Activating specific N2O production pathways to understand emission dynamics in wastewater treatment

• New isotope-validated method disentangles N 2 O formation pathways in WWTPs. • Online stable isotope analysis showed that the hydroxylamine pathway is negligible. • Controlling NH 4 + and DO enabled activation of individual denitrification pathways. • DO, organics and pH effects guide N 2 O emission mitigation. Reducing nitrous oxide (N₂O) emissions from wastewater treatment plants is essential for achieving the sector’s net-zero goals by 2050. Yet the complexity of operating full-scale wastewater treatment obscures our understanding of the mechanisms behind N₂O formation and hence, the development of targeted mitigation strategies. Three main pathways are known to contribute to net N₂O emissions, but their relative and absolute importance shifts over time and is controlled by the interplay of multiple parameters. To date, attempts to experimentally disentangle these mechanisms under conditions representative of full-scale systems have been unsuccessful. This study developed and validated a novel methodology to distinguish major N₂O production mechanisms. The methodology was implemented over one year of operation in two parallel pilot reactors treating dynamically varying municipal wastewater directly extracted from an actual sewer system, thereby closely replicating full-scale conditions. Online isotope analysis was used to validate pathway separation. Stable isotopes showed that the N₂O production by the hydroxylamine pathway is negligible under all studied conditions. Nitrifier denitrification was successfully isolated from heterotrophic denitrification by controlling the concentration of ammonium, nitrite, and dissolved oxygen and became more active at lower dissolved oxygen concentrations. Lower dissolved oxygen, higher organic carbon availability, and lower pH increased N₂O production by heterotrophic denitrification during aeration. These new insights provide a systematic framework for understanding N 2 O dynamics and support the development of mitigation strategies at full-scale.