Biological hydrogen production using dark fermentation has been proposed as an alternative and renewable way of producing hydrogen from biomass. However, theoretically, the conversion of biomass to bio-hydrogen using dark fermentation only utilizes one third of the energy content of the sugars derived from starch or cellulose; the rest being in the form of organic acids also produced by the microorganisms. To provide a viable alternative, the energy contained in the organic acids produced in dark fermentation must also be utilized. This can be done in several ways, one of which is to produce methane in an anaerobic digestion step. The technical and economical feasibility of this two-step biological process is investigated in the present study employing three base cases reflecting the different strategies that can be used when performing dark fermentation: high productivity, high yield, and low productivity-low yield. The glucose concentrations, hydrogen productivities and yields studied herein ranges from 4-20 g/L, 7.25-45.75 mmol H(2)/(lh) and 1.37-3.48 mmol H(2)/mole glucose, respectively. The production of pure methane was included as a reference case to investigate how the production of hydrogen affects the production cost. The cost estimates ranged from 50 to 340 (sic)/GJ for the three base cases and the reference case for the process alternatives investigated. The results show that the capital costs and the nutrients used in the two biological steps are the main contributors to the cost in all base cases and the reference case. Furthermore, increasing the substrate concentration and, surprisingly, decreasing the hydrogen yield, reduces the production cost. (C) 2010 Published by Elsevier Ltd.