臺灣博碩士論文加值系統

本研究將甘蔗渣以1% H2SO4與2%NOH預處理，每一預處理程序為120C加熱40分鐘。這些預處理的蔗渣(SCB)以蔗渣與麥麩4:1的重量比進行固態培養Trichoderma reesei RUT C30生產纖維素酶。在30℃培養6天後，酶活性達到13.70 2.81 FPU / g-DS。以含有纖維素酶活性的固態培養基質(CSCB)在50C直接進行水解反應4小時，之後降溫到40C，接種一株耐高溫酵母Kluyveromyces marxianus進行同步糖化發酵(SSF)生產酒精，得到酒精轉換率(conversion yield)為14.101.84％（w / w）。若補充 5 FPU/g-DS CTec2 或 10 FPU/g-DS 由核能研究所生產的纖維素水解酶，則酒精轉換率分別為30.30.4％（w / w）和27.50.1%（w / w）。若使用葡萄糖或將CSCB進行失活處理再加入15 FPU/g-DS CTec 2進行酒精生產，得到的酒精轉換率為 36.80.4% (葡萄糖)與30.10.5% (CSCB)。本研究證明直接使用CSCB來生產酒精，能夠減少使用商業的纖維素酶，從而降低利用纖維素生產酒精的成本。

Sugarcane bagasse was pre-treated by 1% H2SO4 followed by 2% NaOH. Each pre-treatment process was carried out at 120C for 40 min. The pre-treated sugarcane bagasse (SCB) supplemented with wheat bran in a weight ratio of 4:1 was used as the substrate for cellulase production by solid state fermentation using Trichoderma reesei RUT C30 at 30 C. Cellulase activity of 13.70 2.81 FPU / g-DS was achieved after 6 days’ incubation. 5% (w/v) cellulase containing SCB (CSCB) was directed hydrolyzed at 50C for 4 h, then the hydrolyzed CSCB slurry was cooled to 40C, thermotolerant yeast (Kluyveromyces marxianus) was pitched to the slurry for ethanol production in simultaneous saccharification and fermentation (SSF) process. The yield of ethanol was 14.10.8% (w/w) from CSCB. Ethanol yield of 30.30.4％（w / w）and 27.50.1% (w/w) was achieved from CSCB supplemented with with 5 FPU/g-DS CTec 2 or 10 FPU/g-DS cellulase produced from the Institute of Nuclear Energy Research. The ethanol yield of 36.80.4% from glucose or 30.10.5% from deactivated CSCB plus 15 FPU/g-DS CTec2 was achieved respectively from our ethanol fermentation system. This study proved that direct employ CSCB to produce ethanol can reduced the cost of commercial cellulase.

致謝 II
摘要 III
ABSTRACT IV
目錄 V
圖目錄 X
表目錄 XI
第一章 緒論 1
1.1 研究動機 1
1.2文獻回顧 3
1.2.1 膳食纖維(Dietary fibre) 3
1.2.2 木質纖維素(lignocellulose) 3
1.2.3 麥麩(Wheat bran) 7
1.2.4 蔗渣 7
1.2.5 纖維素水解酵素 8
1.2.5.1 纖維素水解酵素作用機制 9
1.2.6 水解纖維素菌種 11
1.2.7 固態發酵 13
1.2.7.1 固態發酵優缺點 14
1.2.8同步醣化與發酵 14
第二章 材料與方法 17
2.1 實驗儀器 17
2.2 實驗材料 18
2.2.1 菌種 18
2.2.2 藥品 18
2.2.3 固態培養基 19
2.3實驗架構 20
2.3.1 Trichoderma reesei培養 20
2.3.2 同步糖化發酵生產酒精測試 21
2.4 實驗方法 22
2.4.1基質處理 22
2.4.1.2基質水解測試 23
2.4.2 Trichoderma reesei菌種培養 23
2.4.2.1菌種保存 23
2.4.2.2 纖維素水解液製作 23
2.4.2.3 培養T. reesei之平板培養基 24
2.4.2.4 培養T. reesei之液態培養基 24
2.4.2.5 培養T. reesei之固態培養基 24
2.4.3 Kluyveromyces marxianus菌種培養 25
2.4.3.1菌種保存 25
2.4.3.2 K. marxianus平板培養基 25
2.4.3.3 K. marxianus液態培養基 25
2.4.4 發酵槽生產酒精培養基 26
2.4.4.1 alpha-cellulose培養基 26
2.4.4.2 前處理過蔗渣培養基 26
2.4.4.3粗酵素液培養基 27
2.5分析方法 27
2.5.1.1 纖維素水解酵素(cellulase，FPAse)測定(Filter Paper Assay)(Ghose，1987) 27
2.5.1.2 內切纖維水解酵素(Endoglucanase，CMCase)測定(Ghose，1987) 28
2.5.1.3 聚木醣酵素(Xylanase)測定(Tsao et. al. 2000) 29
2.5.1.4 -葡萄糖苷酶(-glucosidase)測定(Tsao et. al. 2000) 29
2.5.2 水解液分析 30
2.5.2.1 DNS還原醣測試方法 30
2.5.2.2 葡萄糖含量 31
2.5.2.3 酒精濃度與水解成份測定 31
第三章 結果與討論 33
3.1蔗渣前處理 33
3.1.1先酸再鹼前處理蔗渣水解測試 33
3.1.2不同前處理蔗渣水解測試 35
3.2 固態發酵生產纖維素酶 37
3.2.1固態培養氮源添加對其酵素的影響 37
3.2.1固態培養碳源添加對其酵素的影響 38
3.2.1放大培養纖維水解酵素活性 39
3.3 生產酒精 40
3.3.1純纖維素生產酒精(alpha-cellulose前處理過蔗渣) 40
3.3.2 以自製固態發酵基質生產酒精 43
3.3.2.1 抑菌實驗 45
3.3.3 自製固態發酵搭配商業用酵素 47
3.3.3.1 自製酵素與商業用酵素的活性比較 47
3.3.3.2 補充不同比例商業用酵素 49
結論 52
參考文獻 53

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