Escherichia coli perform mixed acid fermentation and produce hydrogen gas (H-2) as one of the fermentation end products. E. coli can ferment sugars like glucose, xylose and alcohols like glycerol. It has been shown that E. coli has the ability to utilize pretreated organic waste (BSG or DG) or mixtures of it with glycerol and H-2 can be produced. H-2 evolution was maximum when the concentration of BSG was 4% and DG - 10% yielding 1.4 mmol L-1 H-2. H-2 evolution was prolonged to similar to 24-120 h when mixtures of glycerol and DG or BSG wastes were applied. Moreover, in hycE (lacking large subunit of Hyd-3) or hyfG (lacking large subunit of Hyd-4) single mutants H-2 production was absent compared to wild type suggesting that Hyd-3 and Hyd-4 are responsible for H-2 generation. In addition, multiple mutant enhanced cumulative H-2 production similar to 3-4 fold. Taken together it can be proposed that BSG or DG wastes either together or in mixture with glycerol can be applied to obtain E. coli biomass and produce bio-H-2. The novel data can be used to further control effectively the application of organic waste resources as a feedstock for developing bio-H-2 production technology. (C) 2020 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.