Pathways and Controls of N2O Production in Nitritation-Anammox Biomass

Nitrous oxide (N2O) is an unwanted byproduct during biological nitrogen removal processes in wastewater. To establish strategies for N2O mitigation, a better understanding of production mechanisms and their controls is required. A novel stable isotope labeling approach using N-15 and O-18 was applied to investigate pathways and controls of N2O production by biomass taken from a full-scale nitritation-anammox reactor. The experiments showed that heterotrophic denitrification was a negligible source of N2O under oxic conditions (>= 0.2 mg O-2 L-1). Both hydroxylamine oxidation and nitrifier denitrification contributed substantially to N2O accumulation across a wide range of conditions with varying concentrations of O-2, NH4+, and NO2. The O-2 concentration exerted the strongest control on net N2O production with both production pathways stimulated by low O-2, independent of NO2- concentrations. The stimulation of N2O production from hydroxylamine oxidation at low O-2 was unexpected and suggests that more than one enzymatic pathway may be involved in this process. N2O production by hydroxylamine oxidation was further stimulated by NH4+, whereas nitrifier denitrification at low O-2 levels was stimulated by NO2 at levels as low as 0.2 mM. Our study shows that 15N and 180 isotope labeling is a useful approach for direct quantification of N2O production pathways applicable to diverse environments.