This study aimed to optimize the hydrogen production from various seed sludges (two kinds of sewage sludges (S1, S2), cow dung (S3), granular sludge (S4) and effluent from condensed soluble molasses H-2 fermenter (S5)) and enhancement of hydrogen production via heat treatment for substrate and seed sludge by using the solid residues of biodiesel production (BDSR). Two batch assay tests were operated at a biodiesel solid residue concentration of 10 g/L, temperature of 55 degrees C and an initial cultivation pH of 8. The results showed that the peak hydrogen yield (HY) of 94.6 mL H-2/g volatile solid (VS) (4.1 mmolH(2)/g VS) was obtained from Si when substrate and seed sludge were both heat treated at 100 C for 1 h. However, the peak hydrogen production rate (HPR) and specific hydrogen production rate (SHPR) of 1.48 L H-2/L-d and 0.30 L H-2/g VSS-d were obtained from S2 without any treatment. The heat treatment was found to increase the HY in both the cases of sewage sludges Si and S2. The HY of 89.5 mL H-2/g VS (without treatment) was increased to 94.6 mL H-2/g VS and 82.6 mL H-2/g VS (without treatment) was increased to 85.7 mL H-2/g VS for S1 and S2. The soluble metabolic product (SMP) analysis showed that the fermentation followed mainly acetate butyrate pathway with considerable production of ethanol. The total bioenergy production was calculated as 2.8 and 2.9 kJ/g VS for favorable hydrogen and ethanol production, respectively. The BDSR could be used as feedstock for dark fermentative hydrogen production. Copyright (C) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.