A polycyclic aromatic hydrocarbon contaminated soil remediator based on the combined action mechanism of plants and microorganisms, its preparation method and application

Abstract

The present invention relates to a polycyclic aromatic hydrocarbon (PAH)-contaminated soil remediation agent based on the combined action mechanism of plants and microorganisms, and a preparation method and application thereof. The soil remediation agent is biochar fixed with a PAH-degrading bacterial solution and simulated root exudates; the PAH-degrading bacteria are Pseudarthrobacter scleromae J-1 and Bacillus sp. J-3; the simulated root exudates are a mixture composed of sugars, small molecule acids, and amino acids. The present invention provides two strains of bacteria capable of efficiently degrading PAHs, and then by fixing the PAH-degrading bacterial solution and simulated root exudates on biochar, it overcomes the problems existing in conventional plant-microbial remediation technologies, such as the growth of plants being easily affected by environmental factors, the secretion amount and rate of root exudates being limited by the growth cycle, and the limited range of rhizosphere effects. The remediation agent of the present invention has a 20% to 40% increase in the PAH removal rate compared to single microbial remediation, improving the remediation efficiency of PAH-contaminated soil.

Description

Technical Field

[0001] The present invention relates to a polycyclic aromatic hydrocarbon contaminated soil remediation agent based on the combined action mechanism of plants and microorganisms, its preparation method and application, belonging to the technical field of environmental remediation. Background Art

[0002] Polycyclic aromatic hydrocarbons (PAHs) are a class of typical persistent organic pollutants widely present in the environment, mainly from biomass combustion, industrial emissions, vehicle exhaust, petroleum sources, etc. Most of them have "carcinogenic, teratogenic and mutagenic" effects, and can be transported over long and short distances through air or water, and finally accumulate in the soil, thus endangering the ecosystem and human health. There are significant differences in the content of PAHs in the surface soil of China among different regions, and there are also differences among different land use types in the same region. Therefore, it is necessary to develop suitable soil remediation technologies for areas or sites severely polluted by PAHs to assist in the battle against pollution prevention and control and reduce or eliminate ecological and health risks.

[0003] Scholars at home and abroad have carried out a large number of studies on organic contaminated soil remediation technologies. Phytoremediation combined with microorganisms is one of the most promising remediation technologies, with the advantages of no secondary pollution and no damage to the soil structure and properties. At present, the research on phytoremediation combined with microorganisms for PAHs contaminated soil mainly focuses on two aspects: the combined remediation of plants and specialized degrading bacteria and the combined remediation of plants and mycorrhizal fungi. The combined remediation can accelerate the removal of PAHs in the soil, and the main mechanism lies in the degradation enhancement effect of plant root exudates on degrading microorganisms. Root exudates refer to the general term of various organic compounds secreted by plant roots into the external environment during their life activities. The release amount accounts for 10% - 20% of the annual photosynthesis output, and there are various types. Plant root exudates can provide nutrients for rhizosphere degrading microorganisms, affect the distribution, activity and community diversity of microorganisms, promote the degradation of PAHs by microorganisms, and achieve the rapid reduction of their effectiveness and toxicity. However, the phytoremediation combined with microorganisms technology also has some limitations, such as (1) plant growth is more sensitive to the environment, and the secretion amount and secretion rate of root exudates are significantly affected by the growth cycle and environmental conditions; (2) the action range of this technology is mainly the rhizosphere microenvironment, and the concentration of root exudates shows a gradient decreasing effect with the increase of the distance from the roots, affecting the quantity and distribution of degrading bacteria in the soil, and further affecting the soil remediation effect; (3) most of the microorganisms used are degrading bacterial communities directly screened from contaminated soil, and they are not coupled and optimized with plant root exudates. The above factors affect the application range of the combined remediation technology. Therefore, how to innovate the phytoremediation combined with microorganisms technology and broaden the technical adaptability is of great significance for the remediation of PAHs contaminated soil.

