| Fossil fuel's massive uses cause the environmental pollution and the greenhouse day by day serious, in addition the shortage of energy the threat. They make the clean energy the development and applly into ultimately. Hydrogen is not only the only product of combustion is water, but also has relatively high heat value and more easily converted into other energy forms and so on. So it is considered the cleanest, most development potential of energy sources. Biological hydrogen has mild reaction conditions, low energy consumption, use of solar energy, which can properly resolve the contradiction between energy and environment. Hydrogen energy development and treatment of organic waste water purification of organic combination of hydrogen production by photosynthetic bacteria is a low-cost, low power hydrogen production of green technology. Rhodopseudomonas palustris is an ancient the purple non-sulfur bacteria, which can produce hydrogen under photosynthetic heterotrophic conditions. Recently, it becomes a mode of photosynthetic hydrogen production strain at home and abroad.Hydrogen production metabolic pathway in photosynthetic bacteria, the nitrogenase enzyme catalyzes the hydrogen gas. Uptake hydrogenase catalyzed hydrogen proton and electron and re-supply of nitrogen-fixing enzyme, playing a role in energy recovery. Polyhydroxybutyrate synthase is to restore power in the form of material PHB. In general, H2 decomposition can reduce the amount of hydrogen. Poly-hydroxybutyrate (PHB) can reduce power consumption, reducing the amount of hydrogen produced by nitrogenase.Our aim is to construct a di-mutant bioengineering strain of Rhodopseudomonas palustris CQU01 with phbC (PHB synthase) and hupL) gene knocked-out, and improve the strain's hydrogen produce ability. We applied R. palustris CQU01 isolated from silt as initial strain, for phbC gene knocked out, and R. palustris CQU012 with hupL knocked out which be constructed before in our lab strain as the initial strains.First of all, PCR amplified phbC both sides of the 5 'flank and 3' flank as a pair of homologous exchange during the exchange of arms. These two genes were jointed in the pMD18-T vector and sequenced to verify. The pSUP202 vector contains BamHâ… and Hindâ…¢restriction sites. And correct-sequenced genes 5 'flank and 3' flank were cloned into the BamHâ… and Hindâ…¢-digested pSUP202, generating the previous vector'pSUP202-phbC'. The Emr cassette was cloned into the'pSUP202-phbC', generating the suicide gene vector pYMT. The pYMT was transformed into E. coli S17-1, obtaining the donor bacteria. The donor was mixed with the original strain (receptor bacteria) R. palustris CQU01. The correct integration of the mutants was confirmed by PCR test and subsequent sequence analysis. The single mutant strain R. palustris CQU013 with phbC knocked out and double mutant strain R. palustris CQU014 with phbC-hupL knocked out were obtained.Under the same conditions, the bacterial growth curves and hydrogen production characteristics of the mutant strains showed that the biological characters of the mutant strains had some difference with the wild strain R. palustris CQU01. Two mutant strains of bacteria increased mainly in less than 120h, and the bacteria was not significantly increased. But the wild strain of the bacteria increased until 168h, and then began to decline; Mutant strain has logarithmic growth phase longer than the wild strain. But the maximum biomass was significantly lower than the wild strain. The mutant strains may consume a large number of energy, so strain biomass is lower than the wild strain. Hydrogen production results also show that the two mutant strains have the remarkable enhancement of hydrogen. The same fermentation conditions, The total H2 yield of R. palustris CQU013 and R. palustris CQU014 increased by 31% and 76% respectively comparing to wild strain, which up to 454mL and 604mL per liter fermentation broth in 216 hours. Both the phbC gene and hupL gene affected the yield of hydrogen produce. In particular, the double mutant strain is expected as an effective engineering strain for photosynthetic hydrogen production and microbial biological treatment of industrial wastewater.The studies have shown that hydrogen production capacity of double mutant strain is more than the phbC gene and the single hupL gene mutant. Two genes are involved in the metabolism of photosynthetic hydrogen production. Double mutant can improve the hydrogen production significantly. But the result of bacterial metabolic pathways variability and diversity, there may be other metabolic pathways. So the increase is limited. On R. palustris hydrogen production capabilities, we have to understand the hydrogen metabolic pathway and the mechanism.
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