In this work continuous hydrogen production by Escherichia coli (XL1-BLUE) and its purification by membrane gas separation were studied. Firstly, a kinetic investigation was performed on formate supplemented broth in order to determine exponential growth phase (5-7 h) while the most intense hydrogen fermentation takes place. Furthermore, important process design parameters such as saturation constant and maximal growth rate were calculated (K-s = 0.77 g l(-1), mu(max) = 0.39 h(-1)). Afterward, based on the kinetic study, continuous hydrogen fermentations using cultures of E. coli (XL1-BLUE) were carried out in a CSTR reactor configuration applying various hydraulic retention times (HRT) related to both exponential and stationary growth period (5 h, 7 h, 9 h). The results indicated that highest hydrogen yield (0.26 mmol H-2/mmol formate added) and productivity (5.1 mmol H-2 l(-1) d(-1)) could be achieved by applying HRT = 7 h that does not allow the living cells to reach stationary phase. In addition to hydrogen production, the concentration of bioH(2) by polyimide membrane under different operational circumstances was investigated using pure and mixed gases, as well. The results of single gas experiments indicated that increasing the temperature has positive effect on separation efficiency. Moreover, the influence of retentate and feed flow ratio (Q(R)/Q(F)) was studied applying binary H-2/CO2 gaseous mixture and it was found that polyimide membrane has high potential for H-2 purification since 18% increase in H-2 concentration and 22% decrease in CO2 content could be attained in the permeate by a one-step separation process. Copyright (C) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.