近年來由於清潔再生能源議題發燒，以木質纖維素類的生物量做為料源生產能源已成為一門顯學。不管是早期的同步醣化及共發酵系統(SSCF)或近期的整合型生化反應系統(CBP)，都是為了解決水解酵素生產，纖維素水解及發酵產能的問題，以提高整體纖維素生產能源的效率。本研究以Clostridium屬做為研究對象，找出可以在高溫厭氧條件下進行纖維素水解或醣解產氫的菌株，並研究其生理生化特性。
因此，選用了牛糞系統做為來源，成功的優勢化具有水解濾紙的Clostridium屬菌群，而其菌群結構主要分於Clostridial cluster I，III、XII，XVIII，當中只有Cluster III的Clostridium clariflavum為纖維素水解菌株。所以，針對此優勢化樣本中的纖維素水解菌株進行分離與分子生物鑑定，並命名為Clostridium clariflavum H1。藉由電子顯微鏡觀察此菌株H1會附著濾紙上，當在水解纖維素時會產生黃色物質(YAS)，且環境惡劣時會有內孢子的保護構造產生的特點。此外，菌株H1在55~60℃及初始pH=7.5~8為最佳的生長溫度及pH，而當以纖維雙糖培養觀察其生長曲線時，需要16小時才能適應環境，到第44小時才進入高原期。而在不同碳源廣度測試方面，菌株H1可以利用纖維素類及木聚醣的碳源產生氫氣及乙醇，並有還原醣累積的現象，但直接利用寡醣類的纖維雙糖，蔗糖，及單醣類的葡萄糖，木糖發酵產生氫氣及乙醇的效果不佳。相較於另一株從蔗渣堆肥中分離出的高溫厭氧纖維素水解菌株Thermoanaerobacterium thermosaccharolyticum RCB，其特性卻對木聚醣，寡醣類及單醣類有顯著的利用產生氫氣、乙醇與丁醇，反而對纖維素類的碳源無法產生還原醣累積，及有明顯的產氫氣及醇類的表現。於是進行水解酵素活性測試，其結果顯示菌株H1對於半纖維素類的木聚醣只要一天的時間就以還原醣的濃度增加1.70 g/L的速率達到飽和期，發現其自由型的木聚醣水解酵素活性高達7.45±2.98 U，而固定型的木聚醣水解酵素活型則達2.03±0.30 U/mg，但至於菌株H1針對纖維素類外切型及內切型酵素活性表現不明顯。最後，將菌株H1應用於鹼處理過後蔗渣，稻稈及狼尾草的天然纖維素，發現以水解狼尾草的產生還原醣的濃度最高，以5g/L的初始濃度培養七天後，其還原醣濃度可累積到1.32±0.3 g/L，此時測其水解酵素活性發現也是以木聚醣的水解酵素為優勢約1.91~3.01 U/mg之間，至於外切型及內型的纖維素水解酵素表現依舊不明顯。
本研究已成功地取得扮演纖維素水解酵素生產已進行纖維素水解的菌株及醣解發酵菌株，並了解其生理生化特性資料。這些資料可以供後續實際反應槽操作或其他相關生質能研究的參考。

In the recent years, clear and renewable energy has become an emerging topic to ensure the environment sustainability in the future. That effectively converts lignocellulosic biomass to the energy and reusable solvents already drew much attention, while the biological processes such as the simultaneous saccharification and co-fermentation (SSCF) or the recent consolidated bioprocessing (CBP) were developed to demonstrate the feasibility. Whichever the process was actually required the cellulase production, hydrolysis and fermentation mediated by microbial populations to increase the efficiency of energy recovering. In this research, it was focused on the selected members in the Genus Clostridium with a goal obtain the cellulolytic and fermentative anaerobes at the thermophilic anaerobic conditions, and then preliminary characterization on biochemical and physiological properties of these microorganisms in cellulose hydrolysis were studied accordingly.
To achieve so, the cattle manure was selected as the microbial source, and succeeded in enriching the key microbial populations capable of hydrolyzing filter paper. Based on microbial community analysis, it was found that the various Clostridium groups were present in the enrichment, including Clostridial cluster I, III, XII, and XVIII. Of those, the strain H1 close to Clostridium clariflavum within the cluster III was successfully isolated in the pure culture and then further studied for the cellulolytic capability. The scanning electron microscope observation showed that the strain H1 could attach on the fibers of the filter paper during hydrolyzing and form the terminal endospores in the aged medium environment. In particular, the strain H1 seemed to produce yellow affinity substance (YAS) to enhance the cellulose hydrolysis. Besides, the strain H1 had the optimal growth temperature between 55~60℃ and the optimal initial pH between 7.5~8. Using cellobiose as the growth substrate, the strain H1 needed a lag time of 16 hrs to adapt the environment and then reached the plateau phase in 44 hrs in the growth curve. In addition to cellobiose, the strain H1 could favorably utilize cellulose and xylan to produce hydrogen and ethanol with accumulation of reducing sugar. For example, when the initial concentration of α-cellulose at 5 g / L, it was found the highest concentration of reducing sugar accumulated up to 2.23 ± 0.3 g / L. This might be because the strain H1 had a slower ability on fermenting oligosaccharides like cellobiose, sucrose and monosaccharides like glucose, xylose. However, as compared with the strain Thermoanaerobacterium thermosaccharolyticum RCB isolated from bagasse compost previously in our laboratory, it had a good ability utilizing xylan, oligosaccharides and monosaccharides to produce hydrogen, ethanol and butanol but not good in utilizing cellulose substrates.
Further, the result of the cellulase activity analysis showed that the strain H1 reached the stationary level with the increasing rate of reducing sugar concentration 1.70 g/L/day. It was discovered that the highest activity of cell-free and cell-bound xylanase was 7.45 ± 2.98 U/mg and 2.03 ± 0.30 U/mg, respectively, whereas the expression of exoglucanase and endoglucanase enzyme activities was not prominent. Finally, the strain H1 was applied to hydrolyze the natural cellulose materials like bagasse, rice straw, and napier grass after alkali treatment. The results showed that when the initial concentration of carbon at 5 g / L was hydrolyzed by the strain H1, the highest concentration of reducing sugar could accumulate up to 1.32 ± 0.3 g /L in the culture with xylanase activity of 1.91 ~ 3.01U/mg. The overall result from this study suggested that the strain H1 has a great potential in converting the cellulose feedstock to sugars, which can be further converted to bioenergy using the hydrogen-producing or alcohol-producing specialists such as T. thermosaccharolyticum RCB.
From this research, we succeeded in getting the cellulolytic bacteria which produced the cellulase and the fermentative bacteria, and understand the characterization. It could provide this datum for the actual reactor operation or the reference of other biomass energy.

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