Method for preparing microbial consortium composite remediation agent for synergistic treatment of paes and CD

Abstract

A method for preparing a microbial consortium composite remediation agent for synergistic treatment of PAEs and Cd. The preparation method comprises: preparing a functional microbial consortium; preparing a microbial consortium carrier; and preparing the microbial consortium composite remediation agent. In the involved microbial consortium composite remediation agent, the functional microbial consortium is subjected to cadmium-tolerance acclimation, thereby not only exhibiting broad-spectrum degradation activity against PAEs, but also overcoming the issue of reduced degradation efficiency of single functional strains under the stress of heavy-metal Cd pollution in composite contaminated soil. The involved composite remediation agent is modified with sulfhydryl groups, providing excellent passivation effects on heavy metals such as cadmium. In combination with the functional microbial consortium, the remediation agent can achieve the effect of removing PAEs and Cd from the soil synchronously.

Description

A method for preparing a microbial complex remediation agent that synergistically treats PAEs and Cd Technical Field

[0001] This invention relates to the field of soil pollutant treatment technology, specifically to a method for preparing a microbial composite remediation agent that synergistically treats PAEs and Cd. Background Technology

[0002] There is a problem of combined pollution of phthalic acid esters (PAEs) plasticizers and heavy metal Cd in farmland soils in karst areas.

[0003] To address the aforementioned issues, a more economical and efficient approach is to use immobilized bacterial agents, which are modified into a complex composed of a functional microbial community and its carrier.

[0004] To address the problem of combined pollution from phthalate plasticizers (PAEs) and heavy metal cd (Cd), a bacterial flora is needed that can both tolerate Cd's damaging effects on cells and effectively remove PAEs. Based on the degradation capabilities of bacterial strains, the inventors have constructed a functional bacterial flora capable of efficiently degrading PAEs through a quorum sensing mechanism. Technical issues

[0005] In preparing the vector based on the above-mentioned functional bacterial communities, the inventors discovered the following problems with the current technology:

[0006] For the treatment of PAEs in farmland, both the microbial community and the biochar carrier currently used have shown good effects in adsorbing and removing organic matter. However, their adsorption and passivation capabilities for heavy metals are insufficient, with small binding coefficients and easy desorption.

[0007] To address the aforementioned issues, the inventors attempted to introduce thiol-modified montmorillonite to enhance the passivation ability of the remediation agent against heavy metals. Consequently, they designed a microbial community-fixed composite remediation agent, providing technical support for resolving the problem of excessive combined pollution of phthalate plasticizers and heavy metal Cd in farmland soils in karst areas. Technical solutions

[0008] To achieve the above objectives, the present invention provides a method for preparing a microbial composite remediation agent that synergistically treats PAEs and Cd. The composite remediation agent prepared by this method can not only efficiently degrade PAEs in the soil through functional microbial communities, but also passivate Cd in the soil through a thiolized carrier, thereby achieving the simultaneous removal of PAEs and Cd from the soil.

[0009] The present invention discloses a method for preparing a microbial community complex repair agent that synergistically treats PAEs and Cd, comprising the following steps:

[0010] S1. Preparation of functional bacterial communities;

[0011] S1-1. Acclimation of Cd-tolerant strains: Under the premise of ensuring normal growth of the strains and their ability to degrade PAEs, strains of *Gordonella*, *Rhodococcus*, and *Bacillus* were introduced into a series of Cd-tolerant strains. 2+ Induced acclimatization in LB medium;

[0012] S1-2: The three strains treated in S1-1 were activated in LB medium for 24 h, and OD was adjusted. 600 =1.0;

[0013] S1-3: Mix the three strains treated with S1-2 in a volume ratio of 1:1:1 to obtain a functional bacterial group with PAE degradation and Cd resistance.

[0014] S2. Preparation of microbial vectors;

[0015] S2-1, Preparation of mercaptomontmorillonite;

[0016] S2-2, Preparation of biochar carrier;

[0017] S2-3, Carrier Composite: First, the mercaptomontmorillonite in S2-1 and the biochar carrier in S2-2 are placed in pure water and mixed evenly. Then, after centrifugation, washing, and drying, the microbial carrier is obtained.

[0018] S3. Prepare a microbial community complex repair agent;

[0019] S3-1. Mix the functional bacterial group prepared in S1 with the bacterial carrier prepared in S2 to obtain a mixed system.

[0020] Let n be the multiplier and n∈R + If the amount of bacterial carrier added is 1n g, the amount of functional bacterial group added is [10n, 30n] mL;

[0021] S3-2. First, the mixture obtained in S3-1 is shaken and cultured at 150~200 r / min, constant temperature of 30℃, and in the dark for 24h; then, under aseptic conditions, it is filtered, rinsed, and dried at 25℃ to obtain the microbial complex repair agent.

[0022] Furthermore, the mercaptomontmorillonite in S2-1 is sodium silicate-mercaptomontmorillonite or polyvinyl alcohol-mercaptomontmorillonite.

[0023] Note: The interlayer spacing of montmorillonite structures is generally less than 1 nm when unmodified, which makes it difficult for montmorillonite to be adsorbed and fixed with biochar carriers to form a stable organic-mineral complex structure, which poses a risk of shedding during use; therefore, it is necessary to expand the interlayer spacing of montmorillonite.

[0024] Furthermore, the method for preparing sodium silicate-mercaptomontmorillonite is as follows:

[0025] S2-1-A. Montmorillonite and sodium silicate are added to an ethanol-water solution of 3-mercaptopropyltrimethoxysilane and mixed evenly. The temperature is controlled at 25℃ and the pH is 9.5~10. The mixture is stirred continuously for 6~7 h to obtain thiol-modified microporous montmorillonite, which is denoted as sodium silicate-thiol montmorillonite.

[0026] The mass ratio of montmorillonite, sodium silicate thioglycolate, and 3-mercaptopropyltrimethoxysilane is 1:0.06:0.1.

[0027] The mass concentration of the ethanol-water solution is 95%.

[0028] Let M1 be the sum of the masses of montmorillonite and sodium silicate, and V1 be the sum of the volumes of 3-mercaptopropyltrimethoxysilane and ethanol-water solution. Then M1:V1 = 1.06 g:20 mL.

