臺灣博碩士論文加值系統

本研究方向主要是探討Clostridium acetobutylicum BCRC 10639液態發酵培養製程中，醋酸根離子 (CH3COO-) 以及疊氮化鈉 (NaN3) 的添加對於丁醇 (butanol) 生成之影響性。在小型搖瓶實驗中，首先探討不同碳、氮源濃度對於C. acetobutylicum生產butanol之影響，待基礎培養基確立後，則各別進行CH3COO-及NaN3的添加試驗。
文獻中指出醋酸 (acetate) 的添加有助於防止菌體衰退，並增加溶劑生產穩定性，且從代謝路徑分析發現，添加acetate可經由CoA-transferase的作用進一步將路徑中的丁酸 (butyrate) 轉為butanol。因此在小型搖瓶實驗中，首先討論不同濃度醋酸鈉 (CH3COONa) 添加下，對於C. acetobutylicum生產butanol之影響，待確定出適當濃度後，再以改變不同種類的CH3COO-做進一步探討。
由實驗結果顯示，添加20 mM CH3COONa對於增加butanol產量的效果較為明顯，butanol濃度與對照組相比，發酵48 hr後可從7.89 g/l上升至9.33 g/l，產量約提升18%。由於CH3COONa對於butanol的生成有明顯效益，因而進一步探討不同種類的CH3COO-於20 mM添加對butanol的影響。實驗結果得知，CH3COO-不論是與帶+1價、+2價的金屬離子或是與帶+1價的非金屬離子結合，對於butanol產量的影響都是相近的，顯示出CH3COO-有助於C. acetobutylicum生產butanol，但與其結合之陽離子無關。
根據文獻提出acetate雖可加快butanol生成，但卻有降低菌體生長速率之可能，因此本研究擬進行將添加20 mM CH3COO-培養基，於發酵36 hr後離心移除 CH3COO-培養液，接續置換於50、70、90 g/l葡萄糖 (glucose) 培養基的置換實驗，其目的為使C. acetobutylicum加快進入產butanol階段，再利用置換去除CH3COO-以增進菌體生長，連帶達成提升butanol產量之作用。實驗數據顯示，置換於50 g/l glucose的與對照組 (未置換) 相比，butanol最高產量由9.33 g/l上升至9.99 g/l，產量僅提升7%，而就置換於70、90 g/l glucose而言，產量雖可大幅提升27-40%，但仍有明顯的butanol抑制情形。
由前述實驗結果推斷，CH3COO-無法大量提升butanol產量可能是butyrate濃度不足，因此進一步探討同時添加acetate、butyrate對butanol產量的影響。而實驗數據表示，以發酵24 hr後添加有機酸的效果較好，butanol產量約提昇18%，證實butanol生成除了在acetate的幫助下，還必須要有足夠的butyrate才可使產量明顯提升。
NaN3添加部份，改變不同的NaN3添加濃度及添加時間去抑制hydrogenase活性，探討對C. acetobutylicum生產butanol之影響。實驗結果發現，添加NaN3不僅有助於butanol之生成，還可提升butanol於產物 (solvents) 中所佔比例，其中又以2 mg/l NaN3所生產之butanol產量較佳，butanol濃度由6.7 g/l上升至8.8 g/l，產量約提昇31%。

Butanol is an important intermediate in chemical synthesis and also is regarded as a potential fuel or fuel additive as compared to the traditional additive-alcohol. The objective of this study is to investigate the possible influences of CH3COO- and NaN3 added on butanol fermentation by Clostridium acetobutylicum BCRC 10639. Firstly, the concentrations of carbon source and nitrogen source were tested to determine the optimal ferment medium, then we further discuss the effects of addition CH3COO- and NaN3 on the solventogenesis stage of butanol production.
The results of CH3COO- adding experiments showed 20 mM CH3COONa could increase as high as 18% of butanol concentration. Furthermore, in the experiment of CH3COO- medium replacement suggested that biomass was improved by 62% and the butanol productivity was increased by up to 47%.
The addition of NaN3 in the medium had shown that the production of butanol was enhanced at 2 mg/l NaN3 added, which led to the increase of ratio of butanol/solvents. The butanol concentration was increased from 6.7 to 8.8 g/l under this fermentation conditions.

中文摘要 III
英文摘要 V
目錄 VI
圖目錄 IX
表目錄 X
第一章 緒論 1
第二章 文獻回顧 2
2.1 生質燃料 2
2.2 生物丁醇 —Butanol 4
2.2.1 以發酵程序產butanol之進展 4
2.2.2 Butanol的應用 6
2.3 Butanol生產方式 8
2.3.1 Butanol的合成和代謝 8
2.3.2 生產butanol之菌種 10
2.4 影響butanol發酵之環境因子 12
2.4.1 pH值影響 12
2.4.2 有機酸影響 13
2.4.3 Hydrogenase影響 14
2.5 Acetate的添加效應 15
2.6 NaN3的添加效應 16
第三章 實驗材料與方法 17
3.1 實驗菌株 17
3.2 實驗藥品 17
3.3 實驗儀器 19
3.4 分析方法 20
3.4.1 Biomass分析 20
3.4.2 代謝產物濃度分析 20
3.4.3 Glucose分析方法 20
3.5 實驗方法 21
3.5.1 菌種培養 21
3.5.2 培養基組成 22
3.6 實驗培養條件 24
3.6.1 碳源濃度的影響 24
3.6.2 兩段式碳源添加的影響 24
3.6.3 總氮源濃度的影響 24
3.6.4 添加CH3COONa的影響 25
3.6.5 添加CH3COO-的影響 25
3.6.6 CH3COO-的培養基置換試驗 25
3.6.7 添加acetate和butyrate的影響 26
3.6.8 添加NaN3的影響 26
3.6.9 攪拌式發酵槽實驗 26
第四章 結果與討論 28
4.1 研究架構 28
4.2 碳源濃度之影響 29
4.4 總氮源濃度之影響 35
4.5 CH3COONa添加之影響 37
4.6 CH3COO-添加之影響 40
4.7 CH3COO-的培養基置換試驗 42
4.8 添加acetate、butyrate之影響 44
4.9 NaN3添加之影響 46
4.10 攪拌式發酵槽實驗 48
第五章 結論與未來展望 50
5-1 結論 50
5-2 未來展望 52
參考文獻 53
附錄 57

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