Chronic impacts of oxytetracycline on mesophilic anaerobic digestion of excess sludge: Inhibition of hydrolytic acidification and enrichment of antibiotic resistome.

We evaluated the chronic impact of oxytetracycline (OTC) on performance and antibiotic resistance development during the mesophilic anaerobic digestion (AD) of antibiotic-containing biomass. Mesophilic AD was conducted in a completely stirred tank reactor by constantly feeding municipal excess sludge spiked with increasing concentrations of OTC (0-1000 mg L-1) under a solid retention time of 20 days over a period of 265 days. Results showed that methane generation of mesophilic AD was inhibited when the OTC concentration in digested sludge was increased to around 18,000 mg kg-1 (OTC dose, 1000 mg L-1), due to the inhibition of fermenting and acidogenic bacteria. Metagenomic sequencing and high-throughput quantitative PCR analysis demonstrated that tetracycline resistance genes were the most dominant type (38.47-43.76%) in the resistome, with tetG, tetX, tetM, tetR, tetQ, tetO, and tetL as the dominant resistant subtypes throughout the whole experimental period. The relative abundance of these tet genes increased from 2.10 × 10-1 before spiking OTC (OTC concentration in digested sludge, 8.97 mg kg-1) to 2.83 × 10-1 (p < 0.05) after spiking OTC at a dose of 40 mg L-1 (OTC concentration in digested sludge, 528.52 mg kg-1). Furthermore, mobile genetic elements, including integrons, transposons, and plasmids, were also enriched with the increase in OTC dose. Based on partial canonical correspondence analysis, the contributions of horizontal (mobile element alteration) and vertical (bacterial community shift) gene transfer to antibiotic resistome variation were 29.35% and 21.51%, respectively. Thus, considering the inhibition of hydrolytic acidification and enrichment of antibiotic resistome, mesophilic AD is not suggested to directly treat the biomass containing OTC concentration higher than 200 mg L-1.