Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21536
標題: 以流氏細胞儀偵測厭氧產氫醱酵系統中微生物產氫活性
Detection of the hydrogenase activity of hydrogen-producing bacteria in an anaerobically fermentative continuous culture system by flow cytometry
作者: 余憲忠
Yu, Sian-Jhong
關鍵字: 厭氧醱酵產氫系統;專一性的螢光分子探針;原位反轉錄聚合脢連鎖反應;流式細胞儀
出版社: 生命科學系
摘要: 
在厭氧醱酵產氫系統快速產氫時期,篩選出系統中的主要產氫微生物Clostridium及其他可能協助產氫的微生物並以16S rDNA定序分析法進行鑑定,其中的一株菌株Clostridium beijerinckii L9醱酵系統中的優勢產氫菌。嘗試將所獲得的菌株重新當做菌源並以廢酵母粉培養基進行批次醱酵培養,發現其產氫量及系統穩定度皆有明顯的提升,顯示C. beijerinckii L9非常具有潛力可以發展成適用於廢酵母粉培養基產氫醱酵的微生物製劑,有效的提升產氫系統的產氫效率使不穩定的生物產氫系統達到穩定操作。
比較Clostridium屬細菌的產氫?基因之核甘酸序列,合成一段對C. beijerinckii L9的產氫?基因具有專一性的螢光分子探針PRT-KE6-FITC。使用C. beijerinckii L9細胞進行細胞內原位反轉錄聚合脢連鎖反應之後以PRT-KE6-FITC探針進行原位雜交,在螢光顯微鏡下可以清楚地看到表現出產氫?活性的C. beijerinckii L9能發散出綠色螢光,這是第一次經由有效地偵測功能性基因─產氫?基因的 mRNA來瞭解產氫菌產氫活性的研究,並實際運用在糖蜜醱酵槽中能表現產氫活性菌之菌株偵測上。以流式細胞儀對廢酵母醱酵產氫系統進行偵測的實驗,利用探針EUB338-FITC偵測到約有74.34%為具有活性的真細菌菌體,利用根據Clostridium屬細菌產氫?基因之保守序列合成的螢光分子探針PRT-KE2對產氫?基因所表現的mRNA進行偵測,發現未經原位反轉錄聚合?連鎖反應的細胞試樣中約有25.55%可以判定是具有產氫?基因表現的產氫菌。顯示在醱酵槽產氫活性最佳時具有產氫活性的產氫菌族群至少佔全部有活性的真細菌族群的三分之一左右,證明在產氫良好的厭氧醱酵槽中產氫菌族群是佔有優勢數量的。

Hydrogen-producting microbes or microbes relative to hydrogen production were isolated from the culture at actively hydrogen-producing stage and identified by 16S rDNA sequencing. Clostridium beijerinckii L9 displayed the best hydrogen producting ability, it could replace straw compost as an only inoculum and let the batch culture have equal amount but more stable hydrogen production. This strain therefore seemly has great potentialities to be developed as a microbial preparation for utilizing fermentative waste of beer brewery to carry out anaerobically fermentative hydrogen production.
By comparing the nucleotide sequences of hydrogenase genes of C. beijerinckii L9 and other Clostridium spp., and fluorescein isothiocyanate(FITC)-tagged oligonucleotide probe —PRT-KE6-FITC which specifically hybridized to the hydrogenase mRNA of C. beijerinckii L9 was synthesized. When C. beijerinckii L9 cells were subjected to in situ reverse transcriptase polymerase chain reaction(RT-PCR) and followed by fluorescence in situ hybridization(FISH) with PRT-KE6-FITC as probe, those cells which expressed hydrogenase activity could be bound by PRT-KE6-FITC probe and the be detected by fluorescence microscopy. This was the first time successfully to study the hydrogenase activity of hydrogen-producing bacteria by detecting the mRNA of a functional gene — hydrogenase gene. In the studies using flow cytometry, when the sample from an anaerobically fermentative continuous culture system, which used unsterilized fermentative waste of beer brewery as nutrient, was subjected to FISH with 16S rDNA — specific fluorescent probe EUB338-FITC, 74.34 % of the objects detected by flow cytometry were active eubacterial cells. If fluorescent probe RT-KE2, which was specific to the consensus sequence of different clostridial hydrogenase mRNA, was used, 25.55 % of detected cells could be identified to express hydorgenase activity. These results exhibited that the hydrogen-producing bacteria was at least one of the third of the whole population of active eubacteria in the system.
URI: http://hdl.handle.net/11455/21536
Appears in Collections:生命科學系所

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