## 3 Results and Discussion
### The Effects of Antimicrobials on Changes in the Diversity of Methanogenic Microorganisms  
Among the methanogenic _Archaea_ involved in the AD processes, the ones most often mentioned in the literature are microorganisms from the order _Methanosarcinales_, with the prevalence of two families, _Methanosarcinaceae_ and _Methanosetaceae_[55]. In this experiment, the number of copies of the genes that are characteristic of two families, _Methanosarcinaceae_ (_MSC_ gene) and _Methanosetaceae_ (_MST_ gene), were assayed, excluding in the preliminary study the presence in CS and SS digestate samples of other families from the domain _Archaea_. The families _Methanosataceae_ and _Methanosarcinaceae_ are acetoclastic microorganisms, sensitive to changes in environmental conditions and to the presence of such inhibitors as antimicrobial pharmaceuticals. _Methanosarcinaceae_ continue stable growth at higher doses of acetate [36], unlike _Methanosataceae_, whose growth can be restrained in the presence of this compound [56]. In our research, regardless of the type of antibiotic added to a substrate, a decrease in the number of _MSC_ and _MST_ gene copies was observed in most of the digestate samples from SS and CS substrates (Figure 2C-F). It is significant to notice that the SS digestate samples were dominated by the _MST_ genes (Figure 2E), while the CS digestate was observed to be dominated by _MSC_ genes (Figure 2D), which proves that different groups of methanogens can be responsible for CH4 production, regardless of the substrate applied in AD. The number of copies of the _MSC_ gene in SS samples ranged from 1.19 \(\times\) 10\({}^{5}\) to 6.33 \(\times\) 10\({}^{6}\), whereas the number of the _MST_ gene copies was from 1.80 \(\times\) 10\({}^{6}\) to 6.33 \(\times\) 10\({}^{6}\) in 1 g of digestate (Figure 2C,E). In the control sample, the number of copies of the _MSC_ gene was 7.26 \(\times\) 10\({}^{5}\), whereas the number of the _MST_ gene copies equaled 5.76 \(\times\) 10\({}^{6}\) per gram of digestate. Compared to the control sample, a statistically significant decrease in the number of the _MST_ gene copies was recorded, which may have been due to the selection pressure exerted by the drugs and to the concentrations of VFAs recorded in the AD reactors [56] (Table 2). The research results concerning the number of gene copies justify the claim that despite the presence of antibiotics in digestates from SS and a decline in the number of the _MST_ gene copies, microorganisms from the family _Methanosetaceae_ persisted in a large number until the AD process terminated. These research results are inconsistent with the data reported by other authors [57; 58; 59], who also demonstrated that of the two families analyzed in this experiment, it is _Methanosataceae_ that dominates throughout the anaerobic conversion of sewage sludge biomass. Relative to the control, the CS digestate samples exposed to MET, AMO or SMX demonstrated a considerable decrease in the number of _MST_ and _MSC_ genes. Interestingly, the presence of OXY in the CS samples resulted in a significant rise in the concentration of _MSC_ genes compared with the control, coinciding with a significant increase in the number of the _mcr_A gene copies, which nevertheless did not translate into a higher efficiency of CH4 production, which in fact was considerably decreased due to the exposure to this antibiotic (Figure 2B). The antimicrobial drugs MET and AMO had the strongest impact on the increase in the abundance of methanogens in substrates, which is evidenced by a decrease in the number of the _MST_ gene copies by as much as two orders of magnitude (\(10^{4}\) copies in 1 g of digestate with MET) compared to the control (\(10^{6}\) copies in 1 g of digestate) (Figure 2F). Out of the two analyzed families, _Methanosarcinaceae_ occurred most numerously in the CS digestate, which is evidenced by the high number of the _MSC_ gene copies (from 2.14 \(\times\)\(10^{6}\) to 8.02 \(\times\)\(10^{7}\) copies in 1 g of analyzed digestate).