臺灣博碩士論文加值系統

基於環境保護與資源永續利用的觀念，氫已被視為21世紀的最佳替代能源。利用微生物產氫的方式已漸受重視，其中厭氧微生物在不需要光照下，可將廢棄的有機物分解產氫，具有低成本、減廢與資源回收再利用的優點。因此，本研究主要結合傳統培養和分子生物方法，分離本土性的產氫菌株與建立本土性產氫菌的16S rDNA資料庫，以提供生物產氫反應槽的植種來源與作為分子標誌以監測生物產氫反應槽中菌群的變動。本研究總共分離出231株菌株，其中產氫菌株佔約52.8%。親緣演化分析的結果顯示，篩選出的11株高產氫菌株，有8株與Clostridium之16S rDNA親緣關係相近，且大部分屬於Clostridium的Cluster I，而其餘的3株則分別與Klebsiella和Megasphaera之親緣關係相近。分別由酵母粉為進流基質的生物產氫反應槽污泥和牛糞堆肥所分離出來的產氫菌株FYa102和C008(施，2002)之產氫活性均高於標準菌株Clostridium buryricum，此二菌株之16S rDNA與Clostridium tyrobutyricum的相似度達99%和98%。利用選殖分析的分法，建立以蔗糖和麥粕為進流基質之生物反應槽的16S rDNA clone library。結果顯示，以蔗糖為進流基質的生物反應槽，有76%的clones為與Clostridium之親緣相近的菌群。相反地，以麥粕為進流基質的生物產氫反應槽，只有0.7%的clones與Clostridium之親緣關係相近，而主要由Bulleidia和Calomator親緣相近的菌群構成，是故生物產氫反應槽的微生物社會結構將受進流基質的影響。而利用傳統培養的方法，自以麥粕為進流基質的生物產氫槽的污泥中，並無分離到與Bulleidia和Calomator親緣相近的菌群，卻分離到與Clostridium、Megasphaera、Lactobacillus和Bacillus之親緣關係相近的分離菌株。綜合以上結果，結合傳統培養和分子生物的方法，將可更完整呈現生物產氫反應槽的微生物社會結構，但造成結果上差異的原因，需要更深入的探討。本研究亦利用所獲得厭氧產氫菌的16S rDNA序列當作分子標誌，藉由變性梯度明膠電泳的指紋圖譜探討生物產氫反應槽中微生物社會結構的變動，在本文中亦有詳加討論。

The realization of fossil fuel reserves are limited and have adverse effects on the environment has forced us to search for alternative sources of energy. Hydrogen is a promising candidate as a clean energy source in the future. Among the many processes of hydrogen production, biological production of hydrogen is potentially more attractive, especially if wastewater and organic waste could be used as the raw material for producing hydrogen. In this study, polyphasic approach combined cultivation-dependent with cultivation-independent methods was used to study the microbial communities in bio-hydrogen reactors. Phylogenetic analysis of a bacterial 16S rDNA clone library from the bio-hydrogen reactor with sucrose as influent showed that a majority of the clones was phylogenetic affinity with the genus of Clostridium (76%). Conversely, the bio-hydrogen reactor with wheat husk as influent showed that only 0.7% were phylogenetic affinity with the genus of Clostridium, but a majority were belonged to the genus of Bulleidia (56.7%) and Calomator (32.0%). The data revealed that the microbial community of bio-hydrogen reactors was affected by the substrates of influent. A total of 231 strains were isolated and 52.8% of which were hydrogen producing bacteria. Comparison of the 16S rDNA sequences of the 13 isolates revealed that 8 isolates with high hydrogen producing potential were members of the Clostridium, most closely related to the Clostridium Cluster I. The hydrogen producing bacteria FYa102 and C008 (Shih, 2002) were closely related to the Clostridium tyrobutyricum, possessed higher hydrogen producing potential than the Clostridium butyricum. The microbial diversity of bio-hydrogen reactors was also investigated by denaturing gel electrophoresis (DGGE) approach. The results indicated that the microbial community analyzed by the cultivation-dependent and cultivation-independent methods were not consistent. The 16S rDNA database of the anaerobic hydrogen producing bacteria was also used as molecular marker to monitor the dynamic change of hydrogen producing bacteria in the bio-hydrogen reactors.

