臺灣博碩士論文加值系統

生質能具有取之不盡、用之不竭之優點，尤以生質乙醇為替代能源者，其二氧化碳的淨排放量遠較化石能源為低。其中又以木質纖維素為生產生質乙醇之原料者，尚可避免排擠糧食等問題。台灣一年約產生160 萬公噸農業廢棄物稻桿，其細胞壁主要即由纖維素構成，因此以稻桿生產生質乙醇除可廢棄物再生外，更可避免燃燒等傳統處理方法造成之環境污染；然而纖維素不易分解利用，以木質纖維素生產乙醇仍無法與糖質或澱粉質原料匹敵。針對纖維素生質乙醇生產之問題，運用菌體之固定化技術，除菌體重複使用可減少生產成本外，亦將好氧水解、厭氧發酵合併為單一步驟於同一系統。此法不僅節省操作步驟、設備，並解決且提高單一菌種所無法進行之作用。故以菌體共固定培養技術來轉化纖維素原料生產生質乙醇，極具可行潛力與開發之經濟價值，為亟待開發之技術。
本研究延續先前共固定培養體系，採用本研究室所開發之新型生化反應器，於不同固定化載體及溶氧量下進行生質乙醇之生產探討，亦即於反應器之實驗中監控通氣量與攪拌轉速對溶氧度變化之影響，同時探討好氧區厭氧區溶氧條件之建立，以提昇生質乙醇產量。於搖瓶中採用相異載體固定化菌體之活性變化方面， loofah sponge載體不論於單階段或二階段固定化培養政策時，其活性皆較佳，雖然loofah sponge載體於搖瓶培養過程中遭降解，然而於生化反應器實驗中卻可減緩其降解速度，故其可作為適當之固定化載體。此外，在預培養時間方面，於搖瓶中分別以CMC或稻桿為基質進行探討，其結果顯示，不論以CMC或稻桿為基質者，其整體活性皆於 2 日預培養後達到最高。又於反應器實驗之溶氧量方面，以新式反應器進行探討後發現，以2L / min 通氣量持續通氣者，可確實分隔好厭氧區。另一方面，於反應器相異載體進行生質乙醇生產上，採用loofah sponge載體時可明顯提昇乙醇生產，於 8 小時後其產量可達 825 ppm ；而於通氣量之探討方面，仍以通氣量為2 L /min者為佳；另外，於不同攪拌轉速之探討方面，溶氧量與產量隨攪拌轉速而提昇，攪拌轉速150 rpm 者其溶氧量呈穩定維持，於 8 小時達最高產量 934 ppm，此結果顯示攪拌轉速與通氣量之變化影響乙醇之生產。最後，延續上述最佳條件，以 20 g / L 之CMC為基質進行高濃度培養，其乙醇產量於 8 小時後可達1080 ppm ，而採用10 g / L CMC進行三次重複饋料培養，其結果發現乙醇產量於 9 小時後累積達 2650 ppm ，顯示本生產系統具穩定性與重複生產之潛力。

There were more than 1600 thousand tons of rice straw agricultural wastes produced in Taiwan every year. Produce bioethanol from rice straw not only regenerates the agriculture waste into bioenergy, but also solves the problem of environmental pollution by rice straw burning. Cellulose is the main component of rice straw. However, it is difficult to hydrolyze the lignocellulose to glucose by enzymes, bio-ethanol produced by lignocellulose still expensive when compared to sugar or starch substrate. Using the technology of cell immobilization might lower the cost by repeat immobilized cells utilization; process of cellulose hydrolysis and glucose fermentation combination, and co-culture system.
In this study, we investigated the bio-ethanol production of our new co-culturing immobilized cells system reactor with dissolved oxygen monitored by different immobilization carriers in different aeration or the agitation speed condition. We also study the aerobic/anaerobic zone construction in ethanol production.