[0029] Note: After sodium modification, the original Ca in the interlayer of montmorillonite is reduced. 2+ Na has been + As replaced, montmorillonite particles become smaller, their flake-like characteristics become more prominent, their adsorption capacity is enhanced, and their ion exchange capacity within the intercellular domains is strengthened.

[0030] Furthermore, the method for preparing polyvinyl alcohol-mercaptomontmorillonite is as follows:

[0031] S2-1-B-1, Interlayer Insertion of Organic Matter: First, montmorillonite is dissolved in deionized water to prepare a suspension with a mass concentration of 2-4%. Then, PVA is added to deionized water to prepare a mixed solution with a mass concentration of 2-3%. Next, the solution is added to the suspension at a volume ratio of 1:4 and ultrasonically vibrated for 5-7 days to obtain a colloidal suspension.

[0032] S2-1-B-2, Low-temperature self-assembly: The colloidal suspension obtained from S2-1-B-1 was treated and then frozen at -10℃ for 24 h to obtain large interlayer montmorillonite with expanded interlayer spacing.

[0033] S2-1-B-3, Thiol-modified: The macrometa-montmorillonite in S2-1-B-2 was added to an ethanol-water solution of 3-mercaptopropyltrimethoxysilane and mixed evenly. The temperature was controlled at 25℃ and the pH was 9.5~10. The mixture was stirred continuously for 6~7 h to obtain the mercapto-modified macrometa-montmorillonite, which is denoted as polyvinyl alcohol-mercaptomontmorillonite.

[0034] The mass ratio of interlayer montmorillonite to 3-mercaptopropyltrimethoxysilane is 1:0.1.

[0035] The mass concentration of the ethanol-water solution is 95%.

[0036] Let M2 be the mass of the interlayer montmorillonite, and V2 be the sum of the volumes of 3-mercaptopropyltrimethoxysilane and the ethanol-water solution. Then M2:V2 = 1 g:20 mL.

[0037] Note: The interlayer spacing of the original montmorillonite is not large, which is not conducive to stable bonding with biochar materials in subsequent processes. Therefore, it is necessary to expand the interlayer spacing of each layer in montmorillonite. In the preparation of polyvinyl alcohol-thiol montmorillonite, PVA is inserted into the gaps of montmorillonite, and the gaps of montmorillonite are expanded by low-temperature self-assembly, providing a basis for subsequent bonding.

[0038] The principle of low-temperature self-assembly of PVA-modified montmorillonite is as follows: at low temperature, water freezes to form ice crystals, and at the solid-liquid interface, the solute separates from the ice crystals; the ice crystals further force montmorillonite particles to accumulate in the gaps between the ice crystals and slowly assemble under supramolecular action; finally, after freeze-drying, a large interlayer montmorillonite material with regular pore distribution is obtained.

[0039] Furthermore, the method for inserting organic matter into the interlayer of S2-1-B-1 is as follows:

[0040] S2-1-B-1-1 First, dissolve montmorillonite in deionized water to prepare a suspension with a mass concentration of 2-4%, and stir at 45-50℃ for 3-4 h. Then, add PVA to deionized water to prepare a mixed solution with a mass concentration of 2-3%.

[0041] S2-1-B-1-2, Add the solution to the suspension at a volume ratio of 1:4, and insert the PVA into the montmorillonite gaps according to the following parameters:

[0042] First, sonicate for 3-5 minutes and let stand for 1 hour; then sonicate for 4-5 minutes and let stand for 2 hours; then sonicate for 5 minutes and let stand for 10 hours; then stir at 45-50℃ for 3 hours; finally, seal and sonicate at room temperature for 5-7 days to obtain a colloidal suspension.

[0043] S2-1-B-1-3. Centrifuge the colloidal suspension in S2-1-B-1-2 to obtain the separated product, wash the separated product repeatedly with deionized water, and finally dilute the separated product with deionized water for storage.

[0044] Furthermore, the biochar carrier paired with sodium silicate-mercaptomontmorillonite is biochar particles, and the biochar carrier paired with polyvinyl alcohol-mercaptomontmorillonite is a mesh carbon fiber.

[0045] Explanation: Biochar particles have smaller particle size and larger specific surface area, making them easier to bond with montmorillonite. However, because the bonding surface with montmorillonite is larger, the bonding surface with bacteria becomes smaller, thus reducing the bacterial load. In contrast, reticulated carbon fibers have a smaller specific surface area than biochar particles, but a larger absolute surface area. While one end is bonded to montmorillonite, the remaining part still has room to load a large number of bacteria. However, this reduces the bonding strength with montmorillonite.

[0046] Furthermore, the preparation method of biochar particles is as follows: plant fibers are crushed and ground, then pyrolyzed under an inert atmosphere. After pyrolysis is completed, the product is ground through a 200-mesh sieve to obtain biochar particles.

[0047] The pyrolysis parameters were as follows: the furnace temperature was increased from room temperature to 600℃ at a rate of 4~5℃ / min and held for 2 h.

[0048] Note: Plant fibers such as corn and rice straw can be used without limitation; biochar particles should be as small and uniform in size as possible to improve the bonding strength under load.

[0049] Furthermore, the preparation method of the reticulated carbon fiber is as follows:

[0050] First, lignocellulose with a length of 1-2 mm is immersed in 0.1 mol / L NaOH solution and stirred at 500-550 r / min for 20-25 h; then the lignocellulose is taken out and dried at 80℃, and finally calcined at a gradient temperature. The product after calcination is washed and dried to obtain mesh carbon fiber.

[0051] The temperature gradient is as follows: first, the temperature is increased from 20℃ to 250℃ at a rate of 4~5℃ / min and held for 5~6 min; then, the temperature is increased from 250℃ to 500℃ at a rate of 20~22℃ / min and held for 1~2 h; finally, the temperature is cooled to 20℃ in the furnace.

[0052] Note: Lignocellulose can retain its plant morphology after being fired at high temperatures, forming a network fiber structure with a plant fiber skeleton.

[0053] Furthermore, the method for carrier composite in S2-3 is as follows:

[0054] S2-3-1. The mercaptomontmorillonite obtained in S2-1 and the biochar carrier in S2-2 are placed in pure water and mixed evenly to obtain a mixture; let n be the scaling factor and n∈R. + Then the amount of mercaptomontmorillonite added is 1n g, the amount of biochar carrier added is [3n, 4n] g, and the amount of pure water added is [100n, 110n] mL;

[0055] S2-3-2. Separate the mixture obtained in S2-3-1. After washing, the separated product is dried at 100℃ for 5 h and finally passed through a 200-mesh sieve to obtain the composite carrier.

