Escherichia coli can produce H(2) from glucose via formate hydrogen lyase (FHL). In order to improve the H(2) production rate and yield, metabolically engineered E. coli strains, which included pathway alterations in their H(2) production and central carbon metabolism, were developed and characterized by batch experiments and metabolic flux analysis. Deletion of hycA, a negative regulator for FHL, resulted in twofold increase of FHL activity. Deletion of two uptake hydrogenases (1 (hya) and hydrogenase 2 (hyb)) increased H(2) production yield from 1.20 mol/mol glucose to 1.48 mol/mol glucose. Deletion of lactate dehydrogenase (ldhA) and fumarate reductase (frdAB) further improved the H(2) yield; 1.80 mol/mol glucose under high H(2) pressure or 2.11 mol/mol glucose under reduced H(2) pressure. Several batch experiments at varying concentrations of glucose (2.5-10 g/L) and yeast extract (0.3 or 3.0 g/L) were conducted for the strain containing all these genetic alternations, and their carbon and energy balances were analyzed. The metabolic flux analysis revealed that deletion of ldhA and frdAB directed most of the carbons from glucose to the glycolytic pathway leading to H(2) production by FHL, not to the pentose phosphate pathway. (C) 2009 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.