## 3 Results
### The Long-Term Impact of Antibiotics on CH4 Production and mcrA Gene Abundance
The _mcr_A gene encoding methyl-coenzyme M reductase is specific to methanogens. Changes in the concentration of the _mcr_A gene could point to fluctuations in methanogen activity which is crucial for the AD process. Therefore, the _mcr_A gene is regarded as a specific molecular marker of AD, and its abundance testifies to the presence and activity of methanogens in samples from various environments [30]. In the current study, _mcr_A concentrations in SS and CS samples collected from PB and CB differed across the experimental series. During long-term AD, the average concentrations of the _mcr_A gene were determined at 4.5 \(\times\) 10\({}^{8}\) and 1.3 \(\times\) 10\({}^{8}\) gene copies in 1 g\({}_{\text{D}}\)\({}^{-1}\) of SS samples collected from PB and CB, respectively, and at 7.6 \(\times\) 10\({}^{8}\) and 1.0 \(\times\) 10\({}^{9}\) gene copies in 1 g\({}_{\text{D}}\)\({}^{-1}\) of CS samples collected from PB and CB, respectively (Figure 2). Similar trends in _mcr_A abundance were observed in SS samples collected from PB and CB in successive experimental series (Figure 2). The concentration of _mcr_A increased in the first experimental series, but it declined gradually in the following series (2-6). Significant differences in _mcr_A levels were observed between CS samples collected from PB and CB in the first three experimental series. In these series, _mcr_A concentrations decreased over  
Figure 2: Prevalence of the _mcr_A gene in sewage sludge (SS) and cattle slurry (CS) samples from process (PB) and control bioreactors (CB). The mean values noted in each experimental series were used. time and were significantly higher in PB than in CB. It should be noted that CH\({}_{4}\) yields were comparable in all experimental series in both PB and CB. In CS samples collected from PB and CB, _mcr_A abundance followed a similar increasing trend from the beginning of the fourth experimental series until the end of the experiment. In both substrates, significant differences in _mcr_A concentrations were observed between PB and CB (Table S2). In SS samples, the concentration of the _mcr_A gene and CH\({}_{4}\) yields were higher in control samples. In turn, the efficiency of AD was comparable in CS samples regardless of drug supplementation, and average _mcr_A abundance was significantly higher in CS samples from PB. The scatter plot generated in the principal component analysis (PCA) indicates that CH\({}_{4}\) production in bioreactors containing SS and CS was not correlated with the prevalence of _mcr_A during long-term AD (Figure 3). This long-term experiment confirmed that changes in CH\({}_{4}\) yields could not be reliably measured based on the abundance of the _mcr_A gene, which is characteristic of methanogens. Despite the fact that this gene has been proposed as a bioindicator for monitoring methanogen activity [31], some authors have reported significant differences between _mcr_A abundance and the concentrations of _mcr_A transcripts in fermented substrates [32; 33]. Therefore, the abundance of _mcr_A transcripts should be taken into consideration to improve the reliability of analyses examining AD efficiency at the molecular level.