- 學位論文
利用重組酵母菌發酵纖維廢棄物產製生質酒精之研究
Studies on Bioethanol Production from Cellulosic Waste by Recombinant Saccharomyces
摘要
利用來自生物體的有機物質所製造的生質燃料，作為替代能源使用，為一種可同時解決石化燃料消耗及降低環境污染的方法。其中，生質酒精以不同比例混合汽油作為運輸工具之替代燃料使用，可降低引擎運轉時二氧化碳與其它溫室效應氣體的排放。木質纖維素為自然界中可供製造生質酒精之最大量生質料源，由於木質纖維素大多為廢棄物質，具有低成本及低環境衝擊之特性，因此是一種極具開發潛力之材料。為使纖維酒精製程具有經濟競爭性，所有的纖維料源都必須儘可能轉化成可發酵醣供利用，近年來，酒精的製造主要以微生物發酵生產，然而，傳統酒精發酵菌株囿限於大部分只能利用六碳醣為碳源，進一步阻礙了工業化的發展。利用基因重組工程技術，開發可同時發酵所有存在於木質纖維素中單醣之菌株，為現行研究的主流，此技術之重點在於，重組菌株可同時轉化半纖維素中普遍存在之五碳醣為酒精。本研究重點在於探討重組酵母菌株發酵纖維廢棄物產製生質酒精之效能，第一部分，評估台灣高梁酒粕生產生質酒精的可行性，本研究設計一系列實驗探討高梁酒粕轉化酒精之效率，包括物理處理、不同酸鹼條件下之微波照射預處理、酵素水解及微生物發酵程序。研究結果顯示，金門高粱酒粕(KS)及嘉義高梁酒粕(CS)組成中，以乾基計算分別含有17.2 ± 0.7% 和18.2 ± 0.6% 纖維素、19.0 ± 0.6% 和 21.6 ± 1.0% 半纖維素、18.5 ± 0.8% 和 20.6 ± 1.7% 酸水解木質素以及22.1 ± 0.7% 和23.3 ± 0.4% 的澱粉存在；經由微波照射預處理與酵素水解程序之KS 和CS 的還原醣產率分別為 331.1和341.3 mg/g 乾重；KS與CS的水解產物後續再經Saccharomyces cerevisiae 發酵作用可產酒精分別達0.13 和 0.14 g/g 乾重。此實驗除證明台灣高梁酒粕為一良好之產製生質酒精料源外，此方法中之條件亦可提供為同時富涵木質纖維素與澱粉之料源產製生質酒精之預處理操作程序參考之用。第二部分，利用傳統酒精發酵酵母菌S. cerevisiae為宿主，構築一可同時利用木質纖維水解液中六碳醣與五碳醣為碳源發酵酒精之重組菌株，表現質體構築來自於Pichia stipitis 之木糖代謝外源性基因XYL1、XYL2及XYL3，並利用強力啟動子ADH1及誘導式啟動子GAL1進行調控，經由此質體轉型之菌株PSC207可於合成基礎培養基中生長並且以高粱酒粕水解液為碳源，進行木糖還原酶(XR)、木糖醇脫氫酶(XDH)及木酮糖激酶(XK)之表現，產生酒精，經由分析顯示，PSC207重組菌株酒精產率較未轉形母株YPH499高，可達理論酒精產率之85.5%；小量批次發酵48小時後之木糖消耗量顯示，PSC207過量表現外源性木糖代謝基因情況下，可消耗較多高粱酒粕水解液中之木醣，達母株YPH499的17.6倍，對照菌株PSC107(所攜帶之內源性XYL3基因為來自於S. cerevisiae CBS8066)的1.4倍，並且，代謝途徑中之副產物木糖醇產量並未與對照組有顯著差異。綜合上述結果，本研究所構築之五碳醣與六碳醣共發酵菌株PSC207能有效利用木質纖維水解液中之可發酵醣碳源轉化成酒精，未來應可擴大範圍應用於連續式發酵製程，建立木質纖維酒精生產技術平台，進一步朝經濟規模發展。
並列摘要
Production of biofuel from biomass is one way to reduce both consumption of fossil fuels and environmental pollution. Bioethanol is appropriate for the fuel blends because of its reduction of CO2 and exhaust greenhouse gas emission from the transport sector. Large-scale lignocellulosic biomass types are the most promising feedstock considering its high yields, low costs, and low environmental impacts. Whole feedstock utilization is one of the primaries to make lignocellulosic ethanol processes economically competitive. Recently, biotechnological fermentation process is generally satisfied in worldwide demand of ethanol, and therefore ethanol producing organisms have been developed for commercial processes, but its narrow scope of fermentable carbohydrates has limited its industrial exploitation. The current available technology, which is the engineered microorganisms must ferment all lignocellulosic derived monosaccharides, including hexose and pentose sugars to ethanol. The improved conversion efficiency of ethanol production is based on utilization of a pentose present in hemicellulose, a ubiquitous component of lignocellulosic biomass. This study evaluate the feasibility of using sorghum liquor waste for bioethanol production, we serially investigated the effectiveness of physical treatment, microwave irradiation pretreatment, enzymatic hydrolysis, and fermentation. Composition analysis revealed that Kinmen sorghum liquor waste (KS) and Chiayi sorghum liquor waste (CS) contain approximately 17.2 ± 0.7% and 18.2 ± 0.6% cellulose, 19.0 ± 0.6% and 21.6 ± 1.0% hemicellulose, 18.5 ± 0.8% and 20.6 ± 1.7% acid detergent lignin, and 22.1 ± 0.7% and 23.3 ± 0.4% starch, respectively, on dry weight basis. The reducing sugar yield obtained after microwave irradiation pretreatment and enzymatic hydrolysis of KS and CS were 331.1 and 341.3 mg/g dry weight. The ethanol yields obtained after fermentation of KS and CS hydrolysates with S. cerevisiae were 0.13 and 0.14 g/g dry weight, respectively. This operation of pretreatment may provide a suggestible pattern of utilizing feedstock that contain lignocellulose and starch for ethanolic fermentation. Furthermore, the fermentation performance of the pentose fermenting S. cerevisiae strains in lignocellulose hydrolysate is designed. Through construction of the integrating vectors by employ strong promoter ADH1 and inducible promoter GAL1 to regulate the expression of heterogeneous XYL1, XYL2 and XYL3 genes in recombinant S. cerevisiae. The resulting strains exhibited xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulokinase (XK) activities in a defined mineral medium containing CS hydrolysate and a higher theoretical ethanol yield approximately 80.7 and 85.5% than that obtained by the parental strain YPH499. The recombinant strain PSC207 overexpression of the heterogeneous genes from P. stipitis CBS6045 resulted in the produced less xylitol (0.25 g xylitol/g xylose) but consumption of more xylose than strain PSC107, which carries XYL3 from S. cerevisiae CBS8066. PsXYL3 is a better target for overexpression than ScXYL3 for engineering S. cerevisiae strains capable of fermenting non-detoxified CS hydrolysate.