Construction And Application Studies On Denitrification Engineering Bacteria PNS

Objective: To improve efficiency of denitrification and cut down the cost of denitrification in wastewater treatment, the denitrification engineering bacteria PNS which express cd1-nitrite reductase efficiently was constructed with the technology of gene cloning. The cd1-nitrite reductase is the key enzyme which catalyses the rate-limiting step in the microbial denitrification process of simultaneous nitrification and denitrification.Methods: The experimental methods in this study are as follows:1. The primers were designed according to the nucleotide sequence of nirS gene published by GenBank and multiple cloning site(MCS) of expression vector pQE-30, the nirS fragment was amplified by polymerase chain reaction with the genomic DNA of pseudomonas aeruginosa PAO1 as template. Following, the amplified product was digested by BamHⅠand HindⅢ, forced cloning into pQE-30, transferred into DH5αand the transformant pQE30-nirS-DH5αwas successfully constructed which include the recombination plasmid pQE30-nirS.2. Identified by restriction enzyme analysis and sequencing, the nucleotide sequence of amplified fragment was totally same as what was published by GenBank, and then the recombination plasmid pQE30-nirS was transferred into the expression strain SG13009, so the denitrification engineering bacteria PNS(pQE30-nirS-SG13009) was constructed successfully. Finally the PNS was identified by SDS-PAGE and His-tag in-gel Stain.3. In order to showing the denitrification of PNS in practical wastewater treatment, the expression efficiency of cd1-nitrite reductase in anaerobic condition and low temperature(at 20℃) was analyzed scientifically. At the same time, the induced concentration and induced time of IPTG were optimized rationally.Results: The major results are as follows:1. The nirS gene encoding cd1-nitrite reductase was amplified successfully by PCR, then it was forced cloning into expression vector pQE-30. Finally sequencing and BLAST revealed that the amplified sequence in pQE-30 is 100% identical to the nirS gene published by GenBank.2. The recombination plasmid pQE30-nirS was transferred into the expression strain SG13009, then the denitrification engineering bacteria was constructed successfully. Meanwhile the PNS was identified by SDS-PAGE and His-tag in-gel Stain.3. In anaerobic condition and low temperature, the expression level of cd1-nitrite reductase is also high, at the same time, the percentage of dissoluble cd1-nitrite reductase is higher than that in optimal conditions.Conclusion: We successfully constructed the denitrification engineering bacteria PNS which express cd1-nitrite reductase efficiently, meanwhile, we provided some important parameters which are essential to PNS in practical applications by analyzeing the expression efficiency of PNS in anaerobic condition and low temperature.