Molecular detection of the clostridia in an anaerobic biohydrogen fermentation system by hydrogenase mRNA-targeted reverse transcription-PCR
Molecular detection of the clostridia in an anaerobic biohydrogen fermentation system by hydrogenase mRNA-targeted reverse transcription-PCR
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DOI：
10.1007/s00253-005-0106-7
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发表时间：
2006-05-01
影响因子：
5
通讯作者：
Huang, CC
中科院分区：
文献类型：
--
作者：
Chang, JJ;Chen, WE;Huang, CC
开发了分子生物学方法来监测使用啤酒酵母废物作为发酵底物的厌氧半固体发酵系统中潜在的产氢梭菌。采用变性梯度凝胶电泳和 16S rDNA 基因靶向聚合酶链式反应 (PCR) 分析来确认系统中梭状芽胞杆菌的存在。值得注意的是，通过使用氢化酶基因靶向逆转录（RT）-PCR，从不同的产氢阶段获得了梭菌的可重复核苷酸序列。这些基于 RNA 的信息表明，主要产氢菌株具有特定的巴氏梭菌样氢化酶序列或特定的糖丁酸梭菌样氢化酶序列。 PCR 和 RT-PCR 之间的氢化酶基因靶向序列图谱的比较表明，含有特定巴氏梭菌样氢化酶的细菌菌株在 mRNA 和细菌群体水平上均占主导地位。另一方面，特定的类糖丁酸芽胞杆菌氢化酶携带菌株表达高水平的氢化酶mRNA，但在群体中可能不占优势。此外，定量实时 RT-PCR 分析显示了梭菌氢化酶 mRNA 的表达模式，并可作为系统的活性指标。
Molecular biological approaches were developed to monitor the potential biohydrogen-producing clostridia in an anaerobic semisolid fermentation system that used brewery yeast waste as the fermentation substrate. The denaturing gradient gel electrophoresis with 16S rDNA gene-targeted polymerase chain reaction (PCR) analysis was employed to confirm the existence of clostridia in the system. Remarkably, reproducible nucleotide sequences of clostridia were obtained from different hydrogen production stages by using hydrogenase gene-targeted reverse transcription (RT)-PCR. These RNA-based information suggested that the predominant hydrogen-producing strains possess either a specific Clostridium pasteurianum-like or a specific Clostridium saccharobutylicum-like hydrogenase sequence. Comparison of the hydrogenase gene-targeted sequence profiles between PCR and RT-PCR revealed that the specific C. pasteurianum-like hydrogenase harboring bacterial strains were dominant in both mRNA and bacterial population level. On the other hand, the specific C. saccharobutylicum-like hydrogenase harboring strains expressed high level of hydrogenase mRNA but may not be dominant in population. Furthermore, quantitative real-time RT-PCR analysis showed the expression pattern of the clostridial hydrogenase mRNA and may serve as an activity index for the system.