Insights into the individual and combined effects of Cu(Ⅱ) and Ni(Ⅱ) on anammox: Nitrogen removal performance, enzyme activity and microbial community.

Anaerobic ammonium oxidation (anammox) is an efficient and economical nitrogen removal process for treating ammonium-rich industrial wastewaters. However, Cu(Ⅱ) and Ni(Ⅱ) present in industrial wastewaters are toxic to anaerobic ammonium-oxidizing bacteria (AnAOB). Unfortunately, the effects of Cu(Ⅱ) and Ni(Ⅱ) on anammox have not been thoroughly investigated, especially when Cu(Ⅱ) and Ni(Ⅱ) coexist. This work comprehensively investigated the individual and combined effects of Cu(Ⅱ) and Ni(Ⅱ) on anammox and revealed the inhibitory mechanisms. With the influent NH-N and NO-N concentration of 230 and 250 mg L, the inhibition thresholds on anammox are 2.00 mg L Cu(Ⅱ), 1.00 mg L Ni(Ⅱ) and 1.00 mg L Cu(Ⅱ) + 1.00 mg L Ni(Ⅱ), and higher Cu(Ⅱ) or Ni(Ⅱ) concentrations resulted in sharp deteriorations of nitrogen removal performance. The inhibition of Ni(Ⅱ) on anammox was mainly attributed to the adverse effect on NiR activity, while the inhibition mechanism of Cu(Ⅱ) seemed to be unrelated to the four functional enzymes, but associated with disruption of cellular and organellar membranes. The behavior of extracellular polymeric substances (EPS) contributed to the antagonistic effect between Cu(Ⅱ) and Ni(Ⅱ) on anammox. In addition, the niche of Candidatus Brocadia and Candidatus Jettenia shifted under the Cu(II) and Ni(II) stress, and Candidatus Jettenia displayed greater tolerance to Cu(II) and Ni(II) stress. In conclusion, this research clarified the combined effect and the inhibitory mechanism of multiple heavy metals on anammox, and provide the guidances for anammox process application in treating high-ammonium industrial wastewaters containing heavy metals.