Isolation And Identification Of A Strain Of Acid Tolerant Denitrifying Bacteria And Its Denitrification Characteristics

Ammonium nitrate used as eluant in the process of uranium acid in-situ leaching usually results in seepage of nitrate into aquifer,which causes the nitrate in groundwater environment out of limits after field leaching.Microbial denitrification method is widely used in the treatment of nitrogenous waste water because of its high efficiency and low cost.However,the conditions of denitrifying bacteria?DNB?are generally neutral or alkalescent,acidic or acidulous conditions are relatively rare.In view of the problems of low pH and high nitrate levels in acidic mine groundwater,Acid-resistant denitrifying bacteria was enriched,isolated and acclimated from the aquifers of a sand stone type uranium ore in Xinjiang by selective medium.The strain?G16X-D?was purified by dilution coating and plate scribing and identified by Biolog physiological and biochemical tests and 16 S rDNA gene sequence analysis.The effects of different initial pH,temperature,carbon sources and C/N on the growth and denitrification of G16X-D were studied by facultative anaerobic liquid culture method to optimize the denitrification conditions.The kinetic relationship between different initial nitrate nitrogen concentration and denitrification rate were analyzed.The effects of heavy metal ions(Zn²⁺,Cd²⁺,U⁶⁺)on the removal of nitrate by G16X-D were discussed.In addition,the effects of G16X-D on nitrate nitrogen removal from bioleaching uranium leaching solution were studied under optimized conditions,which provided the theoretical basis for future application.This paper mainly obtains the following results:An acid-resistant denitrifying bacteria G16X-D was obtained by enrichment,separation and purification.Morphological observation,Biolog physiological and biochemical tests and 16 S rDNA gene sequence analysis showed that strain G16X-D belongs to Pseudomonas aeruginosa.Test results of influencing factors showed that the strain G16X-D had high denitrification ability under the condition of pH4.50,and the denitrification occurred mainly in the logarithmic growth phase.The optimal denitrification conditions were as follows: the optimum carbon source was glucose,C/N=3.0 and the temperature was 35 ?.Under this condition,the removal rate of nitrate nitrogen reached 92.7% when the initial nitrate nitrogen concentration was 160 mg·L^-1,the specific growth rate ?=0.0472 h^-1 and the shortest doubling time td=14.68 h.The optimum pH of strain G16X-D was 5.77⁷.70.K_inetic study showed that the denitrification rate reached the maximum value of V_max=2.99 mg·L^-1·h^-1 when the nitrate nitrogen concentration was 350.84 mg·L^-1,and the half-saturation constant K_m=162.50 mg·L^-1,the inhibition constant K_i=757.47 mg·L^-1.Cd²⁺ inhibited the denitrification of G16X-D,and the higher the concentration,the stronger the inhibitory effect.When U⁶⁺ concentration was below 7.50 mg·L^-1,the denitrification activity was enhanced,and had a inhibitory effect when it was more than 7.50 mg·L^-1.A small amount of Zn²⁺ contributes to the denitrification process,with inhibition of more than 50 mg·L^-1.The order of the toxicity of three heavy metal ions to G16X-D was Cd²⁺> U⁶⁺> Zn²⁺.The denitrification of G16X-D was strongly inhibited by undiluted water samples under the condition of optimized denitrifying culture with the microbial leachate of uranium ore as test raw water.The removal rates of nitrate nitrogen were 64.4% and 41.4% respectively after 8 days culture of G16X-D inoculated with 1% in the test water sample with initial nitrate nitrogen concentration of 86.52 mg·L^-1 and 163.52 mg·L^-1 respectively.The best inoculation amount in the wastewater was 7.5%,and 91.2% nitrate nitrogen could be removed by 96 h with the initial nitrate nitrogen concentration of 160 mg·L^-1.G16X-D has achieved good results for the removal of nitrate nitrogen from acidulous mine wastewater of pH4.50.