The two-chamber microbial fuel cell (MFC) was operated in batch mode, using acclimated hydrogen-producing mixed bacteria as the anodic inoculum, artificial sucrose wastewater as the substrate (sucrose concentration 10.0 g/L). The performance of the MFC was analyzed at different anodic pH microenvironments, such as the initial pH of the anolyte of 8.57, 7.3, 7.0 and 6.0, respectively, while anodic pH-controlled of 7.3 and 7.0. It showed that the best performance was obtained when the MFC was carried out at anodic pH-controlled of 7.3. Taking the interaction of factors into consideration, we adopted response surface methodology (RSM) to investigate the effects of sucrose concentration, operating temperature and ferrous sulfate concentration on the performance of MFG. The optimum condition for maximum output voltage of the two-chamber MFC (external resistance 1000 Omega) was thus obtained. Copyright (C) 2014, Hydrogen Energy Publications, LI.C. Published by Elsevier Ltd. All rights reserved.