Biohythane is alternative fuel to replace fossil fuel for car combustion, and biohythane generation could be potential pathway for energy recovery from wastewater treatment. Microbial electrolysis cell (MEC) is electrochemical technique to convert waste to methane and hydrogen gas for biohythane generation, but the feasibility and stability of MEC needs further investigation to assure sustainable energy recovery. System configuration is paramount factor for electrochemical reaction and mass transfer, and this study was to investigate the configuration impact (single vs dual chamber) of MEC for biohythane generation rate and stability. This study showed that dual-chamber MEC could separate methane and hydrogen gas production in the anode and cathode, and combined both together to produce biohythane. To reduce ohmic resistance for higher current, cation exchange membrane (CEM) was removed from dual-chamber to single-chamber MEC. However, free hydrogen diffusion was allowed in the single chamber since CEM was removed. The diffused hydrogen and substrate towards the cathode would favor the methanogen growth, and thus the hydrogen was consumed to reduce the biohythane generation and energy recovery efficiency (i.e., 7.5 × 10-3 reduced to 5.7 × 10-3 kWh kg-1 degraded COD day-1 after converting dual-chamber to single-chamber MEC). Absolute abundance of methanogen in single-chamber MEC was greatly boosted, as Methanosarcina and Methanobacteriale on the anode surface, increased by 132% and 243%, respectively, while the original dual-chamber MEC could maintain Geobacter growth for high current generation. This is the keystone study to demonstrate the importance of dual-chamber MEC for the feasibility and stability for the biohythane generation, building up the foundation to use electrochemical device to convert the organic waste to the alternative biohythane.