This study reports a fermentative hydrogen production by Escherichia coli using cheese whey as substrate. To improve the biohydrogen production, an E. coli Delta hycA Delta lacI strain (WDHL) was constructed. The absence of hycA and lad genes had a positive effect on the biohydrogen production. The strain produced 22% more biohydrogen in a shorter time than the wild-type (WT) strain. A Box-Behnken experimental design was used to optimize pH, temperature and substrate concentration. The optimal initial conditions for biohydrogen production by WDHL strain were pH 7.5, 37 degrees C and 20 g/L of cheese whey. The specific production rate was improved from 3.29 mL H(2)/optical density at 600 nm (OD(600nm)) unit-h produced by WDHL under non-optimal conditions to 5.88 mL H(2)/OD(600nm) unit-h under optimal conditions. Using optimal initial conditions, galactose can be metabolized by WDHL strain. The maximum yield obtained was 2.74 mol H(2)/mol lactose consumed, which is comparable with the yield reached in other hydrogen production processes with Clostridium sp. or mixed cultures. (C) 2009 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.