Significant improvement in biological hydrogen production is achieved by the use of coupled bacterial cells in reverse micellar systems. Two coupled systems (a) Rhodopseudomonas palustris CGA009/Citrobacter Y19, and (b) Rhodobacter sphaeroides 2.4.1/Citrobacter Y19 bacteria have been immobilized separately in aqueous pool of the reverse micelles fabricated by various surfactants (AOT, CBAC and SDS) and apolar organic solvents (benzene and isooctane). The gene for uptake hydrogenase enzyme has been manipulated further for hydrogen generation. Mutants deficient in uptake hydrogenase (Hup(-)) were obtained from R. palustris CGA009 and R. sphaeroides 2.4.1, and entrapped with Citrobacter Y19 in the reverse micellar systems. More than two fold increase in hydrogen production was obtained by the use of Hup(-) mutants instead of wild-type photosynthetic bacteria together with Citrobacter Y19. Addition of sodium dithionite, a reducing agent to AOT/H(2)O/isooctane reverse micellar system with the coupled systems of wild-type photosynthetic bacteria and fermentative bacterium Y19 effected similar increase in hydrogen production rate as it is obtained by the use of mutants. CBAC/H(2)O/isooctane reverse micellar system is used for the first time for hydrogen production and is as promising as AOT/H(2)O/isooctane reverse micellar system. All reverse micellar systems of coupled bacterial cultures gave encouraging hydrogen production (rate as well as yield) compared to uncoupled bacterial culture. (C) 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.