Compound Mutagenesis And Denitrification Of Rhodococcus Pyridinivorans Under LAS Stress

With the acceleration of urbanization and improvement of people’s living standards,the discharge of domestic sewage is increasing day by day.Nitrogen pollution in domestic sewage can cause environmental pollution and threaten human health.Microbial enhanced wastewater treatment is an efficient wastewater denitrification technology.High-efficiency denitrification and degrading bacteria can simultaneously remove organic matter and nitrogen from sewage,and have become a hot spot in the field of water environment research in recent years.However,with the widespread use of household cleaning agents and laundry chemicals in recent years,the content of anionic surfactants in domestic sewage has gradually increased.Straight-chain alkyl benzene sulfonate（LAS）is the chief anionic surfactant.Sufficient studies have shown that the exposure of a large amount of LAS in water bodies will cause toxicity inhibition of the growth and denitrification function of denitrification bacteria,which will bring difficulties to sewage denitrification treatment.Therefore,the selection and breeding of LAS-tolerant denitrification strains has important practical significance.Based on the previous screening of a denitrifying strain Rhodococcus pyridinivorans C-24,the denitrifying strain was subjected to UV-DES compound forward mutagenesis under LAS stress,in order to strengthen the denitrifying strain C-24 in LAS.The ability of adaptability in the environment to improve the growth,metabolic activity and denitrification ability of mutagenized bacteria under LAS stress.Main results of this study were as foll:（1）In order to obtain a denitrifying strain resistant to LAS stress,the heterotrophic nitrifying aerobic denitrifying strain C-24 was used as the starting strain.The lethality rate was calculated to determine the optimal irradiation time.After primary screening and re-screening,the dominant forward mutation strains with low nitrite accumulation,high TN removal rate,and resistance to LAS stress were bred,and they were genetically stable.When the initial NH₄⁺-N concentration was 25 mg/L,50 mg/L and 100 mg/L,the NH₄⁺-N degradation rate of the mutant strain was 98.71%,94.54%and 74.16%,which was 4.4%,4.26%and 5.08%higher than the primitive strain;When the initial NO₃^--N concentration was 25 mg/L,50 mg/L and 100 mg/L,the NO₃^--N degradation rate of the mutant strain was 74.49%,65.59%and 14.06%,which was 13.67%,8.46%and 4.98%higher than the primitive strain.（2）Under LAS stress,the cell growth rate and cell number of the mutant strain were significantly increased.SEM study showed that high concentration stress would cause serious damage and destruction to the cell morphology and cell membrane of the strain.Compared with the original strain,the time for the mutant to reach the highest OD₆₀₀in logarithmic growth period was shortened by 4 hours.At Las concentrations of 0 mg/L,1mg/L,5 mg/L and 9 mg/L,the OD₆₀₀of the mutant cultured for 24h increased by 1.34%,2.60%,9.45%and 13.35%respectively compared with the original strain.（3）The utilization ability of mutant strains to carbon source was studied by ECO-biolog method.The mutant strains improved the utilization of five carbon sources,including alcohols,eaters,amino acids,amines and carboxylic acids.Under the stress of high concentration of LAS,the mutant strains showed more superior carbon metabolism ability.The ability of the mutant strain to utilize amino acids,lipids,alcohols,amines and organic acids as carbon sources was significantly improved by 16.65%,22.81%,16.21%,13.41%and 10.66%compared with the primitive strains.（4）The inhibition kinetics of LAS on nitrification and denitrification of the strain was studied by using the Haldane model.The results showed that the maximum nitrification rate and denitrification rate of the mutant strain under LAS stress could reach1.1808 mg/L/h and 1.0479 mg/L/h,which were 13.80%and 8.81%higher than the primitive strain;the mutant strain had higher K_mvalues of 0.5333 mg/L and 0.5012 mg/L,which were 275.04%and 69.44%higher than the primitive strain;the mutant strain had higher K_ivalues of 35.8106 mg/L and 11.9481 mg/L,which were 10.83%and 82.05%higher than the primitive strain.It showed that the mutagenesis improved the nitrogen removal ability and tolerance ability of the strain under LAS stress.（5）The expression of nitrification and denitrification genes of mutant strains under LAS stress was studied by fluorescence quantitative PCR.q PCR results showed that the denitrogenation gene expression ability of the mutant was improved under Las stress.Under the stress of 5mg/L LAS,the expression of nitrifying genes amo A and nxr A in the mutant strain was 4.75%and 175.42%higher than that in the original strain,and the expression of denitrifying genes nap A,nar G,nir K,nir S,nor B and nos Z in the mutant strain was 11.08%,33.27%,24.23%,14.53%,11.54%and 186.63%higher than that in the original strain.Regression analysis showed that there was a significant correlation between nitrification gene and nitrification rate,denitrification gene and denitrification rate（p<0.05）,and Las stress significantly reduced the expression activity of functional genes of the mutant.（6）The optimum denitrification conditions were determined by single factor test,i.e.temperature 37℃,p H 8,carbon source sodium succinate,rotating speed 160 r/min and carbon nitrogen ratio 11.LAS,carbon source,rotating speed and carbon nitrogen ratio were selected as the optimization factors of response surface.When Las concentration was 2 mg/l,sodium succinate was selected as carbon source,rotating speed was 190 r/min and carbon nitrogen ratio was 11,the maximum degradation rate of total nitrogen could reach 89.26%.The strain was inoculated into the actual sewage and its actual treatment capacity was investigated.After verification,it was found that the removal rates of TN、NH₄⁺-N、NO₃^--N and NO₂^--N by the mutant strain were 91.47%,95.05%,96.67%and95.33%respectively,which were 8.17%,5.11%,11.82%and 4.28%higher than the original strain.