The treatment of wastewater containing sulfides in bioelec-trochemical systems (BES) causes deposition of sulfur on the anode as a result of a solely electrochemical process. In this study, we investigate whether microorganisms can use this sulfur, ratherthan the anode or soluble sulfate, as an electron acceptor for the oxidation of acetate. Our results indicate that microorganisms use electrodeposited sulfur as preferable electron acceptor over the anode and sulfate and produce sulfide irrespective of electrochemical conditions. Bioelectrochemical and biological sulfide generation pathways were studied under different electrochemical conditions. The obtained results show that the sulfide generation rate at open circuit condition (anode potential -235 +/- 5 mV versus standard hydrogen electrode, SHE)was higher in comparison to the electrochemical sulfide generation even at a lower potential of -275 mV (vs SHE), confirming that sulfide is produced through biological processes without any current generation. However, during closed circuit operation, the overall Coulombic efficiency (97% +/- 2%) is not affected as the produced sulfide (originating from the reduction of deposited sulfur) is spontaneously reoxidized to sulfur when a favorable potential is maintained. This confirms the mediator role of sulfur during acetate oxidation in BES. A diagrammatic representation of the mechanism is proposed to characterize the interactions between acetate oxidation and sulfur conversions on the anode.