Biohydrogen production is measured using a variety of techniques, ranging from low cost intermittent gas release methods where yields are usually reduced due to high partial pressures of hydrogen, to expensive respirometers that can eliminate pressure buildup A new large headspace volume technique was developed that reduces the potential for hydrogen gas inhibition without the need for a respirometer. We tested this method with two strains of clostridia, Clostridium acetobutylicum ATCC 824 and its mutant M5 that lacks a megaplasmid responsible for butanol and acetone production, and a mixed culture (heat-treated sludge) The hydrogen yield using MS (2 64 mol-H(2)/mol-glucose) was 47% higher than that of the parent strain (1.79 mol-H(2)/mol-glucose), and 118% larger than that obtained in tests with the sludge inoculum (1.21 mol-H(2)/mol-glucose). The increased yield for M5 was primarily due to a decrease in biomass synthesis (38%) compared to the parent strain Hydrogen yields measured using this new method were on average 14% higher than those obtained using a conventional respirometric method These findings indicate enhanced biohydrogen production from the megaplasmid-deficient mutant of C acetobutylicum ATCC 824, and that an intermittent gas-sampling technique can effectively measure high hydrogen gas by using a large headspace volume (C) 2009 Professor T. Nejat Veziroglu Published by Elsevier Ltd. All rights reserved.