應用纖維素酶生產菌及耐高溫酵母發酵咖啡果殼生產生質乙醇之研究

咖啡為全球最受歡迎的飲料之一，為僅次於石油之第二大貿易商品，每年於全世界之產量約為 163.4億磅。然而於咖啡豆處理過程中所產生之咖啡果殼 (coffee husk) 屬於一種木質纖維素之農業廢棄物，可用來作為生產生質酒精之原料，但其主要由纖維素、半纖維素及木質素所組成，多半不被利用或不易被降解。因此本研究之目的為篩選出具纖維素酶之微生物將咖啡果殼中之纖維素分解成可發酵醣，再應用自篩之耐高溫酵母將其發酵並生成生質酒精。本研究自落葉、土壤及空氣中分離出 40株菌株，其編號分別為 L1-L18、 S1-S18、 AD1-AD4以及 4株實驗室保存菌株 (BM、 BL、 BT及並以剛果紅染色試驗篩選是否具有分解纖維素之能力。結果顯示，菌株 L7、 L9、 AD1、 AD3、 BM、 BL、 BT及 PC具有纖維素分解能力，其中菌株 AD1及 AD3具有較佳之分解能力，其透明環大小分別為 34.50±3.54 mm及 27.80±3.89 mm。後續再將具有纖維素分解能力之菌株接種於含 1%CMC之生長培養基中，並測定其內切葡聚醣酶活性 (Carboxymethyl cellulase assay for endoglucanase, CMCase) 及總纖維素酶活性 (Filter paper assay for cellulase, FPase)。結果顯示，菌株 AD1及 AD3具有最高之 CMCase分別為 0.13 U/mL及 0.12 U/mL 與最高之 FPase分別為 2.41 U/mL及 2.13 U/mL。另外，自人體糞便中篩選出 1株 (T14) 以及蜜餞中篩選出 2株 (1-6及 2-6) 可於 40℃生長之耐高溫酵母將其以 Einhorn’s發酵管進行葡萄糖發酵能力試驗及乙醇耐受性試驗。其中酵母菌 T14為發酵能力及耐乙醇能力較佳之菌株，其產氣量可達 21.50±3.54 mL，並可耐受 10%之乙醇濃度。之後將菌株 AD1、 AD3及 T14進行菌種鑑定，根據鑑定之結果，將菌株 AD1及 AD3命名為 Bacillus pseudomycoides AD1及 Bacillus pseudomycoides AD3，菌株 T14命名為 Candida sp. T14。並測定菌株 AD1及 AD3所生產之胞外纖維素酶之 pH值及溫度穩定性以及於預處理過後之咖啡果殼可獲得之還原醣含量，其結果顯示，在 pH值方面菌株 AD1及 AD3於 pH 4.8之 FPase顯著高於其他 pH值分別為 1.63 U/mL及 1.64 U/mL 在溫度方面則是於 70℃有較好之 FPase，分別為 1.72 U/mL及 1.84 U/mL；而在還原醣含量方面，菌株 AD1於第 20小時有最高之還原醣含量 1.17 g/L，菌株 AD3則是於第 12小時有最高之還原醣含量 0.89 g/L，根據還原醣含量之結果選定菌株 II AD1作為酵素處理咖啡果殼之菌株作為酵素處理咖啡果殼之菌株。。接著以反應曲面法之接著以反應曲面法之Box-Behnken設計設計探討發酵咖啡果殼生產生質乙醇之最適條件，探討發酵咖啡果殼生產生質乙醇之最適條件，結果顯示，結果顯示，咖啡果殼與咖啡果殼與NaOH以以3:10之之比例進行比例進行預預處理處理、、添加添加5%胞胞外外纖維素酶及纖維素酶及接種接種2%酵母酵母菌接種量菌接種量可獲得最好之生質乙醇含量。未來可獲得最好之生質乙醇含量。未來可應用可應用自篩之菌株自篩之菌株Bacillus pseudomycoides AD1及及Candida sp. T14擴大發酵咖啡果殼並生產生質乙擴大發酵咖啡果殼並生產生質乙醇，除可醇，除可增加增加咖啡果殼咖啡果殼之之附加附加價值，也可減少化石燃料之使用，以減緩全價值，也可減少化石燃料之使用，以減緩全球暖化之問題。球暖化之問題。

