## 4 Discussion and Conclusions  
### Comparison of Tolerance to Sodium  
With increased interest in the two-stage anaerobic digester [17], there is a need to extend the study on methanogen tolerance to sodium beyond the single-stage system. Information in literature indicated that the specific sodium inhibitory concentration on  
Figure 5: The increased EPS concentrations as a function of different sodium concentrations at the 6 th and 48 th h of the experiment. Figure 6: The ratio of EPS carbohydrate to EPS protein (EPS\({}_{\mathrm{c}}\)/EPS\({}_{\mathrm{p}}\)) as a function of different sodium concentrations at the 6 th and 48 th h of the experiment. methanogens differed as substrate, reactor configuration, acclimation period, temperature and test method change. Such variation can be seen even in the conventional single-stage system as shown in Table 1[5, 7, 9, 37, 38, 39, 40]. The sodium tolerance of methanogens could be improved by acclimating the biomass to higher sodium conditions [5, 37] or by the presence of other cations through antagonistic/synergistic effects [5]. Step-wise exposure of methanogens to higher sodium concentrations improved their sodium tolerance [38, 39]. The two-stage anaerobic digestion system is potentially more advantageous than the single-stage system with its improved organic solubilization rates, enhanced net energy production and greater pathogen kills [16]. The archaeal community in the methanogenic reactor of a two-stage anaerobic digester may be different and show greater stability to changes in feed conditions compared to the single-stage system [14]. In this study, 2.4 g Na\({}^{+}\) L\({}^{-1}\) sodium did not have an obvious inhibitory effect on the degradation rates of HAc (Figure 1), and this was higher than the 10% sodium inhibitory concentration indicated in Table 1[7, 9, 41]. In the work of [PERSON] et al. [40], a range of 0.7-3 g Na\({}^{+}\)L\({}^{-1}\) leading to 10% inhibition was observed due to the presence of Mg\({}^{2+}\), which overcame the effect of Na\({}^{+}\) inhibition to an extent. As indicated in Figure 1, 3.2 g Na\({}^{+}\) L\({}^{-1}\) showed 17% inhibition on methanogenic activity. This was, not unexpectedly, even lower than the situation at 10% inhibitory sodium concentration with biomass acclimated to higher sodium concentrations [5, 41] and with the presence of other cations [38, 39]. In our preliminary experiment under similar operation conditions (Supplementary Data: Figure S1) without mitigating the effects of other cations, namely, without centrifugation of biomass in Section 2.2, 5.08 g Na\({}^{+}\) L\({}^{-1}\) showed unobservable inhibition on the methanogenic activity of biomass from the second-stage reactor. This value was higher than the widely accepted moderate sodium inhibitory concentration of 3.5-5.0 g Na\({}^{+}\) L\({}^{-1}\) in the single-stage system [42]. The microbial shift within the population in the second-stage reactor could be a result and reason for the higher sodium tolerance. There are contradictory results in the literature on the dominant methanogens in the second-stage reactor of the two-stage anaerobic digester. [PERSON] et al. [14] reported _Methanosaetaceae_ was the dominant methanogens in the second-stage reactor for the treatment of mixed swine slurry and market biowaste. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline \begin{tabular}{c} **Test** \\ **Substrate** \\ \end{tabular}  & **Microorganisms** & **Test System** & \begin{tabular}{c} **Sodium Concentration** \\ **Concentration (g** \\ **Na\({}^{+}\)L\({}^{-1}\))** \\ \end{tabular}  & \ \begin{tabular}{c} **Sodium Concentration** \\ **(g Na\({}^{+}\)L\({}^{-1}\))** \\ **Leading to** \\ **10\% Inhibition** \\ \end{tabular}  & **References** \\ \hline HAc &
\begin{tabular}{c} Mixed culture, \\ presumably \\ _Methanosaetaceae_ \\ Mixed culture, \\ presumably \\ _Methanosaetaceae_ \\ _Met  
However, [PERSON] et al. [43] reported that _Methanosarcinales_ and _Methanomicrobiales_ were mainly affiliated with the methanogenic reactor of a separated system.