An Denitrifying Bacteria Isolated And It's Nitrous Oxide Reductase Gene Cloning In RDB

With the development of industry, the emission of nitrogen oxides(NO_x) to the atmosphere causes serious envitonmental problems, which should be brought under strict control. In the previous study, the rotating drum biofilter (RDB) has been developed and applied as an effective technique for NO pollution control. In order to improve the RDB performance for NO denitrifying removal, the investigation studied the main denitrifying bacteria and its nitrous oxide reductase.A heterotrophic denitrifying strain named ND1 has been isolated from RDB. It was proved to be Gram-negative. In addition, it was able to form dry and wrinkled colonies on the medium, adhere on the agar surface, produce yellow pigments and be motile with a single polar flagellum. It was indentificated as the Pseudomonas stutzeri according to morphological and physiological biochemical characteristics and the 16S rRNA sequence analysis. Under the conditions of pH 7.2 and temperature 30℃, the NO3-N removal efficiency by ND1 was up to 100% in 5 days, while the NO2-N was only 85% by the strain DN1 when under the same conditions.showed that ND1 has the strongest denitrification performance at pH 7.5, while below pH 5 it can not survive. The Cu2+ concentration has no effect on the degradation when lower than 75 mg/L. ND1 showed the low tolerance to salt, when the salt concentration higher than 5% of the medium, it can not grow and showed no obvious need to salinity. The different carbon sources on denitrification effect is obvious. Compered with the methanol, glycerol and sodium bicarbonate, the glucose and sodium succinate were the better carbon source for ND1, and the degradation efficiency can exceed 80%. Unger the invariable Carbon concentration, nitrogen source showed no effect on ND1 degradation rate. Additionally, the performance of ND1 on nitrate degradation was better than nitrite by adding sodium nitrate and sodium nitrite as two different nitrogen source.According to the nosZ gene of Pseudomonas CodeHop, a pair of degenerate primers was designed. A gene fragment of 1691bp was obtained by the temperature gradient PCR and the annealing routine PCR. The gene fragment showed 92% homology Pseudomonas sp. D3-15 nitrous oxide reductase nosZ gene (Genbank registration number AM422883.1). It can be concluded that the bacteria Pseudomonas stutzeri ND1 strains have been successfully cloned and nitrous oxide reductase gene nosZ in part.