全球生質柴油產量逐年攀升，因此其副產物甘油也隨之大量增產而逐漸累積。鑑此，開發甘油的新用途不但能解決甘油過剩之問題，亦可降低生質柴油的整體成本。本研究分別以純菌培養(Clostridium pasteurianum)及混菌培養來進行甘油連續醱酵產丁醇之研究。在純菌培養方面，首先以AnGCB (anaerobic granulated cell bioreactor)反應器進行水力滯留時間(Hydraulic retention time, HRT) (24、12及8 h)及攪拌速率(45及75 rpm)之探討，結果顯示，C. pasteurianum會受到丁醇抑制(約10 g/L)而造成不穩態操作，而較高的攪拌速率(75 rpm)及較低的HRT (12及8 h)操作有助於系統操作之穩定性，於甘油濃度60 g/L、攪拌速率75 rpm及HRT 8 h之條件，獲致丁醇濃度、丁醇產率(butanol yield, BY)、產氫速率(hydrogen production rate, HPR)分別為7.5 g/L、0.15 g BuOH/g glycerol consumed及0.485 mol/L/d。本研究另以CSTR (continuous flow stirred tank reactor)反應器進行HRT (24、12及8 h)及甘油濃度(60及40 g/L)之效應探討，結果顯示丁醇抑制造成系統不穩態操作的現象仍然存在，而較低的HRT及甘油濃度對丁醇抑制似乎略有和緩作用，於甘油濃度60 g/L及HRT 8 h之條件，獲致丁醇濃度、BY、 HPR分別為6.5 g/L、0.12 g BuOH/g glycerol consumed及0.838 mol/L/d，相對於AnGCB系統，雖然CSTR系統獲致較佳的HPR，但其丁醇產量較低。為改善丁醇抑制的影響，本研究進一步以CSTR系統進行真空醱酵之效應探討，結果顯示於真空度700 mmHg (表壓-700 mmHg)時丁醇可有效從醱酵槽內移除而避免丁醇抑制，其冷凝液之丁醇濃度高達52.9g/L，而HPR則提高至1.597 mol/L/d，顯示利用連續真空醱酵具有多重效益。
本研究以CSTR系統進行混菌培養，首先以精甘油分別進行pH、基質濃度、HRT之效應探討。在pH試驗結果顯示pH 5.5時可獲致最佳丁醇濃度、BY分別為6.4 g/L、0.21 g BuOH/g glycerol consumed (於甘油濃度100 g/L及HRT 12 h)，而於高pH 7.0條件下有利於1,3-丙二醇生成。基質濃度(100、60、40及25 g/L)之效應探討顯示於各試程其BY介在0.20-0.23 g BuOH/g glycerol consumed (於pH 5.5及HRT 12 h)，而基質濃度以40 g/L時有最佳利用率85%且丁醇濃度高達7.1 g/L。而HRT效應探討發現HRT由12 h降至4 h時，其主產物由丁醇轉為1,3-丙二醇(於pH 5.5及甘油濃度40 g/L)，顯示低HRT時不利於丁醇生成。進一步以粗甘油濃度25 g/L於pH 5.5及HRT 12 h進行進行連續試驗，結果獲致丁醇濃度、BY、HPR分別為4.9 g/L、0.21 g BuOH/g glycerol consumed、0.404 mol/L/d，其丁醇濃度與精甘油醱酵相近，而其產氫表現略高於精甘油。

The global production of biodiesel has increased year by year; therefore a substantial byproduct glycerol increased and accumulated gradually. In the view of this, the development of new application of glycerol can not only solve the problem of excess of glycerol but also can reduce the overall cost of biodiesel. This study explored the continuous butanol fermentation from glycerol with pure culture (Clostridium pasteurianum) and mixed culture, respectively. In terms of pure culture, first, an anaerobic granulated cell bioreactor (AnGCB) was used to explore the effects of agitation rate (45 and 75 rpm) and hydraulic retention time (HRT; 24, 12 and 8 h) on butanol production. The results showed that the activity of C. pasteurianum was inhibited when butanol concentration arrived 10 g/L; thus the resctors were not operated at steady state. Both higher agitation rate (75 rpm) and lower HRT (12 and 8 h) favored the operational stability of systems. Thus, when the operational conditions were glycerol concentration 60 g/L, agitation rate 75 rpm and HRT 8 h, the results showed that the butanol concentration, butanol yield (BY), hydrogen production rate (HPR) was 7.5 g/L、0.15 g BuOH/g glycerol consumed and 0.485 mol/L/d, respectively. Furthermore, a continuous flow stirred tank reactor (CSTR) was used to explore the effects of HRT (24, 12 and 8 h) and glycerol concentration (60 and 40 g/L) on butanol production. The results showed that the unstable operation of system resulting from inhibition of butanol still existed, and using lower HRT and glycerol concentration could slightly decrease the inhibition of butanol. The butanol concentration, BY, HPR was 6.5 g/L, 0.12 g BuOH/g glycerol consumed and 0.838 mol/L/d, respectively, at glycerol concentration 60 g/L and HRT 8 h. Comparing with the AnGCB system, CSTR system got a better HPR but the butanol yield was lower than the AnGCB. To improve the inhibition of butanol, the effect of vacuum fermentation using a CSTR reactor was studied. When the degree of vacuum was 700 mmHg (a gauge pressure of -700 mmHg), butanol can be effectively removed from the fermentor and improving the inhibition of butanol. The butanol concentration of the condensate was up to 52.9g/L, and HPR increased to 1.597 mol/L/d. These results showed that the use of continuous vacuum fermentation displayed multiple benefits.
In terms of the mixed culture, a CSTR reactor was used to investigate the effects of pH, glycerol concentration and HRT on butanol production form refined glycerol. The results showed that the optimal butanol concentration and BY was 6.4 g/L and 0.21 g BuOH/g glycerol consumed, respectively, when the CSTR reactor was operated at pH 5.5 (at glycerol concentration 100 g/L and HRT 12 h), and the reactor operated at high pH value was favorable to the generation of 1,3-propanediol. Then, the reactor was operated at various concentration of carbon source (refined glycerol 100, 60 40 and 25 g/L), the results showed that BY was between 0.20 and 0.23 g BuOH/g glycerol consumed at pH 5.5 and HRT 12 h. The optimal substrate utilization and butanol concentration was 85% and 7.1 g/L and occurred at glycerol concentration 40 g/L. The results of HRT effect showed that the main product shifted from butanol to 1,3-propanediol when HRT decreased from 12 to 4 h (at pH 5.5 and glycerol concentration 40 g/L), suggesting low HRT was not favorable to butanol production. Furthermore, the continuous butanol fermentation from crude glycerol was conducted at substrate concentration 25 g/L, pH 5.5 and HRT 12 h. The results showed that butanol concentration, BY and HPR was 4.9 g/L, 0.21 g BuOH/g glycerol consumed and 0.404 mol/L/d, respectively. The butanol concentration from crude glycerol was similar to that from refined glycerol; and the HPR from crude glycerol was higher than that from refined glycerol.