In the shaking flask research, the enzyme activities of immobilized cells on different carriers showed that loofah sponge performs well in both one stage and two stage immobilize strategy, but decomposed. The loofah sponge decomposed slower when culturing in the bioreactor Besides, suspended culturing in the shaking flask, 2 days preculture could reach highest value summarized enzyme activity in both carboxymethylcellulose and rice straw substrate. We also found the aerobic/anaerobic zones constructed well under the aeration rate of 2L / min. The loofah sponge immobilization carriers, had yield of ethanol concentration reached 825 ppm after 8 hrs fermentation. The aeration rate of 2 L /min performed better than the others. The dissolved oxygen (DO) was affected by different agitation speeds, the DO value rose with the agitation speed. The agitation speed of 150 rpm could maintain the DO level stably with the ethanol concentration reached 934 ppm after 8hrs fermentation. Higher substrate concentration 20g/L of caboxymethylcellulose(CMC) could achieve the ethanol concentration of 1080 ppm after 8 hrs fermentation. Three repeated batches of substrate concentration 10 g/L CMC could reach the ethanol concentration of 2650 after 9 hrs fermentation. These results showed that our immobilized co-culture-cell bioreactor system had high potential of continuous and stable bio-ethanol production.

指導教授推薦書…………………………………………………………
口試委員會審定書………………………………………………………
授權書 …………………………………………………………………iii
誌謝 ……………………………………………………………………iv
中文摘要 ………………………………………………………………v
英文摘要………………………………………………………………vii
目錄 ……………………………………………………………………ix
圖目錄…………………………………………………………………xvi
表目錄 ………………………………………………………………xxiv
第一章 緒論……………………………………………………………1
1.1 研究動機………………………………………………………1
1.2 研究目的………………………………………………………2
第二章 文獻回顧………………………………………………………3
2.1生質能源利用現況 ……………………………………………3
2.2再生能源 ………………………………………………………4
2.3農業廢棄物稻桿 ………………………………………………6
2.4木質纖維素結構 ………………………………………………9
2.5纖維素乙醇生產………………………………………………10
2.5.1木質纖維素之前處理…………………………………10
