A composite bacteria and its application in polycyclic aromatic hydrocarbon contaminated soil in-situ covering / barrier material

Abstract

The application discloses a kind of composite bacteria and its application in polycyclic aromatic hydrocarbon contaminated soil in situ covering / barrier material, belong to soil pollution remediation and risk management technical field.The composite bacteria of the present application is compounded by Pseudomonas fluorescens PB4, Bacillus licheniformis PS5, Kocuria FB6 and Bacterium parvum JM1, and the four strains have strong synergism and can efficiently degrade polycyclic aromatic hydrocarbon pollutants in soil.The composite bacteria is applied to PAHs contaminated soil in situ covering / barrier material, and the prepared remediation material is prepared by loading the above composite bacteria on modified bioactive carbon, nutrient slow-release microspheres, quartz sand and bentonite as base carrier.The in-situ covering / barrier material has the functions of physical barrier, adsorption and biodegradation of PAHs.The material is laid on the surface layer or around of PAHs contaminated soil, can efficiently block the migration and diffusion of PAHs in deep soil to the upper or around, and realizes long-term remediation and risk management of contaminated soil in situ.

Description

Technical Field

[0001] This invention belongs to the field of in-situ remediation and risk management technology of contaminated soil, specifically involving a composite microorganism and its application in in-situ covering / barrier materials for polycyclic aromatic hydrocarbon (PAH) contaminated soil. It is suitable for long-term stable in-situ remediation and risk management of high-concentration PAHs contaminated soil in sites. Background Technology

[0003] Currently, the remediation of PAH-contaminated soil at sites typically involves either ex-situ treatment or in-situ disposal. While ex-situ treatment can completely remove contamination, it is costly, difficult to implement, and prone to secondary pollution. In-situ disposal usually employs economical and effective methods such as biodegradation and in-situ containment / covering, but biodegradation efficiency is low and time-consuming, especially for soils contaminated with high concentrations of PAHs. In contrast, in-situ cover / covering is a rapid method for treating PAH-contaminated soil at sites. Traditional in-situ cover / covering involves placing clean containment materials (such as gravel, clay, zeolite, bentonite, ash, cement, etc.) on or around the contaminated soil to prevent the migration and diffusion of PAHs. However, traditional cover / covering materials can only achieve PAH adsorption and fixation, and long-term use can easily reach adsorption saturation, posing a risk of secondary PAH release. Novel covering / barrier materials utilize active materials with PAH adsorption and degradation functions (such as activated carbon, zero-valent iron, and degrading bacteria) mixed with sand or clay as covering / barrier materials for in-situ treatment of contaminated soil. These new covering / barrier materials not only isolate contaminated soil from external contact but also degrade PAHs through adsorption, reducing the risk of secondary PAH release.

[0004] Chinese patent CN115672966B proposes an improved method for the combined in-situ covering and electrochemically enhanced biodegradation remediation of organically contaminated soil. The barrier layer is made of a compacted mixture of activated carbon, quartz sand, zero-valent iron, and degrading bacteria screened from contaminated soil, and degradation is enhanced by a periodically polarity-switching electric field. While this novel in-situ covering / isolation material effectively isolates contaminated soil from the external environment and adsorbs and degrades pollutants, it also alters the living environment of microorganisms in the underlying contaminated soil to some extent. For example, oxygen deficiency in the underlying soil reduces the degradation activity of microorganisms, affecting the natural degradation of PAHs in the contaminated soil. This novel combined in-situ covering and electrochemically enhanced biodegradation method for the in-situ remediation of organically contaminated soil in industrial sites solves the problems of oxygen deficiency and low pollutant degradation efficiency in the underlying soil. The method includes two processes: a top covering to block pollutant diffusion and an electric field-enhanced pollutant degradation process. The blocking process is completed by the improved in-situ covering layer, and the degradation process is completed by electrochemical oxidation and biodegradation. However, this method has certain shortcomings under long-term remediation conditions: First, the adaptability of the degrading bacteria is poor, leading to reduced degradation activity; second, the covering material lacks nutrients such as N and P, and the degrading bacteria will experience a decline in biological activity due to the influence of the electric field and the lack of nutrients such as nitrogen and phosphorus during long-term remediation; third, nutrient ions such as nitrogen and phosphorus in the contaminated soil will accumulate towards the positive and negative electrodes due to the electric field, reducing the nutrients in the soil, which in turn leads to a decrease in the number and activity of microorganisms in the soil. Moreover, ion migration will also cause the current of the remediation system to decay, weakening the electrochemical oxidation effect, ultimately resulting in insufficient long-term effectiveness of the overall remediation system. Therefore, to address the above drawbacks, a composite bacteria and a new in-situ covering / barrier material adapted to the electric field-enhanced remediation system are developed. This material possesses the characteristics of long-term slow release of nutrients and synergistic adsorption-degradation, which can overcome technical challenges such as the decline of microbial activity, nutrient loss, and unstable remediation effects, achieving in-situ long-term remediation and risk management of PAHs contaminated soil. Summary of the Invention

