Effects of some key factors on hydrogenotrophic denitrification were investigated in lab-scale bioreactors. Results indicated that optimum biomass loading, pH value, temperature, nitrate loading, and C/N ratio in this study were optical density at 600 nm (OD600) of 0.173, 6.07.0, 35 A degrees C, 105 mg L-1, and 30, respectively. To obtain a better understanding of microbial community in the bioreactors, the 454-pyrosequencing technology was used to characterize the 16S ribosomal RNA (16S rRNA) gene of bacteria in selected samples Y1 and Y2, in which a total of 62,559 effective sequences (36,445 in Y1 and 26,114 in Y2) were obtained. The taxonomic complexities in two samples were compared at phylum/class/genus levels. In total, 41 bacterial phyla, 43 bacterial classes, and 312 genera were detected, where phylum Firmicutes, class Clostridia were most abundant. Genus Proteiniclasticum was predominant among the top 100 genera. This work aims to add some novel insights into hydrogenotrophic denitrification process and its microbial community structures in bioreactors.