## 3 _Introduction_  
Anaerobic digestion is often a cost-effective technology for sludge treatment to reduce biosolids and recover energy [1]. Since the hydrolysis process is the rate-limiting step in the anaerobic digestion of sludge, various pretreatment methods have been proposed to improve the disintegration efficiency of sludge particulates [2]. Among these, alkaline pretreatment with controlled pH is best known for its high efficiency in solubilizing complex substrates [3,4]. However, the sodium ion would be introduced into the anaerobic system during pH regulation if using sodium hydroxide (NaOH) or sodium carbonate (Na\({}_{2}\)CO\({}_{3}\)) [5].  
Anaerobic digestion has been reported to be hindered by the presence of high concentrations of sodium ions. The effect of sodium ions on the activities of methanogensin the single-stage system has been previously elucidated [6; 7; 8]. Although an appropriate amount of sodium has been reported to be essential for methanogen growth [6; 7; 9], high sodium concentrations (5-10 g Na\({}^{+}\) L\({}^{-1}\)) inhibit methanogenic activities and biogas production [6; 9]. The sodium inhibition mechanism on methanogens has been previously explored by researchers.  
[PERSON] and [PERSON] [8] reported the effect of sodium on the chemical and physical characteristics of sludge, e.g., soluble microbial products, extracellular polymeric substances (EPS), turbidity, viscosity and capillary suction time. Other researchers also reported that sodium caused the inhibition of biomass activity [10; 11], which was typically characterized by a decrease in the degradation rates of volatile fatty acids (VFA) [12]. Furthermore, with the presence of sodium in the growth environment, bacteria cells would produce EPS as a protective response [9; 13]. However, these studies focused on the conventional single-stage anaerobic digestion system.  
The microbial communities and their activities in the two-stage anaerobic digester would likely be different from those in the single-stage system [14]. The methanogens in the second-stage reactor of a two-stage anaerobic digester were reported to be more stable than those in the single-stage system [14; 15]. With stage separation, better control of loading could be achieved on the first-stage reactor, consequently, resulting in a more stable growth environment for methanogens in the second-stage reactor [15].  
In the two-stage anaerobic system investigated in this study, the second-stage reactor received effluent from the first-stage reactor wherein thermo-chemical pre-treatment was applied to substantially improve solubilisation of organic particulates. Therefore, the methanogens' dominance, sensitivities, as well as their stress responses to inhibition, would likely be different from those observed in a single-stage system.  
With benefits of improved organic loading rates and process stability [16], the two-stage anaerobic digester has received increased interest for optimizing methane production in recent years [17]. However, the issues caused by sodium loading and toxicity may affect the successful operation of the two-stage anaerobic digester [18]. This study investigates the effect of sodium on the viability and activity of the methanogens from the second-stage reactor of a two-stage anaerobic system, which could help to provide knowledge for the design, operation and optimisation of the two-stage anaerobic digester for applications on the more saline organic wastes.