Klebsiella oxytoca HP1 is a promising candidate for dark hydrogen production due to its high hydrogen production rate (HPR) and aerobic hydrogen production character. In this study, two hydrogenase subunit over-expressed strains, K. oxytoca HP1/hycE and K. oxytoca HP1/hycG, were constructed to further enhance K. oxytoca hydrogen production. Hydrogenase activity, hydrogen molar yield (HMY) and HPR from glucose under anaerobic and aerobic conditions were assessed for the recombinant strains. In K. oxytoca HP1/hycE, the HMY, HPR and hydrogenase activity under anaerobic condition was increased by 90.5%, 93.5% and 2.10 fold, relative to the wild-type strain. In K. oxytoca HP1/hycG, the respective increases were 31.7%, 33.3%, 1.55 fold, relative to the wild-type. Thus, over-expression of hydrogenase subunit may significantly enhance hydrogen production in dark-fermentation strains. The anaerobic HMY of K. oxytoca HP1/hycE reached 3.43 mol-H-2/mol-glucose. Furthermore, under oxygen stress conditions, the natural hydrogen production capacity of wild-type K. oxytoca HP1 was enhanced by over-expression of its hydrogenase subunits. Next, the hycE or hycG gene from K. oxytoca HP1 was transformed into E. coli DH-5 alpha to analyze the oxygen-tolerant role of the hydrogenase subunits. Under oxygen stress the recombinant strains E. coli DH-5 alpha/hycE and E. coli DH-5 alpha/hycG were capable of hydrogen production, unlike the wild-type strain. This indicates that the HycE and HycG subunits of K. oxytoca HP1 hydrogenase are key mediators of K. oxytoca HP1 aerobic hydrogen production. Copyright (C) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.