中文摘要...................................................................... Ⅰ
英文摘要...................................................................... Ⅱ
誌謝.......................................................................... Ⅲ
目錄.......................................................................... Ⅳ
表目錄........................................................................ Ⅶ
圖目錄........................................................................ Ⅷ
第一章 前言................................................................... 1
1-1研究緣起................................................................... 1
1-2研究目的................................................................... 3
1-3研究架構................................................................... 4
第二章 文獻回顧............................................................... 5
2-1全球能源危機............................................................... 5
2-1-1替代能源................................................................. 5
2-1-2氫氣..................................................................... 5
2-2生物產氫................................................................... 6
2-2-1機制種類................................................................. 7
2-2-2為何選擇生物產氫......................................................... 11
2-3厭氧發酵產氫............................................................... 13
2-3-1種類與產氫效能........................................................... 13
2-3-2梭菌屬產氫菌............................................................. 13
2-3-2-1生理特性............................................................... 14
2-4厭氧發酵產氫的影響因子..................................................... 16
2-4-1基質種類................................................................. 16
2-4-2植種來源................................................................. 18
2-5分子生物方法應用於微生物生態的研究......................................... 19
2-5-1聚合酵素鏈鎖反應......................................................... 19
2-5-2變性梯度明膠電泳......................................................... 20
2-5-3限制酵素段片法........................................................... 20
2-5-4螢光原位雜交法........................................................... 21
第三章 材料與方法............................................................. 22
3-1產氫菌的分離............................................................... 22
3-1-1樣本來源................................................................. 22
3-1-2產氫菌優厚培養的篩選與分離............................................... 22
3-2分離菌株生理特性的測試..................................................... 23
3-3生物產氫反應槽菌群結構變化的探討........................................... 24
3-3-1厭氧產氫反應槽的進流基質................................................. 24
3-3-2培養條件................................................................. 24
3-4分析方法................................................................... 24
3-4-1氫氣的分析............................................................... 24
3-4-2揮發酸的分析............................................................. 25
3-4-3醇的分析................................................................. 25
3-4-4DNA萃取.................................................................. 25
3-4-5聚合酵素鏈鎖反應......................................................... 26
3-4-6瓊脂膠體電泳............................................................. 28
3-4-7變性梯度明膠電泳......................................................... 28
3-4-8分子選殖................................................................. 30
3-4-9定序..................................................................... 31
3-4-10親緣關係的分析.......................................................... 31
3-4-11掃描式電子顯微鏡的觀察.................................................. 32
3-5主要儀器................................................................... 33
第四章 結果與討論............................................................. 35
4-1本土性產氫菌株的分離....................................................... 35
4-1-1高活性產氫菌株的篩選與分離............................................... 35
4-1-2分離菌株的親緣演化關係................................................... 37
4-1-3分離菌株的生理特性探討................................................... 43
4-2利用分子生物方法分析生物產氫反應槽的菌群結構............................... 48
4-2-1以蔗糖為基質的生物產氫反應槽的選殖分析................................... 48
4-2-1-1DGGE快速篩選........................................................... 48
4-2-1-2親緣演化分析........................................................... 51
4-2-2以麥粕為基質的生物產氫反應槽的選殖分析................................... 55
4-2-2-1DGGE快速篩選........................................................... 55
4-2-2-2親緣演化分析........................................................... 58
4-3以分子生物方法探討生物產氫反應槽菌群結構的變動............................. 63
4-3-1不同水力停留時間操作下的菌群結構差異..................................... 63
4-3-2不同稀釋倍數下的菌群結構差異............................................. 65
4-3-2-1利用多管發酵法探討以麥粕為基質的生物產氫反應槽的菌群變動............... 65
4-3-2-2利用多管發酵法探討以酵母粉為基質的生物產氫反應槽的菌群變動............. 67
第五章 結論與建議............................................................. 69
5-1結論....................................................................... 69
5-2建議....................................................................... 71
第六章 參考文獻............................................................... 72
附錄.......................................................................... 79
自述.......................................................................... 85

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