[0005] (a) Technical problems to be solved The purpose of this invention is to overcome the shortcomings of existing in-situ cover / barrier remediation technologies for PAH-contaminated soils, such as poor compatibility of degrading bacteria in cover materials, insufficient nutrition, low activity of degrading bacteria, and insufficient long-term remediation effectiveness. This invention provides a composite microbial system with strong compatibility, high degradation efficiency, and good stability, and its application in in-situ cover / barrier materials for PAH-contaminated soils. This invention scientifically constructs a stable degradation composite microbial system and specifically matches it to in-situ cover / barrier materials, achieving synergistic effects among strains, materials, and electric fields. This solves the problem that traditional technologies cannot simultaneously achieve pollution barrier and complete pollutant degradation, providing a green, long-lasting, low-cost, and scalable in-situ remediation solution for PAH-contaminated soils, significantly improving the long-term effectiveness of PAH-contaminated soil remediation and risk management.

[0006] (II) Technical Solution To achieve the above objectives, the main technical solutions adopted by the present invention are as follows:

[0007] The first aspect of this invention provides a composite bacterial strain comprising *Pseudomonas fluorescens* PB4, *Bacillus licheniformis* PS5, *Cocheria spp.* FB6, and *Microbacterium megaterium* JM1. The effective viable count ratio of the four strains is as follows: *Pseudomonas fluorescens* PB4 20%–30%, *Bacillus licheniformis* PS5 25%–35%, *Cocheria spp.* FB6 15%–25%, and *Microbacterium megaterium* JM1 15%–25%. The total effective viable count of the composite bacterial strain is ≥2.0 × 10⁻⁶. 10 CFU / g dry matter;

[0008] The fluorescent Pseudomonas PB4 is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M2011260;

[0009] The Bacillus licheniformis PS5 is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M2011261;

[0010] The Coccidia FB6 strain is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M2013360.

[0011] The Mexican microbacterium JM1 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20211664.

[0012] Preferably, the effective viable count ratio of the four strains is: 25% for Pseudomonas fluorescens PB4, 25% for Bacillus licheniformis PS5, 25% for Coxella spp. FB6, and 25% for Microbacterium gracilistylus JM1.

[0013] A second aspect of this invention proposes an in-situ covering / barrier material for polycyclic aromatic hydrocarbon contaminated soil containing the aforementioned composite bacteria.

[0014] Furthermore, the materials, by weight, include 1-3 parts of compound bacteria, 30-50 parts of modified biological activated carbon, 10-20 parts of nutrient slow-release microspheres, 20-30 parts of quartz sand, and 5-15 parts of bentonite.

[0015] Preferably, the materials, by weight, include 2 parts of compound bacteria, 45 parts of modified biological activated carbon, 15 parts of nutrient slow-release microspheres, 25 parts of quartz sand, and 10 parts of bentonite.

[0016] Furthermore, the modified bio-activated carbon is iron-modified bio-activated carbon; the nutrient slow-release microspheres are chitosan-sodium alginate coated nitrogen and phosphorus composite nutrient microspheres; the quartz sand has a particle size of 60-80 mesh; and the bentonite has a particle size of 200-300 mesh.

[0017] A third aspect of this invention provides a method for preparing an in-situ covering / barrier material for polycyclic aromatic hydrocarbon-contaminated soil, characterized by comprising the following steps:

[0018] S1. Strain activation and propagation: *Pseudomonas fluorescens* PB4, *Bacillus licheniformis* PS5, *Cocheria spp.* FB6, and *Microbacterium megaterium* JM1 were inoculated into their respective culture media and cultured at constant temperature with shaking until the culture medium reached OD. 600 =1, centrifuge to collect bacterial cells, resuspend in sterile water to prepare single bacterial suspension, and mix according to the effective viable cell count ratio to obtain compound bacterial suspension;

[0019] S2. Modification of biological activated carbon: Add pretreated activated carbon to a mixed solution of Fe(NO3)3 and FeSO4 at a solid-liquid ratio of 1g:5-15ml. The Fe content in the mixed solution is calculated as a molar ratio. 2+ Fe 3+ =1:2, ultrasonically treated for 1-2 hours, dried in an 80℃ forced-air drying oven to constant weight, then rinsed with deionized water until the solution is clear, then calcined in a muffle furnace at 300-400℃ for 1-2 hours, and naturally cooled to obtain iron-loaded modified biological activated carbon.

