Pathways and controls of N₂O production in nitritation-anammox biomass

Nitrous oxide (N ₂O) is an unwanted byproduct during biological nitrogen removal processes in wastewater. To establish strategies for N ₂O mitigation, a better understanding of production mechanisms and their controls is required. A novel stable isotope labeling approach using ¹⁵N and ¹⁸O was applied to investigate pathways and controls of N ₂O production by biomass taken from a full-scale nitritation-anammox reactor. The experiments showed that heterotrophic denitrification was a negligible source of N ₂O under oxic conditions (≥0.2 mg O ₂ L ^-1). Both hydroxylamine oxidation and nitrifier denitrification contributed substantially to N ₂O 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 ₂O production with both production pathways stimulated by low O ₂, independent of NO ₂ ^- concentrations. The stimulation of N ₂O 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 ₂O 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 ¹⁵N and ¹⁸O isotope labeling is a useful approach for direct quantification of N ₂O production pathways applicable to diverse environments.