Hydrogen production from crude glycerol by two-stage processes of dark fermentation using Klebsiella sp. TR17 and photo fermentation using Rhodopseudomonas palustris TN1 (Rps. palustris TN1) was investigated in batch experiments. In dark fermentation, the cumulative hydrogen production and hydrogen yield was 64.24 mmol H-2/L and 5.74 mmol H-2/g COD consumed, respectively with 80.21% of glycerol conversion rate. The dark fermentation effluent (DFE) was employed for photo fermentation. Effect of DFE concentrations (0-5 times dilution), with and without supplementation of yeast extract (2.3 g/L) + NaHCO3 (0.63 g/L), and glutamate (2-8 mM) were optimized. The optimal conditions for hydrogen production from Rps. palustris TN1 were 5 times dilution of DFE without the supplement of yeast extract + NaHCO3, and 2 mM glutamate. Under the optimum conditions, the cumulative hydrogen production of 3.12 mmol H-2/L and hydrogen yield of 0.68 mmol H-2/g COD consumed was obtained. The total hydrogen yield of two-stage processes was estimated to be 6.42 mmol H-2/g COD consumed which was 10.4% of the theoretical yield. Copyright (c) 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.