摘要
本研究利用 Bacillus megaterium ATCC 10778來進行維生素B12 之生產，整個培養生產過程分為三階段，第一階段以好氧培養來累積菌體，第二階段則以厭氧培養來合成與累積維生素 B12 之前驅物，第三階段再以好氣培養將維生素 B12 前驅物合成為具生理活性之維生素 B12。本研究採用高效液相層析-紫外光 (HPLC-UV) 分析法及維生素B12 微生物分析法來進行 B. megaterium ATCC 10778 所生產出之維生素 B12 之定量。本研究利用搖瓶培養探討第二階段培養基組成 (三種前驅物之濃度：δ-aminolevulinic acid，簡稱ALA、5,6-dimethylbenzimidazole，簡稱DMB、鈷離子；葡萄糖濃度、氮源濃度及 pH) 及培養策略 (第二階段：厭氧培養期及第三階段：好氧培養期之培養時間) 對維生素 B12 產量之影響。發現含有 9% 葡萄糖濃度、7 % 氮源濃度及 pH 6.8 的 TB 調整培養基可顯著提高B. megaterium ATCC 10778 之維生素 B12 產量。此外在第二階段厭氧培養達30 小時及第三階段好氧培養達 14.5 小時亦可得較佳之維生素 B12 產量。根據上述最適之維生素 B12 生產條件，使用醱酵槽進行放大生產，在第二階段厭氧培養的部分，藉由添加氮氣來達到更佳之厭氧環境，使得最終維生素 B12 之產量達到 338.20 μg/L。
並列摘要
In this study, I used Bacillus megaterium ATCC 10778 to produce vitamin B12 with a three-stage culture strategies. The first stage is to increase the number of bacteria, the second stage is for vitamin B12 precursor synthesis and accumulation under anaerobic condition, and the third stage is for completing the synthetic steps to transform precursors into bioactive vitamin B12 under aerobic condition. I used high performance liquid chromatography - ultraviolet detector method (HPLC-UV) and microbiological method to quantify vitamin B12 produced by B. megaterium ATCC 10778. I evalueted the effect of medium components on vitamin B12 production by suspension culture in Erlenmeyer flask. The concentrations of precursors (ALA, δ-aminolevulinic acid; DMB, 5,6-dimethylbenzimidazole; Co2+), glucose, nitrogen source, and pH value were all analyzed. Meanwhile, different duration of the second stage (anaerobic culture) and the third stage (aerobic culture) were also considered. I found the optimal condition for vitamin B12 production by B. megaterium ATCC 10778 is 9% (w/v) glucose and 7% (w/v) nitrogen source in the modified TB medium under pH 6.8. In addition, 30-hour anaerobic culture and 14.5-hour aerobic culture resulted in the highest vitamin B12 production. Based on the above-mentioned optimal conditions for vitamin B12 production, I scaled up the whole process in the fermenter with nitrogen gas addition to establish a better anaerobic environment in the second stage. The final production of vitamin B12 was up to 338.20 μg/L.