Microflora Analyse In Active Sludge Of Sewage Treatment And Clone And Express Nap Gene Cluster In Pseudomonas

Objectives: To explicate the composition of microflora in active sludge of sewage treatment. To obtain proper strains fitting for genes manipulating to enhance the ability of denitrification. To clone napEFDABC gene cluster from Pseudomonas strain G179 and express it in the strains we had screened. Methods and Results: First of all, we counted the population of bacteria in the active sludge. Using their biochemical and growth properties identify those strains we had isolated. We noticed that dominative bacteria in the active sludge of denitrifying reactor were Pseudomonas (23%), Acinetobacter (12%), Moraxella (16%) and Enterobacter (16%). Chosing two of them for gene improvement based on their features about denitration and resistance to antibiotics. Those two bacteria were identified as Pseudomonas stutzeri and Pseudomonas mendocina. Then, complete napEFDABC gene cluster was amplified by PCR and got a 4.7kb fragment. Which was cloned into broad-host range vector pJMS6α-lac. Restriction enzyme digestion and PCR identification confirmed thatthe recombinant plasmid pJMS6α-nap was constructed successfully. The recombinant plasmid was introduced into those two strains we had got. Two recombinants were screened by 50μg/ml kanamycin. According to identification, we could conclude that the recombinant was what we needed. Finally, we observed the recombinant's ability of degrading nitrate. Equal recombinant remove more nitrate than original bacterium.Conclusion: Expression of napEFDABC can promote original bacterium degrade nitrate.