[0020] S3. Preparation of sustained-release nutrient microspheres: Prepare a 1.5%–2.5% sodium alginate solution, add 2%–5% urea and 1%–3% potassium dihydrogen phosphate, stir evenly to remove bubbles, drop the mixed sol into a 1.0%–2.0% calcium chloride solution, and solidify for 30–60 min to obtain nascent microspheres. Coat the nascent microspheres with a 0.3%–0.8% chitosan solution for 20–40 min, wash and dry to obtain the final product.

[0021] S4. Preparation of covering / barrier materials: Mix modified bio-activated carbon, nutrient slow-release microspheres, quartz sand and bentonite according to the specified ratio, then add the compound bacterial suspension and stir evenly.

[0022] Furthermore, in step S1, the culture temperature is 28–32°C and the culture time is 36–72 h.

[0023] The fourth aspect of this invention proposes the application of the aforementioned polycyclic aromatic hydrocarbon (PAH) contaminated soil in situ covering / barrier material, which is used for in-situ remediation and risk management of PAH-contaminated soil in industrial sites.

[0024] Furthermore, the application method includes the following steps: cleaning the surface debris of PAHs-contaminated soil, leveling the land, and evenly laying the in-situ covering / barrier material on or around the contaminated soil surface with a thickness of 3-8 cm, naturally compacting it to complete the in-situ covering / barrier; and simultaneously laying an intermittently polarity-switching electric field while laying the in-situ covering / barrier material, with the electric field strength controlled at 0.8-1.5 V / cm, to achieve in-situ remediation and risk management of contaminated soil.

[0025] (III) Beneficial Effects 1. This invention provides a composite bacteria for the efficient degradation of PAHs, which is composed of Pseudomonas fluorescens PB4, Bacillus licheniformis PS5, Coxella spp. FB6, and Microbacterium ramosae JM1. The four strains work synergistically and complement each other to cover the degradation needs of PAHs pollutants with different ring numbers.

[0026] 2. This invention specifically addresses the core problems of existing cover / barrier materials, such as lack of nutrient supply, decreased activity of degrading bacteria, and loss of soil nutrients. By adding slow-release nutrient microspheres, nitrogen and phosphorus nutrients can be released at a constant rate over a long period, maintaining high activity of degrading bacteria in the cover / barrier layer. At the same time, it can also compensate for the soil nutrient loss caused by the electric field, ensuring stable long-term remediation effects. It solves the problems of secondary release of pollutants due to poor adsorption performance of cover materials and inactivation of strains due to substrate scarcity.

[0027] 3. This invention uses iron-loaded modified activated carbon and bentonite as adsorbent materials, which greatly improves the adsorption capacity of the covering / barrier material for PAHs. The strong adsorption capacity of modified activated carbon and bentonite can efficiently adsorb PAHs in the soil and prevent pollutant migration. Iron and carbon can enhance electron transfer under the action of an electric field, improve the efficiency of electrochemical oxidation degradation, and achieve a triple synergistic effect of adsorption-electrochemical degradation-microbial degradation.

[0028] 4. The addition of quartz sand and bentonite to this invention can improve the overall structural density and stability of the covering / barrier material, effectively resist electric field disturbances, prevent material loosening, stabilize the repair microenvironment, and solve the problems of structural damage and performance degradation of traditional repair materials under long-term electric field conditions.

[0029] 5. The material of this invention is environmentally friendly and produces no secondary pollution. The raw material cost is low and the preparation process is simple. It can not only achieve physical barrier between contaminated soil and the external environment, blocking the migration and diffusion of PAHs, but also achieve efficient and long-term degradation of PAHs in conjunction with the electric field, greatly reducing the risk of secondary release of pollutants. It is suitable for the remediation of industrial contaminated sites with high concentrations and large areas of PAHs, and has a wide range of applications. Attached Figure Description

[0030] Figure 1 Schematic diagram of a method for horizontally deploying electrodes to cover the top layer in situ;

[0031] Figure 2 A statistical graph showing the changes in soil current;

[0032] The diagram is labeled as follows: 1. Power source, 2. Ammeter, 3. Clean soil, 4. Covering / barrier layer, 5. Contaminated soil, 6. Electrode. Detailed Implementation

[0033] The present invention will be described below with reference to specific embodiments. It should be noted that the following embodiments are examples of the present invention and are only used to illustrate the present invention, but the present invention is not limited to these embodiments.

[0034] The *Pseudomonas fluorescens* PB4 strain used in this invention is deposited at the China Center for Type Culture Collection (CCTCC) on July 19, 2011, with accession number CCTCC NO: M 2011260, and the address of the depository is Wuhan University, Wuhan, China. This strain has been disclosed in Chinese invention patent application number 201110302755.3, publication number CN 103031256 A.