2.5.2 纖維素之水解 ………………………………………14
2.5.2.1 纖維素水解菌 …………………………………14
2.5.2.2 纖維素分解酵素 ………………………………15
2.5.2.3 纖維素分解酵素之產物反饋抑制作用 ………16
2.5.2.4 纖維素水解酵素的誘導 ………………………17
2.5.3 糖類之發酵 …………………………………………18
2.5.4 纖維素乙醇生產之菌株 ……………………………18
2.6 纖維素乙醇生產製程 ………………………………………19
2.7 糖化發酵菌體固定化 ………………………………………20
2.8 生物反應器的應用及種類 …………………………………22
2.8.1 充填型生物反應器 …………………………………23
2.8.2 攪拌式反應器 ………………………………………23
2.8.3 氣舉式反應器 ………………………………………23
2.9 通氣、攪拌對於溶氧量（DO）之影響………………………24
2.10 設計反應器時所需考量之因素……………………………25
2.11 新型纖維素乙醇生產反應器之開發與設計………………27
第三章 材料與方法 …………………………………………………30
3.1實驗材料………………………………………………………30
3.1.1菌株……………………………………………………30
3.1.2 試藥 …………………………………………………30
3.1.3 設備儀器 ……………………………………………31
3.1.4 培養基組成 …………………………………………33
3.1.4.1 Bushnell-Haas selection medium（BHS）…33
3.1.4.2 Zymomonas mobilis 第一級活化培養基……33
3.1.4.3 Zymomonas mobilis第二級活化培養基……34
3.1.5 DNS試劑………………………………………………35
3.1.6 3%海藻酸鈉溶液與0.3M CaCl2 溶液之配製………36
3.2實驗方法………………………………………………………37
3.2.1菌種保存………………………………………………37
3.2.2菌種培養………………………………………………37
3.2.3菌體接種方法…………………………………………38
3.2.4 糖化培養 ……………………………………………38
3.2.5 Z.mobilis 之放大……………………………………38
3.2.6海藻酸鈉包埋…………………………………………38
3.2.7絲瓜纖維固定化………………………………………39
3.2.8稻桿處理………………………………………………39
3.2.9酵素活性測試…………………………………………39
3.2.10產物分析 ……………………………………………40
3.2.10.1 HPLC 分析…………………………………40
3.2.10.2葡萄糖檢測劑(glucose test) ……………41
3.2.10.3 GC 分析……………………………………41
3.2.11菌體濃度測定 ………………………………………42
第四章 結果與討論……………………………………………………43
4.1不同離子交換濃度之聚合物膠體對乙醇生產影響之探討…43
4.2二階段固定化培養政策與最佳固定化載體及預培養天數之探討 ……………………………………………………………44
4.2.1不同載體之二階段固定化培養政策之探討…………45
4.2.1.1以alginate-Ca為載體進行二階段培養政策之活性探討 ………………………………………………48
4.2.1.2 以PU ＃HR-13為載體進行二階段培養政策之活性探討 ………………………………………………50
4.2.1.3以PU ＃HR-08為載體進行二階段培養政策之活性探討 …………………………………………………53
4.2.1.4以loofah sponge為載體進行二階段培養政策之活性探討 ……………………………………………56
4.2.2最佳固定化載體之探討………………………………58
4.2.3最佳預培養天數之探討………………………………63
4.2.3.1以CMC作為基質之預培養天數對菌體酵素活性影響之探討…………………………………………63
4.2.3.2以稻桿作為基質之預培養天數對菌體酵素活性影響之探討 ……………………………………65
4.3 反應器溶氧量之探討 ……………………………………… 69
4.3.1探討轉速對反應器溶氧量之影響 ……………………70
4.3.2中間分隔物固定型式之探討 …………………………73
