Thiosulfate-driven denitrification was investigated in a FBR at pH 4.45–6.90. Denitrification efficiency > 99% was achieved at a pH as low as 5.30. 100% denitrification was sustained even at a pH of 4.75 by continuous CO2 purging. A mixotrophic biofilm dominated by T. denitrificans developed at acidic pH. pH and alkalinity governed FBR performance and microbial dynamics.