## 4 Discussion and Conclusions
### Sodium Inhibition Mechanism in the Second-Stage Reactor  
A high sodium concentration would induce unbalanced osmotic pressure and cause microbial cells to dehydrate [58], which would then lead to cell death and process failure [59; 60]. In this study, the sodium inhibition mechanism on biomass activity was further investigated by ATP measurement and an EPS test. As the currency of energy, ATP can be rapidly destroyed within dead cells [61]. ATP measurements were utilized to indicate the total biomass activity in the anaerobic digestion system [29]. The ATP content of sludge was reported to couple well with other common indicators in the anaerobic system, such as the VFA, biogas and sludge age during different operation stages [62]. In this two-stage anaerobic digester, as indicated in Figure 4, the increase in sodium concentration led to a decrease in the total biomass activity (i.e., the specific ATP concentration). The results from [PERSON] and [PERSON] [63] support this conclusion with a decrease in ATP concentration that was observed after incubating the biomass with an inhibitory sodium concentration in a single-stage anaerobic system. At a high sodium concentration, the lower ATP value might be caused by the excess consumption of ATP to counteract the unbalanced Na\({}^{+}\) concentration gradient across the cell membrane [64]. With the lower biomass activity at a higher sodium concentration, more EPS was produced. As indicated in Figure 5, during the periods of the 6 th and 48 th h, the increased EPS concentrations at higher sodium concentrations (5.1-9.1 g Na\({}^{+}\) L\({}^{-1}\)) were higher than those at low sodium concentrations (1.3-3.2 g Na\({}^{+}\) L\({}^{-1}\)). The results from [PERSON] et al. [13] support this observation that more EPS was produced by bacteria when the NaCl concentrations increased from 0 to 20 g Na\({}^{+}\) L\({}^{-1}\). This increased EPS concentration could be utilized to counteract the osmotic disequilibrium conditions caused by sodium inhibition through supplying carbon and energy and absorbing nutrients, organic pollutants and heavy metals [26]. [PERSON] and [PERSON] [65] also suggested that the increased EPS would be effective in providing a diffusion barrier for the cell walls, alleviating the unbalance of osmotic pressure around the cell membrane and mitigating the cell dehydration under high salinity conditions. The data from Figure 6 indicates that, during the incubation period of the 6 th to 48 th h, the EPS carbohydrate concentrations had a trend to increase at the 48 th h compared with those at the 6 th h in terms of the EPSc/EPSp ratio at all added sodium concentrations. The greater EPS carbohydrate fraction than the EPS protein might suggest that EPS carbohydrate would be crucial in resisting salt-shocking conditions. This could be supported by [PERSON] et al. [13] and [PERSON] and [PERSON] [66], who also observed that, compared to protein, carbohydrate was the main contributor to the accumulated EPS. The lower total biomass activity coupled with increased EPS concentration indicated that, although the produced EPS could be utilized to counteract the osmotic disequilibrium conditions, the residual ATP for the growth and maintenance of microorganism would be lower (Figure 4). This observation suggests that, at a higher sodium concentration (i.e., 9.1 g Na\({}^{+}\) L\({}^{-1}\)) (Figure 1), the methanogenic activity that was completely inhibited may relate to the low total biomass activity. The higher sodium tolerability of 2.4 g Na\({}^{+}\) L\({}^{-1}\) may also be related to the higher total biomass activity of 33 ug g\({}^{-1}\) VSS ATP. The present data may suggest that ATP measurements could serve as a useful indicator for sodium inhibition on microorganisms. This study indicated a higher sodium tolerance in the second-stage of a two-stage anaerobic digester from the aspects of the HAc degradation rate, biomass activity, viable methanogens and microbial shift with a higher abundance of _Thermotoga_, _Clostridium_ and _Methanoculleus_.