In previous comparative studies, Enterobacter spH1 was selected as the best hydrogen and ethanol producer (Knothe, 2010). Here, glycerol fermentation was compared between three other strains: Escherichia coli CECT432, Escherichia coli CECT434 and Enterobacter cloacae MCM2/1. E. coli CECT432 was found to perform best with a H-2 productivity of 69.1 mM (1307 mL/L). A co-culture of this E. coli CECT432 strain with the earlier selected Enterobacter spH1 showed a 3.1-fold higher H-2 productivity (4767 mL/L.) from pure glycerol and higher biomass production. Remarkably, the hydrogen yield per mol of glycerol also increased from 0.61 to 1.26 mol H-2/mol glycerol. The co-culture was also tested using waste glycerol from biodiesel. Waste glycerol was characterized and found to consist of (w/v): glycerol 47.5%, water 40.5%, ash content 4.8% and non-glycerol organic matter (MONG) 7.2%. The amount of total soluble organic carbon (TOC) in the crude glycerol was 317 g/L. A maximum H-2 yield and ethanol yield of 1.53 and 1.21 mol/mol glycerol was obtained on the waste glycerol, respectively. These yields are the highest reported to date using mesophilic strains. The strains metabolized the crude glycerol without any purification step. The ability to produce H-2 without prior purification of the waste glycerol is attractive because it avoids extra costs in the process. (C) 2016 Elsevier Ltd. All rights reserved.