[0056] Explanation: Taking reticulated carbon fiber as an example, carbonized lignocellulose retains the original plant morphology and has a reticulated skeleton. When mixed with polyvinyl alcohol-mercaptomontmorillonite, the reticulated carbon fiber will combine with polyvinyl alcohol-mercaptomontmorillonite and become entangled on its surface. The surface and gaps of the reticulated carbon fiber can provide a large number of attachment sites, providing structural support for the subsequent attachment of strains. Beneficial effects

[0057] (1) The composite remediation agent designed in this invention has a functional microbial community that has been cadmium acclimatized. It not only has a broad spectrum of degradation of PAEs, but also the various degrading strains in the microbial community can form a mutualistic relationship through quorum sensing, which overcomes the problem that the decomposition efficiency of single functional strains in composite polluted soils is reduced and the competitiveness is weak.

[0058] (2) The composite remediation agent designed in this invention has been modified with thiol groups and has an excellent passivation effect on heavy metals such as cadmium. With the cooperation of functional bacteria, it can achieve the effect of simultaneously removing PAEs and Cd in the soil. Attached Figure Description

[0059] Figure 1 is a growth curve of the cadmium-tolerant acclimatized strain in Example 1 from 0 to 48 h; where Gordonia sp. is a species of Gordonia, Rhodococcus sp. is a species of Rhodococcus, and Bacillus sp. is a species of Bacillus.

[0060] Figure 2 shows the cadmium-tolerant strains from Example 1 in the presence of different Cd concentrations. 2+ OD24 h grown in LB medium 600 Value diagram;

[0061] Figure 3 shows the OD values ​​of the cadmium-tolerant acclimatized strain in Example 1 after 24 h of growth in LB medium at different pH values. 600 Value diagram;

[0062] Figure 4 shows the degradation rate of six PAEs by the cadmium-tolerant domesticated strains in Example 1.

[0063] Figure 5 shows the effect of microbial vectors on Cd in Example 4, Comparative Example 2, and Comparative Example 3. 2+ The saturated adsorption capacity plot; where a and b at the error bars are letter markers in the SPSS significant difference analysis method, indicating significant differences in the degradation rate of PAEs by the bacterial agent;

[0064] Figure 6 shows the degradation efficiency of the composite remediation agents after 5 days of cultivation in Examples 4-10 and Comparative Examples 1-3;

[0065] Wherein, a2 is Example 4, a3 is Example 5, a4 is Example 6, a5 is Example 7, a6 is Example 8, a7 is Example 9, a8 is Example 10, b1 is Comparative Example 1, b2 is Comparative Example 2, and b3 is Comparative Example 3;

[0066] The error bars a, b, d, f, ef, e, cd, and bc are all letter markers in the SPSS significant difference analysis method, indicating significant differences in the degradation rate of PAEs by the bacterial agents;

[0067] Figure 7 is a SEM image of the composite repair agent in Example 4;

[0068] Figure 8 shows the degradation efficiency of the composite remediation agent on PAEs in the soil in Example 4;

[0069] Figure 9 shows the passivation efficiency of the composite repair agent for heavy metal Cd in Example 4. The best embodiment of the present invention

[0070] Example 4: The content of this example is based on Example 3, and mainly describes the preparation method under another parameter, including the following steps:

[0071] S1. Preparation of functional bacterial communities;

[0072] S1-1. Acclimation of Cd-tolerant strains: Under the premise of ensuring normal growth of the strains and their ability to degrade PAEs, strains of *Gordonella*, *Rhodococcus*, and *Bacillus* were introduced into a Cd-containing environment. 2+ Induced acclimatization in LB medium;

[0073] S1-2: The three strains treated in S1-1 were activated in LB medium for 24 h, and OD was adjusted. 600 =1.0;

[0074] S1-3: Mix the three strains treated with S1-2 in a volume ratio of 1:1:1 to obtain a functional bacterial group with PAE degradation and Cd resistance.

[0075] S2. Preparation of microbial vectors;

[0076] S2-1-A: 10 g of montmorillonite and 0.6 g of sodium silicate were added to 200 mL of an ethanol-water solution containing 1 g of 3-mercaptopropyltrimethoxysilane with a mass concentration of 95% and mixed evenly. The temperature was controlled at 25℃ and the pH was 9.5. The mixture was stirred continuously for 6 h to obtain thiol-modified microporous montmorillonite, which was denoted as sodium silicate-thiol montmorillonite.

[0077] S2-2, Preparation of biochar particles: Plant fibers are crushed and ground and then pyrolyzed under a nitrogen atmosphere. After pyrolysis, the product is ground through a 200-mesh sieve to obtain biochar particles, i.e., biochar carriers.

[0078] The pyrolysis parameters were as follows: the furnace temperature was raised to 600℃ at a rate of 4℃ / min and held for 2 h. After pyrolysis was completed, the biochar was ground through a 200-mesh sieve to prepare biochar particles.

[0079] S2-3, Carrier Composite:

[0080] S2-3-1. Sodium silicate-mercaptomontmorillonite in S2-1-A and biochar carrier in S2-2 are placed in pure water and mixed evenly to obtain a mixture.

[0081] The amount of sodium silicate-mercaptomontmorillonite added was 1 g, the amount of biochar carrier added was 3 g, and the amount of pure water added was 100 mL.

[0082] S2-3-2. Separate the mixture in S2-3-1. After washing, dry the separated product at 100℃ for 5 h. Finally, pass it through a 200-mesh sieve to obtain the composite carrier, i.e., the microbial carrier.

[0083] S3. Prepare a microbial community complex repair agent;

[0084] S3-1. Mix the functional bacterial group prepared in S1 with the bacterial group carrier prepared in S2 evenly to obtain a mixed system.

[0085] The amount of bacterial carrier added was 5 g, and the amount of functional bacterial group added was 100 mL.

