本研究主要從台灣山區土壤、腐葉及腐木等有機物質中，分離與鑑定其中具有
木糖發酵性之酵母菌，並分析其木糖發酵酒精之產量，以評估酵母菌株於農業廢棄物之木糖發酵之應用潛力。本實驗於台灣地區中10縣市16鄉鎮山區，共採集249個土壤及有機體樣品。利用僅含木糖單一碳源之YNO (yeast nitrogen base - chloramphenicol)篩選性培養基進行木糖利用酵母菌的增殖與分離，再經YMA (yeast extract - malt extract agar) 純化，將純化過的酵母菌利用菌落巨觀、細胞微觀形態及葡萄糖發酵試驗結果，初步篩選出212株具有木糖利用及葡萄糖發酵能力酵母菌。隨後將篩選後酵母菌利用隨機擴增多型性DNA (Randomly amplified polymorphic DNA, RAPD)排除同樣品中之同種菌株，共分離出171株酵母菌。所有分離菌株均以大單元核糖體DNA(LSU ribosomal DNA) D1/D2 區域序列、傳統之形態、生理生化試驗等特徵鑑定出菌種，共鑑定出162株酵母菌經鑑定歸類為20屬70種，其中子囊菌屬者有Barnettozyma,Blastobotrys, Candida, Cyberlindnera, Debaryomyces, Hanseniaspora, Kloeckera,
Lindnera, Meyerozyma, Millerozyma, Ogataea, Pichia, Saturnispora, Scheffersomyces,Schwanniomyces, Wickerhamomyces, Yamadazyma, Yushanniozyma, Zygoascus等19屬，而擔子菌株則僅有Cryptococcus菌屬。70個種中僅有5菌種之酒精產量為0.5 % (w/v)以上，包括Scheffersomyces stipitis, Candida sp. (125), Candida sp. (112), Candida coipomoensis, Candida solani等。另酒精產量為0.1-0.5 %者有 12個種；0.1 %以下者則有64個種。同種之菌株以及親緣關係相近菌種其酒精產量相近。
酒精產量 0.5 % (w/v)以上之五種10 株酵母菌中，以Sch. stipitis BCRC 21775,NN6S71, Candida sp. (125) NN15S71, Candida sp. (112) NF17S80, C. coipomoensis NY1W01, C. solani NF1M81 等六株產量較高，其中Sch. stipitis BCRC 21775 與NN6S71 產量分別為2.0 %與1.888 %，Candida sp. (125) NN15S71 產量為1.561 %，Candida sp. (112) NF17S80 產量為0.619 %，C. coipomoensis NY1W01 產量為0.539%，C. solani NF1M81 產量為0.538 %。其中將Sch. stipitis BCRC 21775 和產量較高之三株Sch. stipitis NN6S71, Candida sp. (125) NN15S71, Candida sp. (112) NF17S80 於30 ℃, 100 rpm 為振盪發酵條件下，其最適發酵時間分別為第60, 40, 60, 72 小時。此外，菌株Candida sp. (125) NN15S71 其產量雖與Sch. stipitis BCRC 21775 相當，但其生長溫度可至42 ℃。因此本研究分離出菌株Candida sp. (125)具有與Sch. stipitis 相同良好發酵能力且可耐高溫，值得進一步探討與分析發酵特性以做為未來木糖發酵生質能源的菌株使用。

The objective of this study is isolation, identification and ethanol of xylose-fermenting yeasts isolated from soil, organic materials in Taiwan. The two stages of selection strategy was applied to screen xylose-fermenting yeast strains: xylose-assimilation (XA) yeasts were enriched and selected from samples with medium YNO (yeast nitrogen base- chloramphenicol), then glucose-fermenting (GF) yeast strains were screened among the XA yeasts. Totally, two hundred and twelve yeast strains with ability of xylose - assimilation and glucose-fermention were isolated from 249 samples collected from forest area distributed in 10 counties and 16 villages in Taiwan. DNA fingerprinting profiles with primer (GTG)5 and cell morphology of the selected yeast strains were applied to eliminate the identical yeast strains in a sample, and one hundred and seventy-one isolated were recovered after the two steps. The yeast strains representing 20 genus, such as Barnettozyma, Blastobotrys, Candida, Cyberlindnera, Debaryomyces, Hanseniaspora, Kloeckera, Lindnera, Meyerozyma, Millerozyma, Ogataea, Pichia, Saturnispora, Scheffersomyces, Schwanniomyces, Wickerhamomyces, Yamadazyma, Yushanniozyma, Zygoascus and Cryptococcus and 70 species were identified based on traditional and molecular approaches including cell and spore morphology, physiology and LSU (large subunit) D1 ⁄ D2 and ITS (Internal transcribed spacers) fragments of ribosomal DNA. The ethanol productivity from 5% D-xylose of all these selected strains were detected. Among the strains of sixty-four species of 70 species, their ethanol productivity were less than 0.1 % (w/v), strains of 12 species ramged from 0.1-0.5 % (w/v), while 10 strains representing 5 species, such as Scheffersomyces stipitis, Candida sp. (125), Candida sp. (112), Candida coipomoensis, Candida solani produced greater than 0.5 % (w/v) of ethanol productivity. The ethanol productivity of the top five strains Scheffersomyces stipitis BCRC 21775, NN6S71, Candida sp. (125) NN15S71, Candida sp. (112) NF17S80, Candida coipomoensis NY1W01, Candida solani NF1M81, were detected after culture at 30 ℃ for 48 hours with continuous shaking 100 rpm in 50 ml medium containing 5 % xylose under microaerobic conditions, their ethanol productivity were 2.0, 1.888, 1.561, 0.619, 0.539 and 0.538 % (w/v), resepectively. The optimum fermentation time of Sch. stipitis BCRC 21775 and NN6S71, C. sp. (125) NN15S71, C. sp. (112) NF17S80 is 60 hours, 40 hours, 60 hours, 72 hours resepectively. Furthermore, Sch. stipitis BCRC 21775 and C. sp. (125) NN15S71 have similar ethanol production,but C. sp. (125) NN15S71 can grow at 42 ℃. According to the results described above, C. sp. (125) NN15S71 with high ethanol productivity from xylose revealed favorable characteristics for xylose fermentation, such as high growth temperature, which could be as a good potential for xylose-fermenting in industry.

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