Competitive inhibition of hydrazine on hydroxylamine oxidation by ammonium-oxidizing bacteria and its stimulative impact on associated N2O production

This work investigated the impact of hydrazine (N2H4), a key intermediate product of the anaerobic ammonium oxidation (anammox) process and a proposed additive to enhance the metabolism of anammox bacteria, on the hydroxylamine (NH2OH) oxidation process mediated by hydroxylamine oxidoreductase (HAO) in ammoniumoxidizing bacteria (AOB). Through batch tests using highly enriched AOB-dominated sludge, we demonstrated that N2H4 competitively inhibited NH2OH oxidation while significantly stimulating nitrous oxide (N2O) production. Molecular docking confirmed the competitive inhibition relationship between NH2OH and N2H4 for the same active sites of HAO, verifying the higher binding affinity of NH2OH (binding energy: -2.3 kcal/mol) compared with N2H4 (binding energy: -1.7 kcal/mol). The interference of N2H4 was further substantiated with dedicated batch tests initiated with NH4+. The competitive inhibition of <= 10 mg-N/L N2H4 was found to induce significant NH2OH accumulation, elevate N2O production and impair partial nitrification efficiency. This work raised not only awareness about the indirect negative impact of the anammox process on the activity and functionality of AOB but also concerns regarding the application of N2H4 to enhance the activity of anammox bacteria, particularly in partial nitrification/anammox systems.