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標題: 以醱酵策略改善基因重組大腸桿菌及梭狀芽孢桿菌之生質丁醇生產能力
Fermentation approach for enhancing 1-butanol production using recombinant Escherichia coli and Clostridium acetobutylicum ATCC 824
作者: 陳尚楷
Chen, Shang-Kai
關鍵字: 醱酵策略;Fermentation approach;大腸桿菌;正丁醇;OGAB;Clostridium acetobutylicum ATCC 824;孢子體;Acetone-Butanol-Ethanol醱酵;Escherichia coli;n-Butanol;OGAB;Clostridium acetobutylicum ATCC 824;Spore;Acetone-Butanol-Ethanolfermentation
出版社: 化學工程學系所
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本研究主要在探討利用不同醱酵策略,進而改善微生物代謝生產生質丁醇的能力。本研究可分為兩部分,其一為:利用經Ordered Gene Assembly in Bacillus subtilis(OGAB)基因工程方法構築之大腸桿菌(E. coli T5),測試其生質丁醇產能表現,並加以改善。實驗結果顯示,大腸桿菌利用外源植入丁醇代謝路徑;(Promoter Pr)-thil-crt-bcd-etfB-etfA-hbd-adhe1-adhe;以Terrific Broth Modified medium加上20 g/L 葡萄糖,於72小時厭氧環境培養下,其丁醇產量與Specific BuOH yield分別為4.5 mg-butanol/L 和 4.5 mg-butanol/g-cell,並藉由不同醱酵參數的設計,探討E. coli T5在不同條件下丁醇生產的能力表現及生理現象。實驗結果指出,在碳源低初始濃度、選擇具較佳還原力的碳源種類、厭氧環境、酸鹼值為6 或加入glutathione,citrate 的條件下均能有效地增加生質丁醇產量,從中,本研究結果推論acetyl-CoA 胞內濃度和細胞內低氧化還原態為E. coli T5丁醇生產重要的關鍵。其二則為以孢子型態保存之原生菌種梭狀芽孢桿菌(Clostridium acetobutylicum ATCC 824)直接進行Acetone-Butamol-Ethanol醱酵(spore-culture),測試其程序可行性,實驗結果顯示,當以LB-F培養基與60 g/L 葡萄糖,且植菌量比例為10.71 %(V/V),經72小時spore-culture批次醱酵,可得丁醇產量12.65±0.85 g-butanol/L、 butanol yield, 0.22±0.02 g-butanol/g-glucose 和butanol productivity, 0.18±0.01 g-butanol/L/h,輔以不同醱酵條件(植菌量比例、初始葡萄糖濃度、Ca+2濃度)的測試探討,發現其最大丁醇產量大都介於10.5-12.5 g-butanol/L,其中,若以100 g/L 葡萄糖進行spore-culture醱酵,可得butanol titer 14.05±0.96 g-butanol/L 、butanol yield 0.23±0.01 g-butanol/g-glucose、butanol productivity 0.20±0.01 g-butanol/L/h 與total A-B-E titer 25.99±1.86 g-ABE/L、total A-B-E yield 0.43±0.01 g-ABE/g-glucose、total A-B-E productivity 0.36±0.03 g-ABE/L/h。

In this study, different fermentation approaches were applied for improving the bio-butanol producing ability of microorganisms. This research can be divided into two parts. One is:Engineered butanologenic Escherichia coli T5 constructed by the OGAB method was used for 1-butanol production. The results showed the feasibility of the artificial butanologenic operon, (Promoter Pr)-thil-crt-bcd-etfB-etfA-hbd-adhe1-adhe, where the 1-butanol titer, specific BuOH yield, and BuOH yield were 4.50 mg/L, 4.50 mg-BuOH/g cell, and 0.35 mg-BuOH/g-glucose, respectively. Fermentation conditions of anaerobic, low initial concentrations of carbon sources, low oxidation state of carbon source, pH of 6, addition of glutathione and citrate, had been shown for efficiently improving the 1-butanol production. The premise behind these fermentation approaches can be categorized into two lines of reasoning, either elevated the availability of acetyl-CoA or lowered the intracellular redox state. By comparing the fermentation conditions tested in this study, pH has been shown to be the most efficiency strategies for 1-butanol production while the replacement of glucose with glycerol provides the highest improvement in butanol yield. The other was using spore-form Clostridium acetobutylicum ATCC 824 for Acetone-Butanol-Ethanol fermentation directally. This kind of fermentation method called “spore-culture”. The results shows the feasibility of spore-culture process where the 1-butanol titer, butanol yield and butanol productivity were 12.65±0.85 g-butanol/L, 0.22±0.02 g-butanol/g-glucose, and 0.18±0.01 g-butanol/L/h, respectively. However, the max 1-butanol titer was 10.5-12.5 g-butanol/L through different fermentation conditions testing, like the inoculum level of spores, the initial glucose concentration used, and the concentration of calcium ion used. Among the fermentation conditions tesing, it could produce 14.687±1.744 g-butanol /L while using 100 g/L Glucose as the carbon source. Another spore-culture fermentation results by using 100 g/L glucose were butanol yield, 0.23±0.01 g-butanol/g-glucose; butanol productivity, 0.20±0.01 g-butanol/L/h; total A-B-E titer, 25.99±1.86 g-ABE/L; total A-B-E yield, 0.43±0.01 g-ABE/g-glucose; and total A-B-E productivity, 0.36±0.03 g-ABE/L/h, respectively. By comparing the fermentation performance between spore-culture method and traditional culture method, spore-culture method has been showed the high potential for Acetone-Butanol-Ethanol fermentation process. 
其他識別: U0005-0108201309151600
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