## 4 Discussion  
### Dynamics of Butyrate Degradation  
In the AD process, acetate is mainly formed during acetogenesis from other VFAs, such as butyrate (Equation (2)) [23]. The electrons produced during the oxidation of butyrate are transferred to protons to generate hydrogen. It must be mentioned that these oxidation reactions are thermodynamically unfavorable and can only proceed when the produced hydrogen is consumed by methanogens to keep a low partial pressure. Finally, methane is produced in methanogenesis from either the oxidation of acetate (Equation (4)) or the reduction of CO\({}_{2}\) by H\({}_{2}\) (Equation (5)), which is the most sensitive step to high VFA concentrations compared to other steps. Acetate can be metabolized to methane in two ways: syntrophic acetate oxidation coupled with hydrogenotrophic methanogenesis (Equations (3) and (5)) and acetoclastic methanogenesis (Equation (4)). \[\mathrm{CH_{3}COO^{-}+4H_{2}O\to 2 HCO_{3}{}^{-}+H^{+}+H_{2}\quad\Delta Go^{ \prime}=+105\ kJ} \tag{3}\]  
\[\mathrm{CH_{3}COO^{-}+H_{2}O\to CH_{4}+HCO_{3}{}^{-}\quad\Delta Go^{\prime}=-3 1\ kJ} \tag{4}\]  
\[4H_{2}+2 HCO_{3}{}^{-}+H^{+}\to CH_{4}+3H_{2}O\quad\Delta Go^{\prime}=-131\ kJ \tag{5}\]  
Excessive acetate (Figure 1A) was probably formed due to biomass hydrolysis and the decomposition of yeast extract (a component of the medium, 0.1 g/L). Acetate could also be produced from H\({}_{2}\) and CO\({}_{2}\) by homoacetogenic bacteria. Since the affinity of homoacetogens for H\({}_{2}\) is lower than that of hydrogenotrophic methanogens, they cannot compete and exhibit low activity in balanced methanogenic communities [38]. However, under unfavorable conditions, e.g., in the presence of inhibitors of methanogenic archaea or at high substrate loads, homoacetogenic bacteria gain an advantage for growth [39]. _Methanothrix_ spp. is the most common acetoclastic methanogen in bioreactors with low acetate concentrations, including those for the treatment of mixed sewage sludge [40; 41; 42]. _Methanosarcina_ spp. is more abundant when acetate concentration in the medium increases, especially in the case of thermophilic communities [43]. Members of the genus _Methanosarcina_ have a high capacity for adaptation to varying environmental conditions, and their increased abundance therefore promotes the stabilization of the community [44]. It was shown previously that the adaptation of the methanogenic community may occur due to the accumulation of _Methanothrix_ spp. [45] and specific aggregation [41], when the concentration of acetate increases gradually. No data are available on the inhibitory concentrations of butyrate used as a sole carbon source. [PERSON] et al. considered the inhibitory effect of butyrate indirect and associated primarily with rapid acetate accumulation in the medium [16]. Our findings (Table 1) are in agreement with the results of [PERSON] et al. [46], who reported halving the methane production in the presence of 220, 170, and 48 mM of acetate, butyrate, and propionate, respectively. The inhibitory effect of acetate was previously shown to be more pronounced than that of butyrate [16; 47]. Literature data show differences in inhibitory concentrations of acetate. [PERSON] et al. reported a decrease in the activity of acetoclastic methanogens at 68 mM acetate [46]. [PERSON] and [PERSON] found that 150 mM acetate concentration caused the destabilization of a large-scale thermophilic reactor fed with SS and OF-MSW [48]. According to [PERSON] et al., inhibitory acetate concentrations varied from 23 to 81 mM [17]. [PERSON] et al. showed that acetate in concentrations from 5 to 100 mM affected the rate of thermophilic methanogenesis: the rate of methane production was higher at lower acetate concentrations [45]. The latter may vary, however, depending on the degree of the community adaptation to experimental conditions, as can be seen from our results. We observed the inhibition of methanogenesis at acetate concentrations above 50 mM in the first transfer.