Bicarbonate (HCO 3-) can be used as an inorganic carbon source for hydrogen-based denitrification (HD). Since HCO3is considered to accelerate the NO2reduction rate, this study is attempted to minimize the hydraulic retention time (HRT) of HD systems using varied amounts of HCO3-: deficient, moderate, and abundant amounts. The results implied that a low NO2amount was removed at unsuitably short HRTs, resulting in poor HD efficiency despite being supplemented with abundant HCO3amounts. HCO3assisted in rapidly acclimatizing the bacteria having nirS gene, causing higher NO2reduction rate and aided in changing the bacterial communities. Thauera spp. were the most dominant bacteria in abundant HCO3conditions, achieving high HD efficiency at 8 to 24 h HRT whereas satisfactory efficiency was achieved in the deficient and moderate HCO3amountsystems through the collaboration of Rhodocyclaceae, Alcaligenaceae, and Xanthomonadaceae as predominant bacteria in the community. A strong correlation between the abundance of nirS gene and Thauera spp. was also found. The findings in this study revealed the importance of using HCO3for the enrichment of H2-oxidizing denitrifiers containing nirS gene in order to reduce NO2accumulation to enhance the HD efficiency.