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Microbial community structure and mRNA-hydrogenase expression of a sucrose-feeding fermentative hydrogen production pilot scale bioreactor
|關鍵字:||pilot-scale CSTR;大體積CSTR;Clostridum;PCR-DGGE;Real time RT-PCR;hydrogenase;梭狀芽孢桿菌;PCR-DGGE;Real time RT-PCR;產氫酵素||出版社:||環境工程學系所||摘要:||
國際上研究生物產氫技術已有數十年的歷史，對於厭氧暗醱酵之研究理論基礎已趨於完備，為了加強其實際應用，發展模場規模之反應槽操作技術並朝向實用化應用實屬必須。中興大學環工系環境生物技術實驗室針對「逢甲大學產氫團隊」所架設之實驗室規模之生物醱酵產氫反應槽進行菌群結構分析，目前已清楚暸解此類系統操作在不同環境下菌相組成及優勢菌種的數量。其結果顯示在不同的操作條件下，系統菌群結構包含了Clostridium sp.、Klebsiella sp.、Bifidobacterium sp.及Streptococcus sp.等菌群存在，且菌種數量多寡與產氫效能的優劣有密切的相關性。因此，本研究乃利用PCR-DGGE、Real-time PCR、Real time RT-PCR等分子生物技術，針對逢甲產氫團隊所架設之具商業規模400 L大體積CSTR厭氧醱酵反應槽進行菌群結構、目標菌群數量、比例及產氫酵素表現分析，以探討對其產氫效能之影響關係，期待能建立產氫關鍵性技術以協助發展啟動具商業規模之生物產氫系統。
由菌群結構分析結果顯示，成功啟動產氫反應槽後，當系統pH值控制為6.0至7.0之間時，其Clostridium族群以C. butyricum及C. pasteurianum同時存在，但當pH值穩定控制在6.0以下時C. pasteurianum成為主要產氫菌，且在系統操作於高HRT下時間過長時，也容易生長出非優勢產氫族群導致系統產氫效能降低；而在縮短HRT至4 hr時，有助於系統將非優勢產氫菌洗出(wash out)使反應槽內菌群結構趨於單純，且再提昇至HRT為8 hr後，系統中菌群結構仍可維持較單純的狀態。從目標菌群數量分析結果可以發現，反應槽操作初期系統中非產氫族群佔了較高的比例，當系統趨於穩定時，Clostridium sp.便成為系統中主要優勢菌種且其數量約為108至109 copies/ng DNA且皆佔總菌數之70 %以上，而次要優勢菌種K. pneumoniae數量約為106至108 copies/ng DNA，且其數量、比例多寡明顯影響產氫速率之高低。比較未成功啟動及成功啟動反應槽的產氫酵素表現結果發現，污泥植種於初始pH值較低的環境下，Clostridium sp.具有較高的產氫基因表現，且以單一C. pasteurianum產氫基因表現較為顯著；而污泥植種於初始pH值過高時，將導致Clostridium sp.產氫基因表現低於偵測極限，待pH調整至中性環境後，系統中C. butyricum及C. pasteurianum的產氫基因表現量相當。
The study of biohydrogen production technology for theory of dark fermentation has been complete in the past decades. The courses of application for this technology will be turned to focus on a pilot-scale operating which was developed for the industrial hydrogen production in the future situation. In our present investigations, the research of microbial community structure in hydrogen production dark fermentation bioreactor, which the high rate hydrogen production system was constructed by Fen Chia University, the results shown that the hydrogen production efficiency were related to the present of four predominant bacterial species, including Clostridium sp., Klebsiella sp., Bifidobacterium sp. and Streptococcus sp.. Base on the viewpoint of bacteria factors, therefore, this study was focus on the microbial community structures and hydrogenase expression of a pilot scale bioreactor under different operating conditions by using molecular biological techniques, including PCR-DGGE, real-time PCR and real time RT-PCR which expect to set up the key technology of biohydrogen fermentation system for commercial application.
According to analysis of microbial composition by PCR-DGGE, the results were shown that the C. butyricum and C. pasteurianum were coexist within pH 6.0-7.0, then if keeping at pH 5.5-6.0, C. pasteurianum was become predominate as hydrogen production species in the system. Meanwhile, the results also indicate that high HRT (12 h) operated in a long time was not benefit for hydrogen production, because it was increasing the substrate competitor existing such as several facultative anaerobes, whereas wash out of those species were present in HRT 4 h with performed the high hydrogen production rate, and keeping simply structure of bacteria composition as only C. pasteurianum surviving even if changes to HRT 8 h again. For the results of bacteria quantification by real-time PCR, high ratio of non-hydrogen production bacteria were existed in the system within initial operation. In the steady state, the amount of Clostridium sp. cell and its ratio per other microbial species were increasing to 108-109copies/ng DNA and over 70% respective. For the K. pneumoniae which the sub-dominant hydrogen production bacteria in the system, the cell count was around 106-108copies/ng DNA through the system operation. To comparing with the hydrogen production efficiency and variation of K. pneumoniae cell count, high ratio of this specie exist following with low hydrogen production rate, this result indicate that K. pneumoniae was a major substrate competitor to the hydrogen production species, C. pasteurianum. This study also uses the RT-qPCR technique to measure the hydrogenase (hydA) gene expression targeting two attentive Clostridium sp. through the system operation. Significant increasing of hydrogenase mRNA expressing of Clostridium sp. was observed in low pH conditions (5.5-6.0), and the high efficiency of hydrogen production was found to correlated with a high hydrogenase mRNA expressing of C. pasteurianum in the system.
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