This study aimed to evaluate the simultaneous and long-term effect of increasing concentrations of three antimicrobials belonging to various classes of antibiotics (β-lactams, fluoroquinolones, and nitroimidazoles) on (1) the efficiency of anaerobic digestion of sewage sludge, (2) qualitative and quantitative changes in microbial consortia responsible for the methane fermentation process, (3) the fate of antibiotic resistance genes (ARGs).. Long-term impact of antimicrobials on the efficiency and the microbial community during anaerobic digestion of sewage sludge

The anaerobic digesters were loaded with substrate supplemented with a mixture of amoxicillin, ciprofloxacin, and metronidazole or only with the substrate (control, K). The study was divided into six experimental series (doses 1 - 6, D1 – D6), depending on the concentration of the antibiotics. Each of the six experimental series lasted an average of 45 days, while the total duration of the experiment was 268 days. A total of 56 samples were collected. All samples taken from the process and control bioreactor were analyzed for methane production and VFA's content. Samples for DNA isolation and sequencing were selected at the beginning, middle, and end of each of the six series, in the case of the process bioreactor (D1-D6). For the same purpose, one sample from the first, fourth and last experimental series was selected from the control bioreactor (K1, K4, K6). Long-term exposure of the sewage sludge to a mixture of metronidazole, amoxicillin, and ciprofloxacin significantly decreased the production of methane and changed the composition of the population of microorganisms. The analysis of the sequencing results also showed that the frequency of readings characteristic for the Archaea, in general, did not translate into the efficiency of biogas production. Moreover, it is probable that the hydrogen-dependent, methylotrophic pathway of methane production may be extremely important in ensuring the stability of anaerobic treatment in the presence of antimicrobial substances.