zonation was tested as a pretreatment method for enhanced biohydrogen production from wheat straw. Ozone pretreatment effectively degraded wheat straw lignin, and the delignification increased with increase in the applied ozone dose. Results of reducing sugar measurement showed that under our experimental conditions ozone pretreatment significantly increased reducing sugar yields. A simultaneous enzyme hydrolysis and dark fermentation experiment was then conducted using a mixed anaerobic consortium, and the results demonstrated that ozone pretreatment significantly increased biohydrogen production. Compared to the untreated one, hydrogen production in the samples ozonated for 15, 30, 45 and 90 min increased 107%, 134%, 158% and 138%, respectively. Slight inhibitory effect on the dark fermentation was observed with the sample ozonated for 90 min, and the inhibitory effect was due to prolonged ozonation. These results proved that enhancement of biohydrogen production from lignocellulosic biomass using ozone as a pretreatment method is technically feasible. Copyright (C) 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.