## 3 Results  
### Dynamics of Processes Involved in Syntrophic Degradation of Butyrate  
A consortium of thermophilic bacteria and archaea adapted to high concentrations of butyrate and acetate was obtained by sequentially transferring the biomass of thermophilically digested sewage sludge into the fresh medium with higher concentrations of butyrate, i.e., 20, 50, 95 and 170 mM, respectively. The dynamics of methane production via syntrophic butyrate degradation followed by the production and degradation of acetate is shown in Figure 1, and the rates of the processes are given in Table 1.  
Experiments started at an initial butyrate concentration of 20 mM. At this concentration, the complete degradation of butyrate with the production of methane as the final product proceeded in three stages (Figure 1A). During the first stage (10 days), butyrate degradation was accompanied by the rapid accumulation of acetate (up to 60 mM). The \begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**Maximum/Average Rates, mmol/(L Day)**} & \multicolumn{4}{c}{**Initial Butyrate Concentration, mmol/L**} \\ \cline{2-5}  & **20** & **50** & **95** & **170** \\ \hline butyrate degradation & 7.1/1.3 & 1.6/1.2 & 10.3/4.5 & 7.5/3.3 \\ acetate production & 15.7/4.8 & 3.6/2.8 & 3.3/3.1 & 7.7/3.8 \\ acetate consumption & 3.65/1.2 & 3.1/1.8 & 10.1/5.8 & 1.03/- \\ methane production from butyrate & 3.3/0.48 & 3.6/2.5 & 21.2/8.3 & 9.4/2.8 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: The rates of processes involved in the syntrophic degradation of butyrate. Figure 1: Dynamics of anaerobic butyrate degradation at different initial concentrations of butyrate: (**A**) 20 mmol/L, (**B**) 50 mmol/L, (**C**) 95 mmol/L and (**D**) 170 mmol/L. amount of acetate produced was not equimolar, i.e., the measured acetate concentration was 1.5 times higher than the one calculated using the Equation (2) [37]:  
\[\text{CH}_{3}\text{CH}_{2}\text{CH}_{2}\text{COO}^{-}+2\text{H}_{2}\text{O} \to 2\text{CH}_{3}\text{COO}^{-}+\text{H}^{+}+2\text{H}_{2}\quad\Delta\text{G} \text{o}^{\prime}=+48\text{ kJ} \tag{2}\]  
The consortium was further transferred to the fresh medium with 50 mM butyrate, after the acetate was consumed completely (Figure 1B). While the rates of butyrate degradation at 50 mM were lower than at 20 mM, the process of methane production was balanced better (Table 1). During the first 20 days of the experiment, 30.2 out of 50 mM butyrate was degraded, and 50.8 mM acetate was accumulated in the medium, which was at least 16% below the maximal theoretically calculated value. This was an indication of coupled processes of syntrophic butyrate degradation and acetoclastic methanogenesis. Hydrogen was detected in the gas phase during the first day of incubation (0.3 mM) and close to the end of the experiment (2.24 mM). No significant effect of H\({}_{2}\) on the activity of syntrophic bacteria was observed. The most efficient butyrate degradation and methane production was observed, when the enrichment culture was further transferred into the medium with 95 mM butyrate (Figure 1C, Table 1). Butyrate decomposition occurred at the highest rate of 10.3 mmol/(L day) and was completed on day 23 of incubation. Hydrogen (up to 0.5 mM) was detected on day 3 and was consumed within 24 h. The highest acetate concentration was 58.8 mM (\(\sim\)47% of the theoretically calculated maximum value), and methane production rate reached the highest value of 21.2 mmol/(L day). This indicated that the microbial community contained active butyrate-degrading syntrophic bacteria, hydrogenotrophic and acetoclastic methanogens and became resistant to high substrate concentrations. Thus, the butyrate in concentrations of up to 100 mM had no inhibitory effect on the enrichment culture. The subsequent increase in butyrate concentration to 170 mM resulted in a decrease in rates of butyrate degradation and methane production (Figure 1D, Table 1).