The application of nitrification–denitrification over nitrite (nitritation–denitritation) with municipal (i.e. diluted and cold (or low-temperature)) wastewater can substantially improve the energy balance of municipal wastewater treatment plants. For the accumulation of nitrite, it is crucial to inhibit nitrite-oxidizing bacteria (NOB) with simultaneous proliferation of ammonium-oxidizing bacteria (AOB). The present study describes the effect of the influent total ammonium nitrogen (TAN) concentration on AOB and NOB activity in two moving bed biofilm reactors operated as sequencing batch reactors (SBR) at 15 °C (SBR I) and 21 °C (SBR II). The reactors were fed with diluted reject water containing 600, 300, 150 and 75 mg TAN L−1. The only factor limiting NOB activity in these reactors was the high concentrations of free ammonia and/or free nitrous acid (FNA) during the SBR cycles. Nitrite accumulation was observed with influents containing 600, 300 and 150 mg TAN L−1 in SBR I and 600 and 300 in SBR II. Once nitrate production established in the reactors, the increase of influent TAN concentration up to the original 600 mg TAN L−1 did not limit NOB activity. This was due to the massive development of NOB clusters throughout the biofilm that were able to cope with faster formation of FNA. The results of the fluorescence in situ hybridization analysis preliminarily showed the stratification of bacteria in the biofilm.

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