指導教授：童心欣臺灣大學：環境工程學研究所鍾亞萱Chung, Ya-HsuanYa-HsuanChung2014-11-302018-06-282014-11-302018-06-282014http://ntur.lib.ntu.edu.tw//handle/246246/264188環境中富含著大量纖維素物質，分解纖維素的微生物透過產生纖維素分解酵素可將纖維素分解，並產生小分子的可溶性醣類，小分子醣類再經過發酵作用後產生發酵產物，發酵產物如氫氣及乙醇經過轉化後成為可利用的能源，若能將含量如此豐富的纖維素資源經適當處理後運用在生物性替代能源的開發上，可有效減輕目前所面臨的能源短缺問題，也能降低對於環境的汙染問題。 纖維素需透過外在力量斷鍵後才能分解成小分子醣類，因此本研究著重在如何增強纖維素被水解的效果。透過馴養大鼠盲腸內的菌群及不同動物的糞便菌群，選擇內含纖維素或 CMC 成分之合適培養基，分別進行批次馴養，藉由改變培養基組成、碳源成分及馴養條件等，找出最佳纖維素分解菌的馴養方式，以養出能穩定降解纖維素之菌群為主要目的。研究發現以初期經過活化前培養的牛糞便、大鼠糞便及大鼠盲腸菌群馴養最為成功，CMC 降解效果優於其他馴養測試，最大降解率都發生在馴養初期，牛糞便菌群的 CMC 最大降解率約為 75.5%；大鼠糞便菌群的 CMC 最大降解率約為 62.6%；大鼠盲腸菌群的 CMC 最大降解率約為 60.1%。 當馴養達到穩定階段後，為了判定是否馴養出具有分解纖維素能力之菌群，利用專一性引子針對內切型及外切型纖維素分解酵素進行測試，確定纖維素分解酵素的存在。根據 NCBI 比對結果，判定序列為Clostridium sp. Strain BNL1100，由於 Clostridium 菌屬具有降解纖維素功能，其中 Clostridium cellulolyticum 菌種的纖維素降解率甚至可高達 85% 以上，是纖維素分解菌的主要菌屬，從這一點可直接判斷馴養系統中纖維素分解菌群的存在，而偵測出外切型及內切型纖維素分解酵素這兩種功能性基因也能間接推斷纖維素分解菌群的存在。 為了找出最適合大鼠盲腸及牛糞便菌群的馴養環境，因此對於不同的溫度及 pH 值進行測試比較，透過降解能力優劣判斷馴養最適溫度及 pH 值。從實驗結果推論大鼠盲腸反應槽及牛糞便反應槽最適生長環境在pH 6.5 及 pH 7.5，而在 pH 7 時，降解率都有下降的趨勢，造成 pH 值在 6.5 至 7.5 之間產生先下降後上升的趨勢變化，可能原因為在此 pH 值範圍內發生菌群改變的現象。最適溫度則介於 35 至 40°C 之間，因此後續反應槽的操作條件可根據實驗結果調整成最適當的溫度及酸鹼值，使菌群利用 CMC 的效果提高。Cellulose is the most abundant polymer material on Earth. Certain microorganisms can produce cellulolytic enzymes and degrade cellulose efficiently. Cellulose is hydrolyzed and turns into glucose with the help of cellulase. Glucose derived from cellulose is fermented into ethanol, hydrogen and other volatile fatty acids. The fermentation products could provide a partial substitution for fossil fuels and other energy. If such abundant cellulose material can be used properly for the development of renewable biomass, it can not only ease the current problem of fossil fuel shortage, but also reduce the environmental pollution. Cellulose is an organic polysaccharide and cannot be degraded easily. The objectives of this study were to enhance the efficiency of cellulose hydrolysis through the enrichment of rat cecal microbiota and fecal microbiota. By changing the medium composition, carbon sources and enrichment condition, the study tried to find out the best way to enrich the cellulolytic bacteria. The results showed that the best degradation efficiency happened in the beginning of the enrichment of rat cecal microbiota, cow fecal and rat fecal microbiota through activation and preculture. The best degradation efficiency of cow fecal microbiota is 75.5%, rat fecal microbiota is 62.6%, and rat cecal microbiota is 60.1%. In order to make sure the presence of cellulolytic microorganism, functional-gene-specific primers were used to detect cellulase functional genes. According to NCBI database, the sequence result was Clostridium sp. Strain BNL1100. Clostridium is known as cellulolytic bacteria, thus the result indicated the existence of cellulolytic microorganisms in the enrichment. By detecting cellulase functional genes, the existence of cellulolytic microorganisms could also be inferred. In order to find the suitable enrichment condition for the rat cecal microbiota and the cow fecal microbiota, different temperatures and pH values were used to evaluate the degradation efficiency. It turned out that the most suitable pH value for the rat cecal reactor and the cow fecal reactor is 6.5 and 7.5. Degradation didn’t report high efficiency at pH 7. The most suitable temperature was at 35C° to 40°C. The operating condition of reactor could be adjusted to the most suitable condition for the cellulolytic microorganism based on the tested pH value and temperature.誌謝 i 摘要 ii ABSTRACT iv 總目錄 vi 表目錄 xii Chapter 1 前言 1 1.1 研究背景 1 1.2 研究目的 3 Chapter 2 文獻回顧 4 2.1 纖維素 4 2.2 纖維素分解酵素 5 2.3 纖維素分解酵素之檢測 8 2.4 可分解纖維素之微生物 9 2.5 馴養 11 2.6 影響反應操作之環境因子 12 2.7 反應槽之設計 14 Chapter 3 實驗與方法 16 3.1 實驗架構 16 3.2 培養基組成 18 3.3 樣品採集與處理 22 3.4 總醣(Total sugar)定量 23 3.5 還原醣(Reducing sugar)定量 24 3.6 酵素活性測試 25 3.7 功能性基因纖維素分解酵素之檢測 26 3.7.1 DNA 萃取 26 3.7.2 Primer收集與測試 27 3.7.3 PCR之最佳化測試 29 3.7.4 PCR 產物之純化 31 3.7.5 TA cloning 32 Chapter 4 結果與討論 34 4.1 批次馴養結果 34 4.1.1 不同培養基對於馴養大鼠盲腸菌群之影響 34 4.1.2 碳源變化對於馴養牛及羊糞便菌群之影響 37 4.1.3 固定碳源對於馴養大鼠盲腸菌群之影響 40 4.1.4 改變馴養方式對於馴養菌群之影響 43 4.1.5 以反應槽馴養牛糞便菌群及大鼠盲腸菌群 49 4.2 功能性基因之測定 52 4.3 溫度及pH值對於降解率及還原醣量之比較 58 Chapter 5 結論與建議 62 5.1 結論 62 5.2 建議 64 5.3 應用 66 參考文獻 67 附錄 701710363 bytesapplication/pdf論文公開時間：2019/09/03論文使用權限：同意有償授權(權利金給回饋學校)纖維素馴養纖維素分解菌纖維素分解酵素總醣還原醣[SDGs]SDG7[SDGs]SDG12thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/264188/1/ntu-103-R01541120-1.pdf