The use of hydrogen as an energy source represents an alternative to reduce the environmental impact since water is the only major by-product in its combustion. It can be used as an on-board fuel for motive power through internal combustion engine or fuel cell that converts chemical energy (an electro-chemical device) and can be applied in several transportation devices. The hybrid system for hydrogen production by combining the dark fermentation followed by photofermentation has great potential. In the current work, the production of hydrogen was investigated in repeated-batch processes using dark fermentation effluent as substrate to pure culture (Rhodopseudomonas palustris) and co-culture purple non-sulfur bacteria (Rhodopseudomonas palustris and Rhodobacter capsulatus). The influence of glucose and milk whey permeate (MWP) on the hydrogen production was studied. The results showed that the system employing the co-culture and glucose kept the culture activity for a long time (679 h) and the amount of accumulated hydrogen was 0.98 +/- 0.02 mol and maximum productivity reached 287.39 +/- 5.75 mmol of H-2/L.day. The findings from alternating feeding between glucose and milk whey permeate, showed higher results for the amount of accumulated hydrogen (1.41 +/- 0.01 mol). Besides, maximum productivity was of 266.60 +/- 5.33 mmol of H-2/L.day. (C) 2018 Elsevier Ltd. All rights reserved.