A microbial fuel cell based on activated sludge from a wastewater treatment plant (WWTP) was implemented as an alternative for glycerol utilization. With a prolonged hydraulic retention time and sludge age, the required conditioning stage was three weeks in order to achieve stable values of the cell current density (external resistance of 100 σ), total suspended solids (TSS) and chemical oxygen demand (COD) in the effluent. Subsequent changes in the glycerol concentration required a shorter period of time to achieve stable values. The influence of the substrate concentration was studied, with Monod-type behavior found for cell performance (cell current density under closed circuit and maximum power output), attaining the maximum power for a glycerol concentration of 1080 mg L^-1, without further improvement at higher concentrations. Under these conditions, electrochemical performance did not rely on substrate availability, but rather on the capability of the microorganism to electrooxidize glycerol. In this system, the anode appeared to be the governing electrode. Its performance significantly varied with the glycerol concentration. Nevertheless, the main barrier in this membrane-based system was ohmic resistance, which should be drastically reduced. © 2013 The Electrochemical Society.