丁二酸生產菌的突變篩選及其醱酵控制之研究

Study on mutation and screening of Actinobacillus succinogens mutant and its fermentation control

莊孟歆(MENG-SHIN CHUANG)

指導教授 ： 李文乾

國立中正大學/工學院/化學工程學系/碩士(2016年)

摘要

本研究以產丁二酸放線桿菌(Actinobacillus succinogens, BCRC 80310)為出發菌株，藉不同的pH調節劑的添加，以饋料醱酵方式提升丁二酸產量。醱酵使用鹼液(5 N NaOH)與混合鹼(4 N NaOH搭配4 N Mg(OH)2)兩種方式來維持pH於6.8，這是因為單純使用NaOH可能會使鈉離子濃度快速上升，造成菌量及丁二酸的的生產速率有明顯的減少。使用混合鹼時，仍可進行再一次的饋料，以提高產物濃度。
本研究亦利用NTG（N-methyl-N′-nitro-N-nitrosoguanidine）處理使細菌突變，得到一株突變菌，編號mu-b7。經批次醱酵，利用pH控制策略改善鈉離子累積的問題，最終丁二酸濃度可達44.57 g/L，生產速率0.75 g/L/hr，丁二酸與醋酸比12.3，丁二酸產率可達1.09 g/g。為了解決突變菌適應較慢的問題，在前培養添加甘油，最終可得丁二酸濃度44.77 g/L，生產速率0.90 g/L/hr，丁二酸與醋酸比14.3，丁二酸產率可達1.13 g/g。除了適應培養外，也利用饋料醱酵方式改善甘油抑制問題，最終丁二酸濃度61.61 g/L，生產速率1.20 g/L/hr，丁二酸與醋酸比12.6，丁二酸產率可達1.07 g/L/hr。

關鍵字

醱酵 ； 丁二酸 ； 甘油

並列摘要

Actinobacillus succinogens (BCRC 80310) was used by the starting strain in this study. The present work studied the influence of pH control on the fermentation of A. succinogens use glycerol as the carbon source. During the bacterial fed-batch fermentation, the pH value was adjusted at 6.8 using 5 N NaOH or a combined liquor of 4 N NaOH and 4 N Mg(OH)2. Results suggest that higher sodium concentration in the fermentation medium because pH titration with higher concentration NaOH could significantly inhibit the cell growth and consequently reduce the production of succinic acid. After N-methyl-N′-nitro-N-nitrosoguanidine (NTG) treatment to induce bacterial mutation, a mutant strain (mu-b7) of A. succinogens was obtained. In the batch fermentation, the pH controlling strategy using combined liquor to adjust pH at 6.7 which was able to solve the problem of Na+ accumulation. Moreover concentration of succinic acid could reach 44.57 g/L, with a production rate of 0.75 g/L/hr and a succinic acid to acetic ratio of 12.3. The succinic acid yield was 1.09 g/g. In the batch fermentation of mutant strain with higher concentration of glycerol, the lag phase time was longer than that of wild type. This problem could be solved by the adaptation of mutant bacterial cells in glycerol in pre-culture stage. Furthermore, the concentration of succinic acid could reach 44.77 g/L, with a production rate of 0.9 g/L/hr and a succinic acid to acetic ratio of 14.3. The succinic acid yield was 1.12 g/g. In addition to adding glycerol in pre-culture, the fed-batch fermentation was also used to reduce the inhibition of glycerol. Finally, the concentration of succinic acid could each 61.61 g/L, with a production rate of 1.2 g/L/hr and the succinic acid to acetic ratio of 12.6. The succinic acid yield was 1.07 g/g.

並列關鍵字

fermentation ； Actinobacillus succinogens ； succinic acid ； glycerol

參考文獻

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