Research Scheme 3
Chapter 2 LITERATURE REVIEW 5
2.1 Current Global Energy Trend 5
2.2 Lignocellulosic Feedstock as Bioethanol Raw Materials 7
2.3 Pretreatment Technologies of Lignocellulosic Feedstock 11
2.3.1 Physical Pretreatment 13
2.3.1.1 Mechanical Comminution 13
2.3.1.2 Extrusion 14
2.3.2 Chemical Pretreatment 15
2.3.2.1 Acid Pretreatment 15
2.3.2.2 Alkaline Pretreatment 16
2.3.2.3 Oxidative Delignification Pretreatment 17
2.3.2.4 Organosolv Pretreatment 18
2.3.3 Physico-Chemical Pretreatment 19
2.3.3.1 Steam Explosion 19
2.3.3.2 Wet Oxidation Pretreatment 21
2.3.3.3 Ammonia Fiber/Freeze Expansion (AFEX) Pretreatment 21
2.3.4 Biological Pretreatment 22
2.4 Enzymatic Hydrolysis of Cellulosic Feedstock 23
2.4.1 Overview of Cellulolytic Enzyme System 23
2.4.2 Factors Affecting Enzymatic Hydrolysis 26
2.5 Cellulosic Ethanol Fermentation 28
2.5.1 Microorganisms for Ethanol Fermentation 28
2.5.2 Metabolism of Bioethanol Fermentation 31
2.5.3 Cellulosic Ethanol Production Strategies 34
2.5.3.1 Separate Hydrolysis and Fermentation (SHF) 35
2.5.3.2 Simultaneous Saccharification and Fermentation (SSF) 36
2.5.3.3 Consolidated Bioprocessing (CBP) 36
2.5.4 Cell Immobilization 38
Chapter 3 MATERIALS AND METHODS 41
3.1 Chemicals and Materials 41
3.2. Equipment 43
3.3 Microorganism Strains and Cultivation Medium 45
3.3.1 Glucose Fermenting Bacterium and Its Cultivation Medium 45
3.3.2 Xylose Fermenting Yeast and Its Cultivation Medium 45
3.4 Analytical Methods 47
3.4.1 Determination of Cellulase Activity Based on Filter Paper Unit (FPU) 47
3.4.2 Determination of Reducing Sugar Concentration Using DNS Test 47
3.4.3 Determination of Cell Concentration 48
3.4.4 Determination of the Concentration of Soluble Component 48
3.4.5 Simulation of Ethanol Production Performance Using Monod-type Kinetic Model 49
3.5 Experimental Methods 50
3.5.1 Acid Pretreatment of Bagasse 50
3.5.2 Alkaline Pretreatment of Bagasse 50
3.5.3 Immobilization of Zymomonas mobilis with Calcium Alginate and Poly vinyl alcohol (PVA) 51
3.5.4 Batch Fermentation of Glucose to Produce Ethanol 52
3.5.5 Batch Co-Fermentation of Glucose and Xylose to Produce Ethanol 53
3.5.6 Fermentation of Glucose to Produce Ethanol with Continuous Stirred Tank Reactor (CSTR) 53
3.5.7 Fed-Batch Strategies for Glucose Fermentation to Produce Ethanol Using Immobilized Cells of Zymomonas mobilis 54
3.5.8 Fed-Batch Strategies for Pentose Fermentation to Produce Ethanol Using Suspended Cells of Pichia stipitis 54
3.5.9 Continuous Xylose Fermentation by Suspended Pichia stipitis Coupled with Membrane Separation 55
3.5.10 Fermentative Production of Cellulosic Ethanol 55
3.5.10.1 Batch SHF and SSF Ethanol Production Using Acid Pretreated Bagasse as the Carbon Source 55
3.5.10.2 Continuous SHcF and SScF Ethanol Production Using Alkaline Pretreated Bagasse as the Carbon Source 56
Chapter 4 RESULT AND DISCUSSION 59
4.1 Optimum Conditions for Batch Ethanol Fermentation Using PVA-Immobilized Zymomonas mobilis 59
4.1.1 Effect of Initial Glucose Loading on Ethanol Production by PVA-Immobilized Zymomonas mobilis 59
4.1.2 Effect of Immobilized Cells Loading on Ethanol Production by PVA-Immobilized Zymomonas mobilis 61
4.1.3 Reusability of Immobilized Cells 63
4.2 Batch Co-Fermentation of Glucose and Xylose Using PVA-Immobilized Zymomonas mobilis and Pichia stipitis 65
4.3 Fed-Batch Strategy for Ethanol Fermentation from Glucose Using PVA-Immobilized Zymomonas mobilis 69
4.4 Continuous Ethanol Fermentation by PVA-Immobilized Zymomonas mobilis 72
4.4.1 Effect of Feeding Medium Composition on Ethanol Fermentation in Continuous System 73
4.4.2 Effect of Glucose Loading Rate on Ethanol Fermentation in Continuous System 75
4.4.3 Effect of Particle Loading on Ethanol Fermentation in Continuous System 77
4.4.4 Optimization of Continuous Ethanol Fermentation by Experiment Design 79
4.5 Ethanol Production from Xylose by Pichia stipitis 83
4.5.1 Continuous Xylose Fermentation using PVA-immobilized Pichia stipitis 83
4.5.2 Fed-Batch Xylose Fermentation using Suspended Pichia stipitis 85
4.5.3 Continuous Xylose Fermentation by Suspended Pichia stipitis Coupled with Membrane Separation 90
4.6 Cellulosic Ethanol Production from Acid-Pretreated Lignocellulosic Feedstock by Immobilized Zymomonas mobilis 95
4.7 Continuous Cellulosic Ethanol Fermentation from Alkaline-Pretreated Lignocellulosic Feedstock by Immobilized Zymomonas mobilis and Suspended Pichia stipitis 103
Chapter 5 CONCLUSIONS 112
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