Abstract: Denitrifying MBBR was used for the treatment of reverse osmosis concentrate with high TN and N ${\mathrm{O}}_x^{-}$ -N (N ${\mathrm{O}}_3^{-}$ -N and N ${\mathrm{O}}_2^{-}$ -N) concentrations, which was generated from Dalton Filtration Reverse Osmosis (DFRO) unit in the high-quality water reclamation process of wastewater treatment plant effluent. The variation of denitrifying MBBR efficiency and the copy number of nitrogen removal genes were studied extensively under four different substrate concentrations. The results show that when the influent N ${\mathrm{O}}_3^{-}$ -N and TN increased within the range of (8.70±6.34)-(24.23±8.69) and (28.43±5.69)-(44.10±7.37) mg/L, respectively, the N ${\mathrm{O}}_3^{-}$ -N and TN removal ratios remained stable with the increase of removal rates, but the N ${\mathrm{O}}_3^{-}$ -N and TN removal rates increased, while the N ${\mathrm{O}}_2^{-}$ -N removal ratios and rates decreased. The N ${\mathrm{O}}_3^{-}$ -N and TN removal ratios and rates decreased when the influent N ${\mathrm{O}}_2^{-}$ -N concentration increased within the range of (10.94±8.51)-(20.94±5.78) mg/L, while the N ${\mathrm{O}}_2^{-}$ -N removal ratios and rates increased. The biofilm on denitrifying MBBR carriers mainly consisted of cocci, bacillus and filamentous bacteria. The copy numbers of nitrogen removal genes in carrier biofilm and suspended sludge increased with the increase of influent N ${\mathrm{O}}_3^{-}$ -N and TN concentration, and the copy numbers of nirK, nirS and Anammox genes also increased with the increase of influent N ${\mathrm{O}}_2^{-}$ -N concentration.