中文摘要
本研究自大雪山、五指山等地區193樣本中，共分離出65株具葡萄糖發酵性之木糖利用酵母菌，經分子鑑定出42株20種、另7株可歸屬為2屬5種，因與目前已描述種有明顯差異而未鑑定出種名。此65菌株併本實驗室104株相關菌株，共同進行親緣關係樹分析比對，並進行酒精發酵及高溫、酒精、水解物耐受性與抑制物酒精發酵等試驗，以篩選出具良好特性之木糖發酵菌。所有試驗菌種經半嫌氣木糖酒精發酵試驗後顯示：酒精轉換率較佳之菌株為Scheffersomyces stipitis LK11S02, LN7S32, TJ6M05, TJ22M11, EN32S01, GA1M06, Candida sp. (125) NN15S71 及Candida shehatae WE1W06，酒精產量皆可達18.1 g/l 以上，酒精轉換率為66.1-94.6%。厭氧木糖酒精發酵試驗，酒精產能最佳之菌株為S. stipitis LK11S02，發酵時間為72小時後，可達22.0 g/l，轉換率為86%。培養時間若延長至168小時產量可達23.4 g/l，轉換率則為92%。高溫耐受性試驗中，LK11S02和NN15S71菌株分別於30℃, 37℃不同溫度下，有最高酒精產量，分別為24.2和9.8 g/l，而LK11S02及其突變株—F906 於35℃高溫下，酒精產量分別為3.7 g/l, 11.7 g/l，突變株F906其酒精產量顯著高於野生菌株。另以甲酸、乙酸、呋喃甲醛、羥甲基糠醛及酒精等抑制物存在下之發酵試驗，結果顯示：S. stipitis LK11S02分別於8 g/l甲酸、2 g/l醋酸、1 g/l呋喃甲醛、1 g/l羥甲基糠醛抑制物存在下，有22.96, 18.30, 13.80, 20.37 g/l之最高酒精產量，而突變株F906, A2609於羥甲基糠醛濃度為0.25-2.5 g/l時，酒精產量只有微幅變化，突變株對羥甲基糠醛耐受性明顯高於野生菌株。過去報告中顯示，S. stipitis 對於氧氣需求非常苛刻，必須在微好氧的環境下進行木糖酒精發酵，而本研究於台灣，拉拉山樣區由土壤分離出之菌株—LK11S02是S. stipitis 目前唯一在厭氧條件下，具有木糖酒精發酵高產能之特性菌株。木糖還原酶於NADH及NADPH之KM值分別為226.87及260.42 μM，由此結果推測於XR-XDH路徑中，輔酶供需達到平衡，不需要額外注入氧氣即可使木糖代謝不中斷所致。並於高濃度水解抑制物中，保有良好木糖酒精發酵特性，可作為實際運用於工業化發展上之潛力菌株。

Abstract
This study explored the characterization of xylose-fermenting yeasts isolated from Taiwan. Sixty-five xylose-assimilating yeasts with glucose- fermenting ability from 193 samples were collected, including leaves, soil, and mushrooms from 9 counties in Taiwan. Fourty-two of these strains can be identified and classified into 20 different species and 7 unrecognized species compared with currently recognized species based on molecular characteristics. The characteristics of these strains and other 104 strains retrieved from our collection were analyzed in terms of phylogenetic relationship, ethanol fermentation, thermotolerance, ethanol-resistant and inhibitor tolerance test. The ethanol fermentation from xylose under semi-anaerobic condition for strains LK11S02, LN7S32, TJ6M05, TJ22M11, EN32S01, GA1M06, NN15S71 and WE1W06 showed at least 18.1 g/l of ethanol production and 66.1-94.6 % of ethanol yield respectively. S. stipitis LK11S02 demonstrated highest ethanol production of 22.0 g/l and yield of 86 % under anaerobic condition for 72 hrs, and 23.4 g/l of ethanol production and 92 % of yield for 168 hrs. LK11S02, NN15S71 had the highest yield of ethanol were 24.21, 9.83 g/l at 30℃ and 37℃ respectively. Furthermore, formic acid, acetic acid, furfural, hydroxymethyl furfural ethanol, were verified as inhibitors in ethanol fermentation. S. stipitis LK11S02 showed the highest ethanol yields of 22.96, 18.30, 13.80, 20.37 g/l under inhibitor presence of 8 g/l formic acid, 2 g/l acetic acid, 1 g/l furfural, 1 g/l HMF respectively. HMF concentration of 0.25-2.5 g/l did not affect ethanol production by the mutant strain F906, A2609. S. stipitis can ferment xylose only under semi-anaerobic condition, which is fasitidous in demanding oxygen concentration. But S. stipitis LK11S02 can produce ethanol from xylose under anaerobic condition, because Km value of xylose reductase with NADH and NADPH revealed 226.87 and 260.42 μM, respectively, indicating NADH is easier to be converted to NAD+ favoring the process of xylitol to D-xylose. So S. stipitis LK11S02 is an excellent candidate for xylose fermentation under anaerobic conditions. Meanwhile strain LK11S02 showed a higher tolerance in the presence of inhibitor, such as formic acid, acetic acid, furfural and HMF.

目錄
頁次
目錄 I
表目錄 III
圖目錄… Ⅳ
中文摘要 V
英文摘要 VII
壹、前言 1
一、發展替代能源的重要性 1
二、生質酒精之發展 1
三、木質纖維素結構及組成 1
四、木質纖維素酒精製程 2
五、生質酒精發酵過程中之抑制因子 4
(一)酒精 7
(二)高溫 7
(三)醋酸 7
(四)甲酸 7
(五)呋喃甲醛 8
(六)羥甲基糠醛 8
六、酒精發酵酵母菌 8
七、木糖發酵酒精代謝途徑 9
八、發酵菌株特性改良 11
九、研究動機 11
十、研究目的 12
貳、材料與方法 14
一、藥品 14
二、培養基 14
三、器材 15
四、儀器及設備 16
五、研究方法 16
(一)樣品採集 16
(二)菌種分離、純化 16
(三)菌株篩選 17
(四)菌種保存 17
(五)木糖杜罕試管發酵試驗 17
(六)分子鑑定 17
(七)親緣關係分析 19
(八)木糖酒精發酵試驗 19
(九)生長耐受性試驗 21
(十)菌株突變 23
(十一)抑制物對酒精發酵影響 23
(十二)酵素動力學 24
(十三)最適發酵條件： 25
參、結果 26
一、菌株收集 26
二、親緣關係分析 30
三、初步篩選 30
四、木糖發酵特性分析 36
五、菌株發酵特性改良 41
六、抑制物對酒精發酵影響 51
七、最適發酵條件 55
八、木糖還原酶對NADH / NADPH之親和力 63
肆、討論 65
一、不同菌株之木糖發酵產量分析 65
二、厭氧狀態下，酒精發酵產量分析 65
三、逆境壓力下，菌株耐受性結果分析 67
伍、結論 70
陸、參考文獻 71

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