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We examined nitrate-dependent Fe2+ oxidation mediated by anaerobic ammonium oxidation (anammox) bacteria. Enrichment cultures of “Candidatus Brocadia sinica” anaerobically oxidized Fe2+ and reduced NO3 − to nitrogen gas at rates of 3.7 ± 0.2 and 1.3 ± 0.1 (mean ± standard deviation [SD]) nmol mg protein−1 min−1 , respectively (37°C and pH 7.3). This nitrate reduction rate is an order of magnitude lower than the anammox activity of “Ca . Brocadia sinica” (10 to 75 nmol NH4 + mg protein−1 min−1 ). A15 N tracer experiment demonstrated that coupling of nitrate-dependent Fe2+ oxidation and the anammox reaction was responsible for producing nitrogen gas from NO3 − by “Ca . Brocadia sinica.” The activities of nitrate-dependent Fe2+ oxidation were dependent on temperature and pH, and the highest activities were seen at temperatures of 30 to 45°C and pHs ranging from 5.9 to 9.8. The mean half-saturation constant for NO3 − ± SD of “Ca . Brocadia sinica” was determined to be 51 ± 21 μM. Nitrate-dependent Fe2+ oxidation was further demonstrated by another anammox bacterium, “Candidatus Scalindua sp.,” whose rates of Fe2+ oxidation and NO3 − reduction were 4.7 ± 0.59 and 1.45 ± 0.05 nmol mg protein−1 min−1 , respectively (20°C and pH 7.3). Co-occurrence of nitrate-dependent Fe2+ oxidation and the anammox reaction decreased the molar ratios of consumed NO2 − to consumed NH4 + (ΔNO2 − /ΔNH4 + ) and produced NO3 − to consumed NH4 + (ΔNO3 − /ΔNH4 + ). These reactions are preferable to the application of anammox processes for wastewater treatment.

Nitrate-Dependent Ferrous Iron Oxidation by Anaerobic Ammonium Oxidation (Anammox) Bacteria