Isolation And Immobilization Application Of A High Efficiency Aerobic Denitrifier

At present, eutrophication of water was more and more serious in our country. The treatment of excessive amounts of ammonia, nitrate, nitrite, phosphate and other nutrients on aquaculture has become a hot spot. Aerobic denitrification bacteria plays an important role in the nitrogen treatment of eutrophication, therefore, screen separation of effective aerobic denitrification strains have the vital significance. Twenty three bacterial strains with a capacity of denitrification were isolated from fish pond in Nanchang (Jiangxi) and Luan (Anhui), of which eight strains have the high rate of denitrification while one strain named HS-N62 has the strongest effect of denitrification. Strain HS-N62 showed 99% 16S rRNA gene sequence similarity to many Pseudomonas strains. Based on the morphological characteristics, physiological and biochemical characteristics, the strain HS-N62 was identified as Pseudomonas sp. The results showed that the denitrifying rate of strain HS-N62 reached 96% in 12 hours with initial nitrate nitrogen concentration of 140 mg/L and no nitrite nitrogen accumulation, and tolerated high levels of nitrate nitrogen.HS-N62 can grow with nitrite nitrogen as the only source of nitrogen and has high removal efficiency. The growth rate and characteristics of HS-N62 were further studied, and the results showed that the optimum growth temperature ranged from 30℃to 37℃, pH value from 6.0 to 8.0 and the optimal C/N ratio 10:1. Meanwhile strain HS-N62 could utilize a variety of carbon sources for growth. The most suitable denitrification condition for strain HS-N62 was confirmed by using a series of the orthogonal test. The optimum combination of strain HS-N62 was as follows:sodium succinate as carbon sources, C/N 10:1, pH 7.0, temperature 35℃. The result with single factor analysis was basically the same. This denitrifying strain HS-N62 also has good ability of phosphorus removal, and the removal rate within 12 hours was up to 67.7% (at initial phosphate concentration 57 mg/L). Analysis of the nitrite reductive gene and the Sequencing result showed that, HS-N62 contained nirK gene, which is highly homologous to the nirK of uncultured bacterium HM628847.This study through improving and optimizing the traditional method of immobili-zation PVA-boric acid, Bacteria and powdered activated carbon were immobilized with sodium alginate and polyvinyl alcohol to make into small balls using SiO2 and CaCO3 embedded to enhance the strength of the small spherical particle. The nitrate nitrogen removal rates of NO3--N, which was determined in the SC medium, reached 57.3% and 87.2% at 12 h and 24 h when the immobilized bacterium HS-N62 was used at the first time. The NO3--N could be degraded completely after another 12 h, and there almost no nitrite nitrogen will be accumulated. This method was easier to prepare PVA ball than other methods and improve the mass transfer performance and the permeability of the ball, also improve the removal rate of nitrate nitrogen. Compared with the SC medium that was treated by embedding immobilized denitrifying bacteria and free denitrifying bacteria, it showed that the former had the higher tolerance and the strong ability of nitrogen removal, under the unfavorable pH and temperature.