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The [FeFe]-hydrogenase of Rhodospirillum rubrum (Rr-HydA; Rru_A0310) was expressed in an Escherichia coli strain co-expressing the hydrogenase maturation proteins HydE, HydF, and HydG from Clostridium acetobutylicum (Ca-HydEFG). Rr-HydA, which purified as a 50 kDa protein, showed an in vitro H2-evolving activity in the presence of dithionite-reduced methyl viologen. The ability of Rr-HydA to carry out H2-evolving activity when heterologously expressed in a Rhodobacter sphaeroides nifDK hupSL mutant containing the genes coding for Ca-HydEFG was examined. Photoheterotrophic H2 evolution was only observed in the presence of Rr-HydB [Rru_A0309 (22 kDa)], which is encoded by a gene located immediately upstream from the gene encoding Rr-HydA. Rr-HydB contains Fe–S cluster(s). Consistently, in a reaction mixture containing spinach ferredoxin oxidoreductase and NADPH, H2 was evolved when both Rr-HydA and Rr-HydB were present together, and the rate of H2 evolution by Rr-HydA was proportional to the level of Rr-HydB until an equimolar ratio of the two proteins was reached. Thus, it is proposed that Rr-HydB acts as Rr-HydA-specific ferredoxin that donates electrons to Rr-HydA through a direct molecular interaction.

Rhodobacter sphaeroides evolves H2 by nitrogenase under photoheterotrophic growth conditions. The maximum accumulation level of H2 increases by up to 60% when the culture medium, in which succinate and ammonium ion are the major carbon and nitrogen sources, is supplemented with ethanol at 0.5% (vol/vol). Such an effect by ethanol is not observed when ammonium ion is omitted from the medium. Ethanol is not used as a carbon source under the conditions examined, and the utilization of succinate and ammonium ion by cell is not affected by ethanol. The nitrogenase activity in the presence of ethanol, which is approximately 60% higher compared with that determined in its absence, is regulated at the level of nif transcription since the similar increase in the β-galactosidase activity of nifH-lacZ fusion was reproducibly observed under the same conditions. Interestingly, nifA transcription also increased similarly in the presence of ethanol. Neither glnB nor glnK interruptions abolished the ethanol-mediated enhancement of H2 evolution. Thus, the presence of ethanol results in the enhancement of nitrogenase expression of R. sphaeroides through the increase of NifA expression in a manner independent of GlnB and GlnK.

Purple non-sulfur photosynthetic bacterium Rhodobacter sphaeroides KD131 wild type (wt) and its PHB synthase deleted-mutant P1 were evaluated for hydrogen (H2) production from acetate and butyrate, the most abundant liquid end products of dark fermentation. In the presence of glutamate (8 mM), 60 mM of acetate and 30 mM of butyrate were degraded down to 41.5% and 24.0%, respectively, and achieved a H2 yield (HY) of 0.65 mol H2/mol acetate- and 2.50 mol H2/mol butyrate-consumed, while 30 mM succinate exhibited an HY of 3.29 mol H2/mol substrate-consumed. The order of HY observed was inversely related to poly-(3-hydroxybutyrate) (PHB) content and pH increase in the broth. When mutant P1 was used, in spite of depressed cell growth and lower substrate degradation compared to those observed in strain KD131 wt, higher H2 production was observed, achieving around two-fold increase of HY in both acetate and butyrate. A pH control to 7.0 during fermentation was effective in increasing substrate degradation and decreasing PHB content, thereby significantly increasing H2 production. When pH was controlled to 7.0, strain KD131 wt evolved more H2 by 2.36 and 1.70 folds in the acetate- and succinate-medium, respectively, compared to those observed in without pH control. The highest H2 production was observed when the mutant P1 was photo-fermented with a pH control to 7.0 in the medium containing acetate-(NH4)2SO4. It seemed that pH control had an effect not only on the depressed production of PHB but also on soluble microbial products and secondary metabolites, which would compete with H2 production in expending reducing power.