[0086] S3-2. First, the mixture obtained in S3-1 was shaken and cultured at 150 r / min, constant temperature of 30℃, and in the dark for 24 h. Then, under aseptic conditions, it was filtered, rinsed, and dried at 25℃ to obtain the microbial complex repair agent. Embodiments of the present invention

[0087] To further illustrate the methods and effects of this invention, the technical solution of this invention will be clearly and completely described below in conjunction with experiments.

[0088] In the following examples, PAEs include six EPA-controlled plasticizers: dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), butyl benzyl phthalate (BBP), di(2-ethylhexyl) phthalate (DEHP), and di-n-octyl phthalate (DOP); all reagents were of analytical grade and purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0089] Example 1: This example describes a method for preparing a microbial composite remediation agent that synergistically treats PAEs and Cd. The microbial carrier is composed of polyvinyl alcohol-thiol montmorillonite and reticulated carbon fiber, and includes the following steps:

[0090] S1. Preparation of functional bacterial communities;

[0091] S1-1. Acclimation of Cd-tolerant strains: Under the premise of ensuring normal growth of the strains and their ability to degrade PAEs, strains of *Gordonella*, *Rhodococcus*, and *Bacillus* were introduced into a Cd-containing environment. 2+ Induced acclimatization in LB medium;

[0092] S1-2: The three strains treated in S1-1 were activated in LB medium for 24 h, and OD was adjusted. 600 =1.0;

[0093] S1-3: Mix the three strains treated with S1-2 in a volume ratio of 1:1:1 to obtain a functional bacterial group with PAE degradation and Cd resistance.

[0094] The acclimatization results are shown in Figures 1-4. Figure 1 shows the growth curve of the cadmium-tolerant acclimatized strain from 0 to 48 hours; where Gordonia sp. represents *Gordonia*, Rhodococcus sp. represents *Rhodococcus*, and Bacillus sp. represents *Bacillus*. Figure 2 shows the growth curve of the cadmium-tolerant acclimatized strain in environments containing different amounts of Cd. 2+ OD24 h grown in LB medium 600 Figure 3 shows the OD values ​​of the cadmium-tolerant acclimatized strains after 24 h of growth in LB medium at different pH values. 600 Value plot; Figure 4 shows the degradation rate of six PAEs by cadmium-tolerant domesticated strains;

[0095] S2. Preparation of microbial vectors;

[0096] S2-1-B-1-1 First, montmorillonite is dissolved in deionized water to prepare a suspension with a mass concentration of 4%, and stirred at 50°C for 4 h. Then, PVA is added to deionized water to prepare a mixed solution with a mass concentration of 3%.

[0097] S2-1-B-1-2, Add the solution to the suspension at a volume ratio of 1:4, and insert the PVA into the montmorillonite gaps according to the following parameters:

[0098] First, sonicate for 5 minutes and let stand for 1 hour; then sonicate for 5 minutes and let stand for 2 hours; then sonicate for 5 minutes and let stand for 10 hours; then stir at 50°C for 3 hours; finally, seal and sonicate at room temperature for 7 days to obtain a colloidal suspension.

[0099] S2-1-B-1-3. Centrifuge the colloidal suspension in S2-1-B-1-2 to obtain the separated product, and wash the separated product repeatedly with deionized water. Finally, dilute the separated product with deionized water at a ratio of 1 g: 50 mL and store it for later use.

[0100] S2-1-B-2, Low-temperature self-assembly: The colloidal suspension obtained from S2-1-B-1 was treated and then frozen at -10℃ for 24 h to obtain large interlayer montmorillonite with expanded interlayer spacing.

[0101] S2-1-B-3, thiol modification: The macrometa-montmorillonite in S2-1-B-2 was added to an ethanol-water solution of 3-mercaptopropyltrimethoxysilane and mixed evenly. The temperature was controlled at 25℃ and the pH was 10. The mixture was stirred continuously for 7 h to obtain the thiol-modified macrometa-montmorillonite, which was denoted as polyvinyl alcohol-thiol montmorillonite.

[0102] The amount of interlayer montmorillonite added was 1 g, and the amount of ethanol-water solution of 3-mercaptopropyltrimethoxysilane added was 25 mL.

[0103] S2-2, Preparation of mesh carbon fibers:

[0104] First, 2 mm long lignocellulose was immersed in 0.1 mol / L NaOH solution and stirred at 550 r / min for 25 h. Then, the lignocellulose was removed and dried at 80℃. Finally, it was calcined at a gradient temperature. The product after calcination was washed and dried to obtain reticulated carbon fibers, i.e., biochar carrier.

[0105] The temperature gradient is as follows: first, the temperature is increased from 20℃ to 250℃ at a rate of 5℃ / min and held for 6 min; then, the temperature is increased from 250℃ to 500℃ at a rate of 22℃ / min and held for 2 h; finally, the temperature is cooled to 20℃ in the furnace.

[0106] S2-3, Carrier Composite:

[0107] S2-3-1. Mix the polyvinyl alcohol-mercaptomontmorillonite in S2-1-B-3 with the biochar carrier in S2-2 in pure water until homogeneous to obtain a mixture.

[0108] The amount of polyvinyl alcohol-mercaptomontmorillonite added was 1 g, the amount of biochar carrier added was 4 g, and the amount of pure water added was 110 mL.

[0109] S2-3-2. Separate the mixture in S2-3-1. After washing, dry the separated product at 100℃ for 5 h. Finally, pass it through a 200-mesh sieve to obtain the composite carrier, i.e., the microbial carrier.

[0110] S3. Preparation of a fixed composite repair agent;

[0111] S3-1. Mix the functional bacterial group prepared in S1 with the bacterial carrier prepared in S2 to obtain a mixed system.

[0112] The amount of bacterial carrier added was 5 g, and the amount of functional bacterial group added was 300 mL;

[0113] S3-2. First, the mixture obtained in S3-1 was shaken and cultured at 200 r / min, constant temperature of 30℃, and in the dark for 24 h. Then, under aseptic conditions, it was filtered, rinsed, and dried at 25℃ to obtain the microbial complex repair agent.

