An ideal separator is essential for efficient power production from air-cathode single-chamber microbial fuel cells (MFCs). In this study, we use different kinds of membranes as separators, including Nafion 117 proton exchange membrane, polyethersulfone and poly(vinylidene fluoride) microfiltration membranes. Temporal variations of cathode performance are monitored during the experiment. Results show that MFCs with microfiltration membranes present higher power output but deterioration is still observed after about 600-h operation. With the utilization of appropriate separators (e.g., polyethersulfone membrane), biofouling, cation fouling and chemical scale fouling of the cathodes are alleviated while reaction fouling seems inevitable. Moreover, it is found that Coulombic efficiency (CE) and energy efficiency (EE) are also related to the cathode performance. Despite relatively high oxygen diffusivity (1.49 x 10(-5) cm(2) s(-1)), CE and EE of the MFC with 0.1 mu m pore-size polyethersulfone membrane can reach 92.8% and 13.7%, respectively, when its average power density registers 403.5 mW m(-2). This phenomenon might be attributed to the finding that the overall substrate consumption rate due to oxygen reduction and respiration is almost constant in the air-cathode MFCs. Oxygen leakage into the electrolyte can be inhibited due to the efficient oxygen reduction reaction on the surface of the cathode. (C) 2014 Elsevier B.V. All rights reserved.