Candida tropicalis ATCC 20962 之十二碳二元酸轉化生產研究

Abstract

許多微生物具有將長鏈正烷類或脂肪酸轉化為二元酸之能力，二元酸轉化平台之相關研究及產程開發是以熱帶假絲酵母 (Candida tropicalis) 為主。本研究以熱帶假絲酵母 (Candida tropicalis) ATCC 20962，進行十二碳二元酸 (DCA12) 之醱酵轉化研究，該醱酵策略分為菌體累積及正十二烷 (n-dodecane) 轉化為二元酸兩個階段。在第一階段之菌體累積，分別於醱酵槽比較批式饋料培養 (fed-batch culture) 與批式培養 (batch culture) 對正十二烷之轉化效率。批式培養後經 96 小時之轉化可得到 65 g/L 之 DCA12，而批式饋料培養結果顯示，單以葡萄糖作為碳源之饋料培養基，無法有效提升生菌數，而饋料培養基中添加酵母抽出物與蛋白腖等有機氮源後，雖能有效提升生菌數，但無 DCA12 產出。當以無機銨鹽作為氮源後此現象獲得改善，經 72 小時之轉化可得到 99 g/L 之 DCA12，產率為 1.37 g/L/h。此外，在第二階段 (轉化階段) 維持高生菌數是獲得十二碳二元酸高產量的必要條件。將菌體濃度提升至批式培養之 3 倍後，並調整葡萄糖供給速率及添加少量微量元素和維生素，經 48 小時之轉化可得到 87.8 g/L 之 DCA12，產率達 1.82 g/L/h。進一步以相同策略將菌體濃度提升至批式培養之 5 倍後，經 158.75 小時之轉化 DCA12 最高濃度可達 130.8 g/L，產率為 0.82 g/L/h。後者 DCA12 產率低於前者，推測為培養環境之溶氧濃度過低 (前者約 40% 飽和度，後者約 20% 飽和度)，限制了十二碳二元酸之轉化效率。菌種改良方面，藉由紫外線照射導致菌株突變後，於搖瓶中培養並篩選二元酸高產量菌株，可獲得產量增加一倍之突變株。

Many microorganisms are able to convert n-alkanes and fatty acids into long chain dicarboxylic acids (DCA), and many researches and bioprocesses using C. tropicalis to produce long chain DCA have been proposed. In this study, the bioprocess is generally divided into two stages, the growth of C. tropicalis ATCC 20962 and bioconversion of n-dodecane (alkane C12) into dodecanedioic acid (DCA12). To get higher DCA productivity, it is required to increase the number of viable cells and bioconversion ability with optimized fermentation conditions. We compared batch culture with fed-batch culture about the accumulation of viable cells and bioconversion of n-dodecane. The production of DCA12 reached 65 g/L in batch culture with 96 h bioconversion. In fed-batch culture, fed medium containing only carbon source (glucose) cannot raise the number of viable cells efficiently. Addition of organic nitrogen source (yeast extract and peptone) in fed medium can effectively increase the number of viable cells, but the cells cannot produce DCA12. Instead, adding inorganic ammonium salts as nitrogen source in fed medium can make 3-fold increase of viable cell compared with batch culture and raise DCA12 production to 99 g/L in 72 h bioconversion (1.37 g/L/h of productivity). Moreover, it is necessary to maintain the large number of viable cells in bioconversion phase for higher DCA production. By adjusting the feeding rate of glucose with supplemental trace elements and vitamins in bioconversion phase, the production of DCA12 reached 87.8 g/L after 48 h bioconversion (1.82 g/L/h of productivity) from 3-fold viable cells. By the same fed-batch strategy to get 5-fold increased of viable cells, the production of DCA12 reached 130.8 g/L after 158.75 h bioconversion (0.82 g/L/h of productivity). We assumed that the major limiting factor of DCA12 productivity is dissolved oxygen concentration in culture medium (the former is 40% and the latter is 20%). For strain improvement, one strain mutated by UV-irradiation has been selected after screening to have 2-fold DCA12 productivity in flask culture.