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In this study, microbial fuel cells (MFCs) – operated with novel cation- and anion-exchange membranes, in particular AN-VPA 60 (CEM) and PSEBS DABCO (AEM) – were assessed comparatively with Nafion proton exchange membrane (PEM). The process characterization involved versatile electrochemical (polarization, electrochemical impedance spectroscopy – EIS, cyclic voltammetry – CV) and biological (microbial structure analysis) methods in order to reveal the influence of membrane-type during start-up. In fact, the use of AEM led to 2–5 times higher energy yields than CEM and PEM and the lowest MFC internal resistance (148 ± 17 Ω) by the end of start-up. Regardless of the membrane-type, Geobacter was dominantly enriched on all anodes. Besides, CV and EIS measurements implied higher anode surface coverage of redox compounds for MFCs and lower membrane resistance with AEM …