## 3 Results
### Real-Time PCR Quantification of Genes Specific to Methanogens  
Methanogenic microorganisms are responsible for the last stage of AD-methanogenesis. Methanogenic members of _Archaea_ can be divided based on their methanogenesis pathways; acetoclastic, hydrogenotrophic, and methylotrophic. Among the methanogens, only the order _Methanosarcinules_ is capable of methane production through all three pathways. Moreover, _Methanosarcinules_ are also the only acetoclastic methanogens that have been identified to date [34; 35; 36]. Bacteria belonging to both _Methanosarcinaceae_ and _Methanosactaceae_ families can perform acetoclastic methanogenesis. In this pathway, acetate is activated to acetyl-CoA, either by the combined action of transacetylase and acetate kinase or by the activity of acetyl-CoA synthetase, in the case of _Methanosarcinaceae_ and _Methanosactaceae_, respectively [37]. The abundance of methanogens can be indicative of AD efficiency [4]. The prevalence of the above methanogens are affected by the composition of the fermented substrate and the presence of inhibitors such as antibiotics [5]. The presence of specific methanogenic inhibitors can promote the activity of some methanogenic _Archaea_ that can perform different pathways to produce methane. In this study, the structure of archaeal communities was dominated by families belonging to the order _Methanosarcinules_, so it can be assumed that acetoclastic, hydrogenotrophic, or methylotrophic pathways could be dominant in CH\({}_{4}\) production. In SS samples from PB and CB, the copy numbers of gene characteristics for _Methanosarcinaceae_ remained stable at 10\({}^{4}\) g\({}_{\text{D}}\)\({}^{-1}\) in most experimental series (Figure 4a). In the last sixth experimental series, _Methanosarcinaceae_-specific genes increased to 10\({}^{6}\) in SS samples from both PB and CB. No significant changes (\(p>0.05\)) in the copy numbers of gene characteristics for _Methanosarcinaceae_ were found during the AD of SS (Table S2). The copy numbers of that gene point to a stable increase in _Methanosarcinaceae_ counts and activity during SS fermentation regardless of antibiotic supplementation [23]. Cattle slurry samples were characterized by significantly higher (\(p<0.05\)) variations in the copy numbers of the gene characteristic for _Methanosarcinaceae_ relative to SS samples (Figure 4b; Table S2). Interestingly, in samples collected in the third, sixth, and seventh experimental series, copy numbers of _Methanosarcinaceae_-specific genes were one order of magnitude higher in samples exposed to antibiotics (10\({}^{5}\), 10\({}^{6}\), and 10\({}^{6}\) g\({}_{\text{D}}\)\({}^{-1}\), respectively) than in control samples (10\({}^{4}\), 10\({}^{5}\), and 10\({}^{5}\) g\({}_{\text{D}}\)\({}^{-1}\), respectively). The presence of higher doses of antibiotics disrupted the structure of methanogens, promoting the growth of microorganisms exhibiting the greatest adaptation. According to the literature, microorganisms belonging to the family _Methanosarcinaceae_ have a high level of metabolic capability [6; 38]. In comparison to _Methanosactaceae_, _Methanosarcinaceae_ are characterizedby faster growth and more effective CH\({}_{4}\) production, also under unfavorable conditions inside bioreactors [11]. Figure 3: Ordination analysis of sewage sludge ((**A**) SS) and cattle slurry ((**B**) CS) samples from process and control bioreactors. Principal component analysis (PCA) of the abundance of _mcrA_ gene, genes characteristics for _Methanosarcinaceae_ (MSC), _Methanosatedaceae_ (MST), and methane production. D—dose ID, C—control. in the last experimental series of SS treatments increased intensely (yellow cluster). The predominance of a given methanogenic family in the substrate is determined by the origin of organic matter [40], the ability to metabolize various substrates by methanogenesis ([PERSON] et al., 2016), and environmental factors such as the presence of inhibitors [41], which may ultimately affect the efficiency of treatment.