Temperature resistant mutants of Rhodobacter capsulatus generated by a directed evolution approach and effects of temperature resistance on hydrogen production

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

Hydrogen production properties of Rhodobacter capsulatus with genetically modified redox balancing pathways

Rhodobacter capsulatus produces molecular hydrogen under the photoheterotrophic growth condition with reduced carbon sources (organic acids). Under this condition, ubiquinol pool is over reduced and excess reducing equivalents are primarily consumed via the reduction of CO2 through the Calvin-Benson-Bassham (CBS) pathway, the dimethylsulfoxide reductase (DMSOR) system or by the reduction of protons into hydrogen gas with the use of nitrogenase to maintain a balanced intracellular oxidation-reduction potential (redox balance). In order to investigate the effect of redox balancing pathways on nitrogenase-dependent hydrogen production, CO2 fixation was blocked by inactivating the phosphoribulokinase (PRK) of CBS pathway in wild type (MT1131), uptake-hydrogenase deficient strain (YO3), and cyt cbb(3) oxidase and uptake-hydrogenase deficient double mutant (YO4) strains. The hydrogen production properties of newly generated strains deficient in the CBB pathway were analyzed and compared with wild type strains. The obtained data indicated that, the total hydrogen production was increased slightly in CBB deficient mutant of YO3 and YO4 (4.7% and 12.5% respectively). Moreover, the maximum hydrogen production rate was increased by 13.3% and 12.7% for CBB deficient mutant of MT1131 and YO3 respectively. It was also observed that under the photoheterotrophic growth condition with ammonium as a nitrogen source, PRK deficient strains gave photoheterotrophically competent ammonium insensitive revertants. Copyright (C) 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved

Draft Genome Sequences of Two Heat-Resistant Mutant Strains (A52 and B41) of the Photosynthetic Hydrogen-Producing Bacterium Rhodobacter capsulatus

The draft genome sequences of two heat-resistant mutant strains, A52 and B41, derived from Rhodobacter capsulatus DSM 1710, and with different hydrogen production levels, are reported here. These sequences may help understand the molecular basis of heat resistance and hydrogen production in R. capsulatus