Molecular detection of the clostridia in an anaerobic biohydrogen fermentation system by hydrogenase mRNA-targeted reverse transcription-PCR.
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.
Chang
Jui-Jen
JJ
Department of Life Sciences, National Chung Hsing University, Taichung, 40227, Taiwan.
Chen
Wei-En
WE
Shih
Shiou-Yun
SY
Yu
Sian-Jhong
SJ
Lay
Jiunn-Jyi
JJ
Wen
Fu-Shyan
FS
Huang
Chieh-Chen
CC
D000595:Amino Acid Sequence; D000693:Anaerobiosis; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification*; D005285:Fermentation; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006859:Hydrogen / Q000378:metabolism*; D006864:Hydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D012333:RNA, Messenger; D012336:RNA, Ribosomal, 16S; D006859:Hydrogen; D006864:Hydrogenase