Aspergillus niger repairing agent for deltamethrin-polluted soil and preparation method of aspergillus niger repairing agent

Abstract

The invention discloses an aspergillus niger repairing agent for deltamethrin-polluted soil and a preparation method thereof, and relates to the field of logistics monitoring, and the preparation method comprises the following steps: preparing a liquid culture system containing deltamethrin, setting deltamethrin concentration gradient ladder domestication and continuous passage, executing domesticated thallus collection and dry thallus mass normalization, and obtaining the deltamethrin-polluted soil. And executing esterase activity detection and activity improvement amplitude confirmation of unit dry thalli. Through gradient domestication and esterase activity threshold screening, a functional flora with high adaptability and high hydrolysis ability to deltamethrin is constructed, and the functional flora is combined with a magnetic response porous carrier and a cross-linked microcapsule structure to realize thallus activity protection, directional release and engineering addition. By synchronously monitoring the change trend of deltamethrin and 3-phenoxy benzoic acid and implementing segmented addition and reactivation utilization, the pollutant removal efficiency is effectively improved, the accumulation risk of intermediate products is reduced, meanwhile, recovery and recycling of a repairing agent are achieved, and the repairing effect, cost control and field operability are considered.

Description

Technical Field

[0001] This invention relates to the field of Aspergillus niger remediation, and in particular to an Aspergillus niger remediation agent for deltamethrin-contaminated soil and its preparation method. Background Technology

[0002] As a widely used pyrethroid pesticide, deltamethrin has a high application frequency in agricultural production and public health. It has certain residual stability and migration risks in the soil environment. With long-term application and improper disposal, the accumulation of deltamethrin in the soil has gradually caused environmental safety and ecological risks. For this type of organic pesticide pollution, bioremediation has gradually become an important technical route for soil remediation due to its characteristics such as in-situ treatment, environmental friendliness and low risk of secondary pollution. Among them, microbial remediation methods, represented by fungi, have received continuous attention.

[0003] In existing bioremediation technologies for deltamethrin-contaminated soil, microbial agents are mostly added in the form of conventional strains or simple carriers. Their adaptability and stable degradation ability to high-concentration deltamethrin environments are limited, making the remediation efficiency susceptible to environmental fluctuations. At the same time, existing microbial agents often lack synchronous monitoring and control methods for key hydrolysis intermediates, which can easily lead to the problem of phased accumulation of intermediates during the degradation process. Furthermore, microbial agents are difficult to effectively recover and reuse after remediation, increasing on-site remediation costs and management difficulties. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by providing an Aspergillus niger remediation agent for deltamethrin-contaminated soil and its preparation method. The aim is to achieve stable adaptation to high-pollution environments by constructing a deltamethrin-gradient-acclimated Aspergillus niger community, combined with esterase activity screening. Through a porous inorganic framework and magnetically responsive structure, the agent achieves structured loading and controllable recovery in the later stages. Furthermore, by monitoring deltamethrin and its key hydrolysis products in stages, and through synergistic application and environmental parameter regulation, the risk of intermediate product accumulation is effectively suppressed, thereby improving the stability, controllability, and engineering applicability of the soil remediation process.

[0005] Therefore, this application provides an Aspergillus niger remediation agent for deltamethrin-contaminated soil and its preparation method, comprising the following steps:

[0006] Step S100: Configure a liquid culture system containing deltamethrin, set up a deltamethrin concentration gradient for acclimatization and continuous subculturing, collect acclimatized bacterial cells and normalize the quality of dry bacterial cells, and perform esterase activity detection and confirmation of activity increase per unit of dry bacterial cells.

[0007] Step S200: Perform solid culture medium preparation and aseptic treatment, set the inoculation and solid culture conditions for the preparation production strain, prepare Aspergillus niger spore elution and spore suspension, set the Aspergillus niger spore concentration and moisture load control, and set the vacuum low temperature drying and Aspergillus niger spore powder preparation.

[0008] Step S300: Configure the selection and particle size range of inorganic porous framework carrier, perform dust removal and cleaning and heat treatment to stabilize the inorganic porous framework carrier, configure iron salt mixed solution and pore wetting adsorption, configure co-precipitation reaction system and generate Fe3O4 magnetic microparticle layer in situ, configure magnetic response inorganic porous carrier particle cleaning, neutralization and drying, and perform Fe3O4 magnetic microparticle layer loading confirmation and magnetic response performance threshold determination.

[0009] Step S400: Prepare a sterile chitosan solution and limit the mass fraction and pH of the chitosan solution; prepare a sterile dialdehyde starch crosslinking agent solution; prepare a composite core loading mixture and limit the component ratio; prepare the composite core loading mixture into the sterile chitosan solution to form chitosan dialdehyde starch crosslinked microcapsules; and clean and solidify them.

[0010] Step S500: Set the extrusion rounding granulation molding and limit the particle size range of composite particles, configure the single particle compressive strength quality control and set the single particle compressive strength threshold, configure the spore survival rate quality control, and configure the deltamethrin initial hydrolysis characterization activity quality control.

[0011] Step S600: Perform on-site sampling and testing of deltamethrin concentration and 3-phenoxybenzoic acid concentration, set the dosage of granulated Aspergillus niger remediation agent, set the remediation environment parameters for coordinated control, and perform phased monitoring of removal rate and 3-phenoxybenzoic acid accumulation trend.

[0012] In some specific embodiments, step S100 specifically includes:

[0013] Step S100.1: Configure a liquid culture system containing deltamethrin, and set up a stepwise acclimatization and continuous subculturing of deltamethrin concentration gradient.

[0014] Step S100.2: Perform acclimatization of bacterial cells and normalization of dry bacterial cell quality, and perform esterase activity detection and activity enhancement confirmation of unit dry bacterial cells.

[0015] In some specific embodiments, step S200 specifically includes:

[0016] Step S200.1: Prepare and sterilize the solid culture medium, set the inoculation and solid culture conditions for the preparation production strain, and prepare Aspergillus niger spore elution and spore suspension.

[0017] Step S200.2: Set the concentration and moisture load control of Aspergillus niger spores, and set the vacuum low temperature drying and Aspergillus niger spore powder preparation.

[0018] Aspergillus niger spore suspension was concentrated by centrifugation to obtain Aspergillus niger spore concentrate. The centrifugation conditions were set at 4000g to 6000g and the centrifugation time was set at 8min to 12min. After centrifugation, the supernatant was discarded, and the precipitate was defined as Aspergillus niger spore concentrate. The water load of the Aspergillus niger spore concentrate was used to control the efficiency of subsequent vacuum low-temperature drying. The solid content of the Aspergillus niger spore concentrate was set at 15% to 25%. When the solid content of the Aspergillus niger spore concentrate was lower than 15%, the centrifugation concentration step was repeated once. When the solid content of the Aspergillus niger spore concentrate was higher than 25%, a sterile protective agent solution was added to dilute it to below 25%.

[0019] Spread the concentrated Aspergillus niger spore slurry evenly onto the drying tray, with a spreading thickness of 2mm to 5mm. Place the drying tray into a vacuum low-temperature drying device, with the drying temperature set to 25℃ to 30℃ and the vacuum pressure set to 10kPa to 20kPa.

