Tracing N(2)O formation in full-scale wastewater treatment with natural abundance isotopes indicates control by organic substrate and process settings

Nitrous oxide (N(2)O) dominates greenhouse gas emissions in wastewater treatment plants (WWTPs). Formation of N(2)O occurs during biological nitrogen removal, involves multiple microbial pathways, and is typically very dynamic. Consequently, N(2)O 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 N(2)O 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 N(2)O monitoring, allow identification of the N(2)O production pathways in a full-scale plug-flow WWTP (Hofen, Switzerland). Heterotrophic denitrification appears as the main N(2)O production pathway under all tested process conditions (0–2 mgO(2)/l, high and low loading conditions), while nitrifier denitrification was less important, and more variable. N(2)O production by hydroxylamine oxidation was not observed. Fractional N(2)O elimination by reduction to dinitrogen (N(2)) during anoxic conditions was clearly indicated by a concomitant increase in site preference, δ(18)O(N(2)O) and δ(15)N(N(2)O). N(2)O reduction increased with decreasing availability of dissolved inorganic N and organic substrates, which represents the link between diurnal N(2)O 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 N(2)O emissions. Our results demonstrate that monitoring of the N(2)O isotopic composition holds a high potential to disentangle N(2)O 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 N(2)O formation or microbial assays to develop efficient N(2)O mitigation strategies.