The anaerobic oxidation of ammonium (anammox) uses nitrite as terminal electron acceptor. The nitrite can cause inhibition to the bacteria that catalyze the anammox reaction. The literature shows a great divergence on the levels of NO2- causing inhibition. Moreover, the conditions influencing the resistance of anammox bacteria to NO2- inhibitory effect are not well understood. This work investigated the effect of the pH and the concentration of nitrite on the activity and metabolism of anammox granular sludge under different physiological conditions. Batch activity tests in a range of pH values were carried out in which either actively metabolizing cells or resting cells were exposed to nitrite in the presence or absence of the electron donating substrate ammonium, respectively. The response of the bacteria was evaluated by analyzing the specific anammox activity, the accumulation of nitric oxide, and the evolution of the ATP content in the biomass. Additionally, the effect of the pH on the tolerance of the biomass to single substrate feeding interruptions was evaluated in continuous anammox bioreactors. The results show that the influence of the pH on the NO2- inhibition of anammox bacteria is greater under non-metabolizing conditions than during active metabolism. The exposure of resting cells to NO2- (100 mg N L-1) at pH values below 7.2 caused complete inhibition of the anammox activity. The inhibition was accompanied by accumulation of the intermediate, nitric oxide, in the gas phase. In contrast, just mild inhibition was observed for resting cells exposed to the same NO2- concentration at pH values higher than 7.5 or any of the pH values tested in assays with actively metabolizing cells. ATP initially increased and subsequently decreased in time after resting cells were exposed to NO2- suggesting an active response of the cells to nitrite stress. Furthermore, bioreactors operated at pH lower than 6.8 had greater sensitivity to NO2- during an ammonium feed interruption than a bioreactor operated at pH 7.1. The results suggest that the ability of resting cells to tolerate NO2- inhibition is seriously impeded at mildly acidic pH values; whereas actively metabolizing biomass is resistant to NO2- toxicity over a wide range of pH values. (C) 2014 Wiley Periodicals, Inc.