[0004] Most of the currently studied organic contaminated soil remediation agents are immobilized bacterial agents. Researchers use immobilization technology to immobilize specific highly degradable bacteria or bacterial communities on suitable carriers by physical, chemical and other methods. The carrier provides a relatively independent microhabitat for the microorganisms, reduces the influence of external factors on the microorganisms, and keeps their biological activities at a relatively high level, so as to ensure the efficient exertion of their degradation effects. However, this technology mainly utilizes the degradation effect of microorganisms and does not utilize the advantages of plant root exudates. Whether plant root exudates can be coupled with exogenous highly degradable bacteria and, under the protection of the carrier, the three play a synergistic role to achieve a better remediation effect is worthy of in-depth exploration. Summary of the Invention

[0005] Aiming at the deficiencies of the existing technology, the present invention provides a polycyclic aromatic hydrocarbon contaminated soil remediation agent based on the combined action mechanism of plants and microorganisms, its preparation method and application. The present invention uses PAHs-degrading bacterial communities as target microorganisms, screens typical components that can promote the degradation of PAHs from the components of PAHs remediation plant root exudates reported and experimentally detected, prepares them into simulated root exudates, loads the degrading bacterial communities and the simulated root exudates on a biochar carrier to prepare a soil remediation agent, and applies it to the removal of PAHs in PAHs-contaminated soil and other contaminated media.

[0006] The technical solution of the present invention is as follows:

[0007] A polycyclic aromatic hydrocarbon contaminated soil remediation agent based on the combined action mechanism of plants and microorganisms, wherein the polycyclic aromatic hydrocarbon contaminated soil remediation agent is a biochar fixedly adsorbed with PAHs-degrading bacterial liquid and simulated root exudates;

[0008] The PAHs-degrading bacteria are Pseudarthrobacter scleromae J-1 and Bacillus sp. J-3;

[0009] The Pseudarthrobacter scleromae J-1 was deposited at the China Center for Type Culture Collection on November 16, 2021. The deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The deposit number is CCTCC NO: M20211342; the Bacillus sp. J-3 was deposited at the China Center for Type Culture Collection on November 16, 2021. The deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The deposit number is CCTCC NO: M 20211343;

[0010] The simulated root exudates are a mixture composed of sugars, small molecule acids and amino acids.

[0011] Preferably according to the present invention, the effective viable count of Pseudarthrobacter scleromae J-1 in the polycyclic aromatic hydrocarbon contaminated soil remediator is 5×10 8 ~5×10 9 CFU / g, and the effective viable count of Bacillus sp. J-3 is 2×10 8 ~5×10 9 CFU / g.

[0012] Preferably according to the present invention, the concentration of sugars in the simulated root exudates is 1.2 - 1.4 g C / L, the concentration of small molecule acids is 0.15 - 0.46 g C / L, and the concentration of amino acids is 0.7 - 1.4 g C / L.

[0013] Preferably according to the present invention, the sugars are a mixture of glucose, sucrose and fructose.

[0014] More preferably, the concentration ratio of glucose, sucrose and fructose is 1:(0.8 - 1):1.

[0015] Preferably according to the present invention, the small molecule acids are a mixture of succinic acid, malic acid and citric acid.

[0016] More preferably, the concentration ratio of succinic acid, malic acid and citric acid is (0.9 - 1):1:1.

[0017] Preferably according to the present invention, the amino acids are a mixture of arginine, serine and valine.

[0018] More preferably, the concentration ratio of arginine, serine and valine is 1:1:(0.8 - 1).

[0019] Preferably according to the present invention, the biochar is prepared by the following method:

[0020] Crush and dry the corncobs, pyrolyze them under oxygen-limited conditions at 200 - 500 °C for 120 - 150 minutes, crush and sieve them after cooling, and take the part with a particle size of 0.25 - 2.5 cm to obtain biochar.

[0021] The preparation method of the above polycyclic aromatic hydrocarbon contaminated soil remediator based on the plant-microorganism combined action mechanism includes the following steps:

[0022] Activate and ferment the PAHs degrading bacteria to obtain a fermentation broth; centrifuge the fermentation broth, collect the bacterial cells and resuspend them in the simulated root exudate solution to obtain a mixed solution; then add biochar to the mixed solution for mixed adsorption and fixation, and after filtration, prepare the polycyclic aromatic hydrocarbon contaminated soil remediator.

[0023] Preferably according to the present invention, the steps for activating and fermenting and culturing the PAHs-degrading bacteria are as follows:

[0024] Inoculate the PAHs-degrading bacteria on a nutrient agar medium and activate and culture at 30 °C for 24 to 48 h; then pick microbial colonies from the nutrient agar medium and inoculate them into a nutrient broth medium, and ferment and culture at 30 °C on a shaker with a rotation speed of 130 to 160 rpm for 16 to 24 h to obtain a PAHs-degrading bacteria fermentation broth;

[0025] Among them, the components of the nutrient agar medium are: beef extract powder 3.0 g / L, peptone 10.0 g / L, NaCl 5.0 g / L, agar 10 g / L, pH 7.0 to 7.4;

[0026] The components of the nutrient broth medium are: beef extract powder 3.0 g / L, peptone 10.0 g / L, NaCl 5.0 g / L, pH 7.0 to 7.4.