[0020] After drying, the dried flakes are aseptically pulverized. The pulverization method is set to low-speed shear pulverization, the pulverization speed is set to 200 rpm to 600 rpm, and the pulverization time is set to 30 s to 120 s. After pulverization, the flakes are sieved through a 60-mesh sieve, and the material that passes through the sieve is defined as Aspergillus niger spore powder.

[0021] Spore counts were determined from Aspergillus niger spore powder using a serial dilution plating method. The incubation temperature was set at 30℃±1℃, the incubation time at 48 h, and the spore count threshold was set at at least 1×10⁻⁶ spores. 9 CFU / g.

[0022] The moisture content of Aspergillus niger spore powder is checked, and the threshold for moisture content determination is set at no more than 8%. The spore powder is also subjected to contamination limit confirmation, which is performed by incubating on nutrient agar plates at 30°C for 48 hours without the appearance of bacterial colonies. Aspergillus niger spore powder that meets the thresholds for spore count, moisture content, and contamination limit is defined as raw material powder for pharmaceutical preparation. This raw material powder is packaged in aluminum foil composite bags, and the relative humidity control threshold inside the aluminum foil composite bags is set at no more than 30%. A batch number for the raw material powder is established, and the batch number is archived in correspondence with the strain number used in pharmaceutical preparation.

[0023] In some specific embodiments, step S300 specifically includes:

[0024] Step S300.1: Select and limit the particle size range of the inorganic porous framework carrier, perform dust removal and cleaning and heat treatment to stabilize the inorganic porous framework carrier, prepare iron salt mixed solution for pore wetting and adsorption, prepare co-precipitation reaction system, and generate Fe3O4 magnetic microparticle layer in situ.

[0025] Step S300.2: Clean, neutralize, dry and shape the magnetic response inorganic porous carrier particles, and perform Fe3O4 magnetic microparticle layer loading confirmation and magnetic response performance threshold determination.

[0026] In some specific embodiments, step S400 specifically includes:

[0027] Step S400.1: Prepare a sterile chitosan solution and limit the mass fraction and pH of the chitosan solution; prepare a sterile dialdehyde starch crosslinking agent solution; prepare a composite core loading mixture and limit the component ratio.

[0028] Step S400.2: Prepare the composite core loading mixture and introduce it into a sterile chitosan solution to form chitosan dialdehyde starch cross-linked microcapsules, followed by cleaning and curing.

[0029] In some specific embodiments, step S500 specifically includes:

[0030] Step S500.1: Set the extrusion rounding granulation molding, limit the particle size range of composite particles, configure the single particle compressive strength quality control, and set the single particle compressive strength threshold.

[0031] Step S500.2: Configure spore survival rate quality control and configure deltamethrin initial hydrolysis characterization activity quality control.

[0032] In some specific embodiments, step S600 specifically includes:

[0033] Step S600.1: Perform on-site sampling and testing of deltamethrin concentration and 3-phenoxybenzoic acid concentration, and set the dosage of granulated Aspergillus niger repair agent.

[0034] Step S600.2: Set up collaborative control of remediation environment parameters, and perform phased monitoring of removal rate and 3-phenoxybenzoic acid accumulation trend.

[0035] Obtain the field water holding capacity (FBC) measurement value; the soil moisture content corresponding to the FBC is defined as θ. FC The soil moisture content contaminated with deltamethrin to be remediated is defined as θ, and the relative field capacity is defined as W. FC (%)=θ / θ FC×100%, the relative field water holding capacity control threshold is set at 55% to 70%, the pH control threshold for the soil contaminated with deltamethrin to be remediated is set at 6.0 to 7.5, the environmental temperature control threshold for remediation is set at 20℃ to 35℃, the turning and maintenance cycle is set at 7 days, and the turning and maintenance is used to maintain the aerobic state.

[0036] Monitoring time points were set at days 0, 7, 14, and 28. Soil deltamethrin concentration C was recorded on day 0. DM,0 Record the soil deltamethrin concentration C on day td. DM,t The removal rate of deltamethrin is defined as η. t .

[0037] The soil 3-phenoxybenzoic acid concentration C was recorded on days 0, 7, and 14. 3PBA,0 C 3PBA,7 and C 3PBA,14 The criterion for the increasing trend of 3-phenoxybenzoic acid accumulation in soil was set as C. 3PBA,14 >C 3PBA,7 And C 3PBA,7 >C 3PBA,0 The additional trigger condition is determined on day 14, and the additional trigger condition is set to η. 14 <70% or meeting the criterion of an increasing trend in the accumulation of 3-phenoxybenzoic acid in the soil, the supplementary dosage is defined as D add .

[0038] The granulated Aspergillus niger remediation agent corresponding to the dosage was added into the soil contaminated with deltamethrin to be remediated and then mixed thoroughly.

[0039] After day 28, an external magnetic field recovery component was obtained. The external magnetic field recovery component was set to a permanent magnet with a surface magnetic induction intensity of 0.3T, the distance between the permanent magnet and the outer wall of the soil container was set to 2cm, and the magnetic response aggregation time was set to no more than 120s. The granulated Aspergillus niger remediation agent product after magnetic response aggregation was separated to obtain recovered particles.

[0040] A reactivation solution was prepared, comprising sterile phosphate buffer and glucose aqueous solution. The pH of the sterile phosphate buffer was set to 7.0 ± 0.1, and the glucose mass fraction was set to 0.5%. The recovered particles were then placed in the reactivation solution for reactivation culture. The reactivation culture temperature was set to 30℃ ± 1℃, the shaking speed was set to 180 rpm, and the reactivation culture time was set to 12 h. After reactivation, the recovered particles underwent spore survival rate verification and deltamethrin initial hydrolysis activity verification. The spore survival rate threshold was set to be no less than 80%, and the deltamethrin initial hydrolysis activity threshold was set to be no less than 0.10 mg·L⁻¹·min⁻¹. -1 ·g -1Particles that are reactivated and meet the spore survival rate threshold and the initial hydrolysis characterization activity threshold of deltamethrin are defined as reusable particles. Reusable particles are returned and the next round of addition is performed. The threshold for the number of reuse cycles is set to be no less than 3 rounds.

[0041] In summary, this application provides an Aspergillus niger remediation agent for deltamethrin-contaminated soil and its preparation method. Through gradient acclimatization and esterase activity threshold screening, a functional bacterial community with high adaptability and high hydrolysis capacity to deltamethrin is constructed. This community is then combined with a magnetically responsive porous carrier and a cross-linked microcapsule structure to achieve bacterial cell activity protection, targeted release, and engineered addition. By simultaneously monitoring the changing trends of deltamethrin and 3-phenoxybenzoic acid and implementing segmented addition and reactivation, the pollutant removal efficiency is effectively improved, the risk of intermediate product accumulation is reduced, and the remediation agent can be recovered and recycled. This approach balances remediation effectiveness, cost control, and on-site operability, making it suitable for engineered remediation applications of deltamethrin-contaminated soil. Attached Figure Description

[0042] Figure 1 This is an overall flow chart of an Aspergillus niger remediation agent for deltamethrin-contaminated soil and its preparation method provided in the embodiments of this application. Detailed Implementation

[0043] Please refer to Figure 1 The present invention illustrates a process of an embodiment of an Aspergillus niger remediation agent for deltamethrin-contaminated soil and its preparation method according to the present disclosure.

