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The aim of the study was to assess the effect of anode materials, namely a carbon nanotube (CNT)-buckypaper and a commercial carbon paper (CP) on the performance of a two-chamber microbial electrolysis cell (MEC), in terms of hydrogen production and main electrochemical characteristics. The experiments were performed using both acetate-based synthetic wastewater and real wastewater, specifically the effluent of a dark fermentative hydrogenogenic reactor (fermentation effluent), using cheese whey (CW) as substrate. The results showed that CP led to higher hydrogen production efficiency and current density compared to the CNT-buckypaper anode, which was attributed to the better colonization of the CP electrode with electroactive microorganisms, due to the negative effects of CNT-based materials on the bacteria metabolism. By using the fermentation effluent as substrate, a two-stage process is developed, where dark fermentation (DF) of CW for hydrogen production occurs in the first step, while the DF effluent is used as substrate in the MEC, in the second step, to further increase hydrogen production. By coupling DF-MEC, a dual environmental benefit is provided, combining sustainable bioenergy generation together with wastewater treatment, a fact that is also reinforced by the toxicity data of the current study. [ABSTRACT FROM AUTHOR]