[0027] Preferably according to the present invention, the mass-to-volume ratio of the biochar to the simulated root exudates is (1 to 2):10, and the unit is: g / mL.

[0028] Preferably according to the present invention, the mixing and adsorption and fixation time of the mixed solution and the biochar is 16 to 24 h.

[0029] According to the present invention, the application of the above soil conditioner in repairing PAHs-polluted soil includes the following steps:

[0030] Add nitrogen-phosphorus agricultural compound fertilizer to the PAHs-polluted soil, adjust the molar ratio of carbon, nitrogen, and phosphorus in the polluted soil to (100 to 120):(5 to 10):1, adjust the soil moisture content to 20 to 50%, loosen the soil, add the soil conditioner according to the mass ratio of 1 to 5%, mix evenly, stir once every week, maintain the soil moisture content at 20 to 50%, and repair at 15 to 40 °C for 50 to 70 days. Detect the content of PAHs in the soil. If the repair target is reached, stop the repair. If the target is not reached, add the second round of soil conditioner to continue the repair until the repair target is reached.

[0031] Those not detailed in the present invention can be carried out according to the prior art.

[0032] The beneficial effects of the present invention are as follows:

[0033] 1. The present invention first provides two strains of bacteria, Pseudarthrobacter scleromae J-1 and Bacillus sp. J-3, which can efficiently degrade PAHs. Then, by immobilizing the PAHs-degrading bacterial solution and simulated root exudates on the organic carrier biochar, it overcomes the problems existing in the conventional plant-microbial combined remediation technology, such as the growth of plants being easily affected by environmental factors, the secretion amount and rate of root exudates being restricted by the growth cycle, and the limited range of rhizosphere effects. The remediation cycle of the remediation agent of the present invention is not restricted by plant growth, has strong soil adaptability, and broad application prospects. Moreover, the simulated root exudates in the present invention are highly coupled with Pseudarthrobacter scleromae J-1 and Bacillus sp. J-3, and a significant synergistic effect is produced after their combination. At the same time, with biochar as the carrier, the remediation of PAHs-contaminated soil is realized. Compared with the single microbial remediation method, the PAHs removal rate of the remediation agent provided by the present invention is increased by 20% - 40%, effectively improving the PAHs removal rate and the remediation efficiency of PAHs-contaminated soil.

[0034] 2. The formula of the root exudates screened by the present invention that can promote the degradation of PAHs by target microorganisms is clear and can be commercially purchased; using agricultural waste such as corncobs as the precursor to prepare the biochar carrier is easy to obtain in large quantities, and at the same time, waste resource utilization is realized. The prepared soil remediation agent has low cost and good remediation effect, and has significant economic and social benefits.

[0035] 3. The present invention prepared simulated root exudates through screening and verification, and then combined them with high-pollutant-degrading microorganisms, producing a synergistic effect and increasing the pollutant removal rate, which broadens the idea for innovative biotechnology and provides guidance for the research and development of PAHs-contaminated soil remediation agents. Description of the Drawings

[0036] Figure 1 It is the mixed standard spectrum of small molecule acids in the root exudates of the PAHs-remediating plant Lolium perenne.

[0037] Figure 2 It is the mixed standard spectrum of amino acids in the root exudates of the PAHs-remediating plant Lolium perenne.

[0038] Figure 3 It is the bar chart of the effect of the sugar components in the simulated root exudates on the degradation of PAHs by the bacterial community.

[0039] Figure 4 It is the bar chart of the effect of the small molecule acid component I in the simulated root exudates on the degradation of PAHs by the bacterial community.

[0040] Figure 5 Bar chart showing the effect of small molecule acid component II in simulated root exudates on the degradation of PAHs by the microbial community.

[0041] Figure 6 Bar chart showing the effect of amino acid component I in simulated root exudates on the degradation of PAHs by the microbial community.

[0042] Figure 7 Bar chart showing the effect of amino acid component II in simulated root exudates on the degradation of PAHs by the microbial community.

[0043] Figure 8 Response surface 3D map of the effect of the interaction of different components on the degradation rate of PAHs;

[0044] In the figure: a, b, and c are the response surface 3D maps of the interactive effects of carbohydrates and small molecule acids, carbohydrates and amino acids, and amino acids and small molecule acids on the total PAHs degradation rate, respectively. Detailed implementation mode

[0045] To make the technical problems, technical solutions, and advantages to be solved by the present invention clearer, the present invention will be further described below through examples, but not limited thereto.

