Study On The Denitrification And Phenol Removal Performance Of Aerobic Denitrification Mutant Strain BR4

According to relevant information released by our country’s environmental protection department,nitrogen pollution has become the main factor of water pollution in our country.At the same time,with the increasing demand for national development and modernization,phenol,which is widely used in the industrial field,as a typical pollutant,has become one of the most important pollutants in our country because of its high toxicity.Aiming at this phenomenon,the strains stored in the laboratory were irradiated with He-Ne laser to screen high-efficiency degrading bacteria.The purpose of the experiment was to improve the degradation performance of nitrate nitrogen and phenol,enhance the biological resistance of strains in toxic environments,and improve the effect of water effluent.The main conclusions are as follows:Using laser mutation breeding method,the degradation performance of phenol and nitrate nitrogen of the strain was investigated and the lethality rate and positive mutation rate were calculated.The optimal parameters of He-Ne laser irradiation were determined as the power was 20 m W and the irradiation time was 15 minutes,and the influence of power density on the mutagenesis of this experiment was excluded.The positive variant strain was subcultured for 8 generations to select the strain with stable genetic performance.Among them,the phenol and nitrate degradation rates of the mutant strain BR4 were 84.28%and82.17%,respectively,at the 8th generation.Compared with the original strain,the fastest degradation rates of nitrate nitrogen and phenol increased by 11.64 and 1.99 mg/（L·h）during the denitrification process of eutrophication,respectively.The degradation rates at 54 h were100%and 97.65%.Therefore,strain BR4 was used as the experimental strain in this study.The activity assay of phenol hydroxylase（PHO）,catechol 1,2-dioxygenase(C₁₂O)and catechol 2,3-dioxygenase(C₂₃O)in the supernatant and crude enzyme liquid of aerobic denitrification medium was carried out.The C₂₃O enzyme activity was not detected in the experiment,and the other two enzymes had higher enzyme activities in the crude enzyme solution.Adding phenol to LB medium for induction and incubation,the specific activities of PHO and C₁₂O enzymes after induction were measured to be about 16 times and 12 times that before induction,respectively.Compared with the original strain,the enzyme activity of the mutant strain BR4 was significantly enhanced during aerobic denitrification.The enzymatic reaction kinetics of PHO and C₁₂O were studied,and the K_mvalues of PHO and C₁₂O were obtained as 23.229 and 39.244μmol/L.The v_maxvalues were 0.119 and 0.115 mol/（L·min）,respectively.Determination of nitrate reductase（NAR）and nitrite reductase（NIR）during aerobic denitrification and degradation of nitrate nitrogen.Among them,NAR has higher enzymatic activity.It was preliminarily speculated that the degradation path of nitrate nitrogen was NO₃^‐‐N reduction to NO₂^‐‐N,and then converted into gaseous nitrogen or assimilated by cells.Experiment to explore the effect of phenol concentration on NAR and NIR enzyme activities.The results showed that the enzymatic activity of NIR was always lower than that of NAR,and the curve of NIR enzymatic activity was steeper and decreased rapidly when the concentration of phenol increased.However,high concentrations of phenol had a stronger inhibitory effect on NIR.Comparing the changes of NAR and NIR enzyme activities during the aerobic denitrification process between the mutant strain and the original strain,it was proved that the enzyme activity was significantly enhanced after the mutagenesis.The enzymatic reaction kinetics of the two enzymes was studied.The K_mvalues of NAR and NIR were 0.63 and 1.1μmol/L.The v_maxvalues were 37.04 and 25.64 mol/（L·min）,respectively.The substrate inhibition model Haldane equation was used to analyze the cell growth and substrate degradation process of the mutagenized strain,and the software was used to fit the data.The₈),K_sand_values of the model equation were 0.43 h^-1,9.39 mg/L and286.76 mg/L.A and B values were 0.95 and 0.03 respectively.It was proved that in the toxic environment of phenol,the inhibition of the growth of the mutagenized strain was less,and the degradation rate of phenol can still be maintained.It proved that the mutant strain has potential value in the application of nitrogen and phenol removal in sewage wastewater.A single factor experiment was used to investigate the effects of temperature,p H,and speed on the denitrification and phenol removal performance of the mutant strains.The best cultivation conditions were 30°C,p H=7.0,and 210 r/min.The BBD response surface optimization analysis was carried out with Design-Expert software,the regression model was significant,and the overall fitting effect was good.The optimal incubation conditions predicted by the model were adjusted reasonably,and the experiment was verified under the conditions of 30°C,p H=7.5,and rotation speed of 230 r/min.The degradation rate of nitrate nitrogen obtained in the experiment was basically close to the predicted value,and the degradation rate of phenol was 100%at this time.Therefore,the verification experimental conditions can be used as the best conditions in the follow-up in-depth research.