本研究探討在生物產氫的系統程序中，暗醱酵厭氧產氫與生物產氫系統的功能性基因（產氫酵素）之間的關係。在研究中，以篩選自厭氧醱酵污泥槽的Clostridium butyricum CGS5為菌種範本。而在暗醱酵產氫系統中，產氫酵素是氫氣生成反應中的主要酵素。因此，在暗醱酵產氫的培養系統中，監測產氫酵素的基因表現是一個可靠的偵測方法。實驗結果印證了生物產氫功能性基因的表現（產氫酵素基因序列來自於Clostridium butyricum CGS5，乃篩選自活性污泥的本土性產氫菌種）與生物產氫效能之間的關係，並且培養在蔗糖為唯一碳源的反應器中。完整的Clostridium butyricum CGS5產氫酵素基因序列是以primer cross-linking和PCR產物定序方式來進行基因序列的解碼。接著，根據由解碼方式所得到的產氫酵素基因序列來設計的PCR和qPCR反應所需要用到的探針引子，並且利用所設計的探針引子和qPCR技術來偵測已經由反轉錄形成的cDNA的產氫酵素RNA的copy number。實驗結果顯示，Clostridium butyricum CGS5的氫氣生成速率與產氫酵素基因表現（也可作為產氫酵素基因cDNA的copy number）有線性正比的關係存在，而生物菌種濃度的曲線趨勢圖與產氫酵素DNA的copy number有相似的趨勢。
在整個產氫厭氧醱酵過程中，不論是產氫酵素DNA和反轉錄的產氫酵素基因（PCR複製反應作用後）的copy number，還是DNA和RNA的核酸總量（PCR複製反應作用前）都隨著CGS5菌種的生長被監測。在PCR複製反應過程後，產氫酵素DNA的copy number在菌種生長對數後半期與菌種生長初期比較結果有100倍以上的增加量，而反轉錄後的產氫酵素基因的copy number在菌種生長對數後半期與菌種生長初期的比較也有500倍的增加。來自mRNA反轉錄而得的產氫酵素的表現量與DNA和RNA的核酸總量的表現相比較則有滯遲的現象發生，然而產氫酵素DNA copy number的增加趨勢與DNA核酸總量有極為相似的地方。以上所討論的分子性質層面或生長的各種因素均與氫氣生成有其對應關係。16S rRNA的反轉錄表現在38小時有最大量的出現，而其出現的時間比產氫酵素的反轉錄量（在48小時）來得早。因此，所有產氫酵素的分子性質與蔗糖基質利用反應、菌種生長和氫氣生成都有相符合的趨勢。這些實驗說明了基因表現量（如反轉錄形成cDNA）可以與氫氣生產效率有相對關係存在，也可能被利用當作程序控制系統的應用工具。
在PCR與qPCR反應過程中，我們利用監測Clostridium butyricum CGS5的產氫基因的方式來尋找添加牛血清蛋白（BSA）以避免PCR反應抑制物之最佳化濃度。在改善PCR偵測限制實驗中，100 ng/l 的BSA濃度比目前所找到的文獻論文中所使用的BSA濃度來的低且較具有效率。在以添加100 ng/l 的BSA來增加PCR效應的PCR實驗中，有添加BSA的反應會使得PCR產物有較亮且多的螢光反應在具有螢光染劑(ethidium bromide)的溶液中、有最低的偵測限制及較大值的Ct值。BSA效應不僅能應用於PCR和qPCR反應中，也可以應用於絕大多數的微生物反應系統上。當添加的BSA濃度大（等）於100 ng/l時，利用qPCR來測量基因複製的copy number可能會有所減少、Ct值可能增加而PCR產物的Tm值則有可能會因此而改變。但是在PCR產物的SYBR Green dissociation的圖形中，以DNA為目標基因的純菌菌種系統或混菌菌種系統，均顯示100 ng/ l的BSA濃度可以減少圖形中peak面積及Tm值的變化量。
最後，RT-PCR和qPCR技術證明也可以被利用在不同的微生物系統的可行性，例如以Caldimonas taiwanensis On1T則可在多酵素共同作用系統中水解澱粉。在Caldimonas taiwanensis On1T的澱粉水解醱酵過程中，從實驗結果也可以看出已熟知的澱粉水解酵素的基因表現與澱粉水解活性的趨勢相符合。以Caldimonas taiwanensis On1T為菌種的澱粉水解系統中，假設已知的澱粉水解酵素是主要的澱粉水解酵素。實驗結果也證明顯示，可以應用新興的分子生物技術（結合RT-PCR和qPCR技術）來擴大微生物應用系統來探討監測基因表現和生物功能之間的關係。

This study was undertaken to identify the relationship between the performance of dark H2 fermentation and expression of the key functional gene (i.e., hydrogenase gene) involved in the bioH2 production process. Clostridium butyricum CGS5 isolated from anaerobic sewage sludge was used as the model strain for this study. Hydrogenase is the key enzyme responsible for bioH2 production in dark fermentation. Therefore, the expression of hydrogenase gene is a good indicator for the performance of a dark H2 fermentation culture. We investigated the correlation between expression of the functional gene (hydA encoding for hydrogenase in C. butyricum CGS5) and bioH2 production activity during batch growth, where an indigenous H2-producing isolate C. butyricum CGS5 used sucrose as the sole carbon source. The full sequence of hydA gene of C. butyricum CGS5 (2174 bps in size) was decoded via primer cross-linking and amplicons sequencing techniques. The copy number of hydA mRNA was determined by reverse transcription and real time quantitative PCR (qPCR) using designed probes according to hydA gene sequence. The results indicated that the hydrogen production rate of C. butyricum CGS5 was proportional to the level of hydA expression (represented by the copy number of hydA cDNA), whereas the biomass concentration profile also followed a similar trend to that of the hydA DNA copies.
