A shorter start-up time and highly negative anode potentials are needed to improve single-chamber air-cathode microbial fuel cells (MFCs). Using a glass fiber separator reduced the start-up time from 10 d to 8 d at 20 degrees C, and from 4 d to 2 d at 30 degrees C, and enhanced coulombic efficiency (CE) from <60% to 89% (20 degrees C) and 87% (30 degrees C). Separators also reduced anode potentials by 20-190 mV, charge transfer resistances by 76% (20 degrees C) and 19% (30 degrees C), and increased CV peak currents by 24% (20 degrees C) and 8% (30 degrees C) and the potential range for redox activity (-0.55 to 0.10 mV vs. -0.49 to -0.24 mV at 20 degrees C). Using a glass fiber separator in an air-cathode MFC, combined with inoculation at a mesophilic temperature, are excellent strategies to shorten start-up time and to enhance anode performance and CE. (C) 2012 Elsevier Ltd. All rights reserved.