Citrobacter amalonaticus Y19 is a unique chemoheterotrophic microorganism capable of carbon monoxide (CO)-dependent hydrogen (H-2) production. To improve its biocatalytic potential, the effects of medium composition on cell growth and H-2 production were investigated. Among several carbon sources tested, maltose resulted in the best CO consumption and H-2 production. Meanwhile, L-cysteine supplementation improved cell growth and H-2 production, suggesting that this amino acid stimulates synthesis of Fe-S clusters. When molybdenum and selenium, which are essential cofactors of formate hydrogen lyase (FHL), were added, formate accumulation was reduced. Optimized medium composition improved the activities of CO-dependent H-2 production (6.3-fold), CODH (22.3-fold) and hydrogenase (2.9-fold). The optimized medium composition also enhanced H-2 production in recombinant C. amalonaticus Y19-cooA, which homologously over expresses CooA, the transcriptional activator of the coo operons. Cells grown under this condition exhibited improved stability in the course of repeated-batch CO-dependent H-2 production under non-growing conditions. Copyright (C) 2016, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.