[0046] Microbial source:

[0047] The PAHs-degrading microbial community in Examples 1 to 6 consists of Pseudarthrobacter scleromae J-1 (strain preservation number: CCTCC NO: M 20211342) and Bacillus sp. J-3 (CCTCC NO: M 20211343). The preservation institution is the China Center for Type Culture Collection, and the address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0048] The components of the nutrient agar medium used in the examples are: beef extract powder 3.0 g / L, peptone 10.0 g / L, NaCl 5.0 g / L, agar 10 g / L, pH 7.0 - 7.4;

[0049] The components of the nutrient broth medium are: beef extract powder 3.0 g / L, peptone 10.0 g / L, NaCl 5.0 g / L, pH 7.0 - 7.4.

[0050] Example 1: Degradation of PAHs by the degrading microbial community

[0051] Pseudarthrobacter scleromae J-1 and Bacillus sp. J-3 were respectively inoculated on nutrient agar medium and activated at 30 °C for 36 h. Then, microbial colonies were picked from the nutrient agar medium and inoculated into 50 mL of nutrient broth medium, and mixed fermentation was carried out at 30 °C on a shaker at a rotation speed of 150 rpm for 20 h. The fermentation broth was centrifuged at 4 °C and 8000 r / min for 10 min to obtain mixed bacteria. The residual carbon source in the bacteria was washed with PBS buffer, and after repeating the washing 2 times, 50 mL of PBS buffer was added to resuspend the bacteria to prepare the bacterial solution required for the degradation system.

[0052] In a laminar flow hood, a PAHs-acetone stock solution (PHE: PYR: BaP = 10:10:1) with a final concentration of 20 mg / L was added to a conical flask containing BH medium, sealed with a sterilized sealing film, and placed in a shaker and shaken for 12 h to volatilize the organic solvent. The degradation bacterial solution was inoculated at a ratio of 10%, and the total volume of the degradation system was 50 mL. A treatment group without inoculating the bacterial solution was set as the blank control. The conical flask was placed in a shaker and oscillated at 150 rpm and 30 °C for 7 d. Three parallels were set for the treatment group. After the degradation was completed, the residual amount of PAHs in the system was measured, and the PAHs degradation rate was calculated.

[0053] Calculation showed that the mixed flora of Pseudarthrobacter scleromae J-1 and Bacillus sp. J-3 had strong degradation ability for PAHs in a liquid shaking flask, and the microbial degradation rate of PAHs within 7 d was 35.8%.

[0054] Example 2: Determination of root exudate components in PAHs-remediating plants

[0055] Through a hydroponic experiment, the root exudates of the PAHs-remediating plant Lolium perenne were collected, freeze-dried, and reconstituted with sterilized distilled water to determine typical small molecule organic acids and amino acid substances.

[0056] An HPLC determination method for 9 small molecule organic acids was constructed, and the chromatogram is as Figure 1 shown. Figure 1 The peaks from left to right in the figure are oxalic acid, tartaric acid, formic acid, malic acid, lactic acid, acetic acid, citric acid, maleic acid, and fumaric acid. The small molecule acids in the root exudates of Lolium perenne were analyzed, and oxalic acid, formic acid, and malic acid were detected, while the other 6 small molecule acids were not detected. Among the 3 detected acids, oxalic acid had the highest concentration, which was 9.4 ± 2.9 mg / g of plant dry weight, accounting for 91% of the total amount of small molecule acids.

[0057] An amino acid analyzer determination method for 16 free amino acids (aspartic acid, threonine, serine, glutamic acid, glycine, alanine, cysteine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, arginine) was constructed. The mixed standard spectrum is as shown in Figure 2 Figure 1. The amino acids in the root exudates of ryegrass were analyzed, and 10 amino acids were detected, namely aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, tyrosine, lysine, and histidine. Among them, the concentrations of valine and glutamic acid were relatively high, 36.4 ± 2.0 and 20.4 ± 8.5 μg / g plant dry weight, respectively, accounting for 43% and 24% of the total amino acids. Cysteine, methionine, isoleucine, leucine, phenylalanine, and arginine were not detected.

