Two challenges for improving the performance of air cathode, single-chamber microbial fuel cells (MFCs) include increasing Coulombic efficiency (CE) and decreasing internal resistance. Nonbiodegradable glass fiber separators between the two electrodes were shown to increase power and CE, compared to cloth separators (J-cloth) that were degraded over time. MFCtestswereconductedusing glass fibermatswith thicknesses of 1.0mm (GF1) or 0.4 mm (GF0.4), a cation exchange membrane (CEM), and a J-cloth (JC), using reactors with different configurations. Higher power densities were obtained with either GF1 (46 ± 4 W/m3) or JC (46 ± 1 W/m3) in MFCs with a 2 cm electrode spacing, when the separator was placed against the cathode (S-configuration), rather than MFCs with GF0.4 (36 ± 1 W/m3) or CEM (14 ± 1 W/m3). Power was increased to 70 ± 2 W/m3 by placing the electrodes on either side of the GF1 separator (single separator electrode assembly, SSEA) and further to 150 ± 6 W/m3 using two sets of electrodes spaced 2 cm a part (double separator electrode assembly, DSEA). Reducing the DSEA electrode spacing to 0.3 cm increased power to 696 ± 26 W/m3 as a result of a decrease in the ohmic resistance from 5.9 to 2.2 Ω. The main advantages of a GF1 separator compared to JC were an improvement in the CE from 40% to 81% (S-configuration), compared to only 20-40% for JC under similar conditions, and the fact that GF1 was not biodegradable. The high CE for the GF1 separator was attributed to a low oxygen mass transfer coefficient (ko ) 5.0 x 10-5 cm/s). The GF1 andJCmaterials differed in the amount of biomass that accumulated on the separator and its biodegradability, which affected long-term power production and oxygen transport. These results show that materials and mass transfer properties of separators are important factors for improving power densities, CE, and long-term performance of MFCs. © 2009 American Chemical Society.