## 3 Results  
### The Long-Term Impact of Antibiotics on CH4 Production and mcrA Gene Abundance  
In PB containing SS, exposure to increasing concentrations of AMO, CIP, and MET significantly decreased the efficiency of CH\({}_{4}\) production (by 5-8%) relative to control (\(p<0.05\)) during long-term AD (Table S2). However, CH\({}_{4}\) production trends were similar in both experimental and control samples of SS (Figure S1). The amount of CH\({}_{4}\) produced from SS during AD was 176 and 182 L kg VS\({}^{-1}\) on average in PB and CB, respectively. Average CH\({}_{4}\) yields were significantly higher in PB than in CB (by 6%; 10 L kg VS\({}^{-1}\)) only in the third experimental series. The greatest differences in the efficiency of biogas production between PB and CB were noted in SS supplemented with AMO, CIP, and MET doses of 2, 0.5, and 0.5 ug mL\({}^{-1}\) (series 2) and 8, 4, and 4 ug mL\({}^{-1}\) (series 4), respectively (Figure 1). No significant differences in CH\({}_{4}\) production during AD were found between PB and CB containing CS (\(p>0.05\)) (Figure 1 and Figure S1, Table S2). In all seven experimental series, the average CH\({}_{4}\) yields were more than twice lower in PB and CB containing CS (87 and 86 L kg VS\({}^{-1}\), respectively) than in PB and CB containing SS. These results indicate that the tested antibiotics did not significantly affect the AD of CS and that CS fermentation was significantly less effective than SS fermentation in terms of CH\({}_{4}\) yields. The organic matter in CS is more difficult to digest than the organic matter in SS [25]. Importantly, in this study, the substrate such as CS was characterized by a particular variability of individual parameters depending on the sampling time during a long-term experiment. The values of vs. and TS were variable, depending on the intensity of precipitation and the season of the year, which resulted in high standard deviations of these values (Table 1). Moreover, both SS and CS represent different environments characterized above all by diverse communities of microbes. Large amounts of bacterial and archaeal populations are involved in the complex microbiological process, such as AD. There are numerous studies indicating the benefits of anaerobic co-digestion of various types of organic waste, among others SS and manure [25; 26; 27]. The literature shows that co-digestion can enable the rise of organic matter levels, improve the activity of individual microorganisms in the anaerobic system and the stability of anaerobic biomass, and finally increase the efficiency of methane production. Figure 1: Heatmap presenting average methane production (L kg VS\({}^{-1}\)) in each experimental series in process (PB) and control bioreactors (CB) containing sewage sludge (SS) and cattle slurry (CS). In a previous study analyzing the influence of high individual antibiotic doses on the AD of SS and CS [5], biogas yields in both tested substrates were significantly lower under exposure to the tested drugs. The accumulation of VFAs in PB also testified to the inhibitory effects exerted by antibiotics on various stages of AD. In the present study, no significant differences in VFA concentrations (\(p>0.05\)) were observed between SS and CS samples collected from PB and CB (Table S3). These results indicate that the mixtures of AMO, CIP, and MET as well as AMO, ENRO, and MET did not exert synergistic effects within the tested range of drug concentrations. According to other authors [28], the AD process is inhibited, and its efficiency declines rapidly only when a threshold concentration of antibiotics has been reached. Moreover, the fact that the differences between the average CH\({}_{4}\) yields in PB and CB containing SS decreased during long-term AD could be attributed to the gradual acclimation of methanogenic microorganisms to antimicrobials [29]. The efficiency of AD was monitored not only by measuring CH\({}_{4}\) and VFA production but was also evaluated at the molecular level.