[0044] like Figure 1 As shown, an Aspergillus niger remediation agent for deltamethrin-contaminated soil and its preparation method include the following steps:

[0045] Step S100: Configure a liquid culture system containing deltamethrin, set up a deltamethrin concentration gradient for acclimatization and continuous subculturing, collect acclimatized bacterial cells and normalize the quality of dry bacterial cells, and perform esterase activity detection and confirmation of activity increase per unit of dry bacterial cells.

[0046] Step S200: Perform solid culture medium preparation and aseptic treatment, set the inoculation and solid culture conditions for the preparation production strain, prepare Aspergillus niger spore elution and spore suspension, set the Aspergillus niger spore concentration and moisture load control, and set the vacuum low temperature drying and Aspergillus niger spore powder preparation.

[0047] Step S300: Configure the selection and particle size range of inorganic porous framework carrier, perform dust removal and cleaning and heat treatment to stabilize the inorganic porous framework carrier, configure iron salt mixed solution and pore wetting adsorption, configure co-precipitation reaction system and generate Fe3O4 magnetic microparticle layer in situ, configure magnetic response inorganic porous carrier particle cleaning, neutralization and drying, and perform Fe3O4 magnetic microparticle layer loading confirmation and magnetic response performance threshold determination.

[0048] Step S400: Prepare a sterile chitosan solution and limit the mass fraction and pH of the chitosan solution; prepare a sterile dialdehyde starch crosslinking agent solution; prepare a composite core loading mixture and limit the component ratio; prepare the composite core loading mixture into the sterile chitosan solution to form chitosan dialdehyde starch crosslinked microcapsules; and clean and solidify them.

[0049] Step S500: Set the extrusion rounding granulation molding and limit the particle size range of composite particles, configure the single particle compressive strength quality control and set the single particle compressive strength threshold, configure the spore survival rate quality control, and configure the deltamethrin initial hydrolysis characterization activity quality control.

[0050] Step S600: Perform on-site sampling and testing of deltamethrin concentration and 3-phenoxybenzoic acid concentration, set the dosage of granulated Aspergillus niger remediation agent, set the remediation environment parameters for coordinated control, and perform phased monitoring of removal rate and 3-phenoxybenzoic acid accumulation trend.

[0051] In some specific embodiments, step S100 specifically includes:

[0052] Step S100.1: Configure a liquid culture system containing deltamethrin, and set up a stepwise acclimatization and continuous subculturing of deltamethrin concentration gradient.

[0053] A liquid culture system containing deltamethrin was prepared under aseptic conditions. The liquid culture system containing deltamethrin included a carbon source, a nitrogen source, inorganic salts, and deltamethrin stock solution. The carbon source was glucose with a mass concentration of 20 g / L. The nitrogen source was peptone with a mass concentration of 5 g / L. The inorganic salts included potassium dihydrogen phosphate and magnesium sulfate heptahydrate with a mass concentration of 1 g / L and a mass concentration of 0.5 g / L. The initial pH of the liquid culture system containing deltamethrin was set to 6.0 ± 0.2.

[0054] The deltamethrin mother liquor was prepared using acetone as a solvent, and the mass concentration of the deltamethrin mother liquor was set at 4 g / L. After the deltamethrin mother liquor was added to the liquid culture system containing deltamethrin, the volume fraction of acetone was set at 0.5% to reduce the uneven dispersion of deltamethrin in the aqueous system.

[0055] Aspergillus niger was inoculated into a liquid culture system containing deltamethrin. The inoculation amount of Aspergillus niger was measured as a spore suspension, and the spore concentration of Aspergillus niger was set at 1×10⁻⁶. 7 The inoculum volume fraction was set at 5% (CFU / mL). The liquid culture system containing deltamethrin was cultured under constant temperature and shaking conditions. The culture temperature was set at 30℃±1℃, the shaking speed was set at 180 rpm, and the single culture time was set at 48h.

[0056] The concentration gradient of deltamethrin was set at four levels: 5 mg / L, 10 mg / L, 20 mg / L, and 40 mg / L, with the order of concentration gradient increase set as 5 mg / L → 10 mg / L → 20 mg / L → 40 mg / L.

[0057] Each grade of deltamethrin concentration gradient was continuously passaged three times. The continuous passage method was set as follows: at the end of 48 hours of culture, Aspergillus niger cells were collected, washed twice with sterile physiological saline, and then a cell homogenate was prepared. The cell homogenate was inoculated into a liquid culture system containing deltamethrin of the same grade concentration at a volume fraction of 10% to form the second and third generation cultures.

[0058] The entry conditions for increasing the deltamethrin concentration gradient from 5 mg / L to 10 mg / L, from 10 mg / L to 20 mg / L, and from 20 mg / L to 40 mg / L were set as follows: at the end of the third generation of culture with the same deltamethrin concentration, the dry mycelial mass concentration of Aspergillus niger was not less than 1.0 g / L, and the mycelial morphology of Aspergillus niger maintained a continuous filamentous aggregate morphology without completely fragmented flocculent sedimentation.

[0059] At the end of the third generation of culture with a 40 mg / L deltamethrin concentration gradient, the acclimatized bacterial cells were collected. The acclimatized bacterial cells were separated from the culture medium by vacuum filtration with a filter membrane pore size of 0.45 μm. The acclimatized bacterial cells were washed twice with sterile phosphate buffer with a pH of 7.0 ± 0.1.

[0060] Step S100.2: Perform acclimatization of bacterial cells and normalization of dry bacterial cell quality, and perform esterase activity detection and activity enhancement confirmation of unit dry bacterial cells.

[0061] The dry cell mass of the acclimatized bacteria is used for subsequent calculation of esterase activity per unit of dry cell. The method for determining the dry cell mass of the acclimatized bacteria is set as follows: the acclimatized bacteria are dried at a constant temperature of 60℃ until the mass is constant. The criterion for constant mass is set as the difference between two consecutive weighings not exceeding 1mg.

[0062] A reaction system for esterase activity detection was constructed, which included crude enzyme extract, phosphate buffer, and p-nitrophenol acetate substrate solution. The pH of the phosphate buffer was set to 7.0±0.1, the final concentration of p-nitrophenol acetate substrate was set to 1 mmol / L, the reaction temperature was set to 30℃±1℃, and the reaction time was set to 10 min.

[0063] The preparation method of the crude enzyme extract was set as follows: the acclimatized bacterial cells and phosphate buffer were mixed at a ratio of 1g dry bacterial cells to 10mL buffer, and the mixture was homogenized for 3min, followed by sonication for 5min. The sonication power was set to 200W, and the pulse mode was set to 2s working and 2s interval. The lysate was centrifuged at 8000g for 10min, and the supernatant was defined as the crude enzyme extract.

