Antibiotics sulfamethoxazole alter nitrous oxide production and pathways in estuarine sediments: Evidenced by the N¹⁵-O¹⁸ isotopes tracing

Estuarine antibiotic residues are profoundly impacting microbial nitrogen (N) cycling and associated N₂O production, but the response of N₂O production pathways to antibiotics remains poorly understood. Here, ¹⁵N–¹⁸O labeling technique combined with molecular methods were used to investigate the impacts of sulfamethoxazole on the contribution of ammonia oxidation (nitrifier nitrification, nitrifier denitrification, and nitrification-coupled denitrification) and heterotrophic denitrification (HD) to N₂O production in estuarine sediments. Results showed that environmental concentration of sulfamethoxazole (4 ng/g) promoted the total N₂O production by 17.1% through nitrifier denitrification. Environmentally relevant (40–4000 ng/g) and irrelevant (40,000 ng/g) concentration of sulfamethoxazole drove nitrification denitrification to gradually lose the dominant role in total N₂O production and ammonia oxidation-derived N₂O, replaced by HD and nitrifier nitrification, while total N₂O production were inhibited. Furthermore, when HD dominated the total N₂O production, the HD-derived N₂O increased by 63.6% with sulfamethoxazole concentration reaching 40,000 ng/g. The mechanistic investigation further showed that nitrifying bacteria were more susceptible to sulfamethoxazole than nitrifying archaea and denitrifiers. The increased expression of nirS gene carried by non-dominant denitrifiers improved the ratio of nirS:nosZ and hence increased HD-derived N₂O under high sulfamethoxazole stresses. Overall, our results provide a comprehensive view into how antibiotics regulate N₂O production and its pathways in estuarine sediments.