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研究生：	蘇怡禎 Su, Yi-Chen
論文名稱：	以溫度轉換策略進行纖維素料源之細菌與酵素水解並進行暗醱酵產氫 BioH2 production from cellulosic hydrolysate prepared via temperature-shift-enhanced bacterial/enzymatic cellulose hydrolysis
指導教授：	張嘉修 Chang, Jo-Shu
學位類別：	碩士 Master
系所名稱：	工學院 - 化學工程學系 Department of Chemical Engineering
論文出版年：	2009
畢業學年度：	97
語文別：	英文
論文頁數：	108
中文關鍵詞：	暗醱酵產氫、Clostridium butyricum CGS5 、Cellulomonas uda 、溫度轉換策略、酸鹼前處理、纖維素水解酵素、纖維素料源、纖維素水解
外文關鍵詞：	Clostridium butyricum CGS5, cellulose hydrolysis, cellulolytic enzymes, Cellulomonas uda, dark hydrogen fermentation, Temperature-shift atrategy, acid/alkaline pretreatment, cellulosic feedstock
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利用纖維素料源生產生質氫氣被視為具發展性及經濟價值的。本研究利用兩階段程序將纖維素料源轉化成氫氣能源。首先以溫度轉換策略來進行纖維素料源之酵素水解生成還原糖，接著以還原糖為基質利用暗氫。其中溫度轉換策略可有效提升纖維素料源酵素水解時之還原糖產量，首先將生產纖維素水解酵素之菌株Cellulomonas uda E3-01培養於35oC條件下，使其大量生產纖維素水解酵素後，利用離心將菌體以及上清液進行分離，接著再將含有酵素之上清液加入新的碳源基質後，提升反應溫度至45oC，以抑制菌株生長及避免還原糖消耗，並藉由升溫來加速纖維素水解。當使用純纖維素 (CMC)、半纖維素 (xylan) 及農業廢棄物 (蔗渣)醱酵程序進行生物產為碳源基質進行纖維素水解時，由溫度轉換策略之實驗結果顯示，此策略確實能有效提升水解液中還原糖之產量，尤其是小分子醣類(單糖、雙糖)的部分。酸 (磷酸) 與鹼 (氫氧化鈉)前處理有助於天然農業廢棄物 (蔗渣) 之酵素水解，尤其是利用磷酸進行前處理，並結合溫度策略水解蔗渣後，可得到較多的葡萄糖與纖維二糖含量。最後，以CMC與xylan進行水解所得之水解液，利用Clostridium butyricum CGS5菌株進行暗醱酵產氫時，其最佳之產氫效率分別為8.8 與6.0 mmol H2/g reducing sugar。利用蔗渣為料源，以溫度轉換策略所得之水解液進行產氫，可得產氫效率為6.0 mmol H2/g reducing sugar。

Biohydrogen from cellulosic feedstock has been considered a promising and economical route of producing bioenergy. Converting inert cellulosic materials to fermentable feedstock via pretreatment/hydrolysis is the key technology towards efficient bioenergy production. In this work, pure (carboxymethyl-cellulose (CMC) and xylan) and natural (bagasse) cellulosic materials were first pretreated by acid and alkaline reagents, and was then hydrolyzed by extracellular cellulase/xylanase produced from an isolated Cellulomonas uda E3-01 strain. A temperature-shift strategy (35oC for cellulolytic enzymes production and 45oC for hydrolysis reaction) was used to increase the reducing sugar (especially, monosaccharide and disaccharide) concentration in the hydrolysates. The CMC, xylan, and bagasse hydrolysates were successfully converted to H2 via dark fermentation with Clostridium butyricum CGS5, attaining a maximum hydrogen yield of 8.8 mmol H2/g reducing sugar (7.9 mmol H2/g substrate), 6.0 mmol H2/g reducing sugar (5.4 mmol H2/g substrate), and 6.0 mmol H2/g reducing sugar (5.4 mmol H2/g substrate).

