Dual isotope labelling combined with multi-omics analysis revealing the N2O source evolution in aerobic biological systems driven by salinity gradient

Salinity is considered a major factor influencing nitrous oxide (N 2 O) emissions from biochemical treatment of high-salinity wastewater, but its mechanism has not been thoroughly investigated. In this study, we investigated the effects of salinity on N 2 O emissions under aerobic conditions. As salinity rose from 0.66 % to 3.66 %, N 2 O emission flux first increased and then decreased, while the emission factor (EF) consistently increased, likely due to significant inhibition of nitrification at 3.66 % salinity. Nitrogen‑oxygen dual isotope labeling experiments demonstrated that the dominant N 2 O production pathway shifted with salinity: from nitrifier nitrification (NN, 36.07 %–40.97 %) at low salinity (0.66 %, 1.66 %), to nitrification-coupled denitrification (NCD, 51.67 %) at 2.66 %, and to nitrifier denitrification (ND, up to 80.81 %) at the salinity of 3.66 %. From the changes in bacterial relative abundances and expressions of 4 key functional genes ( amoA , hao , nor , and nosZ ) revealed by metatranscriptomic sequencing, Nitrosomonas , unclassified Rhodospirillales , and Nitrospira were identified as key contributors to NN, NCD, and ND pathways, respectively, as salinity increased. We also found that the differential expressed genes and metabolites involved in energy metabolism, oxidative phosphorylation, and metabolism of amino acids, pyrimidines, and nucleotides may affect N-cycling bacteria, thereby influencing nitrogen conversion and salinity tolerance as well. This study sheds light on nitrification process in response to salinity stress and offers insights for mitigating greenhouse gas emissions from high-salinity wastewater treatment.