Tracing N₂O formation in full-scale wastewater treatment with natural abundance isotopes indicates control by organic...

Nitrous oxide (N₂O) dominates greenhouse gas emissions in wastewater treatment plants (WWTPs). Formation of N₂O occurs during biological nitrogen removal, involves multiple microbial pathways, and is typically very dynamic. Consequently, N₂O mitigation strategies require an improved understanding of nitrogen transformation pathways and their modulating controls. Analyses of the nitrogen (N) and oxygen (O) isotopic composition of N₂O and its substrates at natural abundance have been shown to provide valuable information on formation and reduction pathways in laboratory settings, but have rarely been applied to full-scale WWTPs. Here we show that N-species isotope ratio measurements at natural abundance level, combined with long-term N₂O monitoring, allow identification of the N₂O production pathways in a full-scale plug-flow WWTP (Hofen, Switzerland). Heterotrophic denitrification appears as the main N₂O production pathway under all tested process conditions (0-2 mgO₂/l, high and low loading conditions), while nitrifier denitrification was less important, and more variable. N₂O production by hydroxylamine oxidation was not observed. Fractional N₂O elimination by reduction to dinitrogen (N₂) during anoxic conditions was clearly indicated by a concomitant increase in site preference, δ¹⁸O(N₂O) and δ¹⁵N(N₂O). N₂O reduction increased with decreasing availability of dissolved inorganic N and organic substrates, which represents the link between diurnal N₂O emission dynamics and organic substrate fluctuations. Consequently, dosing ammonium-rich reject water under low-organic-substrate conditions is unfavorable, as it is very likely to cause high net N₂O emissions. Our results demonstrate that monitoring of the N₂O isotopic composition holds a high potential to disentangle N₂O formation mechanisms in engineered systems, such as full-scale WWTP. Our study serves as a starting point for advanced campaigns in the future combining isotopic technologies in WWTP with complementary approaches, such as mathematical modeling of N₂O formation or microbial assays to develop efficient N₂O mitigation strategies.