Beyond EnzymaticPathways: Contribution of ChemicalReactions to Hydroxylamine Oxidation during Heterotrophic Nitrificationby <i>Alcaligenes faecalis</i>

Abstract The heterotrophic nitrifier Alcaligenes faecalis oxidizes ammonium (NH4+) to dinitrogen gas (N2) via the key intermediate hydroxylamine (NH2OH), accompanied by the formation of nitrite (NO2–), nitric oxide (NO), and nitrous oxide (N2O). Although NH4+ and NH2OH oxidation by A. faecalis were previously attributed to the direct nitrogen formation (dnf) gene cluster, recent evidence suggests that NH2OH oxidation to N2 proceeds through chemical reactions. However, the exact mechanism and relative importance of chemical reactions remain unknown. Here, we characterized the formation of N intermediates in batch incubations of A. faecalis and determined the kinetics of NH2OH-mediated chemical reactions in abiotic tests under varying C/N ratios, oxygen, pH, and matrix conditions. Combined with transcriptomic analyses, we resolved the respective enzymatic and chemical N turnover pathways across cultivation conditions. Distinct formation and removal pathways of N intermediates were observed: NH2OH was oxidized via both biotic and abiotic processes, with abiotic contribution accounting for 23–64% of NH2OH removal under environmentally relevant conditions (C/N = 8–16, 21% O2, pH 7) and increasing to 35–86% at elevated O2 (50%) and alkaline pH (9). NO2– formation was exclusively enzymatic, whereas N2O was produced predominantly through chemical reactions of NH2OH with NO. These findings advance our understanding of inorganic N metabolism of A. faecalis and highlight the previously underestimated role of abiotic reactions in N loss.