Separation,Identification Of Ammonia Oxidizing Bacteria And Construction Of AmoB Gems

Autotrophic Ammonia Oxidizing Bacteria is the main microflora in biological nitrog en removal process, its Ammonium oxidation rate impact nitrogen removal efficiency of wastewater treatment system directly. In this work, autotrophic Ammonia Oxidizing Bact eria was cultured by the methods of enrichment, isolation and purification, using traditio nal microbiological methods and modern molecular biology tools for their identification, then found out amoB gene which has Ammonium oxidation, making use of genetically engineering constructed high efficient Ammonia Oxidizing Bacteria, in order to change t he disadvantage of the long growth cycle and the low nitrogen removal efficiency of au totrophic Ammonia Oxidizing Bacteria.Isolation, identification and phylogenetic analysis of autotrophic Ammonia Oxidizing Bacteria:in this rearch two strains of Ammonia Oxidizing Bacteria were isolated from b iological membrane of aquaculture wastewater treatment facilities,they were named A.P-7 and A.P-8, and were transferred to AOB's liquid separation medium and cultured for 17 days. Quantitative test results showed that the ammonium oxidation efficiency of A.P-7 and A.P-8 can reach 90%. Firstly, the two strains were both primary identified as a species of Pseudomonas on the basis of results of morphological observation, physiologic al and biochemical experiments. Secondly, the two strains were further identified with the methods of PCR amplification, cloning and sequencing. Thirdly,the sequencing results w ere submitted to Genbank for homology search, and combined with comparative and Phy logenetic analysis through MEGA4.0 software.16S rDNA sequence analysis indicated tha t the two strains belonged to Pseudomonas and their homology to Pseudomonas mendoci na was as high as 99%.Construction of amoB reductase GEMs:the A.P-7, which was isolated in this rearch, was used as a starting strain. designed primers according to amoB gene sequence manipulation and the multiple cloning site of plasmid vector pET-28a, and A.P-7's DNA as a template, got amoB genes by PCR amplification. And the genes were cloned into pET-28a after BamHI, Xhol restriction enzyme digestion, then, chemical conversion to the competence of DH5a. Finally, constructed of transformants containing the recombinant plasmid pET-28a-amoB-DH5a. After restriction enzyme digestion and sequencing, the base sequence of PCR amplified products were completely consistent with the amoB gene sequence manipulation of A.P-7. Transformed the recombinant plasmid pET-28a-amoB into expression strain BL21, then, built GEMs of nitrification pET-28a-amoB-BL21 (PAB). Verified the success of amoB reductase expression in PAB or not by SDS-PAGE electrophoresis. To measured the ammonium oxidation effect for PAB, the results showed that PAB's ammonium oxidation rate was 5.06% higher than the A.P-7's.