[0035] The Bacillus licheniformis PS5 strain used in this invention is deposited at the China Center for Type Culture Collection (CCTCC) on July 19, 2011, with accession number CCTCC NO: M 2011261, and the address of the depository is Wuhan University, Wuhan, China. This strain has been disclosed in Chinese invention patent application number 201110415635.4 and publication number CN 103160448 A.

[0036] The *Kocuria* sp. FB6 strain used in this invention is deposited at the China Center for Type Culture Collection (CCTCC) on August 2, 2013, with accession number CCTCC NO: M 2013360, and the address of the depository is Wuhan University, Wuhan, China. This strain has been disclosed in Chinese invention patent application number 201711364798.8 and publication number CN 108160702 A.

[0037] The *Exiguobacterium mexicanum* JM1 strain used in this invention is deposited at the China Center for Type Culture Collection (CCTCC) on December 23, 2021, with accession number CCTCC NO: M 20211664, and the address of the depository is Wuhan University, Wuhan, China. This strain has been disclosed in Chinese invention patent application number 202211208083.4 and publication number CN 115948278A.

[0038] The preparation steps of the composite bacterial solution used in the following examples and comparative examples are as follows:

[0039] Remove the glycerol storage tubes of *Pseudomonas fluorescens* PB4, *Bacillus licheniformis* PS5, *Cocheria spp.* FB6, and *Microbacterium megaterium* JM1 from the -80℃ freezer. Quickly thaw and shake in a 37℃ water bath (20-30 seconds). Pick a small amount of the bacterial suspension and inoculate it onto beef extract peptone agar plates using the streak plate method. Incubate the plates at 28℃ for 3 days, checking purity and activation status. Select healthy colonies for a second inoculation to complete the activation step. Pick single colonies of the above strains from the activated agar plates and transfer them to beef extract peptone liquid agar, incubating at 28℃ with shaking until OD... 600 =1 to terminate the culture, centrifuge, resuspend in sterile water, and then prepare a compound bacterial suspension according to the effective viable count ratio. The effective viable count in the compound bacterial suspension is ≥2.0×10⁻⁶. 10 CFU / ml.

[0040] The formula for the beef extract peptone medium used is as follows: 5 g / L beef extract, 10 g / L peptone, 5 g / L NaCl, pH 7.0, autoclaved at 121℃ for 30 min. No agar powder is added to the liquid medium, and 1.8~2.0% agar powder is added to the plate solid medium.

[0041] To verify the effectiveness of the composite bacteria and its application in in-situ cover / barrier materials for polycyclic aromatic hydrocarbon contaminated soil, the experimental setup used in the following examples is as follows: Figure 1 As shown:

[0042] Figure 1 This is a schematic diagram of an apparatus for an in-situ top-layer covering and horizontally arranged electrode method according to an embodiment of this disclosure, as shown below. Figure 1 As shown, a covering / barrier layer 4, made of iron-loaded modified bio-activated carbon, slow-release nutrient microspheres, quartz sand, bentonite, and PAHs-degrading bacteria, is placed on top of contaminated soil 5. Clean soil 3 is laid on top of the covering / barrier layer 4. Electrodes 6 are laid horizontally on the bottom layer of contaminated soil 5 and the top layer of the covering / barrier layer 4. Electrodes 6 are connected to a DC power supply 1 to form an electric field. The electric field is a periodically polarity-switching electric field. An ammeter 2 is also installed in the electric field.

[0043] The inorganic salt liquid culture medium used in the following examples and comparative examples has the following formulation: MgSO4·7H2O 0.2 g / L; CaCl2·2H2O 0.01 g / L; FeSO4·7H2O 0.005 g / L; K2HPO4 0.4 g / L; MnSO4·H2O 0.02 g / L; NH4NO3 1.0 g / L; Na2HPO4 0.6 g / L, pH 7.2-7.4.

[0044] The PAH-contaminated soil used in the following examples and comparative examples was sourced from a coking site in Shenyang. The total PAH concentration in the soil was 856.22 mg / kg. The concentrations of 16 PAHs that the US EPA prioritizes controlling are shown in Table 1.

[0045] Table 1. PAH content in soil at coking sites

[0046] Note: "-" indicates not detected.

[0047] Example 1: Study on inter-strain antagonism Activated *Pseudomonas fluorescens* PB4, *Bacillus licheniformis* PS5, *Cocheria* FB6, and *Microbacterium megaterium* JM1 were selected and subjected to pairwise plate interaction experiments using the streak method on beef extract peptone agar plates. The plates were incubated at 28°C for 3 days, and the growth of the strains was observed to preliminarily determine whether there was any antagonistic effect among the strains. Table 2 shows that no antagonistic effect was observed among the strains.

