Microscale inhibition of nitrogen removal by sulfamethoxazole in AGS-SBR: Gene-function decoupling and metabolic survival defense

8 mg·L⁻¹ SMX is the threshold, beyond which TN removal severely deteriorates (<30%). • Inhibition stems from AMO/NR activity loss, causing gene-function decoupling. • Thauera abnormally surges under SMX stress but fails to execute actual N-removal. • Microbes shift from N-removal to survival defense, enriching sul1/sul2 ARGs. • SMX activates ABC transporters and quorum sensing for resistance and survival. To investigate the microscale inhibition mechanism of sulfamethoxazole (SMX) on nitrogen removal in Aerobic granular sludge–sequencing batch reactor(AGS–SBRs), system responses were evaluated under gradient SMX concentrations (0–20 mg·L⁻¹). Pollutant sensitivity followed TN > NH₄⁺–N > COD, exhibiting distinct disruption thresholds. Specifically, 8 mg·L⁻¹ emerged as a critical threshold for nitrification inhibition, where NH₄⁺-N removal decreased to 89.88%. As stress intensified to 10 mg·L⁻¹ , TN removal severely deteriorated to below 30%, marking the collapse of denitrification. LC-MS and metagenomic analyses revealed that at low concentrations, SMX was detoxified via S-N bond cleavage and hydroxylation, followed by high-mass coupling polymerization and β-oxidation-driven complete mineralization. However, at 8 mg·L⁻¹ , an enzymatic degradation bottleneck emerged. This accumulation of SMX served as the turning point for microbial succession, significantly inhibiting Nitrosomonas while causing an abnormal abundance surge of Thauera . Under this stress, although nitrogen transformation genes remained abundant, AMO and NR enzymatic activities were suppressed by 12.59% and 33.42%, causing severe gene function decoupling. Consequently, the microbial strategy shifted fundamentally from metabolic degradation to survival defense, evidenced by the marked enrichment of sul1 and sul2 resistance genes, alongside Thauera’s peak contributions to ABC transporters and quorum sensing. These findings provide multidimensional insights for threshold regulation in antibiotic-containing wastewater treatment. Download: Download high-res image (243KB) Download: Download full-size image