[0064] The esterase activity was defined as follows: 1 U is defined as the amount of enzyme that generates 1 μmol of p-nitrophenol per minute under the conditions of 30℃ and pH 7.0. The amount of p-nitrophenol generated was calculated using the absorbance at 405 nm. The conversion method used the p-nitrophenol standard curve, and the concentration points of the standard curve were set to 0, 10, 20, 40, 60, and 80 μmol / L.

[0065] The esterase activity per unit dry bacterial cell is defined as the ratio of the esterase activity value to the mass of dry bacterial cells used in the preparation of the crude enzyme extract. The unit of measurement for esterase activity per unit dry bacterial cell is set as U / g.

[0066] The activity enhancement was confirmed using an unacclimated Aspergillus niger baseline microbial population as a control. After culturing the unacclimated Aspergillus niger baseline microbial population in a liquid culture system without deltamethrin for 48 hours, the esterase activity per unit dry cell was obtained through the following methods: collection of acclimated microbial cells, normalization of dry cell mass, detection of esterase activity per unit dry cell, and confirmation of activity enhancement. The activity enhancement was defined as the increase in esterase activity per unit dry cell of acclimated microbial population relative to that of unacclimated Aspergillus niger baseline microbial population. The threshold for determining the formulation production strain was set at an activity enhancement of not less than 30%. Acclimated microbial populations that met the activity enhancement threshold were defined as formulation production strains.

[0067] The formulation production strains are preserved in the form of spores. The spore preservation system is set as a 20% glycerol aqueous solution, and the preservation temperature of the formulation production strains is set as -80℃. The formulation production strains are assigned a number, and the formulation production strain number is archived in correspondence with the deltamethrin concentration gradient acclimatization record and the esterase activity determination record per unit dry cell.

[0068] In some specific embodiments, step S200 specifically includes:

[0069] Step S200.1: Prepare and sterilize the solid culture medium, set the inoculation and solid culture conditions for the preparation production strain, and prepare Aspergillus niger spore elution and spore suspension.

[0070] Prepare a solid culture medium consisting of wheat bran and corn cob, with a mass ratio of wheat bran to corn cob of 7:3. After adding sterile water, adjust the moisture content of the solid culture medium to 55% ± 2%. Fill the solid culture container with the solid culture medium to a thickness of 2 cm to 4 cm. Perform high-temperature sterilization on the solid culture container at 121°C for 20 min. After cooling the solid culture container to 25°C to 30°C, proceed to the inoculation stage.

[0071] A suspension of spores from the microbial strains used in the formulation production was prepared using sterile physiological saline, and the spore concentration was set at 1×10⁻⁶. 7 CFU / mL up to 5×10 7 CFU / mL, the spore suspension of the formulation production strain was inoculated into the solid culture medium. The inoculation volume fraction of the spore suspension of the formulation production strain was set at 5% ± 1%. After inoculation, the solid culture medium was stirred and mixed. The stirring was set to be 3 to 5 times. The stirring goal was to make the spore suspension of the formulation production strain evenly distributed in the solid culture medium.

[0072] The solid culture container was placed in a constant temperature and humidity culture environment. The culture temperature was set to 28℃ to 32℃, the relative humidity was set to 70% to 85%, and the culture time was set to 72h. During the culture process, the oxygen supply method was set to open the lid for ventilation for 2 to 5 minutes every 12 hours to maintain the aerobic state on the surface of the solid culture medium and promote the formation of Aspergillus niger spores.

[0073] A sterile preservative solution containing trehalose and skim milk powder was prepared. The trehalose mass fraction was set at 5%, and the skim milk powder mass fraction was set at 10%. The sterile preservative solution was prepared with sterile water and filtered through a 0.22 μm filter for sterilization. After solid-state culture, the sterile preservative solution was added to the solid-state culture container. The volume added was sufficient to completely wet the surface of the solid culture medium. The wetting time was set at 10 min. After wetting, Aspergillus niger spores were released by aseptic scraping. The scraping time was set at 3 min to 8 min to obtain an Aspergillus niger spore suspension.

[0074] The Aspergillus niger spore suspension was filtered through two layers of sterile gauze. The filtration target was to remove bran particles and corn cob fibers while retaining Aspergillus niger spores and a small amount of mycelial fragments. After the Aspergillus niger spore suspension was allowed to stand for 5 minutes, the supernatant was taken and proceeded to the concentration step.

[0075] Step S200.2: Set the concentration and moisture load control of Aspergillus niger spores, and set the vacuum low temperature drying and Aspergillus niger spore powder preparation.

[0076] Aspergillus niger spore suspension was concentrated by centrifugation to obtain Aspergillus niger spore concentrate. The centrifugation conditions were set at 4000g to 6000g and the centrifugation time was set at 8min to 12min. After centrifugation, the supernatant was discarded, and the precipitate was defined as Aspergillus niger spore concentrate. The water load of the Aspergillus niger spore concentrate was used to control the efficiency of subsequent vacuum low-temperature drying. The solid content of the Aspergillus niger spore concentrate was set at 15% to 25%. When the solid content of the Aspergillus niger spore concentrate was lower than 15%, the centrifugation concentration step was repeated once. When the solid content of the Aspergillus niger spore concentrate was higher than 25%, a sterile protective agent solution was added to dilute it to below 25%.

[0077] Spread the concentrated Aspergillus niger spore slurry evenly onto the drying tray, with a spreading thickness of 2mm to 5mm. Place the drying tray into a vacuum low-temperature drying device, with the drying temperature set to 25℃ to 30℃ and the vacuum pressure set to 10kPa to 20kPa.

[0078] The vacuum low-temperature drying time was terminated when the moisture content of the Aspergillus niger spore concentrate reached the threshold. The moisture content threshold of the Aspergillus niger spore concentrate was set at 8%. The moisture content determination method was set to take a sample, weigh it, dry it at 105℃ until the mass was constant, and then convert the moisture content.

[0079] After drying, the dried flakes are aseptically pulverized. The pulverization method is set to low-speed shear pulverization, the pulverization speed is set to 200 rpm to 600 rpm, and the pulverization time is set to 30 s to 120 s. After pulverization, the flakes are sieved through a 60-mesh sieve, and the material that passes through the sieve is defined as Aspergillus niger spore powder.

[0080] Spore counts were determined from Aspergillus niger spore powder using a serial dilution plating method. The incubation temperature was set at 30℃±1℃, the incubation time at 48 h, and the spore count threshold was set at at least 1×10⁻⁶ spores. 9 CFU / g.

[0081] The moisture content of Aspergillus niger spore powder is checked, and the threshold for moisture content determination is set at no more than 8%. The spore powder is also subjected to contamination limit confirmation, which is performed by incubating on nutrient agar plates at 30°C for 48 hours without the appearance of bacterial colonies. Aspergillus niger spore powder that meets the thresholds for spore count, moisture content, and contamination limit is defined as raw material powder for pharmaceutical preparation. This raw material powder is packaged in aluminum foil composite bags, and the relative humidity control threshold inside the aluminum foil composite bags is set at no more than 30%. A batch number for the raw material powder is established, and the batch number is archived in correspondence with the strain number used in pharmaceutical preparation.

