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A two-stage fermentation process consisting of dark and photo-fermentation periods was carried out in a batch reactor. In the first stage, glucose was fermented in the dark stage using Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564; CSN1-4) to produce acetate, CO2 and H2. The acetate produced in the first stage is fermented to H2 and CO2 by Rhodobacter sphaeroides NCIMB 8253 for further hydrogen production in the second, illuminated stage. The yield of hydrogen in the first stage was about 3.10 mol H2 (mol glucose)(-1) at a glucose concentration of 10 g L(-1), pH 6 +/- 0.2 and 37 degrees C and the second stage yield was about 1.10-1.25 mol H2 (mol acetic acid)(-1) at pH 6.8 +/- 0.2 and 32 degrees C, without removal of the Clostridium CSN1-4. The overall yield of hydrogen in the two-stage process, with glucose as the main substrate was higher than single-stage fermentation.

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