[0114] Example 2: The content of this example is based on Example 1, and mainly describes the preparation method under another parameter, including the following steps:

[0115] S1. Preparation of functional bacterial communities;

[0116] S1-1. Acclimation of Cd-tolerant strains: Under the premise of ensuring normal growth of the strains and their ability to degrade PAEs, strains of *Gordonella*, *Rhodococcus*, and *Bacillus* were introduced into a Cd-containing environment. 2+ Induced acclimatization in LB medium;

[0117] S1-2: The three strains treated in S1-1 were activated in LB medium for 24 h, and OD was adjusted. 600 =1.0;

[0118] S1-3: Mix the three strains treated with S1-2 in a volume ratio of 1:1:1 to obtain a functional bacterial group with PAE degradation and Cd resistance.

[0119] S2. Preparation of microbial vectors;

[0120] S2-1-B-1-1 First, montmorillonite is dissolved in deionized water to prepare a suspension with a mass concentration of 2%, and stirred at 50°C for 4 h. Then, PVA is added to deionized water to prepare a mixed solution with a mass concentration of 2%.

[0121] S2-1-B-1-2, Add the solution to the suspension at a volume ratio of 1:4, and insert the PVA into the montmorillonite gaps according to the following parameters:

[0122] First, ultrasonically vibrate for 3 minutes and let stand for 1 hour; then ultrasonically vibrate for 4 minutes and let stand for 2 hours; then ultrasonically vibrate for 5 minutes and let stand for 10 hours; next, stir at 45°C for 3 hours; finally, seal and ultrasonically vibrate at room temperature for 5 days to obtain a colloidal suspension.

[0123] S2-1-B-1-3. Centrifuge the colloidal suspension in S2-1-B-1-2 to obtain the separated product, and wash the separated product repeatedly with deionized water. Finally, dilute the separated product with deionized water at a ratio of 1 g: 50 mL and store it for later use.

[0124] S2-1-B-2, Low-temperature self-assembly: The colloidal suspension obtained from S2-1-B-1 was treated and then frozen at -10℃ for 24 h to obtain large interlayer montmorillonite with expanded interlayer spacing.

[0125] S2-1-B-3, thiol modification: The macrometa-montmorillonite in S2-1-B-2 was added to an ethanol-water solution of 3-mercaptopropyltrimethoxysilane and mixed evenly. The temperature was controlled at 25℃ and the pH was 9. The reaction was stirred continuously for 6 h to obtain the thiol-modified macrometa-montmorillonite, which was denoted as polyvinyl alcohol-thiol montmorillonite.

[0126] The amount of interlayer montmorillonite added was 1 g, and the amount of ethanol-water solution of 3-mercaptopropyltrimethoxysilane added was 20 mL.

[0127] S2-2, Preparation of mesh carbon fibers:

[0128] First, 1 mm long lignocellulose was immersed in 0.1 mol / L NaOH solution and stirred at 500 r / min for 20 h. Then, the treated lignocellulose was separated and dried at 80℃. Finally, it was calcined at a gradient temperature. After calcination, the product was washed and dried to obtain reticulated carbon fibers, i.e., biochar carrier.

[0129] The temperature gradient is as follows: first, the temperature is increased from 20℃ to 250℃ at a rate of 4℃ / min and held for 5 min; then, the temperature is increased from 250℃ to 500℃ at a rate of 20℃ / min and held for 1 h; finally, the temperature is cooled to 20℃ in the furnace.

[0130] S2-3, Carrier Composite:

[0131] S2-3-1. Mix the polyvinyl alcohol-mercaptomontmorillonite in S2-1-B-3 with the biochar carrier in S2-2 in pure water until homogeneous to obtain a mixture.

[0132] The amount of polyvinyl alcohol-mercaptomontmorillonite added was 1 g, the amount of biochar carrier added was 3 g, and the amount of pure water added was 100 mL.

[0133] S2-3-2. Separate the mixture in S2-3-1. After washing, dry the separated product at 100℃ for 5 h. Finally, pass it through a 200-mesh sieve to obtain the composite carrier, i.e., the microbial carrier.

[0134] S3. Prepare a microbial community complex repair agent;

[0135] S3-1. Mix the functional bacterial group prepared in S1 with the bacterial group carrier prepared in S2 evenly to obtain a mixed system.

[0136] The amount of bacterial carrier added was 5 g, and the amount of functional bacterial group added was 100 mL.

[0137] S3-2. First, the mixture in S3-1 was shaken and cultured at 150 r / min, constant temperature of 30℃, and in the dark for 24 h. Then, under aseptic conditions, it was filtered, rinsed, and dried at 25℃ to obtain the microbial complex repair agent.

[0138] Example 3: This example describes a method for preparing a microbial composite remediation agent that synergistically treats PAEs and Cd. The microbial carrier is composed of sodium silicate-thiomethylmontmorillonite and biochar particles. The steps are as follows:

[0139] S1. Preparation of functional bacterial communities;

[0140] S1-1. Acclimation of Cd-tolerant strains: Under the premise of ensuring normal growth of the strains and their ability to degrade PAEs, strains of *Gordonella*, *Rhodococcus*, and *Bacillus* were introduced into a Cd-containing environment. 2+ Induced acclimatization in LB medium;

[0141] S1-2: The three strains treated in S1-1 were activated in LB medium for 24 h, and OD was adjusted. 600 =1.0;

[0142] S1-3: Mix the three strains treated with S1-2 in a volume ratio of 1:1:1 to obtain a functional bacterial group with PAE degradation and Cd resistance.

[0143] S2. Preparation of microbial vectors;

[0144] S2-1-A: 10 g of montmorillonite and 0.6 g of sodium silicate were added to 200 mL of an ethanol-water solution containing 1 g of 3-mercaptopropyltrimethoxysilane with a mass concentration of 95% and mixed evenly. The temperature was controlled at 25℃ and the pH was 10. The mixture was stirred continuously for 7 h to obtain thiol-modified microporous montmorillonite, which was denoted as sodium silicate-thiol montmorillonite.

[0145] S2-2, Preparation of biochar particles: Plant fibers are crushed and ground and then pyrolyzed under a nitrogen atmosphere. After pyrolysis, the product is ground through a 200-mesh sieve to obtain biochar particles, i.e., biochar carriers.

[0146] The pyrolysis parameters are as follows: the furnace temperature is raised to 600℃ at a rate of 5℃ / min and held for 2 h. After pyrolysis is completed, the biochar is ground through a 200-mesh sieve to prepare biochar particles.

