牛瘤胃分離出一株具有高乳酸、琥珀酸及微量乙酸之生產菌的鑑定與研究

Isolation and identification of a lactic acid, succinic acid and acetic acid producing strain from bovine rumen

徐華嬬(Hua-Ju Hsu)

指導教授 ： 李文乾

國立中正大學/工學院/化學工程學系/碩士(2014年)

摘要

琥珀酸與乳酸在於化工產業中是兩種重要的有機酸，能作為藥物、食品添加物、及清潔劑等許多產品的中間體或主要成分。本研究針對生產菌種的篩選、培養基條件探討、以及後續代謝途徑的重組等方面，探討如何有效提高有機酸產率，以利於未來工業化醱酵製造有機酸。
　　本研究選擇牛瘤胃中多類菌種作為菌株篩選的來源，以富馬酸鈉塩作為唯一碳源，並利用莫能菌素抑制雜菌的生長，進而藉由選擇性平板培養法進行初篩挑選出 118 株，接著以 TLC 薄層色層分析法，進一步複篩，最後分離出一株編號 No. 8 菌株。此篩選菌株能夠同時產生琥珀酸、乳酸、及其他微量酸類副產物，並經由 16S rRNA 序列的系統發育之序列分析，顯示該菌株可能為大腸桿菌（Escherichia coli）之突變株，與已知之 E. coli 系列的相似性達 99 %。另外，本實驗亦針對乳酸與乙酸代謝途徑上的基因進行 PCR 的擴增，包含 L-ldhA、D-ldhA、mgsA 與 pta 等基因，目的是為了再次確認此菌這些特定基因的序列與其他 E. coli 的差別。
　　藉由針對 50 g/L 葡萄糖濃度醱酵槽的培養下探討，HPLC 分析醱酵產物，此 No. 8 菌株所產生的琥珀酸產量，平均的最終濃度為 6.58 g/L，其產率為 18.2 wt %。同時，本研究也發現，菌株所代謝出的乳酸產量，最終濃度為 23.69 g/L，產率為 65 wt %。在研究中嘗試著利用廉價的甘油
作為主要碳源下，以取代昂貴的葡萄糖，並進行馴化與醱酵，結果顯示此菌株無法在有甘油環境下生長。

關鍵字

大腸桿菌 ； 瘤胃 ； 乳酸 ； 琥珀酸

並列摘要

Succinic acid and lactic acid are two important organic acids in the chemical industry. They can be used as the main ingredient or intermediate products, such as drugs, food additives, cleaners. In this study, we focused on the selection of microorganisms, optimization of media conditions, and recombination of microorganism metabolic pathways to improve concen- tration of succinic acid or lactic acid.
Rumen contains a variety of microorganism, so this study used the rumen contents from the cattle as a source. The enrichment culture medium contains the monensin, which can inhibit a part of rumen microorganisms, like protozoan and methanogenus. Under anaerobic conditions, 118 strains producing organic acid were isolated with yellow loop as selective marker presented in the bromocresol green medium. Finally, a strain No. 8 that can produce succinic acid, lactic acid, and other acids was named as the strain No. 8. The 16s rRNA sequence analysis placed strain No. 8 within the family of E. coli. The most closely related members of the family E. coli have a 16s rRNA similarities of 99 % to strain No. 8. In addition, we had also amplified the genes for the metabolic pathways of lactate and acetate, included L-ldhA, D-ldhA, mgsA and pta genes, to check whether here are mutations in these genes causing, a strain No. 8 with a higher yield of lactic acid and succinic acid.
Results of the batch culture in 5L fermentor showed that strain No. 8 could produce 56.58 g/L succinate from 50 g/L glucose, with final average yield of 18.2 % (w/w). Simultaneously, this strain could also produce lactate at 23.69 g/L, with final average yield of 65 % (w/w). We attempted to change carbon source in the culture medium by replacing glucose with inexpensive glycerol. Results showed that strain No. 8 could not metabolize glycerol. Furthermore, this study works on the recombinant DNA of No. 8. We tried to remove target genes by the method of gene knock-out.

並列關鍵字

E. coli ； Rumen ； Lactic acid ； Succinic acid

參考文獻

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