[0058] Example 3: Effects of simulating root exudate components on the degradation of PAHs by microbial communities

[0059] According to the measurement results of Example 2 and the typical root exudate components of PAHs-remediating plants, the inventor selected three types of components, namely sugars, small molecule acids, and amino acids, to construct simulated root exudates, and studied the effects of these three types of components on the degradation of PAHs by microbial communities, in order to obtain a simulated root exudate formula that can synergistically act with PAHs-degrading microbial communities. The sugar component was glucose, fructose, and sucrose. Two treatment groups were set for the small molecule acid component. The small molecule acid component I treatment group contained succinic acid, malic acid, and citric acid, and the small molecule acid component II treatment group contained succinic acid, malic acid, and oxalic acid. Two treatment groups were set for the amino acid component. The amino acid component I treatment group contained arginine, serine, and glutamic acid, and the amino acid component II treatment group contained arginine, serine, and valine.

[0060] The effects of the above five component treatment groups on the degradation of PAHs by microbial communities were investigated respectively. The material compositions and concentration gradients of each treatment group are set as shown in Table 1. Among them, the concentrations of the sugar component in the degradation system were set to 0.04 g C / L, 0.2 g C / L, 1 g C / L, and 5 g C / L, denoted as sugar-0.04, sugar-0.2, sugar-1, and sugar-5, respectively. The concentrations of the small molecule acid components I and II in the degradation system were both set to 0.04 g C / L, 0.2 g C / L, 1 g C / L, and 2 g C / L, denoted as small molecule acid I / II-0.04, small molecule acid I / II-0.2, small molecule acid I / II-1, and small molecule acid I / II-2, respectively. The concentration gradients of the amino acid components I and II in the degradation system were the same as those of the small molecule acid components, denoted as amino acid I / II-0.04, amino acid I / II-0.2, amino acid I / II-1, and amino acid I / II-2, respectively. The control group was a degradation system without adding simulated root exudates (ARE components) and only containing the degrading microbial community, denoted as microbial community.

[0061] Table 1 Simulated Root Exudates

[0062]

[0063]

[0064] The degrading bacterial community was inoculated on nutrient agar medium and activated at 30 °C for 36 h. Then, microbial colonies were picked from the nutrient agar medium and inoculated into 50 mL of nutrient broth medium, and fermented and cultured at 30 °C on a shaker at a rotation speed of 150 rpm for 20 h. The fermentation broth was centrifuged at 4 °C and 8000 r / min for 10 min to obtain a mixed bacterial mass. The residual carbon source in the bacterial mass was washed with PBS buffer, and after repeating the washing 2 times, 50 mL of PBS buffer was added to resuspend it to prepare the degrading bacterial liquid required for the PAHs degradation system.

[0065] In a laminar flow hood, a PAHs-acetone stock solution with a final concentration of 20 mg / L (PHE:PYR:BaP = 10:10:1) was added to a conical flask containing BH medium, sealed with a sterilized sealing film, and placed in a shaker and shaken for 12 h to volatilize the organic solvent. The degrading bacterial liquid was inoculated at a ratio of 10%, and different concentrations of simulated root exudate components were added thereto. The total volume of the degradation system was 50 mL. The conical flask was placed in a shaker and oscillated and cultured at 150 rpm and 30 °C, and the degradation period was 7 d. Three parallels were set for the treatment group. After the degradation was completed, the residual amount of PAHs in the system was measured, the PAHs degradation rate was calculated, and the bacterial density value (OD600) was measured.

[0066] Calculation showed that when the sugar component was 1 g C / L, the promotion of the bacterial community for PAHs degradation was optimal, and the PAHs degradation rate within 7 d was 48.6%( Figure 3 ), and the OD600 value was 2.10. When the small molecule acid components were 0.04 g C / L and 0.2 g C / L, both small molecule acid I and small molecule acid II showed a promoting effect on PAHs degradation. Especially when it was 0.2 g C / L, the PAHs degradation rate was significantly higher than that of the control group, being 46.3% and 42.8% respectively( Figure 4 and 5 ), and the OD600 value was in the range of 1.2 - 1.3. When the amino acid components were 1 g C / L and 2 g C / L, both amino acid component I and II had an obvious promoting effect on PAHs degradation, and the PAHs degradation rates were 49.6% - 52.4% and 50.7% - 51.3% respectively( Figure 6 and 7 ), and the OD600 value was in the range of 1.5 - 1.8.

[0067] Example 4: Optimization of Simulated Root Exudates

[0068] Based on the results of Example 3, a composite formula was selected with a sugar component (glucose: sucrose: fructose = 1:1:1, 1 g C / L), a small molecule acid component (succinic acid: malic acid: citric acid = 1:1:1, 0.2 g C / L), and an amino acid component (arginine: serine: valine = 1:1:1, 1 - 2 g C / L). Using the degradation rate of PAHs in the degradation system as the response value, the Box - Behnken Design model of DesignExpert software was applied to conduct a simulation experiment design of optimizing the formula combination with 3 factors and 3 levels, in order to obtain a better ARE combination formula. The experimental design is shown in Table 2.