[0147] S2-3, Carrier Composite:

[0148] S2-3-1. Sodium silicate-mercaptomontmorillonite in S2-1-A and biochar carrier in S2-2 are placed in pure water and mixed evenly to obtain a mixture.

[0149] The amount of sodium silicate-mercaptomontmorillonite added was 1 g, the amount of biochar carrier added was 4 g, and the amount of pure water added was 110 mL.

[0150] S2-3-2. Separate the mixture in S2-3-1. After washing, dry the separated product at 100℃ for 5 h. Finally, pass it through a 200-mesh sieve to obtain the composite carrier, i.e., the microbial carrier.

[0151] S3. Prepare a microbial community complex repair agent;

[0152] S3-1. Mix the functional bacterial group prepared in S1 with the bacterial group carrier prepared in S2 evenly to obtain a mixed system.

[0153] The amount of bacterial carrier added was 5 g, and the amount of functional bacterial group added was 100 mL.

[0154] S3-2. First, the mixture in S3-1 was shaken and cultured at 150 r / min, constant temperature of 30℃, and in the dark for 24 h. Then, under aseptic conditions, it was filtered, rinsed, and dried at 25℃ to obtain the microbial complex repair agent.

[0155] Example 4: The content of this example is based on Example 3, and mainly describes the preparation method under another parameter, including the following steps:

[0156] S1. Preparation of functional bacterial communities;

[0157] S1-1. Acclimation of Cd-tolerant strains: Under the premise of ensuring normal growth of the strains and their ability to degrade PAEs, strains of *Gordonella*, *Rhodococcus*, and *Bacillus* were introduced into a Cd-containing environment. 2+ Induced acclimatization in LB medium;

[0158] S1-2: The three strains treated in S1-1 were activated in LB medium for 24 h, and OD was adjusted. 600 =1.0;

[0159] S1-3: Mix the three strains treated with S1-2 in a volume ratio of 1:1:1 to obtain a functional bacterial group with PAE degradation and Cd resistance.

[0160] S2. Preparation of microbial vectors;

[0161] S2-1-A: 10 g of montmorillonite and 0.6 g of sodium silicate were added to 200 mL of an ethanol-water solution containing 1 g of 3-mercaptopropyltrimethoxysilane with a mass concentration of 95% and mixed evenly. The temperature was controlled at 25℃ and the pH was 9.5. The mixture was stirred continuously for 6 h to obtain thiol-modified microporous montmorillonite, which was denoted as sodium silicate-thiol montmorillonite.

[0162] S2-2, Preparation of biochar particles: Plant fibers are crushed and ground and then pyrolyzed under a nitrogen atmosphere. After pyrolysis, the product is ground through a 200-mesh sieve to obtain biochar particles, i.e., biochar carriers.

[0163] The pyrolysis parameters were as follows: the furnace temperature was raised to 600℃ at a rate of 4℃ / min and held for 2 h. After pyrolysis was completed, the biochar was ground through a 200-mesh sieve to prepare biochar particles.

[0164] S2-3, Carrier Composite:

[0165] S2-3-1. Sodium silicate-mercaptomontmorillonite in S2-1-A and biochar carrier in S2-2 are placed in pure water and mixed evenly to obtain a mixture.

[0166] The amount of sodium silicate-mercaptomontmorillonite added was 1 g, the amount of biochar carrier added was 3 g, and the amount of pure water added was 100 mL.

[0167] S2-3-2. Separate the mixture in S2-3-1. After washing, dry the separated product at 100℃ for 5 h. Finally, pass it through a 200-mesh sieve to obtain the composite carrier, i.e., the microbial carrier.

[0168] S3. Prepare a microbial community complex repair agent;

[0169] S3-1. Mix the functional bacterial group prepared in S1 with the bacterial group carrier prepared in S2 evenly to obtain a mixed system.

[0170] The amount of bacterial carrier added was 5 g, and the amount of functional bacterial group added was 100 mL.

[0171] S3-2. First, the mixture obtained in S3-1 was shaken and cultured at 150 r / min, constant temperature of 30℃, and in the dark for 24 h. Then, under aseptic conditions, it was filtered, rinsed, and dried at 25℃ to obtain the microbial complex repair agent.

[0172] Example 5: This example describes a method for preparing a microbial complex repair agent that synergistically treats PAEs and Cd. This example is based on Example 4 with changes to the conditions; all other contents are the same except for the following:

[0173] In S3-1: the amount of bacterial carrier added is 5 g, and the amount of functional bacterial group added is 50 mL.

[0174] Example 6: This example describes a method for preparing a microbial complex repair agent that synergistically treats PAEs and Cd. This example is based on Example 4 with changes to the conditions; all other contents are the same except for the following:

[0175] In S3-1: the amount of bacterial carrier added is 5 g, and the amount of functional bacterial group added is 150 mL.

[0176] Example 7: This example describes a method for preparing a microbial community complex repair agent that synergistically treats PAEs and Cd. This example is based on Example 4 with changes to the conditions; all other contents are the same except for the following:

[0177] In S3-2: constant temperature 25℃.

[0178] Example 8: This example describes a method for preparing a microbial complex repair agent that synergistically treats PAEs and Cd. This example is based on Example 4 with changes to the conditions; all other contents are the same except for the following:

[0179] In S3-2: constant temperature 35℃.

[0180] Example 9: This example describes a method for preparing a microbial complex repair agent that synergistically treats PAEs and Cd. This example is based on Example 4 with changes to the conditions; all other contents are the same except for the following:

[0181] In S3-2: Incubate under light-protected conditions with shaking for 48 h.

[0182] Example 10: This example describes a method for preparing a microbial community remediation agent that synergistically treats PAEs and Cd. This example is based on Example 4 with changes to the conditions; all other contents are the same except for the following:

[0183] In S3-2: Incubate under light-protected conditions with shaking for 72 h.

[0184] Comparative Example 1: This example describes a method for preparing a microbial complex repair agent that synergistically treats PAEs and Cd. This example is based on Example 4 with changes to the conditions. Except for the following, all other contents are the same:

[0185] Use *Gordonella* spp. instead of functional bacteria.

[0186] Comparative Example 2: This example describes a method for preparing a microbial complex repair agent that synergistically treats PAEs and Cd. This example is based on Example 4 with changes to the conditions. Except for the following, all other contents are the same:

[0187] Corn stalk biochar carbonized at 600℃ was used instead of microbial carriers.