4.3.3通氣量影響反應器溶氧量之探討……………………77
4.4 反應器最佳載體之探討 ……………………………………81
4.4.1作為對照組的水解菌共懸浮培養之乙醇生產………82
4.4.2以2.5×10×10 ㎝3塊狀PU ＃HR-08為載體之菌體共固定化培養………………………………………………85
4.4.3以250個 1㎝3切塊PU ＃HR-08為載體之菌體共固定化培養 …………………………………………………87
4.4.4以2.5×10×10 ㎝3塊狀PU ＃HR-13為載體之菌體共固定化培養………………………………………………89
4.4.5以250個 1㎝3切塊PU ＃HR-13為載體之菌體共固定化培養 …………………………………………………91
4.4.6以2.5×10×10 ㎝3塊狀loofah sponge為載體之菌體共固定化培養 ……………………………………………93
4.4.7以250個 1㎝3切塊loofah sponge為載體之菌體共固定化培養 ………………………………………………95
4.4.8以alginate-Ca（平均直徑3.8㎜）為載體之菌體共固定化培養 ………………………………………………98
4.4.9以alginate-Ca（平均直徑2.8㎜）為載體之菌體共固定化培養………………………………………………100
4.4.10在改良式新型反應器中以不同載體進行共固定化生產 之綜合比較 …………………………………………103
4.5反應器以loofah sponge載體進行共固定化生產之最佳通氣量探討 ………………………………………………………104
4.5.1於通氣量 1L / min 下進行共固定化生產之探討…105
4.5.2於通氣量 3L / min 下進行共固定化生產之探討…107
4.5.3以loofah sponge為載體於改良式新型反應器中不同通氣下之綜合比較………………………………………109
4.6反應器以loofah sponge載體進行共固定化生產之最佳攪拌速度探討 ……………………………………………………110
4.6.1於攪拌速度50 rpm下進行共固定化生產之探討……111
4.6.2於攪拌速度100 rpm下進行共固定化生產之探……113
4.6.3於攪拌速度於150 rpm下進行共固定化生產之探討115
4.6.4於攪拌速度於200 rpm下進行共固定化生產之探討117
4.6.5以loofah sponge為載體於改良式新型反應器中不同攪拌速度之綜合比較……………………………………119
4.7於改良式新型反應器中以高濃度基質（20 g / L CMC）進行共固定乙醇之生產 …………………………………………120
4.8重複三批次菌體共固定化培養之乙醇生產 ………………123
4.9反應器以稻桿作為基質之以loofah sponge載體進行共固定化培養生產乙醇之探討………………………………………126
4.9.1於改良式新型反應器中以一般濃度基質（10 g / L 稻桿）共固定化培養進行乙醇之生產…………………127
4.9.2於改良式新型反應器中以高濃度基質（20 g / L 稻桿）共固定化培養進行乙醇之生產………………………129
4.9.3於改良式新型反應器中以稻桿為基質進行重複三批次菌體共固定化培養生產乙醇…………………………132
第五章 結論………………………………………………………… 135
參考文獻 …………………………………………………………… 138
附錄……………………………………………………………………145

圖目錄
圖 1 纖維素之組成……………………………………………………11
圖 2 纖維素分解酵素之產物反饋抑制 …………………………… 17
圖 3 改良式新型反應器 …………………………………………… 29
圖 4 DNS呈色原理…………………………………………………36
圖 5 CaCl2與BaCl2離子交換濃度聚合物膠體固定化菌體之乙醇生產濃度變化……………………………………………………44
圖6 二階段固定化培養政策於不同載體之生質變化量……………47
圖 7 以alginate-Ca為載體固定化水解菌時，3種水解酵素之活性變化 ………………………………………………………………50
圖 8以PU ＃HR-13為載體固定化水解菌時，3種水解酵素之活性變化 ………………………………………………………………52
圖 9以PU ＃HR-08為載體固定化水解菌時，3種水解酵素之活性變化 ………………………………………………………………55
圖 10以loofah sponge為載體固定化水解菌時，3種水解酵素之活性變化 ……………………………………………………………57