[0069] Table 2 Response surface factor level design

[0070]

[0071] The experimental results are shown in Table 3. Using the degradation rate of PAHs as the response value, the quadratic regression equation for the degradation effect among the sugar addition concentration (A), the small molecule acid addition concentration (B), and the amino acid addition concentration (C) is:

[0072] Y (PAHs degradation rate) = +56.12 + 1.99×A + 2.05×B + 0.91×C + 0.28×A×B + 0.35×A×C – 3.68×B×C – 5.86×A 2 – 2.18×B 2 - 2.06×C 2 .

[0073] Table 3 Response surface experimental results

[0074]

[0075] Table 4 is the analysis of variance of the regression equation with the degradation rate of PAHs as the response value. The P - value is significant, and the equation has an insignificant total lack - of - fit value, indicating that the model fitting effect is better, and the optimization result can be obtained based on the response surface.

[0076] Table 4 Analysis of variance with the degradation rate of PAHs as the response value

[0077]

[0078]

[0079] From Figure 8It can be seen that when the ARE component composed of a sugar addition concentration of 1.38 g C / L, small molecule acids of 0.40 g C / L, and amino acids of 0.76 g C / L is added to the degradation system, the highest degradation rate can be achieved, and the predicted total PAHs degradation rate is 57.1%. To verify whether the established regression model is accurate and reliable, three groups of repeated experiments were carried out according to the optimal ratio conditions obtained from the above response surface analysis for verification. Finally, the PAHs degradation rate was 58.7% ± 1.4%, and there was no significant difference from the predicted value, indicating that the prediction model is reasonable and reliable.

[0080] Example 5: Preparation of a polycyclic aromatic hydrocarbon contaminated soil remediator based on the combined action mechanism of plants and microorganisms

[0081] A preparation method of a polycyclic aromatic hydrocarbon contaminated soil remediator based on the combined action mechanism of plants and microorganisms includes the following steps:

[0082] (1) Inoculate PAHs-degrading bacteria on a nutrient agar medium, activate and culture them at 30 °C for 36 h, then pick microbial colonies from the nutrient agar medium and inoculate them into 50 mL of a nutrient broth medium, and ferment and culture them at 30 °C on a shaker at a rotation speed of 150 rpm for 20 h. Centrifuge the fermentation broth at 4 °C and 8000 r / min for 10 min to obtain a mixed bacterial mass. Wash the residual carbon source in the bacterial mass with PBS buffer, repeat the washing 2 times, and then add 50 mL of PBS buffer to resuspend it to obtain a PAHs-degrading bacteria solution.

[0083] (2) Mix the PAHs-degrading bacteria solution with a simulated root exudate solution to obtain a mixed solution; then add biochar to the mixed solution at a mass-volume ratio of 1:10 (g / mL) for mixing and adsorption fixation for 20 h, and after filtration, prepare a polycyclic aromatic hydrocarbon contaminated soil remediator.

[0084] Among them, the concentration of sugars in the obtained polycyclic aromatic hydrocarbon contaminated soil remediator is 1.38 g C / L, and the concentration ratio of glucose, sucrose, and fructose is 1:1:1; the small molecule acids are 0.40 g C / L, and the concentration ratio of succinic acid, malic acid, and citric acid is 1:1:1; the amino acids are 0.76 g C / L, and the concentration ratio of arginine, serine, and valine is 1:1:1.

[0085] The biochar is prepared by the following method: Crush and dry corn cobs, carry out oxygen-limited pyrolysis at 400 °C for 130 minutes, cool and then crush and sieve, and take the part with a particle size of 2 cm as the biochar carrier.

[0086] Example 6: Experiment for measuring the PAHs removal rate of the soil remediator

[0087] Collect soil samples from farmland, air-dry them naturally, crush, mix well, add PAHs stock solution to make the final concentration 50 mg / kg. After volatilizing the solvent, weigh 20 g of PAHs-contaminated soil into multiple 40-ml brown EPA bottles respectively, and then conduct 5 treatments with three parallels for each treatment: Treatment 1: Natural attenuation, only PAHs-contaminated soil, as a blank control; Treatment 2: Add the soil remediation agent prepared in Example 5 to the PAHs-contaminated soil, with an addition amount of 1 g; Treatment 3: Add simulated root exudates, with an addition amount equal to that of the simulated root exudates used when preparing 1 g of the soil remediation agent in Treatment 2; Treatment 4: Add PAHs-degrading bacteria solution, with an addition amount equal to that of the bacteria solution used when preparing 1 g of the soil remediation agent in Treatment 2; Treatment 5: Add biochar carrier, with an addition amount equal to that of the carrier used when preparing 1 g of the soil remediation agent in Treatment 2. Adjust the moisture content to 20-50%, and culture at 25 °C for 20 d. Take soil samples, perform Soxhlet extraction with dichloromethane as the solvent for 24 h, re-dilute with methanol after rotary evaporation, determine the PAHs content by HPLC, and calculate the PAHs removal rate.

