Hydrogen (H-2) is a promising alternative energy carrier which can be produced biologically. Rhodobacter capsulatus, a non-sulfur purple photosynthetic bacterium, can produce H-2 under nitrogen-limited, photoheterotrophic conditions by using reduced carbon sources such as simple organic acids. Outdoor closed photobioreactors; used for biological H-2 production are located under direct sunlight, as a result; bioreactors are exposed to temperature fluctuations during day time. In this study to overcome this problem, temperature-resistant mutants (up to 42 degrees C) of R. capsulatus were generated in this study by a directed evolution approach. Eleven mutant strains of R. capsulatus DSM 1710 were obtained by initial ethyl methane sulfonate (EMS) mutagenesis of the wild-type strain, followed by batch selection at gradually increasing temperatures up to 42 degrees C under respiratory conditions. The genetic stability of the mutants was tested and eight were genetically stable. Moreover, H-2 production of mutant strains was analyzed; five mutants produced higher amounts of H-2 when compared to the DSM 1710 wild-type strain and three mutants produced less H-2 by volume. The highest H-2- producing mutant (B41) produced 24% more H-2 compared to wild type, and the mutant with lowest H-2-production capacity (A52) generated 7% less H-2 compared to the wild type. These results indicated that heat resistance of R. capsulatus can be improved by directed evolution, which is a useful tool to improve industrially important microbial properties. To understand molecular changes that confer high temperature-resistance and high hydrogen production capacity to these mutants, detailed transcriptomic and proteomic analyses would be necessary. Copyright (c) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.