Effects of sulfamethoxazole on coupling of nitrogen removal with nitrification in Yangtze Estuary sediments

Coupling of nitrogen removal processes with nitrification (NR_n) are vital synergistic nitrogen elimination mechanisms in aquatic environments. However, the effects of antibiotics on NR_n are not well known. In the present work, 20-day continuous-flow experiments combined with ¹⁵N tracing techniques and quantitative PCR were performed to simulate the impact of sulfamethoxazole (SMX, a sulfonamide antibiotic) with near in situ concentration on NR_n processes in sediments of Yangtze Estuary. Results showed that SMX with near in situ concentration significantly decreased NR_n, NR_w (uncoupling of nitrogen removal processes with nitrification) and actual nitrogen removal rates via inhibiting nitrogen transformation functional genes (AOB, narG, nirS, nosZ) and anammox 16S rRNA gene, while the coupling links between nitrification and nitrogen removal processes were not broken by the exposure. The proportion of NR_n in total nitrogen removal processes decreased by approximately 10% with SMX addition, due to the different inhibition on AOB, denitrifying genes and anammox 16S rRNA gene. N₂O production and nitrite accumulation remarkably increased with SMX addition under simultaneous nitrification and denitrification, and they strongly correlated with each other. The more severely inhibition on nirS gene (13.6-19.8%) than Nitrospira nxrB gene (0.3-8.2%) revealed that the increased nitrite accumulation with SMX addition mainly occurred in heterotrophic denitrification, suggesting that the increased N₂O production was dominated by the heterotrophic nitrite reduction. Moreover, we estimated that the ratio of external inorganic N eliminated by actual nitrogen removal can upgrade to 6.4-7.4% under circumstances of no inhibition by SMX. This study revealed the effects of SMX with near in situ concentration on NR_n processes and illustrated the microbial mechanism on functional genes level. Our results highlighted the inhibitory effects of SMX on NR_n may contribute to reactive N retention and N₂O production in estuarine and coastal ecosystems.