Molecular ecological insights into the synergistic response mechanism of nitrogen transformation, electron flow and antibiotic resistance genes in aerobic activated sludge systems driven by sulfamethoxazole and/or trimethoprim stresses

Abstract

The prevalence of antibiotics poses a serious challenge to biological nitrogen removal in wastewater. In this study, the effects of sulfamethoxazole and/or trimethoprim (15 mg/L∼30 mg/L) on treatment performance, nitrogen transformation and antibiotic resistance genes (ARGs) were investigated in aerobic activated sludge systems to elucidate the metabolic mechanism under high antibiotic stress. 15 mg/L single antibiotic stress improved total nitrogen removal performance due to the persistence of nitrifiers and enrichment of denitrifiers, with an optimum removal efficiency of 96.5%. Up-regulation of all denitrifying genes, coupled with enhanced electron transfer of Complex II and III, contributed to the emergence of aerobic denitrification. The increased expression of antioxidant genes also alleviated intracellular pressure. Whereas combined antibiotic stress induced the significant down-regulation of denitrifying bacteria and genes (nirKS and nosZ), and suppressed the electron supply for denitrification by restraining genes related to Complex Ⅰ and energy supply by tricarboxylic acid cycle, driving the collapse of activated sludge system, with ammonia and total nitrogen removal efficiencies dropping to below 40% and 20%, respectively. The dominant genera in system changed from TM7a to Thiothrix and Sphaerotilus with increasing antibiotic concentration and type. Moreover, antibiotic stress promoted a slight enrichment of ARGs, especially those encoding efflux mechanisms. Cooperative relationships (> 93%) dominated among ARGs, and Klebsiella was identified as the crucial host. ARGs regulating antibiotic efflux were more likely to be co-expressed with functional genes. These results may provide a theoretical basis for establishing promising strategies to mitigate antibiotic-caused process deterioration.