[0082] In some specific embodiments, step S300 specifically includes:

[0083] Step S300.1: Select and limit the particle size range of the inorganic porous framework carrier, perform dust removal and cleaning and heat treatment to stabilize the inorganic porous framework carrier, prepare iron salt mixed solution for pore wetting and adsorption, prepare co-precipitation reaction system, and generate Fe3O4 magnetic microparticle layer in situ.

[0084] An inorganic porous framework carrier is obtained, which is set as diatomaceous earth carrier or palygorskite carrier. The diatomaceous earth carrier or palygorskite carrier is subjected to sieving and classification to obtain inorganic porous framework carrier particles. The particle size range of inorganic porous framework carrier particles is set to 0.20 mm to 1.00 mm. Powder particles with a particle size of less than 0.20 mm are rejected, and particles with a particle size of more than 1.00 mm are crushed and then re-sieved.

[0085] The inorganic porous framework carrier particles undergo dust removal and cleaning treatment using sterile deionized water with stirring. The stirring speed is set to 200 rpm to 400 rpm, the cleaning time is set to 5 min per cycle, and the number of cycles is set to 3. After the dust removal and cleaning treatment is completed, a filtration and dehydration treatment is performed using a 0.45 μm pore size filter membrane.

[0086] Inorganic porous framework carrier particles were subjected to heat treatment to form pores and stabilize them. The heat treatment temperature was set to 200℃ to 300℃, the heat treatment time was set to 2h, and the heat treatment heating rate was set to 5℃ / min to 10℃ / min. After the heat treatment, the particles were naturally cooled to 25℃ to 30℃ to obtain heat-treated inorganic porous framework carrier particles.

[0087] A mixed iron salt solution was prepared, consisting of ferric chloride hexahydrate and ferrous sulfate heptahydrate, with a molar ratio of ferric chloride hexahydrate to ferrous sulfate heptahydrate of 2:1. The concentration of ferric chloride hexahydrate in the mixed iron salt solution was set to 0.40 mol / L, and the concentration of ferrous sulfate heptahydrate was set to 0.20 mol / L. The solvent for the mixed iron salt solution was set to deionized water, and the dissolution temperature of the mixed iron salt solution was set to 25℃ to 35℃.

[0088] Heat-treated inorganic porous framework carrier particles are added to an iron salt mixed solution to form an impregnation and adsorption system. The solid-liquid ratio in the impregnation and adsorption system is set to 1:10. The impregnation and adsorption system is subjected to ultrasonic impregnation treatment with an ultrasonic power of 150W to 250W and an ultrasonic time of 5min to 10min. After ultrasonic impregnation treatment, stirring adsorption treatment is performed with a stirring speed of 300rpm to 600rpm and a stirring adsorption time of 20min to 40min.

[0089] The adsorption system is heated to the coprecipitation reaction temperature, which is set to 70°C to 85°C. A precipitant solution is prepared, which is either an ammonia solution or a sodium hydroxide solution. The mass fraction of the ammonia solution is set to 10% to 15%, and the mass fraction of the sodium hydroxide solution is set to 2% to 4%.

[0090] The precipitant solution was added dropwise to the impregnation and adsorption system at a rate of 1.0 mL / min to 3.0 mL / min. The pH of the impregnation and adsorption system was monitored and adjusted simultaneously, with a pH threshold of 9.0 to 10.0. The impregnation and adsorption system was kept under stirring at a speed of 500 rpm to 800 rpm. The reaction time of the impregnation and adsorption system within the pH threshold range was set to 30 min to 60 min.

[0091] Fe3O4 magnetic microparticle layers are generated in situ on the surface of inorganic porous framework carrier particles and at the pore inlet position to obtain magnetically responsive inorganic porous carrier particle slurry.

[0092] Step S300.2: Clean, neutralize, dry and shape the magnetic response inorganic porous carrier particles, and perform Fe3O4 magnetic microparticle layer loading confirmation and magnetic response performance threshold determination.

[0093] The magnetically responsive inorganic porous carrier particle slurry underwent solid-liquid separation, with the separation method set to magnetic separation or filtration separation. The magnetically responsive inorganic porous carrier particles were then subjected to deionized water washing and neutralization treatment, with the number of washing cycles set to 4 to 6. The washing termination criterion was set to the pH of the washing supernatant reaching 7.0±0.2. After washing, ethanol replacement dehydration treatment was performed, with the ethanol volume fraction set to 70% and the ethanol replacement time set to 10 min.

[0094] The magnetically responsive inorganic porous carrier particles were subjected to low-temperature drying and shaping. The drying temperature was set to 45℃ to 55℃ and the drying time was set to 6h to 10h to obtain dried magnetically responsive inorganic porous carrier particles.

[0095] The loading of Fe3O4 magnetic microparticles in the dried magnetically responsive inorganic porous carrier particles was verified. The loading of the Fe3O4 magnetic microparticle layer was determined using the mass difference method. The mass difference was defined as the mass of the dried magnetically responsive inorganic porous carrier particles minus the mass of the heat-treated inorganic porous framework carrier particles. The calculated loading of the Fe3O4 magnetic microparticle layer was set as follows:

[0096]

[0097] In the formula: This indicates the loading amount of Fe3O4 magnetic microparticles in the layer, expressed as a percentage (m). dry The mass m of the dry magnetic response inorganic porous carrier particles is represented by the number of particles. heat This indicates the mass of the heat-treated inorganic porous framework carrier particles.

[0098] The loading threshold of Fe3O4 magnetic microparticle layer is set to 5% to 15%. When the loading of Fe3O4 magnetic microparticle layer is less than 5%, a supplementary deposition cycle of coprecipitation reaction system construction and in-situ generation of Fe3O4 magnetic microparticle layer is performed once. When the loading of Fe3O4 magnetic microparticle layer is greater than 15%, a deionized water strong stirring and peeling treatment is performed for 5 min and then retested.

[0099] The dried magnetically responsive inorganic porous carrier particles were subjected to a magnetic response performance threshold determination, which used the magnetic response recovery time as the criterion. The magnetic response recovery time was defined as the time it took for the magnetically responsive inorganic porous carrier particles to accumulate in the liquid phase to the side of the magnetic field under the action of an applied magnetic field. The applied magnetic field condition was set as a permanent magnet with a surface magnetic induction intensity of 0.3T, and the distance between the permanent magnet and the outer wall of the liquid phase container was set to 2cm. The magnetic response recovery time threshold was set to no more than 60s. The dried magnetically responsive inorganic porous carrier particles that met the magnetic response recovery time threshold were defined as magnetically responsive structural carriers. A batch number for the magnetically responsive structural carriers was established, and the batch number of the magnetically responsive structural carriers and the batch number of Aspergillus niger spore powder were archived separately.

[0100] In some specific embodiments, step S400 specifically includes:

[0101] Step S400.1: Prepare a sterile chitosan solution and limit the mass fraction and pH of the chitosan solution; prepare a sterile dialdehyde starch crosslinking agent solution; prepare a composite core loading mixture and limit the component ratio.

