Study On The Isolation And Identification Of The Heterotrophic Nitrification And Aerobic Denitrification Strain JB4 And Nitrogen Removal Characteristics

Studies in this thesis obtained a strain with heterotrophic nitrification and aerobic denitrification at high efficiency using heterotrophic nitrification and aerobic denitrification culture medium and the gradient dilution plate method and the streak plate method combined with Griess reagent method and the BTB method for separation and purification many times,almost non-existent nitrate nitrogen and nitrite nitrogen accumulation.And the strain was identified as Bacillus sp., named Bacillus sp.JB4.On this basis, the physical and chemical factors on the growth and denitrification ability have been studied.Also strain JB4 denitrification path was inferenced preliminarily through the determination of intermediate and final products and the means of molecular.To get a further evaluation of the engineering application value for strain JB4,the organisms were immobilized and then used to purify the wastewater containing nitrogen to provide reference and basis for practical wastewater treatment of heterotrophic nitrification and aerobic denitrifying bacteria in future.Based on the above,some conclusions were given as follows:1. Strain JB4 had a good heterotrophic nitrification and aerobic denitrification ability.Ammonia nitrogen removal rate was 77.78% within 12h in the heterotrophic nitrification medium with the initial concentration as 65.45 mg/L and in the process not the accumulation of nitrate nitrogen but small amounts of nitrate nitrogen accumulation;nitrate removal rate was 86.07% within 12h in the aerobic denitrification medium with initial concentration as 442 mg/L and the accumulation of nitrate nitrogen was less than 1.83 mg/L;strain JB4 didn't grow in the medium of hydroxylamine as the only nitrogen source and hydroxylamine wasn't produced in the process of removing ammonia nitrogen.It was shown that nitrate reductase gene (narG) was observed in the genome of strain JB4,ammonia oxygenase (amoA) and hydroxylamine oxidase genes (had) was't found through the sequencing for the sub-genes of amoA,hao and narG.In conclusion, the hypothetical metabolism path of strains JB4 was that Ammonia nitrogen was directly oxidized into nitrate nitrogen without accumulation of hydroxylamine and nitrate nitrogen was reducted into nitrite nitrogen and then the nitrite nitrogen was converted into nitrogen and the nitrogen was discharged from wastewater finally.2. By investigating the physical and chemical factors on the growth and nitrogen removal ability of strain JB4, the results showed the appropriate conditions for nitrogen removing was that:the temperature was 35?,shaking speed was 180r/min,inoculum concentration of bacterium suspension was 3%,sodium succinate was the best carbon source,C/N was 8,pH of ammonia nitrogen removal was 7.0,pH of nitrate nitrogen removal was 9.0.Ammonia nitrogen removal rate of strain JB4 was from76.95% to 84.58%, nitrate nitrogen removal rate was from 85.61% to 91.34% within 12h in the process.And by the test of orthogonal experiment,the optimum culture conditions of strain JB4 were given as orders:temperature 35?,shaking speed of 200 r/min,the substrate concentration (heterotrophic nitrification was 50 mg/L,aerobic denitrification was 200 mg/L),inoculum concentration (heterotrophic nitrification was 2%,the aerobic denitrification was 3%).Under the optimal conditions,the ammonia nitrogen(initial concentration as 50 mg/L) was degradated to 7.49 mg/L within 12h, the removal rate reached 85.02%;the nitrate nitrogen (initial concentration as 200 mg/L) was degradated to 16.38 mg/L,removal rate reached 91.81%.The results showed that through conditions optimization,nitrogen removal rate of strain JB4 was improved.3.Immobilized microorganisms using Bacillus sp.JB4 with 4% PVA and 2% SA were prepared. And the effects of Immobilized microorganisms on purifying the artificial wastewater and livestock wastewater were observed. Results showed that ammonia nitrogen and nitrate removal rate of Immobilized microorganisms were 88.28% and 94.57% respectively in the artificial wastewater purifying;various nitrogen forms and CODcr were removed to a certain extent and the ammonia nitrogen,nitrate nitrogen,nitrite nitrogen and CODcr removal rates were above 60% except total nitrogen in livestock wastewater repair experiments;the nitrogen removal rate of immobilized microorganisms was better than the free strain JB4 in the process of wastewater purifying.Therefore strain JB4 had the certain application prospect in the wastewater purifying.