Isolation Of Anaerobic Fermentative Hydrogen-producing Bacteria And Construction Of Mutant Library Of Hydrogen-producing Bacteria By Inserting Transponson

Enlarging species resources of hydrogen-producing bacteria is of importance in bio-hydrogen field. We studied the hydrogen-producing characteration of mixed culture and pure bacterium, and constructed mutant library of hydrogen producing bacterium by screening for wild type and transposon mutagenesis of hydrogen-producing bacteria. The aim of this research was to harvest efficient hydrogen-producing microorganisms This research can lay a theoretical foundation for construction of engineering strain that can utilize organic contents in the high-salt organic wastewater to generate hydrogen energy.1. Effect of culture conditions on hydrogen producing of mixed cultureThe effects of pretreatment, initial pH and substrate species on hydrogen producing of mixed culture were determined. Denaturing gradient gel electrophoresis (DGGE) was used for analyzing composition of microbial community. Four different pretreatment methods, including acid treatment, heat-shock, base treatment as well as freezing and thawing, acid pretreatment significantly promoted the hydrogen production by sludge and provided the highest efficiency of hydrogen production among the four methods. Acid pretreatment was favorable to enrich the dominant hydrogen-producing bacterium, i.e. Clostridium sp. and Bacillus sp. The dominant bacteria of mixed culture pretreated by heat shock were Clostridium sp. and Enterococcus sp. However, besides hydrogen-producing bacteria Clostridium sp., much non-hydrogen-producing Lactobacillus sp. was also found in the sludge pretreated with base, freezing and thawing methods. Hydrogen production of mixed culture was evidently affected by different initial pH. At the optimal initial pH of 7.0, hydrogen production potential of mixed culture was the highest, gradually decreasing at the following pH sequence of pH 6.5, pH 7.5, pH 6.0, pH 8.0, pH 5.5, pH 5.0, pH 4.5, and pH 4.0. The dominant bands obtained on DGGE of 16S rRNA gene were identical at various initial pH values. These bands were cloned and sequenced. NCBI blast indicated that the segment of 16S rRNA gene was high identical to that of Clostridium sp. (98%). Mixed culture was cultivated by five different substrates (glucose, sucrose, lactose, starch and peptone). The highest hydrogen production potential of mixed culture was observed when using sucrose as substrate. The dominant bacteria were Clostridium sp.and Bacillus sp.. 2. Identification of hydrogen-producing bacteria and optimization of hydrogen productionIsolation of wild type hydrogen-producing bacteria is necessary means for enlarging species resource of hydrogen-producing microorganism. Two strain hydrogen-producing bacteria were isolated from sludge collected in the intertidal zone of a bathing beach in Tianjin. Bacteria with high hydrogen-producing ability were isolated and identified using light microscopic examination, physiologic and biochemical characterization and 16S rRNA gene sequence analysis. The isolated bacteria were designated Bacillus sp.B2 and Clostridium sp. T7, respectively. Hydrogen production of two strains was measured under marine condition, companied with an initial glucose concentration of 20g/l and an initial pH value of 7.0. The maximum hydrogen production was 1.65±0.048 mol H2/mol glucose for Bacillus sp.B2, whereas 1.79±0.062 mol H2/mol glucose for Clostridium sp. T7.3. Construction of mutant library of Pantoea agglomerans and screening of high hydrogen-producing mutantsTransposon mutagenesis is an effective approach for harvesting efficient hydrogen-producing bacteria. A Tn7-based transposon was randomly inserted into genomic DNA of Pantoea agglomerans BH18 in vitro. Mutants were screened by Kanr and amplification of the inserted sequences. 340 colonies were randomly selected on the plate with 40μg/ml kanamycin, and in which 331 transposon insertion mutants were obtained by amplification of inserted sequences. Capacity of mutants was 4.35×108/ml. Using hydrogen production as screening index, a highly effective hydrogen producing strain TB5 was screened among 331 mutants. Its hydrogen content and hydrogen production were 1.1 and 1.52 times as much as that of wild type, respectively. The optimum pH of hydrogen production for strain TB5 was 7.0, whereas 6.0 for wild type. The ability for utilizing starch as hydrogen producing substrate was investigated. 17 strains with higher hydrogen production than wild type were obtained among 331 mutants.