Oxygen dependent nitric oxide (NO) and nitrous oxide (N₂O) dynamics during aerobic ammonia oxidation

Ammonia-oxidizing microorganisms (AOM) produce atmospherically active gases, such as nitric oxide (NO) and nitrous oxide (N₂O), as intermediates and byproducts of ammonia oxidation, which contribute to ozone depletion and climate change, respectively. While individual AOM groups have been studied for NO or N₂O production separately, a direct, real-time comparison of oxygen (O₂) consumption and depletion alongside NO and N₂O dynamics across all three groups ammonia-oxidizing bacteria and archaea [AOB and AOA] and complete ammonia oxidizers [comammox] under comparable growth conditions has not been reported. Using microsensors, we simultaneously measured O₂, NO, and N₂O during ammonia oxidation by three terrestrial model AOM species: Nitrosomonas europaea (AOB), Nitrososphaera viennensis (AOA), and Nitrospira inopinata (comammox). Key comparative findings are: 1) N. europaea produced a sharp NO peak (∼150 nM) followed by high N₂O accumulation (13 μM) under hypoxia, consistent with nitrifier denitrification. 2) N. viennensis showed a transient NO peak (181 nM) only after O₂ depletion, followed by elevated N₂O production (33 μM), likely involving NO-dependent hybrid formation, and 3) N. inopinata maintained ultra-low NO (<10 nM) and low N₂O (≤1 μM) throughout the experiment, with no evidence of nitrifier denitrification. The NO scavenger 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) completely inhibited AOA but not AOB, revealing fundamental differences in NO turnover and metabolic dependence between the two groups. All three groups had comparable apparent half-saturation constants for O₂ (K_m(app),O2 = 1.6–3.9 μM), but ammonia affinities varied widely (K_m(app),NH4: AOA 0.086 μM, comammox 0.22 μM and AOB 51.5 μM). Comammox produce substantially less NO and N₂O than AOB while maintaining high ammonia affinity, making it a promising candidate for energy-efficient, low-emission wastewater treatment. These findings clarify niche differentiation among AOM and provide a quantitative framework for understanding and mitigating greenhouse gas emissions from nitrification.