[0048] Table 2. Antagonistic effects between strains

[0049] Note: "+" indicates the presence of a sterile area and an antagonistic effect between the two bacteria; "-" indicates the absence of a sterile area and no antagonistic effect between the two bacteria.

[0050] After preliminary determination that there was no antagonistic effect among the strains, a series of compound bacteria were prepared (Table 3). The amplified bacterial suspension was inoculated into beef extract peptone liquid medium, and the initial total number of viable bacteria in each treatment was 3.36 × 10⁻⁶. 4 The compound bacteria were added at CFU / ml in equal proportions to the effective viable count of single strains. After incubation at 28℃ and 180 rpm for 3 days, the number of microorganisms in each culture medium was determined by the spread plating method. Table 3 shows that when two, three, or four strains were cultured in equal proportions, the number of microorganisms was significantly higher than that of a single strain. The highest number of microorganisms was observed when four strains were cultured together, ranging from 3.25 to 18.66 times the number of microorganisms cultured with a single strain, indicating a synergistic effect in the growth of the compound bacteria.

[0051] Table 3 Comparison of Microbial Quantities

[0052] Example 2 Evaluation of the degradation effect of the compound bacteria Shake-flask degradation experiment: The bacterial suspensions of PB4, PS5, FB6, JM1, PB4+JM1 (1:1), PB4+PS5+JM1 (1:1:1), and PB4+PS5+FB6+JM1 (1:1:1:1) prepared after propagation were diluted with sterile water to prepare suspensions with a viable count of 2×10⁻⁶.9 Take 1 ml of a CFU / ml bacterial suspension and inoculate it into 100 ml of inorganic salt liquid medium containing PAHs, so that the viable bacterial count in the medium is 2 × 10⁻⁶. 7 The PAHs in the solution were derived from contaminated soil and were at the same concentration as those in the contaminated soil. The solution was placed on a shaker and cultured in the dark at a temperature of 28℃ and a rotation speed of 180 r / min. Each treatment was repeated three times. After 14 days of culture, the concentration of PAHs in the culture medium was measured and the average degradation rate of PAHs was calculated. The results are shown in Table 4.

[0053] Soil degradation experiment: The soil used in the experiment was PAH-contaminated soil from a coking site in Shenyang. Seven treatments were designed, including PB4, PS5, FB6, JM1, PB4+JM1 (1:1), PB4+PS5+JM1 (1:1:1), and PB4+PS5+FB6+JM1 (1:1:1:1). A proliferated bacterial suspension was added to the soil to achieve an effective viable bacterial count of 8.54 × 10⁻⁶. 8 The soil was kept at CFU / g dry soil moisture content of 20%, and inorganic salt culture medium was added every 5 days. The contaminated soil with the added bacterial suspension was placed in a soil remediation experimental device and incubated in an incubator at 28℃ for 60 days. After the experiment, the PAH content of the soil samples was measured, and the results are shown in Table 5.

[0054] Extraction of PAHs from culture medium: Refer to the National Environmental Protection Standard of the People's Republic of China (HJ478-2009).

[0055] PAHs extraction from soil: 2g of diatomaceous earth was added to a 40 mL extraction tank, followed by a mixture of 5g freeze-dried soil sample (passed through a 70-mesh sieve) and 2g of quartz sand. Finally, 2g of diatomaceous earth was added as a capping agent. The extraction temperature was 100℃, the extraction pressure was 100 bar, and hexane and acetone were mixed in a 1:1 (V / V) ratio as the extraction solvent. The extraction instrument operating procedure was as follows: heating time 5 min, holding time 5 min, purging time 2 min, 2 cycles, washing for 1 min, and nitrogen purging for 2 min. After extraction, the 1:1 (V / V) hexane and acetone mixture was completely evaporated using a Bucher parallel evaporator. Then, 10 mL of chromatographically pure acetonitrile was used to elute the PAHs for subsequent analysis.

[0056] PAHs determination: Quantitative analysis was performed by high-performance liquid chromatography (HPLC) using a variable wavelength fluorescence detector (FLD, Waters 2475) and a Waters-specific PAHs column (250 mm × 4.6 mm × 5 µm particle size). The sample solution (10 µL) was injected into the HPLC system via an autosampler at a column temperature of 25 °C. Gradient elution was used, with the following procedure: 60% water and 40% acetonitrile for 2 min, followed by 100% acetonitrile concentration over 12 min at a flow rate of 1.0 mL / min. -1 The concentration of PAHs was quantitatively calculated by using retention time and peak height, combined with a mixed standard solution of PAHs as an external standard.