[0102] Chitosan powder was obtained, with a degree of deacetylation of not less than 80%. Chitosan powder was added to an acidifying solvent to form a chitosan solution. The acidifying solvent was set to an aqueous solution of glacial acetic acid with a volume fraction of 1.0%. The mass fraction of chitosan in the chitosan solution was set to 1.0% to 2.0%. The chitosan solution was stirred and dissolved at 25°C to 30°C for 2 to 4 hours. The pH of the chitosan solution was adjusted to 5.0 ± 0.2. The chitosan solution was then filtered through a 0.22 μm filter to obtain a sterile chitosan solution.

[0103] Dialdehyde starch crosslinking agent powder was obtained. The dialdehyde starch crosslinking agent powder was added to sterile deionized water to form a dialdehyde starch crosslinking agent solution. The mass fraction of dialdehyde starch crosslinking agent in the dialdehyde starch crosslinking agent solution was set to 0.3% to 0.8%. The aldehyde content of the dialdehyde starch crosslinking agent solution was confirmed by hydroxylamine titration. The aldehyde content threshold was set to be not less than 2.5 mmol / g. The dialdehyde starch crosslinking agent solution that met the aldehyde content threshold was sterilized by 0.22 μm filtration to obtain a sterile dialdehyde starch crosslinking agent solution.

[0104] A magnetically responsive structural carrier was obtained. The magnetically responsive structural carrier was then contacted with sterile phosphate buffer to form a wetting magnetically responsive structural carrier. The pH of the sterile phosphate buffer was set to 7.0±0.1, and the wetting time was set to 10 min to 20 min. The raw material powder for the formulation was obtained and the wetting magnetically responsive structural carrier was added. The mass ratio of the wetting magnetically responsive structural carrier to the raw material powder for the formulation was set to 10:1 to 20:1. The mixing method was set to low-shear stirring, and the stirring time was set to 3 min to 8 min, resulting in a spore-loaded magnetically responsive structural carrier.

[0105] The enzyme-inducing solidified material was obtained. The enzyme-inducing solidified material was defined as solidified particles containing esterase-inducing substrate. The particle size range of the enzyme-inducing solidified material was set to 0.5 mm to 2.0 mm. The amount of enzyme-inducing solidified material added was set to 10% to 20% based on the mass of the spore-loaded magnetic response structure carrier, thus obtaining the enzyme-inducing composite carrier.

[0106] Buffered oxygen-supplying microparticles were obtained. These microparticles were defined as a mixture of calcium carbonate microparticles and magnesium peroxide microparticles. The mass ratio of calcium carbonate microparticles to magnesium peroxide microparticles was set to 4:1. The amount of buffered oxygen-supplying microparticles added was set to 3% to 8% based on the mass of the enzyme-inducing composite carrier, resulting in a composite core-loaded mixture.

[0107] Step S400.2: Prepare the composite core loading mixture and introduce it into a sterile chitosan solution to form chitosan dialdehyde starch cross-linked microcapsules, followed by cleaning and curing.

[0108] The composite core loading mixture is added to a sterile chitosan solution to form a coating dispersion system. The solid-liquid ratio of the coating dispersion system is set to 1:8 to 1:15, the temperature of the coating dispersion system is set to 25℃ to 30℃, the stirring speed of the coating dispersion system is set to 200rpm to 400rpm, and the pre-coating time is set to 5min to 12min to obtain chitosan pre-coated composite particles.

[0109] A sterile dialdehyde starch crosslinking agent solution was added to chitosan pre-coated composite particles to form a crosslinking reaction system. The amount of sterile dialdehyde starch crosslinking agent solution added was set to 5% to 15% based on the volume of the sterile chitosan solution. The reaction time of the crosslinking reaction system was set to 20 min to 40 min. The pH of the crosslinking reaction system was maintained at 5.0 ± 0.2 to form chitosan dialdehyde starch crosslinked microcapsules.

[0110] Chitosan-dialdehyde starch cross-linked microcapsules were separated from the liquid phase using magnetic separation. The microcapsules were then washed with sterile phosphate buffer, with the washing termination criterion set at a pH of 7.0 ± 0.2 for the supernatant. The microcapsules were cured at 25°C to 30°C for 30 to 60 minutes. Spore encapsulation efficiency was then verified using the following method:

[0111]

[0112] In the formula: E(%) represents the spore encapsulation efficiency, N add N represents the total number of spores added when the composite kernel loading mixture enters. cap The total number of spores obtained by dissolving the shell layer of chitosan-dialdehyde starch cross-linked microcapsules in a 1.0% (v / v) glacial acetic acid aqueous solution and counting them is defined as the total number of spores obtained by dissolving the shell layer of the chitosan-dialdehyde starch cross-linked microcapsules. The spore encapsulation efficiency threshold is set to be no less than 85%. Chitosan-dialdehyde starch cross-linked microcapsules that meet the spore encapsulation efficiency threshold are defined as intermediates of microencapsulated repair agents.

[0113] In some specific embodiments, step S500 specifically includes:

[0114] Step S500.1: Set the extrusion rounding granulation molding, limit the particle size range of composite particles, configure the single particle compressive strength quality control, and set the single particle compressive strength threshold.

[0115] A microencapsulated repair agent intermediate is obtained, and an extrusion spheroidization granulation device is acquired. The extrusion spheroidization granulation device includes a screw extrusion unit and a spheroidization shaping unit. The microencapsulated repair agent intermediate enters a mixing granulation system, which includes the microencapsulated repair agent intermediate, microcrystalline cellulose powder, and starch powder. The mass ratio of the microencapsulated repair agent intermediate to microcrystalline cellulose powder to starch powder is set at 5:3:2. Sterile deionized water is added to the mixing granulation system for moisture content adjustment, and the moisture content of the mixing granulation system is set to 25% to 35%. The mixing granulation system then enters the screw extrusion unit to form extruded strips. The die diameter of the rod extrusion unit is set to 1.0 mm to 1.5 mm. The extruded strip enters the rounding and shaping unit to form composite particles. The rotation speed of the rounding and shaping unit is set to 600 rpm to 1200 rpm, and the rounding and shaping time is set to 3 min to 8 min. The composite particles are screened and graded to obtain the target composite particles. The particle size range of the target composite particles is set to 1 mm to 3 mm. The target composite particles enter the low-temperature drying and curing process. The low-temperature drying and curing temperature is set to 35℃ to 45℃, and the low-temperature drying and curing time is set to 4 h to 8 h. The moisture content verification threshold of the target composite particles is set to be no higher than 10%.

[0116] A single-particle compressive strength tester was used. The loading mode of the single-particle compressive strength tester was set to constant-rate compression loading, and the loading speed was set to 1 mm / min. Thirty target composite particles were randomly selected from the target composite particles as strength inspection samples. Each sample was subjected to a crush test and the crushing force was recorded. The crushing force was defined as the peak load when the target composite particle underwent structural rupture. The single-particle compressive strength was defined as the crushing force value. The threshold for single-particle compressive strength was set to be no less than 10 N. When the single-particle compressive strength was less than 10 N, the moisture content of the mixed granulation system was increased by 5 percentage points and the extrusion and rounding granulation process was repeated once.

