On-line analysis of N 2 O isotopic composition during biological nitrogen removal in wastewater treatment to disentangle production and reduction processes

Nitrous oxide (N 2 O) is a potent greenhouse gas, and emissions from wastewater treatment plants (WWTPs) represent a significant and highly variable source. Understanding the dynamics in microbial pathways of N 2 O formation and reduction during biological nitrogen removal is essential for targeted mitigation strategies. Stable isotope analysis of N 2 O ( δ 15 N α , δ 15 N β , δ 18 O, and 15 N site preference) provides a powerful tool to disentangle and quantify N 2 O production and reduction processes, yet conventional analytical approaches lack temporal resolution. Here, we present the first long-term application of an off-axis integrated cavity output spectrometer for real-time N 2 O isotopic analysis at a pilot-scale WWTP over one year of operation. We developed a dynamic dilution system and implemented correction protocols for drift, N 2 O mole fraction dependence, and gas matrix effects on isotopic results, achieving uncertainties of 0.8 ‰ ( δ 15 N α ), 1.1 ‰ ( δ 15 N β ), 0.8 ‰ ( δ 15 N bulk ), 0.5 ‰ ( δ 18 O) and 1.1 ‰ ( 15 N site preference). Representative datasets demonstrate the system's capability to (i) identify dominant N 2 O production pathways under standard WWTP operation, (ii) quantify N 2 O reduction in relation to dissolved oxygen concentration, and (iii) trace nitrogen transformation during low-level 15 N-labelling experiments. Our results indicate nitrifier or heterotrophic denitrification as the main source of N 2 O, and that N 2 O reduction efficiency is strongly controlled by oxygen availability. This study highlights the potential of laser spectroscopy for continuous isotopic monitoring in real-world engineered systems and provides practical guidelines for uncertainty reduction and data interpretation. More specifically, our work forms a foundation for further investigations of the operational factors controlling N 2 O formation and N 2 O reduction in biological WWTPs and other complex anthropogenically-perturbed settings.