[0057] Table 4. PAH concentrations in culture media under different treatments

[0058] Table 5. PAHs concentrations in soils under different treatments

[0059] As shown in Tables 4 and 5, the combined bacterial treatments PB4+JM1, PB4+PS5+JM1, and PB4+PS5+FB6+JM1 exhibited significantly higher degradation effects on PAHs than the single bacterial treatments PB4, PS5, FB6, and JM1, with the PB4+PS5+FB6+JM1 treatment showing the best effect. In the shake-flask degradation experiment, after 14 days, the total PAH removal rate of the PB4+PS5+FB6+JM1 treatment reached 34.47%, which was 11.34%, 16.01%, 17.06%, and 14.04% higher than the single bacterial treatments PB4, PS5, FB6, and JM1, respectively, and 7.50% and 3.49% higher than the combined bacterial treatments PB4+JM1 and PB4+PS5+JM1, respectively. In the soil remediation experiment, after 60 days of treatment, the total PAH removal rate of the PB4+PS5+FB6+JM1 treatment reached 59.18%, which was 29.45%, 29.24%, 25.84%, and 27.64% higher than that of the single-strain PB4, PS5, FB6, and JM1 treatments, respectively, and 17.51% and 11.23% higher than that of the compound strain PB4+JM1 and PB4+PS5+JM1 treatments, respectively.

[0060] Example 3: Preparation of in-situ covering / barrier materials This embodiment provides an in-situ covering / barrier material for PAHs-contaminated soil. The material comprises: 2 parts of a compound microorganism, 45 parts of modified bio-activated carbon, 15 parts of nutrient-release microspheres, 25 parts of quartz sand, and 10 parts of bentonite. The compound microorganism includes *Pseudomonas fluorescens* PB4, *Bacillus licheniformis* PS5, *Cocheria spp.* FB6, and *Microbacterium ramosa* JM1. The effective viable count ratio of the four strains is: *Pseudomonas fluorescens* PB4 20%–30%, *Bacillus licheniformis* PS5 25%–35%, *Cocheria spp.* FB6 15%–25%, and *Microbacterium ramosa* JM1 15%–25%. Preferably, the effective viable count of each of the four strains is 25%, with a total effective viable count ≥ 2.0 × 10⁻⁶. 10 CFU / g dry matter.

[0061] The iron-loaded modified bio-activated carbon is prepared by activating bio-activated carbon with hydrochloric acid and then soaking it in a mixed solution of Fe(NO3)3 and FeSO4. Specifically, the steps include: adding bio-activated carbon that has passed through an 80-mesh sieve to a 1.0 mol / L hydrochloric acid solution at a solid-liquid ratio of 1:10 (g / ml), activating at room temperature for 4 hours, washing with deionized water until neutral, drying at 80℃, pulverizing and passing through a 200-mesh sieve to obtain modified activated carbon; adding the pretreated activated carbon to a mixed solution of Fe(NO3)3 and FeSO4 at a solid-liquid ratio of 1:10 (g / ml). 2+ Fe 3+ The mixture was stirred and soaked at room temperature for 24 hours (molar ratio 1:2), pre-dried at 60℃ and then dried at 105℃ to constant weight. It was then calcined in a muffle furnace at 600℃ for 2 hours and naturally cooled to obtain iron-loaded modified bio-activated carbon.

[0062] The slow-release nutrient microspheres are chitosan-sodium alginate-coated nitrogen and phosphorus composite nutrient microspheres. The preparation method is as follows: first, prepare a 2% sodium alginate solution, then add 3% urea and 2% potassium dihydrogen phosphate, stir evenly to remove bubbles, and then drop it into a 2% calcium chloride solution. After solidification for 45 minutes, the nascent microspheres are obtained. The nascent microspheres are then coated with 0.5% chitosan for 30 minutes, washed and dried.

[0063] The quartz sand has a particle size of 60 mesh, and the bentonite has a particle size of 200 mesh.

[0064] Iron-loaded modified bio-activated carbon, slow-release nutrient microspheres, quartz sand, bentonite, and PAHs-degrading composite bacterial suspension were mixed according to mass fraction. To ensure the activity of the composite bacteria, inorganic salt culture medium was added during the mixing process to make the moisture content of the covering / barrier layer 4 20%. After thorough mixing, the in-situ covering / barrier material was obtained.

[0065] Example 4: Verification of the effectiveness of in-situ covering / barrier materials To verify the effects of the compound bacteria in Examples 1-3 and their application in covering / barrier materials, a small-scale in-situ covering / barrier experiment was conducted in the laboratory on contaminated soil. Four different covering / barrier materials were used in four treatments, as detailed in Table 6.

[0066] Table 6 Composition of Covering / Barrier Materials for Different Treatments

[0067] The PAHs-contaminated soil used in this embodiment is PAHs-contaminated soil from a coking site in Shenyang. A DC electric field was applied to improve the remediation efficiency.

