Scaling up microbial fuel cells (MFCs) requires the development of compact reactors with multiple electrodes. A scalable single chamber MFC (130 mL), with multiple graphite fiber brush anodes and a single air-cathode cathode chamber (27 m(2)/m(3)), was designed with a separator electrode assembly (SEA) to minimize electrode spacing. The maximum voltage produced in fed-batch operation was 0.65 V (1,000 Omega) with a textile separator, compared to only 0.18 V with a glass fiber separator due to short-circuiting by anode bristles through this separator with the cathode. The maximum power density was 975 mW/m(2), with an overall chemical oxygen demand (COD) removal of > 90% and a maximum coulombic efficiency (CE) of 53% (50 Omega resistor). When the reactor was switched to continuous flow operation at a hydraulic retention time (HRT) of 8 h, the cell voltage was 0.21 +/- 0.04 V, with a very high CE = 85%. Voltage was reduced to 0.13 +/- 0.03 V at a longer HRT = 16 h due to a lower average COD concentration, and the CE (80%) decreased slightly with increased oxygen intrusion into the reactor per amount of COD removed. Total internal resistance was 33 Omega, with a solution resistance of 2 Omega. These results show that the SEA type MFC can produce stable power and a high CE, making it useful for future continuous flow treatment using actual wastewaters.