[0088] After calculation: the PAHs removal rate of Treatment 1 is 17.4%, that of Treatment 2 is 67.3%, that of Treatment 3 is 25.7%, that of Treatment 4 is 40.1%, and that of Treatment 5 is 21.6%. Treatment 1 with natural attenuation serves as a blank control, and the removal of PAHs mainly comes from physical and chemical effects such as volatilization, adsorption, aging, etc. and biodegradation by indigenous microorganisms; there is natural attenuation in Treatments 2 to 5, and the difference between the PAHs removal rates of each of them and the removal rate of Treatment 1 is the contribution of the added exogenous substances in Treatments 2 to 5 to the removal of PAHs. The contribution of the soil remediation agent in Treatment 2 is 49.9%, the contribution of the simulated root exudates in Treatment 3 is 8.3%, the contribution of the PAHs-degrading bacteria in Treatment 4 is 22.7%, and the contribution of the carrier in Treatment 5 is 4.2%. The contribution of the soil remediation agent to the removal of PAHs (49.9%) is greater than the sum of the contributions of the simulated root exudates, degrading bacteria, and carrier acting alone (8.3% + 22.7% + 4.2% = 35.2%), indicating that in the prepared soil remediation agent, the simulated root exudates, degrading bacteria, and carrier have a synergistic effect on the degradation of PAHs.

[0089] Example 7: Application effect experiment of soil remediation agent in PAHs-contaminated soil

[0090] Conduct a pot soil experiment using the soil remediation agent prepared in Example 5.

[0091] The specific method is as follows: Collect soil samples from farmland, air-dry them naturally, crush, mix them evenly, add PAHs stock solution to make the final concentration 80 mg / kg, and after volatilizing the solvent, conduct three treatments respectively: Treatment 1: Weigh 1.0 kg of contaminated soil, add 25 g of soil remediation agent and 15 g of fertilizer (nitrophosphate ammonium); Treatment 2: Weigh 1.0 kg of contaminated soil and add 25 g of soil remediation agent; Treatment 3: Weigh 1.0 kg of contaminated soil. Put the contaminated soil after the above three groups of treatments into flowerpots respectively, adjust and maintain the moisture content at 20 - 50%, stir once every week, and place at room temperature for 2 months. For the soil samples, conduct Soxhlet extraction with dichloromethane as the solvent for 24 h, re-dilute to volume with methanol after rotary evaporation, determine the PAHs content by HPLC, and calculate the PAHs removal rate.

[0092] After 2 months of remediation, the PAHs removal rate of Treatment 1 is 89.1%, the PAHs removal rate of Treatment 2 is 62.4%, and the PAHs removal rate of Treatment 3 is 38.6%. It shows that when applying the soil remediation agent prepared by the present invention, its remediation ability can be further enhanced and the PAHs removal rate can be improved after being used in combination with fertilizers such as nitrophosphate ammonium.

[0093] Example 8: Application effect test of soil remediation agent in petroleum-contaminated soil

[0094] Use the soil remediation agent prepared in Example 5 to remediate the soil with 2.6% oil content in Gudao Oil Region of Shengli Oilfield.

[0095] The specific method is as follows: Take 10 m 3 of oil-containing soil for ex-situ remediation. Stir the oil-containing soil evenly, and then divide it into 2 parts for 2 treatments. Treatment 1: The oil-containing soil is used as a blank control; Treatment 2: Add 5% of the remediation agent and 1% of the fertilizer (nitrophosphate ammonium) based on the soil weight to the oil-containing soil and stir evenly. Keep the soil moisture content at 20 - 50%, turn the soil once every week, regularly take samples to determine the PAHs content, and conduct remediation for 2 months. Use organic solvent Soxhlet extraction and HPLC determination to determine the PAHs content, and calculate the PAHs removal rate.

[0096] After 2 months of remediation, the PAHs removal rate of Treatment 1 is 10.2%, and the PAHs removal rate of Treatment 1 is 51.1%. It shows that the soil remediation agent and remediation method provided by the present invention can effectively remove PAHs pollutants in petroleum-contaminated soil, and the remediation effect is remarkable.