[0068] The experimental setup used is as follows Figure 1 The apparatus is 33cm high, 10cm long, and 8cm wide. Contaminated soil that has passed through a 2mm sieve is mixed with an inorganic salt liquid culture medium to achieve a soil moisture content of 20% (w / w). The mixture is then placed into the experimental apparatus and compacted. Electrodes 6 are placed at the bottom when the contaminated soil 5 is added. A 5cm thick covering / barrier layer 4 is placed on top of the contaminated soil 5. Electrodes 6 and a 5cm thick clean soil 3 are placed on top of the covering / barrier layer 4. The electrodes 6 at the bottom of the contaminated soil 5 and the electrodes 6 on top of the covering / barrier layer 4 are connected to a DC power supply 1 to form an electric field. The electric field is a polarity-switching electric field with unequal time intervals. The method of application is that the electrodes 6 at the bottom of the clean soil 3 and the contaminated soil 5 act as the cathode and anode, respectively, for 10 minutes. Then the polarity is reversed, and the electrodes at the bottom of the clean soil 3 and the contaminated soil 5 act as the anode and cathode, respectively, for 5 minutes. This periodic switching process is carried out continuously during the experiment. The applied electric field is 1.2V / cm, and water is added once every 3 days.

[0069] The experiment lasted 35 days. Samples were collected from the cover / barrier layer 4, contaminated soil 5, and clean soil 3. PAHs and microbial changes in the soil samples were measured, and soil current changes were monitored during the experiment.

[0070] The extraction and determination of PAHs in the sample were the same as in Example 2.

[0071] The number of microorganisms in the samples was determined by real-time PCR: Total genomic DNA was extracted from the lyophilized samples using the Fast DNA® spin kit for soils (MP Biomedicals). Using 8F / 518R primers, real-time quantitative fluorescence PCR was performed in a 25 μL reaction system using the SYBR® Premix Ex Taq™ II kit and SYBR green I to estimate the bacterial count. The amplification program was 95℃ pre-denaturation for 30 s, 95℃ denaturation for 15 s, 60℃ annealing for 30 s, and 72℃ extension for 45 s, for a total of 40 cycles, followed by a final extension at 72℃ for 5 min.

[0072] The results of the change of soil current over time are shown in the figure. Figure 2 During the experiment, the control system automatically measured the current every 5 minutes. As shown in the graph, the daily average current generally decreased with increasing treatment time. However, the current increased slightly every 3 days, which is related to the water replenishment every 3 days during the experiment. After water replenishment, and under the polarity-switching electric field, soil ions and nutrients within the nutrient microspheres migrated and redistributed, thus contributing to the maintenance of soil current. Overall, the current in treatment A decreased slowly, by only 5.39 mA, indicating that the application of microorganisms and slow-release nutrient microspheres was beneficial for maintaining the device's current.

[0073] Table 7 shows the results of PAH and microbial count measurements in the cover / barrier layer 4, contaminated soil 5, and clean soil 3 after 35 days.

[0074] Table 7. Changes in polycyclic aromatic hydrocarbon concentrations, total amounts, and microbial abundance in clean soil, cover / barrier layer, and contaminated soil.

[0075] * :3.23×10 10 The number of microorganisms added to the barrier layer for treatments A and C is 0, and the number of microorganisms added to the barrier layer for treatments B and D is 0.

[0076] The results in Table 7 show that no polycyclic aromatic hydrocarbons (PAHs) were detected in clean soil 3 during the experiment, indicating that the covering / barrier layer 4 effectively prevented PAHs from migrating upwards. Treatment A showed the best barrier effect with covering / barrier layer 4, exhibiting the lowest concentration and total amount of PAHs, indicating effective degradation of PAHs in the covering / barrier layer 4. Treatment A also showed the best PAH removal rate in contaminated soil 5, with a total PAH concentration of 398.45 mg / kg and a total PAH removal rate of 53.46%, demonstrating that applying an electric field is beneficial for PAH removal in contaminated soil 5. Regarding microbial numbers, the number of microorganisms in treatment A's covering / barrier layer 4 was significantly higher than the initial amount, indicating that the nutrient-release microspheres provided sufficient nutrients for microbial growth, promoting microbial development. The number of microorganisms in treatments A and B of contaminated soil 5 was also higher than in treatments C and D, indicating that the nutrient-release microspheres, under the influence of the electric field, also provided nutrients to the microorganisms in the contaminated soil, promoting their growth.

[0077] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other way. Any person skilled in the art can make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A compound bacteria, characterized in that, The compound bacteria include *Pseudomonas fluorescens* PB4, *Bacillus licheniformis* PS5, *Cocheria spp.* FB6, and *Microbacterium megaterium* JM1. The effective viable count ratio of the four strains is as follows: *Pseudomonas fluorescens* PB4 20%–30%, *Bacillus licheniformis* PS5 25%–35%, *Cocheria spp.* FB6 15%–25%, and *Microbacterium megaterium* JM1 15%–25%. The total effective viable count of the compound bacteria is ≥2.0 × 10⁻⁶. 10 CFU / g dry matter; The fluorescent Pseudomonas PB4 is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M2011260; The Bacillus licheniformis PS5 is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M2011261; The Coccidia FB6 strain is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M2013360. The Mexican microbacterium JM1 is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M20211664.

