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標題: 以分子生物技術解析與建構厭氧產氫系統
Analysis and construction of a biohydrogen -producing system using molecular biological techniques
作者: 張瑞仁
Chang, Jui-Jen
關鍵字: 產氫酶反轉錄聚合酶連鎖反應
DGGE(denaturing gradient gel electrophoresis), Clostridium
RT-PCR (reverse transcriptase polymerase chain reaction)
real-time PCR
in situ RT-PCR
FISH (fluorescence in situ hybridization)
flow cytometry
energy bug.
出版社: 生命科學系所
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摘要: 本研究從以廢草堆肥為植種源並以啤酒廠廢酵母粉為培養基質,進行批次厭氧醱酵產氫培養系統中取樣,以16S rRNA變性梯度膠體電泳( DGGE ) 技術進行菌相解析的結果,發現梭狀芽孢桿菌屬(Clostridium)細菌為系統內數量上佔優勢的菌群之一。進一步自培養菌液萃取RNA,針對Clostridium屬的產氫酶(hydrogenase)基因進行反轉錄聚合酶連鎖反應(RT-PCR)與即時定量聚合酶連鎖反應(real time PCR),所得的結果顯示產氫酶基因序列和C. pasteurianum 或C. saccharobutylicum相似的兩種Clostridium屬細菌是系統中的產氫優勢菌,且產氫酶基因的表現與產氫狀況相符。另外,利用對產氫酶基因具有專一性的螢光分子探針進行螢光原位雜交(FISH),再用流式細胞儀計數偵測,結果也顯示產氫酶基因序列和C. pasteurianum相似的菌株是系統中數量上與產氫活性上的優勢菌種,而與C. saccharobutylicum相似的菌株則僅在產氫活性的表現上較為優勢,其數量在系統中並不多。進一步以此序列當作為篩選能源微生物線索,從系統中篩選出產氫酶基因序列和C. saccharobutylicum相似的菌株,並經菌種鑑定後命名為C. butyricum M1。將此株菌和另從系統中分離得到的兩株優勢菌(C. beijerinckii L9 與B. thermoamylovorans I)進行共培養醱酵產氫,結果發現具有比原本的系統更高的產氫效益。以本研究利用的分子生物技術所發展出來的方法,再配合原位反轉錄聚合酶連鎖反應(in situ PCR),已經開發出一套以產氫酶基因活性(能源微生物活性)作為指標來評估產氫系統效能的工具。將此工具應用到一個容量為1000公升,以糖蜜作為基質的大型醱酵產氫系統進行微生物產氫活性偵測,再根據偵測結果進行系統操作策略的改良,發現的確是對系統的穩定化操作有很大的幫助。
16S rRNA targeted DGGE was used to analyze the microbial community structure of our fermentative hydrogen-producing system using brewery yeast waste as substrate and straw compost as inoculum. Experimental results indicated that clostridia was the most quantitatively predominant bacteria population in the system. In this study, we developed an approach to monitor quantitatively and qualitatively the clostridial hydrogenase mRNA that may be substantially progressing in hydrogen production by using RT-PCR and real-time PCR. Comparing the results of this study with the results obtained from active microbe by FISH and flow cytometry analysis, it was found that the predominant hydrogen-producing clostridia possess either C. pasteurianum-like or C. saccharobutylicum-like hydrogenase gene. The strain possessing C. saccharobutylicum-like hydrogenase and expressing high level of hydrogenase mRNA but might not be dominant in population was isolated. The strain was designated as C. butyricum M1 after identifying by 16S rDNA sequenceing. When C. butyricum M1 was co-cultured with two other predominant hydrogen producers (C. beijerinckii L9 and Bacillus thermoamylovorans I) which were also isolated from this system, high hydrogen productivity was obtained. We have developed one set of (molecular biological) methodology that can use hydrogenase gene as an energy bug bio-index to evaluate the efficiency of a hydrogen-producing system. This methodology has been applied to a 1000-liter hydrogen-producing system using molasses as substrate and provide appeared to substantial information for improving the system operation.
其他識別: U0005-3107200614404700
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