Pathways and Controls of N2O Production in Nitritation–Anammox Biomass

Nitrous oxide (N 2 O) is an unwanted byproduct during biological nitrogen removal processes in wastewater. To establish strategies for N 2 O mitigation, a better understanding of production mechanisms and their controls is required. A novel stable isotope labeling approach using 15 N and 18 O was applied to investigate pathways and controls of N 2 O production by biomass taken from a full-scale nitritation-anammox reactor. The experiments showed that heterotrophic denitrification was a negligible source of N 2 O under oxic conditions (≥0.2 mg O 2 L –1 ). Both hydroxylamine oxidation and nitrifier denitrification contributed substantially to N 2 O accumulation across a wide range of conditions with varying concentrations of O 2, NH 4 +, and NO 2 – . The O 2 concentration exerted the strongest control on net N 2 O production with both production pathways stimulated by low O 2, independent of NO 2 – concentrations. The stimulation of N 2 O production from hydroxylamine oxidation at low O 2 was unexpected and suggests that more than one enzymatic pathway may be involved in this process. N 2 O production by hydroxylamine oxidation was further stimulated by NH 4 +, whereas nitrifier denitrification at low O 2 levels was stimulated by NO 2 – at levels as low as 0.2 mM. Our study shows that 15 N and 18 O isotope labeling is a useful approach for direct quantification of N 2 O production pathways applicable to diverse environments.