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Nitrous oxide (NO) is an unwanted byproduct during biological nitrogen removal processes in wastewater. To establish strategies for NO mitigation, a better understanding of production mechanisms and their controls is required. A novel stable isotope labeling approach using N andO was applied to investigate pathways and controls of NO production by biomass taken from a full-scale nitritation-anammox reactor. The experiments showed that heterotrophic denitrification was a negligible source of NO under oxic conditions (≥0.2 mg O L). Both hydroxylamine oxidation and nitrifier denitrification contributed substantially to NO accumulation across a wide range of conditions with varying concentrations of O, NH, and NO. The O concentration exerted the strongest control on net NO production with both production pathways stimulated by low O, independent of NO concentrations. The stimulation of NO production from hydroxylamine oxidation at low O was unexpected and suggests that more than one enzymatic pathway may be involved in this process. NO production by hydroxylamine oxidation was further stimulated by NH, whereas nitrifier denitrification at low O levels was stimulated by NO at levels as low as 0.2 mM. Our study shows that N andO isotope labeling is a useful approach for direct quantification of NO production pathways applicable to diverse environments.

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