Characterization Of The Removal Of Phenol And Nitrate Nitrogen Simultaneously By Aerobic Denitrifying Bacteria Pseudomonas Sp.BN5

Phenol is a typical pollutant in wastewater produced by coking plants,oil refineries,plastic factories,pharmaceutical factories,etc.As one of the key pollutants to control in China,phenol is highly toxic to the environment and organisms,and denitrification of wastewater efficiently has been the goal of research.In traditional nitrogen removal process,the nitrogen needs to be nitrified under aerobic environment,and then denitrified under anaerobic condition.The discovery of simultaneous heterotrophic nitrification and aerobic denitrifying bacteria brings us new ideas.It is a kind of strain that can simultaneously perform heterotrophic nitrification and denitrification under aerobic condition in the same reactor to remove nitrogen and organics,which increase the reaction efficiency greatly and also save the treatment costs.In this paper,the heterotrophic nitrification and aerobic denitrification strain Pseudomonas sp.BN5 isolated by our laboratory has been studied.The phenol and nitrate nitrogen were used as the sole carbon source and the sole nitrogen source respectively.The effects of temperature,pH and shaking speed on the performance of the strain were analyzed by single factor method to obtain the optimum culture conditions and to analyze the characteristics of substrate removal and cells`growth.Haldane equation was used to fit the growth kinetics of cells inhibited by substrates.The effects of phenol with different initial concentration on cell growth,key enzymes NAR and NIR activity were studied.The nitrogen balance analysis was used to demonstrate the pathway of substrate removal.The strain was made into immobilized cells using sodium alginate,and Plackett-Burman design,steepest climbing,and Box-Behnken response surface design were used to analyze the significant influencing factors and their optimal values during immobilization.A mutant strain was screened by ultraviolet mutagenesis and its performance was determined.The main conclusions are as follows:The single factor method was used to study the changes in the performance of strains for removing phenol and nitrate nitrogen at different temperatures,pH and shaking speed.When the initial concentration of phenol was 420 mg/L,the maximum phenol degradation rate of strain BN5 during logarithmic growth phase was 17.01 mg phenol/?L·h?,the maximum nitrate nitrogen removal rate was 1.59mg NO₃^--N/?L·h?,and the degradation rates of phenol and nitrate nitrogen were100.00%and 93.31%respectively at 72 h.At 30 h,the maximum accumulation rate of nitrite nitrogen was 6.58 mg/L and was subsequently removed.The cell growth kinetics was fitted using the Haldane equation.The fitting equation was?=0.34S/?12.88+S+S²/202.59?,R²=0.989,which means the fit was good.The strain has the maximum cell growth rate when the initial phenol concentration was 51.08 mg/L.The crude enzyme solution was extracted from strain BN5 and then the experiment was carried out at an initial phenol concentration of 420,570,and 720 mg/L respectively.The results showed that the growth rate decreased with the increase of phenol concentration,but the final nitrate nitrogen degradation rate increased from 93.31%to 98.31%,and the final cell concentration increased from 205.75 mg/L to 213.84 mg/L.The activities of nitrate reductase and nitrite reductase decreased,and nitrite reductase got stronger inhabitation.Immobilized cells were prepared using sodium alginate,and significant influence factors were obtained by Plackett-Burman design.The test center was determined by the steepest climbing experiment.And finally,the optimum values were determined by Box-Behnken design:liquid-solid ratio is 5.00:1,bacterial-cement ratio is 1:24.95,sodium alginate concentration is 4.20%,and the other six insignificant factors are:culture temperature 30?,initial phenol concentration420 mg/L,shaker speed 180 r/min,CaCl₂ concentration 0.3 mol/L,initial pH=7.0,immobilization time is 2 h.Using UV mutagenesis through three repeated screenings,a mutant strain BN5-3 was obtained,which was 20%faster than the original strain and the maximum degradation rate was increased by 2.35%on phenol degradation,the nitrate removal rate also increased by 4.15%and the maximum removal rate got 10.06%quicker than the original one.After 10generations of culture,the degradation rate of phenol was still up to 98.19%,means that the genetic stability was good.