The characteristics of electricity generation and COD removal of dual-chamber microbial fuel cells (MFCs) operated with alkaline substrates were studied. Substrates with constant pH of either 7 or 9 as well as varying pH in a cycle of 7-8-9-8-7 were used. MFCs operated with these substrates were denoted as MFC-pH7, MFC-pH9 and MFC-pHV, respectively. The experimental results indicate that the MFC-pHV can generate the highest performance of 2554 159 mW/m(2). Cyclic voltammetry (CV), active biomass and electrochemical impedance spectroscopy (EIS) measurements were conducted and these results suggested that the MFC-pHV had the highest electrochemical activity per unit biomass and the lowest internal resistance, which together contributed to the improved power output of the MFC-pHV. In addition, compared with the other two MFCs operated at fixed pH values, the COD removal efficiency of the MPC-pHV was improved due to the stronger adaptation to the varying pH-environment. Copyright (C) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.