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研究生:林鼎翔
研究生(外文):Ting-Hsiang Lin
論文名稱:提高Pichia stipitis 生產纖維酒精的木糖發酵效率
論文名稱(外文):Improving efficiency in xylose fermentation for the production of cellulosic ethanol by Pichia stipitis
指導教授:黃雪莉黃雪莉引用關係
指導教授(外文):Shir-Ly Huang
學位類別:博士
校院名稱:國立中央大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:英文
論文頁數:121
中文關鍵詞:纖維酒精木糖發酵去毒化稻稈商業化
外文關鍵詞:cellulosic ethanolxylose fermentationdetoxificationrice strawcommercialization
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在纖維酒精的製程當中,由於可放大的特性,稀酸前處理被認為是眾多前處理程序中最有效率且最具商業可行性的的前處理技術,稀酸前處理可將大部分的半纖維素分解為木醣並溶於半纖維水解液中,在這同時也有有許多分解於葡萄糖、木糖和木質素的發酵抑制物(inhibitor),如:呋喃甲醛(furfural)、羥甲基糠醛(Hydroxymethylfurfural) 和酚類化合物(phenolic compound) 也會被釋放至半纖維水解液中,這一些發酵的抑制物導致菌株在半纖維水解液的發酵表現不佳,同時也影響了纖維酒精的商業化可行性。有鑑於此,提升半纖維水解液中木醣的發酵效率被視為生質酒精商業化相當重要的工作。在本篇論文的第一部分主要是測試馴化的菌株Pichia stipitis 在100L 發酵槽進行木糖發酵生產酒精的可行性,實驗發現當通氣量設定為0.05 vvm 且使用過鹼化去毒化水解液時可得到最佳發酵效率為 0.44±0.02 gp/gs ,使用氨水中和以及氫氧化鈉中和的實驗組分別也可得到0.39±0.01 gp/gs and 0.34±0.01 gp/gs 的酒精生產效率,然而如果考量到過鹼化時所造成的木醣濃度的損失時,可發現使用氨水中和水解液的實驗所得到的酒精總量是和使用過鹼化法的酒精總量是接近相同的,因此氨水中和法可以取代傳統的去除化程序達到簡化木糖發酵的程序,此結果驗證了使用稻稈的木糖發酵成纖維酒精的商業化潛力。本論文的第二部分,發現了水解後的稻稈 (Hydrolyzed rice straw, HRS) 添加於木糖發酵反應中可以提高其酒精的產量,實驗發現發酵時在使用氫氧化鈉中和的水解液中加入HRS後可提升酒精生產效率至0.45 gp/gs ,HRS 同時也應用在合板、蔗渣和竹子的半纖維水解液中,發現菌株的酒精轉化效率也可被提高20%~51% ,證實HRS 這項技術可以被應用在不同的纖維原料的纖維酒精製程中,研究中也針對了HRS 對於菌株可能產生的影響進行推論和研究。綜合以上所述,本研究中利用兩種不同的策略改進了長久以來被認為是一個纖維酒精製程中低效率的木糖發酵程序,利用水解液的調理和改善發酵條件明了木糖發酵在放大規模操作的可行性,水解後的稻稈成功的應用實例驗證了改善木糖發酵效率的科技技術,添加HRS 的實驗也被拿來做測試及改善不只是稻稈還包括蔗渣、合板以及竹子的半纖維水解液都拿來與水解後的稻稈做測試及改善發酵效率的測試實驗。根據本篇論文研究成果,木糖發酵將不會被再被視為一個低效率且被放棄的製程,反而會被視為一個可以提高纖維酒精產量以及商業化潛力的程序。
Among the procedure of cellulosic ethanol, diluted acid pretreatment is considered as most effective and scalable pretreatment among various conditions of pretreatment, duo to its low cost of construction and scalability. When applying diluted acid pretreatment, most part of hemicellulose is dissolved into hemicellulosic hydrolysate as xylose. At the same time, various inhibitors, such as furfural, hydroxymethylfurfural and phenolic compound degraded from xylose, glucose and lignin are also be generated. These fermentative inhibitors hinder the performance of microorganism in hemicellulosic hydrolysate and further the commercialization of lignocelulosic ethanol. Improving efficiency of xylose fermentation is regarded as a critical for commercialization of lignocellulosic ethanol. In first part of thesis, adapted Pichia stiptis was used to demonstrate the feasibility of bioethanol production in 100-L fermentation using rice straw hydrolysate. The results indicated the highest ethanol yield of 0.44±0.02 gp/gs, when the aeration rate was at 0.05 vvm for overliming-detoxified hydrolysate. Hydrolysate conditioning with ammonia and NaOH also gave 0.39±0.01 gp/gs and 0.34±0.01 gp/gs of ethanol yield under the same aeration rate, respectively. However, actual ethanol yield from the fermentation of hydrolysate with ammonia neutralization was similar to that with overliming detoxification after considering the xylose loss resulted from conditioning processes. The results demonstrate the potential in the industrialization of xylose fermentation for bioethanol production from rice straw. In the second part of this research, hydrolyzed rice straw (HRS) was found to increase ethanol yield of xylose fermentation. In NaOH-neutralized hydrolyzate, HRS could induce ethanol yield up to 0.45 gp/gs. With the addition of HRS in hemicellulosic hydrolysate from plywood, bagasse and bamboo, the ethanol yields were increased for 20~51%. Xylose fermentation, usually regarded as low efficient process within the production procedure of lignocellulosic ethanol, was improved by two different strategies proposed in the present studies. The conditioning method of hydrolysate and optimized condition of fermentation proved the feasibility of xylose fermentation in pilot-scale operation. HRS was applied successfully to demonstrate technology improving efficiency of xylose fermentation. Fermentation of hemicellulosic hydrolysate from not only rice straw, but also bagasse, plywood and bamboo were also tested and improved with addition of HRS. According to the results mentioned in this dissertation, the xylose fermentation would not be seen as less efficient and abandoned process, but be taken into account for elevating the production and potential of commercialization of lignocellulosic ethanol.