圖11 loofah sponge 於培養過程分解 ………………………………58
圖 12 Exoglucanase、Endoglucanase於不同載體之固定化菌體活性 60
續 圖 12 beta-Glucosidase、綜合加總活性於不同載體之固定化菌體活性 ………………………………………………………………61
圖13 Exoglucanase 、Endoglucanase 、beta-Glucosidase、綜合加總活性於不同載體之固定化菌體單位活性變化……………………62
圖14 搖瓶中以CMC為基質Exoglucanase 、Endoglucanase 、beta-Glucosidase於不同預培養天數之固定化菌體活性變化…64
圖15 搖瓶中以CMC為基質於不同預培養天數下進行培養時，其生質量與最高加總活性之比較……………………………………65
圖16 搖瓶中以稻桿為基質Exoglucanase 、Endoglucanase 、beta-Glucosidase於不同預培養天數之固定化菌體活性變化…67
圖17 搖瓶中以稻桿為基質於不同預培養天數下進行培養時，其生質量與最高加總活性之比較………………………………………68
圖18 新型反應器之架構圖與實體圖…………………………………69
圖19 實驗進行中好氧區之溶氧量DO值與厭氧區之溶氧量（第1次探討）……………………………………………………………71
圖 20實驗進行中好氧區之溶氧量DO值與厭氧區之溶氧量（第2次探討） …………………………………………………………72
圖21 alginate-Ca膠體粒子膨脹碎裂，堵塞分隔物推擠至厭氧區中…72
圖 22 實驗進行中好氧區之溶氧量DO值與厭氧區之溶氧量（第3次探討）……………………………………………………………73
圖 23 實驗進行中好氧區之溶氧量DO值與厭氧區之溶氧量（第4次探討）……………………………………………………………74
圖 24 alginate-Ca膠體粒子膨脹推動分隔物，未進入厭氧區…… 75
圖 25 實驗進行中好氧區之溶氧量DO值與厭氧區之溶氧量（第5次探討） …………………………………………………………76
圖 26 實驗進行中好氧區之溶氧量DO值與厭氧區之溶氧量（第6次探討） …………………………………………………………77
圖 27 實驗進行中好氧區之溶氧量DO值與厭氧區之溶氧量（第7次探討）……………………………………………………………78
圖 28改良式新型反應器………………………………………………79
圖 29實驗進行中好氧區之溶氧量DO值與厭氧區之溶氧量（第8次探討）……………………………………………………………80
圖 30實驗進行中好氧區之溶氧量DO值與厭氧區之溶氧量與酸鹼值（第9次探討） …………………………………………………80
圖 31 反應器好氧區水解菌以共懸浮培養時，3種水解酵素之預培養活性、主發酵活性變化、預培養溶氧量與酸鹼值變化、主發酵溶氧量與酸鹼值變化及反應物暨產物 ………………………84
圖 32 反應器好氧區水解菌以整塊PU ＃HR-08為載體進行共固定化培養時，3種水解酵素之預培養活性、主發酵活性變化、預培養溶氧量與酸鹼值變化主發酵溶氧量與酸鹼值變化及反應物暨產物濃度變化 ……………………………………………86
圖 33 反應器好氧區水解菌以切塊PU ＃HR-08為載體進行共固定化培養時，3種水解酵素之預培養活性、主發酵活性變化、預培養溶氧量與酸鹼值變化、主發酵溶氧量與酸鹼值變化及反應物暨產物濃度變化 ……………………………………………88
圖34 反應器好氧區水解菌以整塊PU ＃HR-13為載體進行共固定化培養時，3種水解酵素之預培養活性、主發酵活性變化、預培養溶氧量與酸鹼值變化、主發酵溶氧量與酸鹼值變化及反應物暨產物濃度變化 …………………………………………………90
圖 35 反應器好氧區水解菌以切塊PU ＃HR-13為載體進行共固定化培養時，3種水解酵素之預培養活性、主發酵活性變化、預培養溶氧量與酸鹼值變化、主發酵溶氧量與酸鹼值變化及反應物暨產物濃度變化 ………………………………………………… 92
圖 36 反應器好氧區水解菌以整塊loofah sponge為載體進行共固定化培養時，3種水解酵素之預培養活性、主發酵活性變化、預培養溶氧量與酸鹼值變化、主發酵溶氧量與酸鹼值變化及反應物暨產物濃度變化 ……………………………………………94
圖 37 反應器好氧區水解菌以切塊loofah sponge為載體進行共固定化培養時，3種水解酵素之預培養活性、主發酵活性變化、預培養溶氧量與酸鹼值變化、主發酵溶氧量與酸鹼值變化及反應物暨產物濃度變化 …………………………………………… 97
