This study evaluated the interactive effect of Cu(II) and Zn(II) on anaerobic ammonium oxidation (anammox) activity using response surface methodology with a central composite design. A regression model equation was developed and validated to predict the normalized anammox activity (NAA) of anammox granules exposed to various heavy metal concentrations. The joint inhibitory effect tended to exacerbate initially and reversed as the concentrations increased and then moderated again. The most severe inhibition, resulting in a NAA of 20.1%, occurred at Cu(II) and Zn(II) concentrations of 16.3 and 20.0mgL(-1), respectively. Notably, the cumulative toxicity was mitigated with the aid of intermittent exposure acclimatization. Additionally, pre-exposure to Cu(II) in the absence of substrates strongly inhibited anammox activity. However, the presence of NO2(-) significantly enhanced Cu(II) inhibition. Therefore, such conditions should be avoided to minimize the disturbance of the anammox process.
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