Claims

1. A polycyclic aromatic hydrocarbon (PAH)-contaminated soil remediator based on the combined action mechanism of plants and microorganisms, characterized in that, the PAH-contaminated soil remediator is biochar fixed with PAH-degrading bacteria liquid and simulated root exudates; The PAHs-degrading bacteria are Pseudomonas sp. ( Pseudarthrobacter scleromae ) J-1 and Bacillus sp. ( Bacillus ) J-3; The Pseudobacterium ( Pseudarthrobacter scleromae ), J-1, was deposited at the China Center for Type Culture Collection on November 16, 2021. The deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The deposit number is CCTCC NO: M20211342; The Bacillus ( Bacillus sp.), J-3, was deposited at the China Center for Type Culture Collection on November 16, 2021. The deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The deposit number is CCTCC NO: M 20211343; the simulated root exudates are a mixture composed of sugars, small molecule acids and amino acids; The effective viable count of Pseudomonas sp. ( Pseudarthrobacter scleromae ) J-1 in the polycyclic aromatic hydrocarbon contaminated soil remediation agent is 5×10 8 ~5×10 9 CFU / g, and the effective viable count of Bacillus sp. ( Bacillus sp.) J-3 is 2×10 8 ~5×10 9 CFU / g.

2. The PAH-contaminated soil remediator based on the combined action mechanism of plants and microorganisms according to claim 1, characterized in that, in the simulated root exudates, the concentration of sugars is 1.2 - 1.4 g C / L, the concentration of small molecule acids is 0.15 - 0.46 g C / L, and the concentration of amino acids is 0.7 - 1.4 g C / L.

3. The PAH-contaminated soil remediator based on the combined action mechanism of plants and microorganisms according to claim 1, characterized in that, the sugars are a mixture of glucose, sucrose and fructose; the concentration ratio of glucose, sucrose and fructose is 1:(0.8 - 1):

1.

4. The PAH-contaminated soil remediator based on the combined action mechanism of plants and microorganisms according to claim 1, characterized in that, the small molecule acids are a mixture of succinic acid, malic acid and citric acid; the concentration ratio of succinic acid, malic acid and citric acid is (0.9 - 1):1:

1.

5. The PAH-contaminated soil remediator based on the combined action mechanism of plants and microorganisms according to claim 1, characterized in that, the amino acids are a mixture of arginine, serine and valine; the concentration ratio of arginine, serine and valine is 1:1:(0.8 - 1).

6. The PAH-contaminated soil remediator based on the combined action mechanism of plants and microorganisms according to claim 1, characterized in that, the biochar is prepared by the following method: Crush and dry corn cobs, pyrolyze them under low oxygen at 200 - 500 °C for 120 - 150 minutes, cool and then crush and sieve, and take the part with a particle size of 0.25 - 2.5 cm to obtain biochar.

7. The preparation method of the PAH-contaminated soil remediator based on the combined action mechanism of plants and microorganisms according to claim 1, characterized in that, it includes the following steps: Activate and ferment the PAH-degrading bacteria to obtain a fermentation broth; centrifuge the fermentation broth, collect the bacterial cells and resuspend them in the simulated root exudate solution to obtain a mixed solution; then add biochar to the mixed solution for mixed adsorption and fixation, and after filtration, prepare the PAH-contaminated soil remediator.

8. The preparation method according to claim 7, characterized in that, the mass-volume ratio of the biochar to the simulated root exudates is (1 - 2):10, unit: g / mL; the time for the mixed solution and the biochar to be mixed and adsorbed and fixed is 16 - 24 h.

9. Use of the polycyclic aromatic hydrocarbon contaminated soil remediation agent based on the plant-microbial joint action mechanism according to any one of claims 1 to 6 in the remediation of PAHs contaminated soil, characterized in that, it comprises the following steps: Adding nitrogen-phosphorus compound fertilizer for agricultural use to the PAHs contaminated soil, adjusting the molar ratio of carbon, nitrogen, and phosphorus in the contaminated soil to (100~120):(5~10):1, adjusting the soil moisture content to 20~50%, loosening the soil, adding the soil remediation agent at a mass ratio of 1~5%, mixing evenly, stirring once every week, maintaining the soil moisture content at 20~50%, repairing for 50~70 days under the condition of 15~40 °C, detecting the content of PAHs in the soil, if the repair target is reached, stop the repair, if the target is not reached, add the second round of soil remediation agent to continue the repair until the repair target is reached.

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