The influence of concentration of glycerol, inoculum and total nitrogen on hydrogen generation, in batch dark fermentation process in the presence of digested sludge (at 37 degrees C and at initial pH = 6) was studied. Changes in substrate and products concentrations were modeled with modified Gompertz equations (correlation coefficient R(2) = 0.9015). The 1,3-propandiol, butyric acid, acetic acid, lactic acid and ethanol were found as the main liquid metabolites. Maximal substrate yield for hydrogen was 0.41 mol H(2)/mol glycerol and was obtained for medium containing 10 g/l of glycerol with the lowest amount of inoculum 1.16 g volatile suspended solid (VSS)/l. Increase of glycerol concentration from 5 to 30 g/l resulted in much better hydrogen generation, namely from 0.345 to 0.7151 H(2)/l. Further increase of glycerol concentration did not cause any changes. The H(2):CO(2) ratio in biogas in system with the highest substrate yield was always 1. The initial concentration of glycerol does not influence the rate of hydrogen generation. The increase of initial concentration of inoculum from 1.2 to 11.6 gVSS/l results in the decrease of specific hydrogen yield. Nitrogen concentration in medium does not influence the hydrogen production. (C) 2009 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.