Photo-fermentational hydrogen production of Rhodobacter sp. KKU-PS1 isolated from an UASB reactor

Background In this study, the detection of nifH and nifD by a polymerase chain reaction assay was used to screen the potential photosynthetic bacteria capable of producing hydrogen from five different environmental sources. Efficiency of photo-hydrogen production is highly dependent on the culture conditions. Initial pH, temperature and illumination intensity were optimized for maximal hydrogen production using response surface methodology with central composite design. Results Rhodobacter sp. KKU-PS1 (GenBank Accession No. KC478552) was isolated from the methane fermentation broth of an UASB reactor. Malic acid was the favored carbon source while Na-glutamate was the best nitrogen source. The optimum conditions for simultaneously maximizing the cumulative hydrogen production (Hmax) and hydrogen production rate (Rm) from malic acid were an initial of pH 7.0, a temperature of 25.6°C, and an illumination intensity of 2500 lx. Hmax and Rm levels of 1264 ml H2/l and 6.8 ml H2/L-h were obtained, respectively. The optimum initial pH and temperature were further used to optimize the illumination intensity for hydrogen production. An illumination intensity of 7500 lx gave the highest values of Hmax (1339 ml H2/l) and Rm (12.0 ml H2/L-h) with a hydrogen yield and substrate conversion efficiency of 3.88 mol H2/mol malate and 64.7%, respectively. Conclusions KKU-PS1 can produce hydrogen from at least 8 types of organic acids. By optimizing pH and temperature, a maximal hydrogen production by this strain was obtained. Additionally, by optimizing the light intensity, Rm was increased by approximately two fold and the lag phase of hydrogen production was shortened.

Analogue-resistant mutants of Azotobacter chroococcum derepressed for nitrogenase activity and early ammonia excretion having potential as inoculants for cereal crops.

Spontaneous mutants resistant to methionine sulfoximine (Msx), methyl alanine (Mal) and methyl ammonium chloride (Mac) were derived from A. chroococcum strain A103. Msx and Mal-resistant mutants expressed 1.73 to 10.98% of the fully derepressed nitrogenase activity when grown in Burk's medium containing ammonium acetate. Mac-resistant mutants did not express nitrogenase activity in ammonium acetate supplemented medium. The mutants excreted ammonia even after 2 days of growth and some mutants excreted more ammonia as compared to the parent. Selected mutants were inoculated on wheat (Triticum aestivum) and barley (Hordeum vulgare) under field conditions. Majority of the derepressed mutants increased grain yield of wheat and barley varying from 1.2 to 33.3%. However, host-dependent effects on grain yield were observed with different mutants. Two mutants, Mal 27 and Mac 19 showed significant increase in grain yields of both the crops. The results suggest that metabolic analogue-resistant mutants of Azotobacter have potential for use as a biofertilizer for cereal crops.

Expression of Rhizobium meliloti symbiotic promoters in Azorhizobium caulinodans.

Plasmids containing Rhizobium meliloti symbiotic promoters P1 (promoter of nifHDK) and P2 (promoter of fixABCX) when mobilized into the cells of Azorhizobium caulinodans strain IRBG 46 showed strong expression of these promoters under free-living microaerobic as well as symbiotic conditions. Under free-living conditions microaerobiosis (3% or less O2) was found to be sufficient to activate these promoters; expression being higher at 1% than at 3% O2 concentration. Under symbiotic conditions the expression was much more stronger-with bacteroids in stem nodules showing higher expression than those in root nodules. Under both the conditions expression of the promoters in the native R. meliloti strain Rm102F34 was lower than that in the A. caulinodans strain IRBG 46. The results suggest a functional homology of these promoters in the heterologous background of A. caulinodans.

Cloning and sequencing of the nitrogenase structural genes nifHDK of Herbaspirillum seropedicae

The nitrogenase structural genes (nifHDK) of the endophytic diazotroph Herbaspirillum seropedicae were isolated from a genomic bank by plate hybridization. Sequence analysis of the DNA showed a consensus promoter region upstream from the nifH gene containing a -24/-12 type promoter together with NifA- and integration host factor (IHF)- binding sites. The derived protein sequences of NifH, NifD and NifK contained conserved cysteine residues for binding iron-sulfur clusters and the iron-molybdenum cofactor. These protein sequences showed the strongest similarities to the nifHDK gene products of the symbiotic diazotroph Bradyrhizobium japonicum (93.5 per cent, 91.3 per cent and 83.3 per cent, respectively), the plant-associated diazotrophAzospirillum brasilense (90.0 per cent, 83.7 per cent and 75.1 per cent, respectively) and to Thiobacillus ferrooxidans (91.0 per cent, 83.4 per cent and 81.1 per cent, respectively) of the same phylogenetic group of the protobacteria.