Biohydrogen production through dark fermentation is a promising technology for generating renewable energy, while using microalgal biomass as a third generation feedstock can further increase the sustainability of the process. In the present study, Scenedesmus obliquus was used as model microalga substrate for studying the impact of operational parameters in batch dark fermentation trials using a strain of Enterobacter aerogenes bacteria. (i) The initial gas-liquid ratio in the bioreactor (from 13 to 8.2) was tested, resulting in higher bioH(2) yields for ratios above 5. (ii) Different bacterial growth, inoculation procedures and fermentation media were tested in combined experiments. The best conditions were chosen by maximising bioH(2) yield and minimising production time and costs. (iii) The autoclave sterilization effect on sugar extraction and bioH(2) yield was tested for different microalga concentrations (2.5-50 g/L) with best results attained for 2.5 g/L (81.2% extraction yield, 40.9 mL H-2/g alga). For the best operational conditions, fermentation kinetics were monitored and adjusted to the Modified Gompertz model, with t(95) (time required for bioH(2) production to attain 95% of the maximum yield) below 4.5 h. The maximum hydrogen production was higher when using wet algal biomass enabling the energy consuming biomass drying step to be skipped. (C) 2017 Elsevier Ltd. All rights reserved.