[0188] Comparative Example 3: This example describes a method for preparing a microbial complex repair agent that synergistically treats PAEs and Cd. This example is based on Example 4 with changes to the conditions. Except for the following, all other contents are the same:

[0189] The microbial vector is not thiolated.

[0190] Experimental Example 1: This experimental example was used to determine the effect of the bacterial culture vectors in Example 4, Comparative Examples 2 and 3 on Cd. 2+ The saturated adsorption capacity was determined by the following steps: 0.1 g of the bacterial culture carrier was added to 20 ml of 600 mg / L Cd solution containing 0.1 mol / L KNO3. 2+ The solution was prepared at a solid-liquid ratio of 1 g:200 ml, and the pH was adjusted to 6.0. It was then incubated at 25°C with shaking for 1 h, allowed to stand for 16 h, centrifuged for 30 min, and the supernatant was used to determine Cd. 2+ The concentration and experimental results are shown in Figure 5.

[0191] The results show that the bacterial culture vector in Example 4 is effective against Cd. 2+ The saturated adsorption capacity of the composite remediation agent prepared in this invention is significantly greater than that of comparative examples 2 and 3 (P<0.05), indicating that compared with composite remediation agents based on biochar carriers and composite remediation agents without thiolation treatment, the composite remediation agent prepared in this invention has a higher adsorption capacity for the heavy metal Cd. 2+ The adsorption capacity is significantly improved, and the passivation efficiency is obvious.

[0192] Experimental Example 2: This experimental example was used to determine the degradation efficiency of the composite remediation agent in Examples 4-10 and Comparative Examples 1 and 2 on PAEs in an inorganic salt system. The specific steps were as follows: Under sterile conditions, 0.2 g of the composite remediation agent was weighed and added to 20 ml of inorganic salt culture medium containing 6 kinds of PAEs with a total concentration of 30 mg / L. After incubation at 150 rpm and 30℃ in the dark for 5 days, the degradation rate of ΣPAEs was measured and calculated. The experimental results are shown in Figure 6.

[0193] The results show that the composite remediation agent in Example 4 had the highest total degradation rate of the six PAEs, reaching 86.70%. From the degradation rates of ΣPAEs by the composite remediation agents in Examples 5-10, it can be seen that the optimal preparation conditions for the immobilized bacterial agent are a bacterial-to-substrate ratio of 1 g:20 ml, a temperature of 30°C, and light-protected shaking culture for 24 h. As shown in Comparative Example 1, the composite remediation agent prepared by the functional bacterial community significantly increased the degradation capacity of ΣPAEs compared to the functional single bacteria.

[0194] Experimental Example 3: The composite repair agent in Example 4 was observed using a scanning electron microscope in this experiment. The experimental results are shown in Figure 7.

[0195] The results show that the SEM bio-electron microscopy of the composite repair agent in Example 4 shows that a large number of functional strains are attached to its surface and pore structure, and the strains are attached to the composite material in an aggregated or dispersed state, indicating that the prepared composite repair agent can effectively enrich and fix functional bacterial groups.

[0196] Test Example 4: This test example is used to determine the degradation efficiency of the composite remediation agent in Example 4 on PAEs and the passivation efficiency of heavy metal Cd in contaminated soil.

[0197] The composite remediation agent in Example 4 was applied to soil contaminated with PAEs and heavy metal Cd at a dosage of 1%. After dark incubation for 50 days, the content of PAEs and the occurrence form of heavy metals were measured. The experimental results are shown in Figures 8 and 9. Industrial applicability

[0198] As shown in Figure 8, when the composite remediation agent was applied to the contaminated soil in Example 4, the ΣPAEs content decreased from 2.56 mg / kg to 1.17 mg / kg, with a degradation rate of 54.14%, demonstrating high PAEs degradation efficiency. As shown in Figure 9, the exchangeable Cd content in the soil treated with the composite remediation agent decreased by 37.06%, while the carbonate-bound, iron-manganese oxide-bound, and residual Cd contents increased by 19.26%, 13.00%, and 12.55%, respectively. This indicates that the application of the composite remediation agent promoted the transformation of soil Cd from the highly active exchangeable state to the stable carbonate-bound, iron-manganese oxide-bound, and residual states, demonstrating strong heavy metal passivation efficiency.

Claims

1. A method for preparing a microbial community complex repair agent that synergistically treats PAEs and Cd, characterized in that, Includes the following steps: S1. Preparation of functional bacterial communities; S1-1, Acclimation of Cd-tolerant strains: Strains of *Gordonella*, *Rhodococcus*, and *Bacillus* were subjected to gradient concentrations of Cd. 2+ Induced acclimatization in LB medium; The strains of the genus *Gordonium* are *Gordonia* sp., the strains of the genus *Rhodococcus* are *Rhodococcus* sp., and the strains of the genus *Bacillus* are *Bacillus* sp.; S1-2: The three strains treated in S1-1 were activated in LB medium for 24 h, and OD was adjusted. 600 =1.0; S1-3: Mix the three strains treated with S1-2 in a volume ratio of 1:1:1 to obtain a functional bacterial group with PAE degradation and Cd resistance. S2. Preparation of microbial vectors; S2-1, Preparation of mercaptomontmorillonite; S2-2, Preparation of biochar carrier; S2-3, Carrier Composite: First, the mercaptomontmorillonite in S2-1 and the biochar carrier in S2-2 are placed in pure water and mixed evenly. Then, after centrifugation, washing, and drying, the microbial carrier is obtained. S3. Prepare a microbial community complex repair agent; S3-1. Mix the functional bacterial group prepared in S1 with the bacterial carrier prepared in S2 to obtain a mixed system. Let n be the multiplier and n∈R + The amount of the bacterial carrier added is 1 n g, and the amount of the functional bacterial group added is 20 n mL. S3-2. First, the mixture obtained in S3-1 is cultured by shaking at 150~200 r / min, constant temperature of 30℃, and in the dark for 24 h. Then, under sterile conditions, it is filtered, rinsed, and dried at 25℃ to obtain the microbial complex repair agent.

