耐鹽性酒精發酵酵母菌之篩選與研究

Isolation and characterization of halotolerant yeasts with alcohol fermentation ability

林佩蓉(Pei-Rong Lin)

指導教授 ： 徐源泰

國立臺灣大學/生物資源暨農學院/園藝學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.02072

摘要

生質酒精(Bioethanol)係屬於再生能源(renewable energy)，目前發展趨勢之一為纖維酒精(cellulosic ethanol)之開發。基於環境保護(environmental protection)與資源之妥善利用等立場，欲開發高鹽含量之農產加工廢棄物、低度利用之海洋資源及鹽鹼化土地所種植之作物等產製纖維酒精，則製程中則需應用具耐鹽性之酒精發酵酵母菌。
本研究於2010年6月間，分別於台灣北部地區採集甜味種楊桃醃漬液，中部地區採集酸味種楊桃醃漬液及酸菜醃漬液。利用YPD固態培養基將醃漬液中之酵母菌株進行分離及純化後，共得168株酵母菌分離株。利用漸進式鹽度篩選及酒精生成呈色試驗篩選出59株酵母菌分離株，進行培養及細胞形態之觀察，並由各醃漬分離株中隨機選取十數株以26S rDNA之D1/D2區域進行定序鑑定，將其序列與National Center for Biotechnology Information (NCBI)之GenBank資料庫進行比對，結果顯示所得之分離菌株屬於Wickerhamomyces、Pichia及Candida屬。其中與Pichia subpelliculosa序列相似度高達99%之酵母菌分離株tiger 3，以高效液相層析儀(High Performance Liquid Chromatography)分析酒精生成之能力為分離株中最佳者，並進一步探討其分離株之生理生化特性與最適之酒精發酵環境試驗。於酒精發酵之環境試驗結果中得知tiger 3之最適發酵溫度為30℃，最適發酵鹽度為7%。於上述環境下之最適發酵pH值為7，其發酵96小時之酒精生成量為28.16 ± 0.76 g/L。故欲構築tiger 3為利用含鹽物料之纖維酒精菌株，其製程設計較適合併用氨水前處理。研究結果顯示tiger 3為一極具發展潛力之纖維酒精發酵酵母菌株。

關鍵字

耐鹽性 ； 酒精發酵 ； 酵母菌 ； 生質酒精 ； 纖維酒精

並列摘要

Lignocellulosic biomass is recognized as potentially sustainable source for the production of bioethanol, this innovative technology based on cellulosic biomass bioconversion to ethanol requires feedstock and microbial process. There are some agricultural processing residues, marine resources and saline crops with highly salt concentration, which are the potential as a feedstock if we want to use these kinds of material to produce cellulosic ethanol, we certainly need to find yeast strain which could tolerant high salt concentration.
168 yeast strains were isolated from three different kind of pickled liquid in Brassica juncea, sweet carambola, sour carambola, and respectively. 59 isolates were selected through progressive salt concentration assay and enzymatic screening of ethanol production assay. About 10 to 20 isolates randomly picked from each pickled liquids according to their analysis of 26S rDNA D1/D2 domain sequence by NCBI GenBank, these strains could belong to Candida, Pichia, Wickerhamomyces respectively.
Tiger 3, identity index was 99% with Pichia subpelliculosa, which shows the greatest ethanol production ability by HPLC. Also the physiological and biochemical characteristics of tiger 3 were measured. In the experiment of optimal alcohol fermentation conditions, the optimal temperature is 30℃, the optimal salt concentration is 7%. Under those conditions, the optimal fermentation pH value is 7, the ethanol production is 28.16 ± 0.76 g/L after 96 hours fermentation period.

並列關鍵字

halotolerant ； alcohol fermentation ； yeast ； bioethanol ； cellulosic ethanol

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