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Rhodobacter sphaeroides is a purple non-sulfur bacterium which photoheterotrophically produces hydrogen from organic acids under anaerobic conditions. A gene coding for putative chlorophyll a synthase (chlG) from cyanobacterium Prochlorococcus marinus was amplified by nested polymerase chain reaction and cloned into an inducible-expression plasmid which was subsequently transferred to R. sphaeroides for heterologous expression. Induced expression of chlG in R. sphaeroides led to changes in light absorption spectrum within 400-700 nm. The hydrogen production capacity of the mutant strain was evaluated on hydrogen production medium with 15 mM malate and 2 mM glutamate. Hydrogen yield and productivity were increased by 13.6 and 22.6%, respectively, compared to the wild type strain. The results demonstrated the feasibility of genetic engineering to combine chlorophyll and bacteriochlorophyll biosynthetic pathways which utilize common intermediates. Heterologous expression of key enzymes from biosynthetic pathways of various pigments is proposed here as a general strategy to improve absorption spectra and yield of photosynthesis and hydrogen gas production in bacteria.

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Photofermentative production of hydrogen is a promising and sustainable process; however, it should be coupled to dark fermentation to become cost effective. In order to integrate dark fermentation and photofermentation, the suitability of dark fermenter effluents for the photofermentative hydrogen production must be demonstrated. In this study, thermophilic dark fermenter effluent (DFE) of sugar beet thick juice was used as a substrate in photofermentation process to compare wild-type and uptake hydrogenase-deficient (hup (-)) mutant strains of Rhodobacter capsulatus by means of hydrogen production and biomass growth. The tests were conducted in small-scale (50 mL) batch and large-scale (4 L) continuous photobioreactors in indoor conditions under continuous illumination. In small scale batch conditions, maximum cell concentrations were 0.92 gdcw/L c and 1.50 gdcw/L c, hydrogen yields were 34 % and 31 %, hydrogen productivities were 0.49 mmol/(L c·h) and 0.26 mmol/(Lc·h), for hup (-) and wild-type cells, respectively. In large-scale continuous conditions, maximum cell concentrations were 1.44 gdcw/L c and 1.87 gdcw/L c, hydrogen yields were 48 and 46 %, and hydrogen productivities were 1.01 mmol/(L c·h) and 1.05 mmol/(L c·h), for hup (-) and wild-type cells, respectively. Our results showed that Rhodobacter capsulatus hup (-) cells reached to a lower maximum cell concentration but their hydrogen yield and productivity were in the same range or superior compared to the wild-type cells in both batch and continuous operating modes. The maximum biomass concentration, yield and productivity of hydrogen were higher in continuous mode compared to the batch mode with both bacterial strains.