How anammox process resists the multi-antibiotic stress: Resistance gene accumulation and microbial community evolution

The effects of multiple antibiotics on the anaerobic ammonia oxidation (anammox) process were investigated. The resistance of the anammox system to high-concentration antibiotics was also demonstrated through gradual acclimation experiments. Inhibition of the anammox process (R1) occurred when the concentrations of erythromycin (ERY), sulfamethoxazole (SMX) and tetracycline (TC) were 0.1, 5.0 and 0.1 mg L-1, respectively. The nitrogen removal efficiency (NRE) of R1 was reduced from 97.2% to 60.7% within 12 days and then recovered to 88.9 ± 9.5% when the nitrogen loading declined from 4.52 ± 0.69 to 2.11 ± 0.58 kg N m-3 d-1. Even when the concentrations of ERY, SMX and TC were as high as 1.0, 15.0 and 1.0 mg L-1, respectively, R1 maintained stable operation. The increases in the abundance of antibiotic resistance genes (ARGs) and in extracellular polymeric substances (EPS) content showed that the anammox process alleviated stress from multiple antibiotics mainly by producing ARGs and secreting EPS. The molecular docking simulation results illustrated the potential binding sites between ammonium transporter and different antibiotics. The upregulation of functional gene expression and the stable abundance of Candidatus Kuenenia in R1 compared with that in the control suggested that the R1 reactor generally maintained more stable long-term operation. This work provides a new understanding of the application of the anammox process to treat wastewater containing multiple antibiotics.