Full length obtains of hydA and phylogenetic analysis of bio-hydrogen production new species of Clostridium based on efficient hydA degenerate primers

Abstract In fermentative bio-hydrogen production, bacteria from genus Clostridium are considered to be efficient candidates. Studies on bacteria isolation and functional gene analysis of Clostridium make contribution to the H2 production efficiency improvement. In this study, six degenerate primers were designed based on [FeFe]-hydrogenase (hydA) gene sequences from representative biohydrogen producers of Clostridium, and employed to obtain the hydA gene open reading frames (ORFs) of C. guangxiense and C. neuense. Their phylogenetic relationships were also evaluated with hydA gene partial sequences. The ORFs of hydA gene from strains C. guangxiense ZGM211T and C. neuense G1T were obtained with the same length of 1719 bp and theoretical pI 8.09, but different stop codons of TAA and TAG, and Mw of 63.03 and 63.13 kDa, respectively. The phylogenetic tree constructed with hydA partial gene sequences was very similar to that of full-length 16S rRNA gene. It more accurately reflected the phylogenetic relationship of C. guangxiense, C. neuense and related species, and suggested that the hydA gene could be an assistant or reference to 16S rRNA gene in the phylogenetic relationship investigation of bio-hydrogen producers within genus Clostridium.