2. The compound bacteria according to claim 1, characterized in that, The effective viable count ratios of the four strains were as follows: Pseudomonas fluorescens PB4 25%, Bacillus licheniformis PS5 30%, Coxella spp. FB6 25%, and Microbacterium gracilistylus JM1 20%.

3. An in-situ covering / barrier material for polycyclic aromatic hydrocarbon contaminated soil containing the composite bacteria described in any one of claims 1-2.

4. The in-situ covering / barrier material for polycyclic aromatic hydrocarbon contaminated soil according to claim 3, characterized in that, The materials, by weight, include 1-3 parts of compound bacteria, 30-50 parts of modified biological activated carbon, 10-20 parts of nutrient slow-release microspheres, 20-30 parts of quartz sand, and 5-15 parts of bentonite.

5. The in-situ covering / barrier material for polycyclic aromatic hydrocarbon contaminated soil according to claim 3, characterized in that, The materials, by weight, include 2 parts of compound bacteria, 45 parts of modified biological activated carbon, 15 parts of nutrient slow-release microspheres, 25 parts of quartz sand, and 10 parts of bentonite.

6. The in-situ covering / barrier material for polycyclic aromatic hydrocarbon contaminated soil according to claim 3, characterized in that, The modified bio-activated carbon is iron-modified bio-activated carbon; the nutrient slow-release microspheres are chitosan-sodium alginate coated nitrogen and phosphorus composite nutrient microspheres; the quartz sand has a particle size of 60-80 mesh; and the bentonite has a particle size of 200-300 mesh.

7. A method for preparing an in-situ covering / barrier material for polycyclic aromatic hydrocarbon contaminated soil according to any one of claims 3-6, characterized in that, Includes the following steps: S1. Strain activation and propagation: *Pseudomonas fluorescens* PB4, *Bacillus licheniformis* PS5, *Cocheria spp.* FB6, and *Microbacterium megaterium* JM1 were inoculated into their respective culture media and cultured at constant temperature with shaking until the culture medium reached OD. 600 =1, centrifuge to collect bacterial cells, resuspend in sterile water to prepare single bacterial suspension, and mix according to the effective viable cell count ratio to obtain compound bacterial suspension; S2. Modification of biological activated carbon: Add pretreated activated carbon to a mixed solution of Fe(NO3)3 and FeSO4 at a solid-liquid ratio of 1 g: 5–15 ml. The Fe content in the mixed solution is calculated as a molar ratio. 2+ Fe 3+ =1:2, ultrasonically treated for 1-2 hours, dried in an 80℃ forced-air drying oven to constant weight, then rinsed with deionized water until the solution is clear, then calcined in a muffle furnace at 300-400℃ for 1-2 hours, and naturally cooled to obtain iron-loaded modified biological activated carbon. S3. Preparation of sustained-release nutrient microspheres: Prepare a 1.5%–2.5% sodium alginate solution, add 2%–5% urea and 1%–3% potassium dihydrogen phosphate, stir evenly to remove bubbles, drop the mixed sol into a 1.0%–2.0% calcium chloride solution, and solidify for 30–60 min to obtain nascent microspheres. Coat the nascent microspheres with a 0.3%–0.8% chitosan solution for 20–40 min, wash and dry to obtain the final product. S4. Preparation of covering / barrier materials: Mix modified bio-activated carbon, nutrient slow-release microspheres, quartz sand and bentonite according to the specified ratio, then add the compound bacterial suspension and stir evenly.

8. The preparation method according to claim 6, characterized in that, In step S1, the culture temperature is 28–32℃ and the culture time is 36–72h.

9. The application of an in-situ covering / barrier material for polycyclic aromatic hydrocarbon contaminated soil as described in any one of claims 3-6, characterized in that, The material is used for in-situ remediation and risk management of PAH-contaminated soil in industrial sites.

10. The application according to claim 9, characterized in that, The application method includes the following steps: cleaning the surface debris of PAHs-contaminated soil, leveling the land, and evenly laying the in-situ covering / barrier material on or around the contaminated soil with a thickness of 3-8 cm, and naturally compacting it to complete the in-situ covering / barrier; while laying the covering / barrier material in situ, an electric field with intermittent polarity switching is set up, and the intensity of the electric field should be controlled between 0.8 and 1.5 V / cm to achieve in-situ remediation and risk management of contaminated soil.

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