2. The method for preparing a microbial community complex repair agent for synergistic treatment of PAEs and Cd as described in claim 1, characterized in that, The mercaptomontmorillonite mentioned in S2-1 is sodium silicate-mercaptomontmorillonite or polyvinyl alcohol-mercaptomontmorillonite.

3. The method for preparing a microbial community complex repair agent for synergistic treatment of PAEs and Cd as described in claim 2, characterized in that, The method for preparing the sodium silicate-thiol montmorillonite is as follows: S2-1-A. Montmorillonite and sodium silicate are added to an ethanol-water solution of 3-mercaptopropyltrimethoxysilane and mixed evenly. The temperature is controlled at 25℃ and the pH is 9.5~10. The mixture is stirred continuously for 6~7 h to obtain thiol-modified microporous montmorillonite, which is denoted as sodium silicate-thiol montmorillonite. The mass ratio of montmorillonite, sodium silicate, and 3-mercaptopropyltrimethoxysilane is 1:0.06:0.

1. The mass concentration of the ethanol-water solution is 95%. Let M1 be the sum of the masses of montmorillonite and sodium silicate, and V1 be the sum of the volumes of 3-mercaptopropyltrimethoxysilane and ethanol-water solution. Then M1:V1 = 1.06 g:20 mL.

4. The method for preparing a microbial community complex repair agent for synergistic treatment of PAEs and Cd as described in claim 2, characterized in that, The method for preparing the polyvinyl alcohol-mercaptomontmorillonite is as follows: S2-1-B-1, Interlayer Insertion of Organic Matter: First, montmorillonite is dissolved in deionized water to prepare a suspension with a mass concentration of 2-4%. Then, PVA is added to deionized water to prepare a mixed solution with a mass concentration of 2-3%. Next, the solution is added to the suspension at a volume ratio of 1:4 and ultrasonically vibrated for 5-7 days to obtain a colloidal suspension. S2-1-B-2, Low-temperature self-assembly: The colloidal suspension obtained from S2-1-B-1 was frozen at -10℃ for 24 h to obtain large interlayer montmorillonite with expanded interlayer spacing; S2-1-B-3, Thiol-modified: The macrometa-montmorillonite in S2-1-B-2 was added to an ethanol-water solution of 3-mercaptopropyltrimethoxysilane and mixed evenly. The temperature was controlled at 25℃ and the pH was 9.5~10. The mixture was stirred continuously for 6~7 h to obtain the mercapto-modified macrometa-montmorillonite, which is denoted as polyvinyl alcohol-mercaptomontmorillonite. The mass ratio of interlayer montmorillonite to 3-mercaptopropyltrimethoxysilane is 1:0.

1. The mass concentration of the ethanol-water solution is 95%. Let M2 be the mass of the interlayer montmorillonite, and V2 be the sum of the volumes of 3-mercaptopropyltrimethoxysilane and the ethanol-water solution. Then M2:V2 = 1 g:20 mL.

5. The method for preparing a microbial community complex repair agent for synergistic treatment of PAEs and Cd as described in claim 4, characterized in that, The method for inserting organic matter between the intermediate layers in S2-1-B-1 is as follows: S2-1-B-1-1 First, dissolve montmorillonite in deionized water to prepare a suspension with a mass concentration of 2-4%, and stir at 45-50℃ for 3-4 h. Then, add PVA to deionized water to prepare a mixed solution with a mass concentration of 2-3%. S2-1-B-1-2, Add the solution to the suspension at a volume ratio of 1:4, and insert PVA into the montmorillonite gaps according to the following parameters: First, sonicate for 3-5 minutes and let stand for 1 hour; then sonicate for 4-5 minutes and let stand for 2 hours; then sonicate for 5 minutes and let stand for 10 hours; then stir at 45-50℃ for 3 hours; finally, seal and sonicate at room temperature for 5-7 days to obtain a colloidal suspension. S2-1-B-1-3. Centrifuge the colloidal suspension in S2-1-B-1-2 to obtain the separated product, wash the separated product repeatedly with deionized water, and finally dilute the separated product with deionized water for storage.

6. The method for preparing a microbial community complex repair agent for synergistic treatment of PAEs and Cd as described in claim 2, characterized in that, The biochar carrier paired with the sodium silicate-mercaptomontmorillonite is biochar particles, and the biochar carrier paired with the polyvinyl alcohol-mercaptomontmorillonite is a mesh carbon fiber.

7. The method for preparing a microbial community complex repair agent for synergistic treatment of PAEs and Cd as described in claim 6, characterized in that, The preparation method of the biochar particles is as follows: plant fibers are crushed and ground, and then pyrolyzed under an inert atmosphere. After the pyrolysis is completed, the product is ground through a 200-mesh sieve to obtain biochar particles. The parameters for the pyrolysis are: the furnace temperature is increased from room temperature to 600℃ at a rate of 4~5℃ / min and maintained for 2 h.

8. The method for preparing a microbial community complex repair agent for synergistic treatment of PAEs and Cd as described in claim 6, characterized in that, The method for preparing the reticulated carbon fiber is as follows: First, lignocellulose with a length of 1-2 mm is immersed in 0.1 mol / L NaOH solution and stirred at 500-550 r / min for 20-25 h; then the lignocellulose is taken out and dried at 80℃, and finally calcined at a gradient temperature. The product after calcination is washed and dried to obtain mesh carbon fiber. The gradient temperature is as follows: first, the temperature is increased from 20℃ to 250℃ at a rate of 4~5℃ / min and held for 5~6 min; then, the temperature is increased from 250℃ to 500℃ at a rate of 20~22℃ / min and held for 1~2 h; finally, the temperature is cooled to 20℃ in the furnace.

9. The method for preparing a microbial community complex repair agent for synergistic treatment of PAEs and Cd as described in claim 1, characterized in that, The method for carrier composite in S2-3 is as follows: S2-3-1. The mercaptomontmorillonite from S2-1 and the biochar carrier from S2-2 are placed in pure water and mixed thoroughly to obtain a mixture; let n be the scaling factor and n∈R. + The amount of mercaptomontmorillonite added is 1n g, the amount of biochar carrier added is 3n g, and the amount of pure water added is 100n mL. S2-3-2. Separate the mixture obtained in S2-3-1. After washing, the separated product is dried at 100℃ for 5 h and finally passed through a 200-mesh sieve to obtain the composite carrier.

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