Beyond Enzymatic Pathways: Contribution of Chemical Reactions to Hydroxylamine Oxidation during Heterotrophic Nitrification by Alcaligenes faecalis

The heterotrophic nitrifier Alcaligenes faecalis oxidizes ammonium (NH 4 + ) to dinitrogen gas (N 2 ) via the key intermediate hydroxylamine (NH 2 OH), accompanied by the formation of nitrite (NO 2 – ), nitric oxide (NO), and nitrous oxide (N 2 O). Although NH 4 + and NH 2 OH oxidation by A. faecalis were previously attributed to the direct nitrogen formation ( dnf ) gene cluster, recent evidence suggests that NH 2 OH oxidation to N 2 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 NH 2 OH-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: NH 2 OH was oxidized via both biotic and abiotic processes, with abiotic contribution accounting for 23–64% of NH 2 OH removal under environmentally relevant conditions (C/N = 8–16, 21% O 2 , pH 7) and increasing to 35–86% at elevated O 2 (50%) and alkaline pH (9). NO 2 – formation was exclusively enzymatic, whereas N 2 O was produced predominantly through chemical reactions of NH 2 OH 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.