Dynamics of N₂O production pathways analyzed by ¹⁵N¹⁸O isotope labeling

Nitrous oxide production associated with biological nitrogen transformations can contribute substantially to the CO2 footprint of both man-made and natural systems, but the pathways and regulation of N2O production are poorly understood. We developed a 15N/18O dual isotope labelling technique to distinguish and quantify these pathways in mixed communities. The use of 18O-O2 permits differentiation of hydroxylamine oxidation and nitrifier-denitrification driven N2O production by ammonium oxidizing bacteria. We analysed N2O production pathways during biological nitrogen removal at Lynetten wastewater treatment plant. Under anoxia, N2O accumulated due to denitrification, but N2O accumulation was ~3 and 1.7 times higher at 30 and 100 µM O2, respectively. Oxic N2O production was dominated by nitrifier-denitrification, reaching 73% of the total with the remainder due to hydroxylamine oxidation. Our results demonstrate three active pathways of N2O production, each with different environmental controls. The dual 15N/18O isotope labelling approach can contribute to the development of strategies to minimise N2O emissions from man-made and natural systems.