Chang Jui-Jen, Chen Wei-En, Shih Shiou-Yun, Yu Sian-Jhong, Lay Jiunn-Jyi, Wen Fu-Shyan, Huang Chieh-Chen
Department of Life Sciences, National Chung Hsing University, Taichung, 40227, Taiwan.
Appl Microbiol Biotechnol. 2006 May;70(5):598-604. doi: 10.1007/s00253-005-0106-7. Epub 2005 Oct 11.
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.
开发了分子生物学方法，以监测使用啤酒酵母废料作为发酵底物的厌氧半固体发酵系统中潜在的产氢梭菌。采用针对16S rDNA基因的聚合酶链反应（PCR）分析的变性梯度凝胶电泳来确认系统中梭菌的存在。值得注意的是，通过使用针对氢化酶基因的逆转录（RT）-PCR，从不同的产氢阶段获得了可重复的梭菌核苷酸序列。这些基于RNA的信息表明，主要的产氢菌株具有特定的巴氏梭菌样或特定的丙酮丁醇梭菌样氢化酶序列。比较PCR和RT-PCR之间针对氢化酶基因的序列图谱发现，携带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，携带特定丙酮丁醇梭菌样氢化酶的菌株表达高水平的氢化酶mRNA，但在群体中可能不占主导地位。此外，定量实时RT-PCR分析显示了梭菌氢化酶mRNA的表达模式，并可作为该系统的活性指标。
