Separation,Identification Of Autotrophic Nitrobacteria And Construction Of NorB Gems

Autotrophic nitrobacteria is the main microflora in biological nitrogen removal process, its nitrification rate impact nitrification effect and nitrogen removal efficiency of wastewater treatment system directly. In this work, autotrophic nitrobacteria was cultured by the methods of enrichment, isolation and purification, using traditional microbiological methods and modern molecular biology tools for their identification, then found out norB gene which has nitrification, making use of genetically engineering constructed high efficient nitrobacteria, in order to change the disadvantage of the long growth cycle and the low nitrogen removal efficiency of autotrophic nitrobacteria.Isolation, identification and phylogenetic analysis of autotrophic nitrobacteria:Two strains of autotrophic nitrobacteria N3 and N4 were isolated from aerobic activated sludge of A2/O treatment process, transferred to liquid separation medium and cultured for 14 days. Test results indicated that the nitrification efficiency of the two strains can reach 100%. The two strains were primary identified as a species of nitrobacteria on the basis of results of morphological observation, physiological and biochemical experiments.The strains were further identified with the methods of PCR amplification, cloning and sequencing, the sequencing results were submitted to Genbank for homology search, combined with comparative and phylogenetic analysis through MEGA software.16S rDNA sequence analysis showed that the two strains belonged to nitrobacteria and their homology to Nitrobacter Winogradskyi was as high as 98%.Construction of norB reductase GEMs:In this rearch, the N3 was used as a starting strain and its DNA as a template, designed primers according to nitrification reductase norB gene sequence manipulation and the multiple cloning site of plasmid vector pET-28a, got norB genes through PCR amplification. Later, cloned into pET-28a vector after EcoRI, Hindâ…¢restriction enzyme digestion, then, chemical conversion to the competence of DH5a cloning strain, finally, constructed of transformants containing the recombinant plasmid pET-28a-norB. After restriction enzyme digestion and sequencing, the base sequence of PCR amplified products were completely consistent with the norB gene sequence manipulation of N3. Transformed the recombinant plasmid pET-28a-norB into expression strain BL21, then, built GEMs of nitrification pET-28a-norB-BL21 (PNB). Verified the success of norB reductase expression in PNB or not using of SDS-PAGE electrophoresis, obtained a protein band of 57KDa, which was consistent with the expected size. To measured the nitrification effect for the recombinant genetic engineering bacteria PNB, the results showed that PNB's nitrification rate was 12.7% higher than the N3.