摘要 I
Abstract III
目錄 VI
表目錄 XI
圖目錄 XIII
第一章 緒論 1
1-1 前言 1
1-2 研究動機與目的 2
1-3 研究架構 3
第二章 原理及文獻回顧 5
2-1 生質能源 5
2-1-1生質能概述 5
2-1-2 生質柴油 6
2-2甘油 8
2-2-1 甘油之性質 8
2-2-2 甘油之純化 8
2-2-3 粗甘油前處理 9
2-2-4 甘油之應用 10
2-3 丁醇 12
2-3-1 丁醇的基本性質及用途 12
2-3-2 丁醇製備方法 13
2-3-2-1 化學合成法 13
2-3-2-2 生物合成法 14
2-4 丁醇菌種及代謝路徑 16
2-4-1 Clostridium菌屬之特性 16
2-4-2丁醇之代謝路徑 18
2-5氫能 22
2-5-1氫氣製備方法 22
2-5-2甘油暗醱酵產氫 22
2-6 甘油厭氧醱酵之環境因子 25
2-6-1 水力滯留時間 25
2-6-2 pH 25
2-7 甘油醱酵系統 27
2-7-1 CSTR 27
2-7-2 AnGCB 27
2-8 生物丁醇移除技術 29
2-8-1氣體沖提(gas stripping) 29
2-8-2滲透蒸發薄膜(pervaporation) 29
2-8-3真空醱酵(Vacuum fermentation) 30
2-9 分子生物技術於甘油醱酵之應用 31
2-9-1 16S rDNA 及 functional gene 32
2-9-2 聚合酶鏈鎖反應 33
2-9-3 變性梯度明膠電泳 35
2-9-4 菌相分析 37
第三章 材料與方法 38
3-1 甘油醱酵產丁醇&;氫氣 38
3-1-1 菌種來源 38
3-1-1-1 純菌來源 38
3-1-1-2 混菌來源 38
3-1-2 甘油來源 38
3-1-3 粗甘油前處理 39
3-1-4 培養基配方 39
3-1-5 實驗裝置圖 40
3-1-5-1 AnGCB之產丁醇操作 40
3-1-5-2 CSTR之產丁醇操作 41
3-1-5-3 真空醱酵產丁醇之裝置圖 42
3-2 氣體組成分析 44
3-3液體組成分析 46
3-4細胞濃度分析 47
3-5菌相分析 47
3-5-1 DNA萃取 48
3-5-2 聚合酶鏈鎖反應 49
3-5-3 變性梯度明膠電泳 52
3-5-4 Acrylamide/Bis 膠體之DNA純化 53
3-5-5 親緣分析 53
第四章 結果與討論 54
4-1 C. pasteurianum以AnGCB系統於不同攪拌速率及HRT之醱酵產丁醇試驗 54
4-2 C. pasteurianum以CSTR系統產丁醇試驗 61
4-2-1 C.pasteurianum於不同HRT下產丁醇評比 61
4-2-2 AnGCB與CSTR於不同HRT之評比 63
4-2-3 C. pasteurianum於不同基質濃度下產丁醇試驗 67
4-3 C. pasteurianum以CSTR系統於真空醱酵產丁醇試驗 70
4-3-1 C. pasteurianum於不同壓力下之產丁醇評比 70
4-3-2 真空醱酵之優劣 72
4-4 pH值對CSTR系統混菌培養精甘油產丁醇之影響 76
4-5 基質濃度對CSTR系統混菌培養精甘油產製丁醇之影響 83
4-6 不同HRT對CSTR系統混菌培養精甘油產製丁醇之影響 88
4-7 CSTR系統混菌培養粗甘油產製丁醇 93
4-8 C. pasteurianum以AnGCB系統於pH 5.5之醱酵產丁醇試驗 99
第五章 結論與未來展望 101
5-1 結論 101
5-1-1純菌系統 101
5-1-2混菌系統 101
5-2 未來展望 102
參考文獻 104
中文參考文獻 111
致謝 113

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