Research On Remediation Agent For PAHs-contaminated Soils Based On The Combined Roles Of Plants And Microbes

Polycyclic aromatic hydrocarbons(PAHs)are a typical class of persistent organic compounds,which are listed as priority environmental pollutants by countries around the world due to their carcinogenic,teratogenic and mutagenic toxicological effects.Since soil is the "sink" of PAHs accumulation in the environment,the problem of PAHscontaminated soil is becoming more and more severe.Therefore,it is necessary to remediate PAHs-contaminated soil.The plant-microorganism combined remediation technology has received extensive attention due to its advantage of low cost and environmental friendlyness.The combined remediation is based on the synergistic effect of plants and degrading microorganisms.Plant root exudates can provide nutrients for degrading microorganisms in the rhizosphere to facilitate degradation of pollutants,or achieve co-metabolism with pollutants to enhance remediation.However,the exudation of plant root is influenced by the plant growth cycle and environmental conditions,and the rhizosphere effect decreases with the distance from the root increases.Therefore,it is difficult to keep the remediation efficiency stable in a wide range for a long time.Based on the mechanism of plant-microorganism combined remediation,the PAHscontaminated soil remediation agent was invented to innovate the remediation technology and broaden the technical adaptability.PAHs-degrading strains were screened from the actual contaminated substrates.The strains with high-efficiency degradation potential were screened via enzyme efficiency evaluation,which were used in the construction of high-efficiency degradation microflora.The effect of artificial root exudates(ARE)and their components on the degradation of PAHs by the constructed microflora were investigated.,The formulation and the application concentration of ARE were optimized.The PAHs remediation agent was prepared by immobilizing PAHs-degrading microflora and ARE onto biochar as the carrier through adsorption.The remediation effect of the prepared agents on two types of contaminated soils with different properties were investigated and the application feasibility was evaluated.The main conclusions obtained are as follows:(1)Sixty-four purified strains were isolated from contaminated substrates,of which43 were sequenced successfully and identified as 22 different species of bacteria.Nine strains were obtained from petroleum substrates and they were Brucella sp.(P-1),Klebsiella sp.(P-2),Acinetobacter sp.(P-3),Azospirillum sp.(P-4),Sphingopyxis sp.(P-5),Ochrobactrum sp.(P-6),Bacillus sp.(P-7),Pseudoxanthomonas sp.(P-8)and Escherichia sp.(P-9).Thirteen strains were obtained from PAHs substrates,including Pseudarthrobacter sp.(J-1),Ensifer sp.(J-2),Bacillus sp.(J-3),and Paraburkholderia sp.(J-4),Pseudomonas sp.(J-5),Stenotrophomonas sp.(J-6),Cupriavidus sp.(J-7),Sphingomonas sp.(J-8),Acinetobacter sp.(J-9),Rhodococcus sp.(J-10),Pararhizobium sp.(J-11),Advenella sp.(J-12)and Gordonia sp.(J-13).Among these 22 strains,18 strains can utilize high-molecule-weight PAHs(pyrene and benzo [a] pyrene)as carbon source and 10 strains can degrade two or more kinds of PAHs.(2)Ten PAHs-degrading strains of high efficiency were selected from 22 strains using the enzymatic liquid degradation-enzymatic efficiency evaluation method.They were P-1,P-2,P-3,P-5 and P-6 obtained from the PAHs substrates and J-1,J-2,J-3,J-4and J-5 obtained from the PAHs substrates.Twenty-six bacterial bacterial floras were constructed with 5 strains from PAHs substrates in different combinations.Their PAHsdegrading ability was detected and 2 bacterial flora with high PAHs-degrading efficiency were screened.They were bacterial flora A containing strain J-2,J-3,J-4 and J-5 and bacterial flora B containing J-1,J-2,J-3,J-4 and J-5.The other 5 strains obtained from petroleum substrates were constructed as bacterial flora C.The PAHs degradation rates by the 3 bacterial flora in PAHs-liquid medium(50 mg/L)in 7 days were 10.0%,17.0%and 16.1%,respectively.The residual concentration of total PAHs in the system decreased to 33.8 mg/L(flora A),31.2 mg/L(flora B),and 31.4 mg/L(flora C),and the removal rates of PAHs reached 32.4%,37.6% and 37.2%,respectively.The degradation ability of bacterial flora B and C was better than that of bacterial flora A.(3)The effect of ARE with different components and concentrations on the degradation of PAHs by bacterial flora A,B and C were studied.Low concentrations of ARE(0.05 g/L and 0.1 g/L)did not promote the degradation of PAHs while high concentrations of ARE(1 g/L or 10 g/L)significantly promoted the degradation of PAHs.The effect of components contained in ARE at 10 g/L was also studied.Carbohydrate and low-molecular acids can promote the PAHs degradation by bacterial bacterial flora A,B and C to different extent while the amino acids only promoted the degradation by bacterial flora C.The effect of carbohydrate components was not significantly different from that of the complete ARE,indicating that the traditional ARE combined with the three components did not show an advantage.Therefore,it was optimized with single factor and response surface analysis on the basis of the original ARE formula and concentration.Taking bacterial flora B as the target bacterial flora,the optimized formula of ARE and the molar concentration ratio of each component were: carbohydrate components(glucose: sucrose: fructose = 1: 1: 1),low molecular acid components(succinic acid :malic acid : citric acid = 1: 1: 1)and amino acid components(arginine : serine : valine =1: 1: 1).The optimized ARE concentrations were 1.31 g C/L of carbohydrates,0.29 g C/L of low molecular acids and 1.26 g C/L of amino acids.After addition of optimized ARE at the optimal concentration,the degradation rates of PAHs by bacterial flora B reached 38.9%(phenanthrene),35.7%(pyrene),39.9%(benzo [a] pyrene)and 36.7%(total PAHs),respectively,which were 20.1%(phenanthrene),10.7%(pyrene),3.7%(benzo [a] pyrene)and 13.8%(total PAHs)higher than traditional ARE,respectively.(4)Three kinds of PAHs remediation agents were prepared and added into two kinds of polluted soils with different physical and chemical properties with the ratio of 2.5%and 5%.All three remediation agents promoted the removal of PAHs to different extent compared to the control.Among them,the remediation agent with the best performance was prepared as follows.Bacterial flora B was activated and cultured.The cells were collected,re-suspended in ARE solution with optimized formula.The corncob biochar as the carrier was then added with the ratio of 1:10(w/v).The vials were shaken for 16 hours and the agent was obtained by filtration.The total PAHs removal rates of farmland soil in Jinan,Shandong Province was 76.5%(initial pollution level is 64.3mg/kg)and paddy soil in Yongkang,Zhejiang Province was 51.4%(initial pollution level is 89.9mg/kg)with the addition of this agent at the ratio of 5% in 20 days,indicating the feasibility of prepared PAHs soil remediation agent.