Microbial electrolysis system (MES) is a promising technology which integrates microorganisms or other bio-catalysts with an electrochemical system to generate electricity or chemicals including energy carriers such as methane. Compared with the combined anaerobic digestion and CO2 biomethanisation system, MES in principle requires lower energy input for methane production especially when coupled with organic material oxidation. This study investigated the simultaneous CH4 production and organic substance degradation in MES, using synthetic swine wastewater as the model substrate. The results demonstrated that the applied cathodic potential played a crucial role for CH4 production in the biocathode and influenced to a great extent the reactions and operational conditions in the bioanode. The performance of the anode, therefore, could not be controlled exclusively by the organic substance loading applied to this compartment. Abiotic anodes were then evaluated for the organic degradation to alleviate the impact of acidification on anode performance. Although the boron doped diamond (BDD) showed higher rate of organic substance degradation compared to platinum due to its high oxygen overpotential, O2 evolution was still observed as a side reaction during the glucose oxidation process. A middle chamber for O2 removal using N2 stripping was then introduced to control the O2 transfer to the biocathode: the limited reduction in cathodic O2 concentration was achieved at the expense of the complex middle chamber operation and the increased internal resistance. The three-chamber MES therefore resulted in a lower volumetric CH4 production, when compared to the dual-chamber cell. The high pH of the cathodic chamber in both dual-chamber and three-chamber cells indicated an opportunity for plant nutrient recovery, based on a theoretical model simulation.