關鍵字

咖啡果殼；纖維素酶；耐高溫酵母；生質酒精

並列摘要

Coffee is one of the most popular beverages in the world and the second most traded commodity after petroleum, with an annual worldwide production of approximately 16.34 billion pounds. However, the coffee husk produced in the process of coffee bean processing belongs to a kind of lignocellulose agricultural waste, which can be used as the raw material for the production of bioethanol, but it is mainly composed of cellulose, hemicellulose and lignin Most of them are not used or not easily degraded. Therefore, the purpose of this study is to screen out microorganisms with cellulase to decompose cellulose in coffee husks into fermentable sugars, and then use self-screened thermotolerant yeast to produce bioethanol. In this study, 40 strains were isolated from fallen leaves, soil and air, numbered L1-L18, S1-S18, AD1-AD4 and 4 laboratory-preserved strains (BM, BL, BT and PC), and use Congo Red staining test to screen whether it has the ability to decompose cellulose. The results showed that strains L7, L9, AD1, AD3, BM, BL, BT and PC had cellulolytic ability, among which strains AD1 and AD3 had better ability to decompose, and their clear zone were 34.50±3.54 mm and 27.80±3.89 mm. Subsequently, the cellulolytic strains were inoculated into a growth medium containing 1% CMC, and their endoglucanase activity (CMCase) and total cellulase activity (FPase) were determined. The results showed that strains AD1 and AD3 had the highest CMCase of 0.13 U/mL and 0.12 U/mL, respectively; and the highest FPase of 2.41 U/mL and 2.13 U/mL, respectively. In addition, 1 strain (T14) was screened from human feces and 2 strains (1-6 and 2-6) were selected from the preserved fruit. The thermotolerant yeast that can grow at 40°C were tested for glucose fermentation ability in Einhorn's fermentation tube and ethanol tolerance test. Among them, yeast T14 is a strain with better fermentation ability and ethanol tolerance, its gas production can reach 21.50±3.54 mL, and it can tolerate 10% ethanol concentration. Then the strains AD1, AD3 and T14 were identified. According to the identification results, the strains AD1 and AD3 were named Bacillus pseudomycoides AD1 and Bacillus pseudomycoides AD3, and the strain T14 was named Candida sp. T14. The pH value stability IV and temperature stability of the extracellular cellulase produced by the strains AD1 and AD3 and the reducing sugar content available in the coffee husks after pretreatment were determined. The results showed that the FPase of strains AD1 and AD3 at pH 4.8 was significantly higher than other pH values, 1.63 U/mL and 1.64 U/mL, respectively; in terms of temperature, it had better FPase at 70°C, 1.72 U/mL and 1.84 U/mL, respectively; and In terms of reducing sugar content, strain AD1 had the highest reducing sugar content of 1.17 g/L at the 20 hours, and strain AD3 had the highest reducing sugar content of 0.89 g/L at the 12 hours. According to the results of reducing sugar content, strains were selected AD1 as an enzyme-treated strain of coffee husks. Then, the Box-Behnken design of the reaction surface method was used to explore the optimal conditions for fermenting coffee husks to produce bioethanol. The results showed that the ratio 3:10 of coffee husk to NaOH treatment, the 5% of extracellular cellulase added, and the 2% of yeast inoculated can obtain the best biomass ethanol content. In the future, the self-screened strains Bacillus pseudomycoides AD1 and Candida sp. T14 can be used to expand the fermentation of coffee husks and produce bioethanol, which can not only increase the value of coffee husks, but also reduce the use of fossil fuels to alleviate the problem of global warming .