[0117] Step S500.2: Configure spore survival rate quality control and configure deltamethrin initial hydrolysis characterization activity quality control.

[0118] A spore counting detection system was obtained using the serial dilution plating method. The incubation temperature was set at 30℃±1℃, and the incubation time was set at 48h. The intermediate of the microencapsulated repair agent was obtained, and the spore count of the intermediate was determined. The spore count of the intermediate was defined as the number of colony-forming units per unit mass of the intermediate, denoted as N. mid The target composite particles were obtained and their spore count was determined. The spore count of the target composite particles was defined as the number of colony-forming units per unit mass of the target composite particles, denoted as N. pel The spore survival rate calculation is set as follows:

[0119]

[0120] In the formula: V(%) represents the spore survival rate, N pel N represents the number of spores in the target composite particle. mid This indicates the number of spores in the intermediate microencapsulated repair agent. The spore survival rate threshold is set to be no less than 80%. When the spore survival rate is less than 80%, the rotation speed of the rounding and shaping unit is reduced by 200 rpm and the extrusion rounding and granulation process is repeated once.

[0121] A hydrolysis characterization reaction system for deltamethrin was obtained. The hydrolysis characterization reaction system for deltamethrin included sterile phosphate buffer and deltamethrin standard solution. The pH of the sterile phosphate buffer was set to 7.0±0.1, the initial mass concentration of the deltamethrin standard solution was set to 20 mg / L, the temperature of the hydrolysis characterization reaction system was set to 30℃±1℃, and the shaking speed was set to 180 rpm.

[0122] The target composite particles were added to the deltamethrin hydrolysis characterization reaction system, and the mass of the target composite particles added was denoted as m. pel (g), m pel The target concentration was set to 1.00g. The deltamethrin concentration detection module used high-performance liquid chromatography (HPLC) to obtain the deltamethrin mass concentration. The deltamethrin mass concentration at 0 min after adding the target composite particles was recorded as C0, and the deltamethrin mass concentration at 10 min after adding the target composite particles was recorded as C10. 10 The initial hydrolysis activity of deltamethrin per unit mass particle was calculated as follows:

[0123]

[0124] In the formula: A0 represents the initial hydrolysis characterization activity of deltamethrin per unit mass of particles, and the unit of measurement is mg·L. -1 ·min -1 ·g -1 C0 represents the mass concentration of deltamethrin at 0 min, C 10 This indicates the mass concentration of deltamethrin over 10 minutes, in m. pel This indicates the mass of the target composite particles added.

[0125] The initial hydrolysis activity threshold for deltamethrin was set as A0 ≥ 0.10 mg·L⁻¹. -1 ·min -1 ·g -1 When the initial hydrolysis characterization activity of deltamethrin is lower than the threshold, the mass ratio of the microencapsulated repair agent intermediate in the mixed granulation system is increased by 5 percentage points and the extrusion and spheroidization granulation process is repeated once. The target composite particles that meet the single-particle compressive strength threshold, spore survival rate threshold and initial hydrolysis characterization activity threshold of deltamethrin are defined as granulated Aspergillus niger repair agent finished products.

[0126] In some specific embodiments, step S600 specifically includes:

[0127] Step S600.1: Perform on-site sampling and testing of deltamethrin concentration and 3-phenoxybenzoic acid concentration, and set the dosage of granulated Aspergillus niger repair agent.

[0128] Soil contaminated with deltamethrin to be remediated was obtained. A deltamethrin concentration detection module and a 3-phenoxybenzoic acid concentration detection module were also acquired. The deltamethrin concentration detection module used high-performance liquid chromatography (HPLC) to determine the deltamethrin concentration in the soil. The soil deltamethrin concentration was defined as C0. DM The 3-phenoxybenzoic acid concentration detection module uses high-performance liquid chromatography (HPLC) to obtain the soil 3-phenoxybenzoic acid concentration in the soil contaminated with deltamethrin to be remediated. The soil 3-phenoxybenzoic acid concentration is defined as C3. 3PBA .

[0129] To obtain the granulated Aspergillus niger remediation agent, the dosage of the granulated Aspergillus niger remediation agent is defined as D based on the unit soil mass, and the dosage segmentation rule is set as: C. DM When ≤1mg / kg, D=2g / kg, 1 <C DM When ≤10mg / kg, D=5g / kg, C DM When the concentration is >10mg / kg, D = 8g / kg. The granulated Aspergillus niger remediation agent is mixed with the soil contaminated with deltamethrin to be remediated. The mixing time is set to 10min to 20min.

[0130] Step S600.2: Set up collaborative control of remediation environment parameters, and perform phased monitoring of removal rate and 3-phenoxybenzoic acid accumulation trend.

[0131] Obtain the field water holding capacity (FBC) measurement value; the soil moisture content corresponding to the FBC is defined as θ. FC The soil moisture content contaminated with deltamethrin to be remediated is defined as θ, and the relative field capacity is defined as W. FC (%)=θ / θ FC ×100%, the relative field water holding capacity control threshold is set at 55% to 70%, the pH control threshold for the soil contaminated with deltamethrin to be remediated is set at 6.0 to 7.5, the environmental temperature control threshold for remediation is set at 20℃ to 35℃, the turning and maintenance cycle is set at 7 days, and the turning and maintenance is used to maintain the aerobic state.

[0132] Monitoring time points were set at days 0, 7, 14, and 28. Soil deltamethrin concentration C was recorded on day 0. DM,0 Record the soil deltamethrin concentration C on day td. DM,t The removal rate of deltamethrin is defined as η. t The calculation is set as follows:

[0133]

[0134] The soil 3-phenoxybenzoic acid concentration C was recorded on days 0, 7, and 14. 3PBA,0 C 3PBA,7 and C 3PBA,14 The criterion for the increasing trend of 3-phenoxybenzoic acid accumulation in soil was set as C. 3PBA,14 >C 3PBA,7 And C 3PBA,7 >C 3PBA,0 The additional trigger condition is determined on day 14, and the additional trigger condition is set to η. 14 <70% or meeting the criterion of an increasing trend in the accumulation of 3-phenoxybenzoic acid in the soil, the supplementary dosage is defined as D add The calculation setting for the supplementary dosage is as follows:

[0135] D add =0.40×D

[0136] The granulated Aspergillus niger remediation agent corresponding to the dosage was added into the soil contaminated with deltamethrin to be remediated and then mixed thoroughly.

[0137] After day 28, an external magnetic field recovery component was obtained. The external magnetic field recovery component was set to a permanent magnet with a surface magnetic induction intensity of 0.3T, the distance between the permanent magnet and the outer wall of the soil container was set to 2cm, and the magnetic response aggregation time was set to no more than 120s. The granulated Aspergillus niger remediation agent product after magnetic response aggregation was separated to obtain recovered particles.