Rhodobacter sphaeroides KCTC 12085 is a natural isolate which shows a higher ability of photoheterotrophic H 2 production compared with a laboratory strain R. sphaeroides 2.4.1. The H 2 production by R. sphaeroides is observed only under light conditions. In order to render R. sphaeroides to produce H 2 fermentatively as well, a 4.4-kb Rhodospirillum rubrum DNA encompassing a gene coding for pyruvate-formate lyase (PFL) with its putative maturation protein, and a 29.4-kb R. rubrum gene cluster including formate-hydrogen lyase (FHL) complex, its maturation proteins, a transcriptional activator for FHL, and Fe-only hydrogenase with its maturation proteins were cloned and mobilized into R. sphaeroides KCTC 12085. The recombinant R. sphaeroides evolved H 2 during dark fermentative growth. The H 2 production by the recombinant R. sphaeroides under photoheterotrophic conditions was elevated twofold to show 4 mol H 2 /mol of glucose compared with 2 mol H 2 /mol of glucose by its parental strain carrying empty vector. Interestingly, addition of hypophosphite, an inhibitor of PFL, to the recombinant R. sphaeroides under photoheterotrophic conditions still resulted in 3 mol H 2 /mol of glucose, suggestive of active H 2 production not only by nitrogenase but also by Fe-only hydrogenase, which are insensitive to hypophosphite. The results further suggest the photoheterotrophic H 2 production by FHL unless hypophosphite is present. Thus, the H 2 production by the recombinant R. sphaeroides under photoheterotrophic conditions appears to be mediated by the concerted actions of FHL, Fe-only hydrogenase, and nitrogenase. The H 2 -evolving activity by the recombinant R. sphaeroides containing Fe-only hydrogenase of R. rubrum appears to depend on the presence its maturation proteins, suggestive of lack of such proteins in R. sphaeroides .

The genes coding for two PII-like proteins, GlnB and GlnK, which play key roles in repressing the nitrogenase expression in the presence of ammonium ion, were interrupted from the chromosome of Rhodobacter sphaeroides. The glnB–glnK mutant exhibits the less ammonium ion-mediated repression for nitrogenase compared with its parental strain, which results in more H2 accumulation by the mutant under the conditions. Rhodospirillum rubrum produces H2 by both nitrogenase and hydrogenase. R. rubrum containing the recombinant pRK415 with an insert of hydC coding for its own Fe-only hydrogenase showed twofold higher accumulation of H2 in the presence of pyruvate under photoheterotrophic conditions, which was not observed in the absence of pyruvate. The same was true with R. rubrum containing the recombinant pRK415 cloned with hydA coding for Fe-only hydrogenase of Clostridium acetobutylicum. Thus, Fe-only hydrogenase requires pyruvate as an electron donor for the production of H2.

Increase in levels of photosynthetic spectral complexes by maintaining the plasmids harboring DNA encoding puhA, pufBA, or pucBAC in trans in Rhodobacter sphaeroides resulted in decrease of the photoheterotrophic production of H2. However, removal of B875 or B800–850 light-harvesting (LH) complexes affected H2 production differently. Lack of B875 complex following in-frame deletion of pufBA (mutant PUF1) not only slowed photoheterotrophic growth but also decreased H2 production, indicative of the essential requirement of the complex for LH process. However, the pucBA-deleted mutant, PUC1 lacking of B800–850 complex, increased H2 production in comparison with its parental cell by approximately twofold, given irradiated with light (10 W/m2) saturating the growth of wild type. The H2 production of PUC1 did not increase in proportion to the light intensity, which is also observed with wild type. Thus, we suggest that light is not limited for the H2 production of the cells under the experimental conditions employed in this work, but the cellular energy to be used for the formation of B800–850 complex may flow into the metabolism leading to the H2 production in PUC1.

Rhodobacter sphaeroides KD131 and its mutant strain lacking uptake hydrogenase (Hup - ) and PHB synthase (Phb - ) have been studied on H 2 production and cell growth under different culture conditions. Both strains started producing H 2 from the middle of the logarithmic growth phase and continued until the cell concentration leveled out. The rates of H 2 production were 1.32 and 3.34 ml H 2 /mg-dcw for the wild-type and Hup - /Phb - mutant strain, respectively, at the optimum conditions. Malate and lactate were better carbon sources than starch, sucrose or glycerol. Approximately 60% of acetic acid was degraded in 48 h by the wild-type strain and pH increased to 9.4. However, the Hup - /Phb - mutant strain did not grow well and degraded only 19% of acetic acid. The pH ranges of 7.0 were the optimum for the cell growth and pH 7.5 for the H 2 production. Both strains grew and produced hydrogen under the irradiance of 12–120 W/m 2 , but cell growth was inhibited over 400 W/m 2 .