- 學位論文
乳酸及琥珀酸生產菌的突變篩選與發酵培養之研究
Mutation and Cultivation of Actinobacillus Succinogenes and Lactic Acid-producing Strain for the Production of Succinic Acid and Lactic Acid
摘要
乳酸和琥珀酸是聚合物、藥物及化學品的重要中間體。此外，它們可以被用來作為食品添加劑和食物補充。然而，進行乳酸或琥珀酸之發酵時，菌體以會生產醋酸此副產物，造成後續純化困難與碳源浪費。例如，在McKinlay學者之研究當中，在AM3培養基之條件下，A. succinogenes 130Z野生菌的醋酸轉化率約為0.213 g/g葡萄糖。此琥珀酸生產菌A. succinogenes 130Z在含有30g / L的葡萄糖搖瓶中，醋酸轉化約0.114g/g葡萄糖。本實驗室將A. succinogenes 130Z野生菌及乳酸生產菌透過NTG突變與含氟乙酸鈉之洋菜培養基篩選，得到一A. succinogenes突變菌株及乳酸生產突變菌。 此乳酸生產突變株能夠生產在30 g/L葡萄糖之培養基中，其醋酸轉化率約為0.067g/g葡萄糖。利用發酵槽設備將乳酸生產突變菌株進行培養，在含85g/L葡萄糖之發酵液(0.9 L)條件下，乳酸最後濃度約為50.62 ± 2.04g/L，轉化率約0.68 ± 0.067g/g葡萄糖。在研究中也發現，此突變菌株在CO2曝氣之下，僅在TSB medium與特定發酵培養基能夠大幅生長。然而改變培養條件，來探討乳酸產率提升之實驗中，多數條件都讓突變菌株無法快速增生或無法生長，並且乳酸產率也大幅下降。在未來的研究中，將透過馴化手段，將突變菌種可適應各類發酵條件的改變，以提高琥珀酸及乳酸的轉化率與產量。 野生型A. succinogenes發現能夠在含85g/L葡萄糖之發酵液(0.9 L)條件下生產乙酸轉化率為0.067 g/g葡萄糖。在發酵槽中，此A. succinogenes可產生約35.18 g/L的琥珀酸，對應的琥珀酸產量約0.75 g/g葡萄糖。
並列摘要
Lactic acid and succinic acid are the important intermediates for polymers, pharmaceuticals and chemicals. Also, they can be used as a food additive and dietary supplement. However, fermentation of lactic acid or succinic acid can lead to the production of acetic acid (acetate) as the byproduct, causing difficulty in subsequent purification and waste of carbon source. For example, succinic acid-producer A. succinogenes 130Z can accumulate acetate at approximately 0.114 g/g-glucose in shake flasks containing 30 g/L glucose. In this study, the wild-type A. succinogenes and lactic acid-producing strain were induced to mutation using NTG and mutants were screened on the sodium fluoroacetate-containing agar. A lactic acid-producing mutant strain was isolated and found able to produce acetate at only 0.067 g/g-glucose in the culture medium of 30 g/L glucose. In the fermenter, this lactic acid-producing mutant strain could produce approximately 50.62 ± 2.04 g/L lactic acid in the fermentation medium containing 85 g/L glucose initially, corresponding to the lactic acid yield of approximately 0.68 ± 0.07 g/g glucose. We also found that this mutant strain could grow significantly under CO2 aeration only in TSB medium and some specified fermentation media. Many tested culture conditions led to low proliferating rates and low lactic acid yields. The wild-type A. succinogenes was found able to produce acetate at 0.067 g/g-glucose in the culture medium of 85 g/L glucose. In the fermenter, this succinic acid-producing mutant strain could produce approximately 35.18 g/L succinic acid in the fermentation medium containing 85 g/L glucose initially, corresponding to the succinic acid yield of approximately 0.75 g/g glucose. Keywords: succinic acid, Actinobacillus succinogenes, mutation, acetate, sodium fluoroacetate, lactic acid