The use of synthetic wastewater containing carbon sources of different complexity (glycerol, milk and starch) was evaluated in single chamber microbial electrolysis cell (MEG) for hydrogen production. The growth of an anodic syntrophic consortium between fermentative and anode respiring bacteria was operationally enhanced and increased the opportunities of these complex substrates to be treated with this technology. During inoculation, current intensities achieved in single chamber microbial fuel cells were 50, 62.5, and 9 A m(-3) for glycerol, milk and starch respectively. Both current intensities and coulombic efficiencies were higher than other values reported in previous works. The simultaneous degradation of the three complex substrates favored power production and COD removal. After three months in MEG operation, hydrogen production was only sustained with milk as a single substrate and with the simultaneous degradation of the three substrates. The later had the best results in terms of current intensity (150 A m(-3)), hydrogen production (0.94 m(3) m(-3) d(-1)) and cathodic gas recovery (91%) at an applied voltage of 0.8 V. Glycerol and starch as substrates in MEG could not avoid the complete proliferation of hydrogen scavengers, even under low hydrogen retention time conditions induced by continuous nitrogen sparging. (C) 2014 Elsevier Ltd. All rights reserved.