圖38 反應器好氧區水解菌以alginate-Ca（平均直徑3.8㎜）為載體進行共固定化培養時，3種水解酵素之預培養活性、主發酵活性變化、預培養溶氧量與酸鹼值變化、主發酵溶氧量與酸鹼值變化及反應物暨產物濃度變化 …………………………………99
圖39 反應器好氧區水解菌以alginate-Ca（平均直徑2.8㎜）為載體進行共固定化培養時，3種水解酵素之預培養活性、主發酵活性變化、預培養溶氧量與酸鹼值變化、主發酵溶氧量與酸鹼值變化及反應物暨產物濃度變化 …………………………………102
圖40 反應器好氧區水解菌以不同載體進行共固定化培養時，其生質量、最高加總活性與最高乙醇濃度之比較……………………103
圖41反應器好氧區水解菌於通氣 1L / min 下進行共固定化生產時，3種水解酵素之預培養活性、主發酵活性變化、預培養溶氧量與酸鹼值變化、主發酵溶氧量與酸鹼值變化及反應物暨產物濃度變化 ……………………………………………………………106
圖42反應器好氧區水解菌於通氣 3L / min 下進行共固定化生產時，3種水解酵素之預培養活性、主發酵活性變化、預培養溶氧量與酸鹼值變化、主發酵溶氧量與酸鹼值變化及反應物暨產物濃度變化 ……………………………………………………………108
圖43反應器好氧區水解菌於不同通氣下進行進行共固定化培養時，其生質量、最高加總活性與最高乙醇濃度之比較……………109
圖44反應器好氧區水解菌於攪拌速度50 rpm 下進行共固定化生產時，3種水解酵素之預培養活性、主發酵活性變化、預培養溶氧量與酸鹼值變化、主發酵溶氧量與酸鹼值變化及反應物暨產物濃度變化 ………………………………………………………112
圖45反應器好氧區水解菌於攪拌速度100 rpm 下進行共固定化生產時，3種水解酵素之預培養活性、主發酵活性變化、預培養溶氧量與酸鹼值變化、主發酵溶氧量與酸鹼值變化及反應物暨產物濃度變化 ………………………………………………………114
圖 46 反應器好氧區水解菌於攪拌速度150 rpm 下進行共固定化生產時，3種水解酵素之預培養活性、主發酵活性變化、預培養溶氧量與酸鹼值變化、主發酵溶氧量與酸鹼值變化及反應物暨產物濃度變化 ………………………………………………116
圖47反應器好氧區水解菌於攪拌速度200 rpm 下進行共固定化生產時，3種水解酵素之預培養活性、主發酵活性變化、預培養溶氧量與酸鹼值變化、主發酵溶氧量與酸鹼值變化及反應物暨產物濃度變化 ………………………………………………………118
圖48 反應器好氧區水解菌於不同攪拌速度下進行進行共固定化培養時，其生質量、最高加總活性與最高乙醇濃度之比較 ……119
圖49 反應器好氧區水解菌於高濃度基質（20 g / L CMC）下進行共固定化生產時，3種水解酵素之預培養活性、主發酵活性變化、預培養溶氧量與酸鹼值變化、主發酵溶氧量與酸鹼值變化及反應物暨產物濃度變化 …………………………………………122
圖50反應器好氧區水解菌進行重複三批次共固定化生產時，3種水解酵素之預培養活性、主發酵活性變化、預培養溶氧量與酸鹼值變化、主發酵溶氧量與酸鹼值變化及反應物暨產物濃度變化 ………………………………………………………………125
圖51 反應器好氧區水解菌（T-A）以稻桿為基質（10 g / L）進行共固定化生產時，3種水解酵素之預培養活性、主發酵活性變化、預培養溶氧量與酸鹼值變化、主發酵溶氧量與酸鹼值變化及反應物暨產物濃度變化…………………………………………128
圖52 反應器好氧區水解菌（T-A）以稻桿為基質（20 g / L）進行共固定化生產時，3種水解酵素之預培養活性、主發酵活性變化、預培養溶氧量與酸鹼值變化、主發酵溶氧量與酸鹼值變化及反應物暨產物濃度變化 …………………………………………131
圖53 反應器好氧區水解菌（T-A）進行重複三批次共固定化生產時，3種水解酵素之預培養活性、主發酵活性變化、預培養溶氧量與酸鹼值變化、主發酵溶氧量與酸鹼值變化及反應物暨產物（ethanol）濃度變化…………………………………………134

表目錄
表1 Bushnell-Haas selection medium 之成分……………………33
表2 Zymomonas mobilis 第一級活化培養基之成分………………… 34
表3 Zymomonas mobilis 第二級活化培養基之成分………………… 35
表4 DNS試劑之成分……………………………………………………36

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