The aim of this study was to determine the possibility of applying psychrophilic bacteria for hydrogen production in cheese whey and glucose biofermentation process with initial organic load reached 1 g COD/L. Depending on the experimental variant, analyses were carried out for psychrophilic bacteria representing Proteobacteria- Rahnella aquatilis, Raoutella terrigena and Firmicutes - Carnobacterium maltaromaticum, Clostridium algidixylanolyticum. Tested microorganisms were isolated from underground water and from demersal lake water. Regardless of the organic substrate used in experiments, the most effective rate of hydrogen production was obtained for Rahnella aquatilis and Clostridium algidixylanolyticum strains. The significantly lower effectiveness of H-2 production was observed in the process of glucose fermentation by strains Raoultella terrigena and Carnobacterium maltaromaticum. The concentration of H-2 in biogas ranged from 32.61% to 43.21%. The quantity of H-2 production during cheese whey fermentation ranged from 20.1 to 58.1 mL H-2/g CODfed, while during glucose fermentation ranged from 16.3 to 52.1 mL H-2/g CODfed. Taking into consideration the quantity of biogas and its H-2 concentration obtained from various organic substrates during fermentation in psychrophilic conditions, the results of this study may be considered promising for the perspective application. (C) 2017 Elsevier Ltd. All rights reserved.