摘要	I
Abstract	II
Acknowledgement	III
Contents	IV
List of Tables	VI
List of Figures	VII
Chapter 1 Introduction	1
1 Motivation and purpose	1
2 Outline of the thesis	3
Chapter 2 Literature review	5
1 Strain of cellulose hydrolysis	5
2 Lignocellulose	9
2.1 Cellulose	11
2.2 Hemicellulose	11
2.3 Lignin	12
3 Cellulose pretreatment/hydrolysis	13
3.1 Physical pretreatment	13
3.1.1 Mechanical comminution	13
3.1.2 Pyrolysis	14
3.2 Physicochemical pretreatment	14
3.2.1 Steam explosion	14
3.2.2 Ammonia fiber explosion (AFEX)	15
3.2.3 Carbon dioxide explosion	16
3.3 Chemical pretreatment	16
3.3.1 Ozonolysis	16
3.3.2 Acid hydrolysis	17
3.3.3 Alkaline hydrolysis	18
3.3.4 Organosolv Process	19
3.4 Biological pretreatment	20
3.4.1 Bacterial hydrolysis	21
3.4.2 Enzymatic hydrolysis	21
4 Hydrogen production	24
4.1 Physiochemical hydrogen production	25
4.1.1 Thermo chemical method	25
4.1.2 Electrochemical method	26
4.2 Biological hydrogen production	26
4.3 Hydrogen gas production by dark fermentation	30
5 Biological hydrogen production from cellulosic hydrolysate	33
Chapter 3 Materials and methods	36
1 Chemicals and Material	36
2 Equipments	38
3 Bacterial strains and cultivation medium	39
3.1 The cellulase-producing bacteria and culture condition	39
3.2 Hydrogen-producing anaerobic bacteria and culture condition	40
4 Analytical methods	41
4.1 Determination of reducing sugar concentration by DNS method	41
4.2 Determination of total sugar concentration by phenol-sulfuric acid	42
4.3 Determination of protein concentration	43
4.4 Determination of soluble products by HPLC	44
4.5 Determination of gas products by GC	44
4.6 Determination of cellulase activity	44
4.6.1 Exo-glucanase activity	45
4.6.2 Xylanase activity	45
4.6.3 Endo-glucanase activity	45
4.6.4 β-glucosidase activity	46
4.7 Cellulose enzyme assay (Congo Red)	46
4.8 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Zymography	46
5 Experimental methods	48
5.1 Pretreatment method	48
5.1.1 NaOH pretreatment	48
5.1.2 H3PO4 pretreatment	48
5.2 Temperature shift method	49
5.3 Optimal condition for producing cellulase and xylanase	49
5.4 Optimal condition for enzymatic hydrolysis	50
5.5 Biohydrogen production from cellulosic hydrolysate	50
Chapter 4 Results and discussion	51
1 Cellulase-producing microorganism	51
1.1	Optimum reaction temperature of enzyme produced by Cellulomonas uda E-03	52
1.2 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Zymography	53
2 Optimal condition for producing cellulase and xylanase	54
2.1 Effect of dissolved oxygen on cellulase/xylanase production	54
2.2 Effect of temperature, agitation rate and pH on cellulase/xylanase production	56
2.3 Effect of carbon source (CMC) concentration on cellulase/xylanase production	61
3 Effects of sugar inhibition on cellulase and xylanase production	64
3.1 Effect of glucose concentration on enzyme production	64
3.2 Effect of xylose concentration on enzyme production	65
4 Optimal condition for cellulose enzymatic hydrolysis by temperature-shift method	66
4.1 Profiles of reducing sugar production, and cellulolytic enzyme activity during temperature-shift operations	66
4.2 Effect of pH on enzymatic hydrolysis	70
4.3 Effect of agitation rate on enzymatic hydrolysis	73
4.4 Effect of carbon source (CMC) concentration on enzymatic hydrolysis	74
4.5 Effect of carbon source (Xylan) concentration on enzymatic hydrolysis	78
4.6 Pretreatment of bagasse by alkaline and acid methods	82
4.6.1 SEM analysis	82
4.6.2 Effect of pretreatment method on enzymatic hydrolysis of bagasse	84
5 Effects of sugar inhibition on cellulase and xylanase activities during enzyme hydrolysis of cellulosic materials	88
5.1 Effect of glucose inhibition on endoglucanase activity during enzymatic hydrolysis of CMC	88
5.2 Effect of xylose inhibition on xylanase during enzymatic hydrolysis of xylan	89
6 Bio-H2 production from cellulosic hydrolysate by temperature-shift strategy	91
6.1 Bio-H2 production from CMC hydrolysate	91
6.2 Bio-H2 production from xyaln hydrolysate	93
6.3 Bio-H2 production from bagasse hydrolysate	95
Chapter 5 Conclusions	98
References	99
Appendix curriculum vitae	107
                                    

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