Characterization of Fe-hydrogenase genes diversity and hydrogen-producing population in an acidophilic sludge

According to the DNA sequences of six Fe-hydrogenase genes (FHG) of Clostridium species retrieved from the GenBank, a set of primers specific for Fe-hydrogenase genes were identified from their common conserved regions. The length of DNA fragments amplified using these two primers averaged 313 bps. This primer set was then used to investigate the FHG diversity in an acidophilic rice-degrading sludge by methods based on polymerase chain reaction (PCR). Eight new Fe-hydrogenase gene fragments were identified from the sludge, as a result. Similarity based on amino acids among the 14 hydrogenase genes (8 newly found plus 6 known ones) was 39-97%, which is comparable to the similarity of 41-82% among the 6 known hydrogenase genes alone. The low similarity indicates a great diversity on Fe-hydrogenase among the Clostridium species. The primer set was then used to monitor the change of hydrogen-producing microbial population in a batch reaction using the technique of quantitative real-time polymerase chain reaction (qRT-PCR) with SYBR Green I as the fluorescent reagent. Results showed that the hydrogen producers had an average generation time of 4.2h, and a production rate of 7.0 x 10(16) H2-molecule cell(-1)h(-1).