Table of contents
中文摘要 II
Abstract III
Table of contents V
List of figure VIII
List of table X
Chapter 1 Literature review 1
1.1 Environmental issue and government policy 1
1.2 The first and secondary generation bioethanol 3
1.3 Lignocellulosic material 5
1.3.1 Cellulose 6
1.3.2 Hemicellulose 7
1.3.3 Lignin 8
1.4 Methods of pretreatment for lignocellulose 12
1.4.1 Physical pretreatment 13
1.4.2 Chemical pretreatment 13
1.4.3 Physicochemical pretreatment 16
1.4.4 Biological pretreatment 18
1.5 Enzymatic hydrolysis 18
1.6 Fermentation 21
1.7 Challenge of bio-ethanol production from xylose 24
Chapter 2 Motivation and specific aim 27
2.1 Motivation 27
2.2 Specific aims 27
Chapter 3 Pilot-scale ethanol production from rice straw hydrolysate by xylose fermenting Pichia stipites 29
3.1 Introduction 29
3.2 Materials and Methods 32
3.2.1 Microorganisms and inoculum preparation 32
3.2.2 Preparation of hemicellulosic hydrolysates from rice straw 32
3.2.3 Conditioning of hemicellulosic hydrolysates 33
3.2.4 Hydrolysate fermentation 34
3.2.5 Analytical methods 34
3.3 Results and Discussion 35
3.3.1 Composition of rice straw hydrolysates 35
3.3.2 Favorable effect of aeration rates on the fermentation of rice straw hydrolysates 37
3.3.4 Evaluation of the optimum conditioning method for hydrolysate fermentation 38
Chapter 4 The addition of hydrolyzed rice straw in xylose fermentation by Pichia stipitis to increase bioethanol production at the pilot-scale 43
4.1 Introduction 43
4.2 Material and Methods 45
4.2.1 Microorganisms and inoculum preparation 45
4.2.2 Preparation of hemicellulosic hydrolyzates from rice straw 45
4.2.3 Conditioning of hemicellulosic hydrolyzates 46
4.2.4 Preparation of hydrolyzed solid from rice straw 46
4.2.5 Hydrolyzate fermentation 47
4.2.6 Pilot-scale fermentation 48
4.2.7 Analytical methods 48
4.3 Results and Discussion 49
4.3.1 Effect of HRS in various circumstances 49
4.3.2 How is ethanol yield enhanced by HRS ? 52
4.3.3 Extended application of HRS 55
4.4. Conclusion 58
Chapter 5 Influence caused by hydrolyzed rice straw 59
5.1 Introduction 59
5.2 Material and method 60
5.2.1Microorganisms and inoculum preparation 60
5.2.2 Preparation of hemicellulosic hydrolyzates from rice straw 60
5.2.3 Preparation of hydrolyzed solid residue from lignocellulosic biomass 61
5.2.4 Hydrolysate fermentation 61
5.2.5 Analytical methods 62
5.3 results and discussion 64
5.3.1 microscopy observation of P. stipitis on hydrolyzed rice straw 64
5.3.2 Distribution of cell size 64
5.3.3 The protein expression profile 65
5.3.4 Comparison with different hydrolyzed lignocellulosic biomass 67
5.4 Conclusion 69
Chapter 6 Conclusion 71
6.1 Conclusion 71
6.2 Main contribution 72
6.3 Recommendation for further research 73
Reference 74
Appendix 105


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