Copy numbers of the hydrogenase gene (hydA) and mRNA transcripts (cDNA hydA) (after amplification) as well as total DNA and RNA (before amplification) were measured over the course of the growth of strain CGS5. After amplification, the copy number of hydA increased 1000 folds during late exponential growth phase after normalization to the copy number at time 0, and cDNA from mRNA transcripts of hydA also increased 500 folds after normalization. The mRNA transcripts of hydA lagged behind the increase of total DNA and RNA, and increased in hydA more similar than those of total DNA. Increases in both of these parameters corresponded to hydrogen production. Transcripts of 16S ribosomal RNA reached a maximum value earlier (38 h) than did those of hydA (47 h). All molecular characteristics matched those for sucrose utilization, growth and hydrogen production. The experiment results indicated that transcription as measured by cDNA can be related to hydrogen production and possesses the potential to be used as tool for process control.
Detection of hydA genes of Clostridia using designed primer sets for C. butyricum were optimized by the addition of bovine serum albumin (BSA) to PCR and qPCR reactions. A 100 ng/ l of BSA concentration which is lower than previously reported in the literature were found to be most effective on improving the detection limitation. The brightness of amplicons with 100 ng/ l BSA increased in ethidium bromide treated gels, the minimum detection limit with BSA was at least one log greater and cycle threshold (Ct) values were higher than those without BSA in qPCR, indicating improved detection of target DNA for most samples tested. Although amplicon visualization was improved at BSA concentrations ≥100 ng/l, gene copy numbers detected by qPCR were less, CT values increased and Tm values were altered. SYBR Green dissociation curves of qPCR products of DNA from pure culture or sludge samples showed that 100 ng/l of BSA reduced the variability of peak areas and Tm values.
Finally, the reverse transcription and qPCR techniques were also applied to another bacterial system (Caldimonas taiwanensis On1T) possessing the ability to hydrolyze starch under a multi-amylase system. The results showed that the expression of a known amylase gene (Amy gene) seemed to match the trend of starch hydrolysis performance (or enzyme activity) during the fermentation time course. This indicated that the target amylase played a major role in starch hydrolysis activity of the Caldimonas taiwanensis On1T strain. The results also demonstrated the potential of using the proposed molecular biological techniques (i.e., combination of RT-PCR and qPCR) in monitoring the correlation between gene expression and biological function.

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