Construction Of Rhodopseudomonas Palustris HupL-and Research On Characteristic Of Hydrogen-production

It is more and more serious of the energy crisis and environmental pollution in the world today. Worldwide attention has been got on development and utilization of the renewable, clean energy. Hydrogen energy makes it ideal for future alternative energy sources due to its reproducibility and cleanness. The reaction condition of biological hydrogen-producing technology is mild and low energy-consumption. Photosynthetic bacteria are effective and feasible to produce hydrogen which can use solar energy and deal with organic wastewater. Rhodopseudomonas palustris is a modal photosynthetic bacterial strain which has been commonly used both inland and abroad.There are two R. palustris strains separated from silt and mud water in this text. R. palustris hupL deficient mutant was constructed by gene knock-out. The characteristics of hydrogen-producing and growth were researched contrastively between the wild and mutant with a view to improve access to sewage treatment for providing quality bacteria resources. The results are as follows:â‘ Screening, Purification, identification and finally obtained a patent strain of R. palustris. The sludge sampling from the bottom of the lake and riverside were enriched on the enrichment medium, and purified on the isolation medium. We have obtained two strains named R. palustris CQU01 and R. palustris CQU02 respectively through physiology and biochemistry and molecular biology methods. 16S rDNA molecular identification results showed that the two separately with the R. palustris strain: LW-RE and R. palustris strain: TUT3620 has a 97% and 98% similarity.â‘¡The R. palustris hupL targeting suicide vector was constructed and identified. Application of R. palustris CQU01 as the initial strain, the gene fragments hupS, hupC on both sides of hupL were amplified as the exchange arm in a double exchange. These two genes were jointed in the pMD18-T vector and sequenced to verify. The pSUP202 vector contains Salâ… and Hindâ…¢restriction sites, correct-sequenced genes hupS and hupC were cloned into the Salâ… and Hindâ…¢-digested pSUP202, generating the previous vector'pSUP202-hupSC'. A Kmr cassette was excised from pUC4K by Sse8387I. The'pSUP202-hupSC'was digested by Sse8387I and then dephosphorylated. The Kmr cassette was cloned into the'pSUP202-hupSC', generating the suicide gene vector pBPZ.â‘¢R. palustris hupL mutant was constructed and identified. The pBPZ was transformed into E. coli S17-1, obtaining the donor bacteria. The donor was mixed with the original strain (receptor bacteria) R. palustris CQU01 by 1: 3 at the logarithmic growth phase. The pBPZ was into the role of R. palustris CQU01 after conjunction. The purpose Km-resistant strains were screened in RCVBN plate under pressure of Km. The positive strains were bred 20 times at intervals of a 2-generation PCR detection to verify the stability of mutant strain. The usage of methylene blue on the color reaction measured for enzyme uptake hydrogenase activity indicated good stability of R. palustris CQU012 and its hydrogen activity decreased significantly.â‘£Research on characteristics of growth and hydrogen production. The wild and mutant strains of bacteria growth and hydrogen production were compared respectively. Growth characteristics of the two were no significant difference except that R. palustris CQU012 has a little longer logarithmic growth phase. There was a markedly difference between the wild and mutant strains on the volume of hydrogen-producing that R. palustris CQU01 produced a total of 350ml of hydrogen around per 1L culture after 216h and under the same conditions, R. palustris CQU012 generated a total of about 530ml of hydrogen, the total hydrogen production was about 50% improved. These two strains produced only a spot of hydrogen after 216h.The greatly improved hydrogen production capacity, no significant differences in growth characteristics and the stability in genetic characteristics of the mutant strain made it expected to have an applied value. One hand it can alleviate the energy crisis leaning on the biological sources of hydrogen, on the other hand it can provide fine bacteria resources for the organic sewage treatment.