This study investigated the potential of a fluidized-bed biofilm dominated by Thiobacillus denitrificans to sustain thiosulfate-driven denitrification under increasingly acidic conditions. A fluidized-bed reactor (FBR) performing denitrification via thiosulfate (S2O32-) oxidation of a nitrate-contaminated synthetic wastewater was first operated under decreasing feed pH values from 7.00 to 5.25. Denitrification efficiency > 99% was observed even at feed and effluent pH of 5.75 and 5.30, respectively. At lower feed pH values, the denitrification efficiency decreased rapidly due to inorganic carbon deficiency. The addition of a carbonation unit continuously feeding anaerobic grade CO2 to the FBR biofilm allowed to investigate denitrification at pH values lower than 5.0. This new configuration, i.e. FBR with a carbonation unit, was able to sustain a complete and stable denitrification even at pH as low as 4.75. Denaturing gradient gel electrophoresis (DGGE) showed the evolution of the denitrifying biofilm during the FBR operation, resulting in a robust and high-performing mixotrophic consortium of chemolithotrophic and heterotrophic bacteria dominated by T. denitrificans. Batch activity tests performed at three different stages of the FBR operation (feed pH 7.0, 6.0 and 5.25) showed that low pH cultivation enhanced the denitrification activity (mg N/g VS d) of the FBR biofilm at acidic pH values. (C) 2016 Elsevier B.V. All rights reserved.