[0138] A reactivation solution was prepared, comprising sterile phosphate buffer and glucose aqueous solution. The pH of the sterile phosphate buffer was set to 7.0 ± 0.1, and the glucose mass fraction was set to 0.5%. The recovered particles were then placed in the reactivation solution for reactivation culture. The reactivation culture temperature was set to 30℃ ± 1℃, the shaking speed was set to 180 rpm, and the reactivation culture time was set to 12 h. After reactivation, the recovered particles underwent spore survival rate verification and deltamethrin initial hydrolysis activity verification. The spore survival rate threshold was set to be no less than 80%, and the deltamethrin initial hydrolysis activity threshold was set to be no less than 0.10 mg·L⁻¹·min⁻¹. -1 ·g -1 Particles that are reactivated and meet the spore survival rate threshold and the initial hydrolysis characterization activity threshold of deltamethrin are defined as reusable particles. Reusable particles are returned and the next round of addition is performed. The threshold for the number of reuse cycles is set to be no less than 3 rounds.

[0139] In practical application, a liquid culture system containing deltamethrin was constructed under aseptic conditions. Glucose was used as the carbon source at a concentration of 20 g / L, and peptone as the nitrogen source at a concentration of 5 g / L. Potassium dihydrogen phosphate (1 g / L) and magnesium sulfate heptahydrate (0.5 g / L) were added as inorganic salts. The initial pH of the culture system was maintained at 6.0 ± 0.2. Deltamethrin was added as a stock solution of acetone at a concentration of 4 g / L, and the volume fraction of acetone in the system was controlled at 0.5%. Aspergillus niger was inoculated as a spore suspension at a concentration of 1 × 10⁻⁶ spores. 7 The inoculum concentration was 5% (CFU / mL), the culture temperature was controlled at 30℃±1℃, the shaking speed was maintained at 180 rpm, and the single culture cycle was set to 48 h. The concentration of deltamethrin was increased stepwise in the order of 5 mg / L, 10 mg / L, 20 mg / L, and 40 mg / L. Each concentration gradient was passaged 3 times. The criteria for entering the next gradient were that the dry cell mass concentration was not less than 1.0 g / L and the mycelial morphology maintained a continuous aggregate structure. After completing the 40 mg / L gradient culture, the acclimatized bacteria were normalized to dry cell mass, and the esterase activity per unit dry cell was determined using the p-nitrophenol acetate substrate system. Bacterial groups with an increase in esterase activity of not less than 30% were selected as the production strains for the formulation.

[0140] The selected microbial strains for formulation production were propagated using solid-state culture. The solid-state culture medium consisted of wheat bran and corn cob in a mass ratio of 7:3. Sterile water was added to adjust the moisture content to 55% ± 2%, and the packing thickness was controlled within the range of 2–4 cm. The medium was sterilized at 121°C for 20 min. After cooling the culture medium to 25–30°C, spore suspension was inoculated, and the spore concentration was controlled at 1 × 10⁻⁶. 7 –5×10 7 The inoculum concentration was maintained at 5% ± 1% CFU / mL. After inoculation, the mixture was stirred 3–5 times to ensure uniform distribution. The solid-state culture environment was controlled at 28–32℃ and relative humidity at 70–85%. The culture cycle was set at 72 hours, with ventilation every 12 hours for 2–5 minutes to maintain aerobic conditions. After culture, a sterile protectant solution containing 5% trehalose and 10% skim milk powder was added to the culture medium. After soaking for 10 minutes, spores were scraped off. The resulting spore suspension was filtered through gauze and centrifuged to concentrate the solid content to 15–25%. The concentrated slurry was vacuum-dried at 25–30℃ and 10–20 kPa until the moisture content was no higher than 8%. It was then pulverized and sieved through a 60-mesh sieve to obtain spores with a count of no less than 1 × 10⁻⁶. 9 CFU / g Aspergillus niger spore powder.

[0141] Diatomaceous earth or palygorskite was selected as the inorganic porous framework carrier and screened and graded to stabilize the particle size within the range of 0.20–1.00 mm. After being washed three times with sterile deionized water, the carrier was heat-treated at 200–300℃ for 2 hours to stabilize the pore structure. The heat-treated carrier was then added to a mixed iron salt solution consisting of ferric chloride hexahydrate and ferrous sulfate heptahydrate, with a fixed molar ratio of 2:1 and a total solid-liquid ratio of 1:10. The impregnation system was ultrasonically treated at 150–250 W for 5–10 minutes with simultaneous stirring. Adsorption was performed for 20–40 min, followed by raising the system temperature to 70–85 °C. A precipitant was added dropwise at a rate of 1.0–3.0 mL / min under continuous stirring to maintain the pH of the system stably within the range of 9.0–10.0. The reaction time was controlled at 30–60 min, thereby generating a magnetic microparticle layer in situ on the surface of the support and at the pore inlet. The resulting support was washed and neutralized to pH 7.0±0.2 and dried at 45–55 °C for 6–10 h to stabilize the magnetic microparticle layer loading at 5–15% and the magnetic response recovery time at no more than 60 s.

[0142] Chitosan powder with a degree of deacetylation of not less than 80% was dissolved in a 1.0% (v / v) aqueous solution of glacial acetic acid, with the chitosan mass fraction controlled at 1.0–2.0%. The solution was stirred and dissolved at 25–30°C for 2–4 hours, and the pH was precisely adjusted to 5.0 ± 0.2 before filtration for sterilization. Dialdehyde starch crosslinking agent was dissolved in sterile water at a mass fraction of 0.3–0.8%, with its aldehyde content confirmed by hydroxylamine titration to be not less than 2.5 mmol / g. The magnetically responsive structural carrier was moistened with phosphate buffer for 10–20 min and then mixed with Aspergillus niger spore powder at a mass ratio... A spore loading carrier is formed by mixing in a 10:1–20:1 ratio. Simultaneously, an enzyme-inducing immobilizer with a particle size of 0.5–2.0 mm is added at a ratio of 10–20%, along with buffer oxygen-supplying microparticles composed of calcium carbonate and magnesium peroxide in a 4:1 ratio, with the addition ratio controlled at 3–8%. The resulting composite core loading mixture is added to a chitosan solution and pre-coated at 200–400 rpm for 5–12 min. Then, a dialdehyde starch crosslinking agent is added, and the crosslinking reaction is maintained for 20–40 min to form crosslinked microcapsules with a spore encapsulation efficiency of not less than 85%.

[0143] The microencapsulated repair agent intermediate was introduced into an extrusion spheronization granulation system and mixed with microcrystalline cellulose powder and starch powder at a mass ratio of 5:3:2. The moisture content of the mixture was controlled at 25–35%. The mixture was extruded into strips through a screw with a die diameter of 1.0–1.5 mm and spheronized at 600–1200 rpm for 3–8 min. The strips were then sieved to obtain target particles with a particle size of 1–3 mm. The particles were dried at 35–45℃ for 4–8 h to ensure a moisture content of no more than 10%. The compressive strength of the finished particles was tested at a loading speed of 1 mm / min, and the compressive strength threshold was consistently maintained at no less than 10 N. The spore survival rate of the particles was also tested, and the survival rate was consistently maintained at no less than 80%. The initial hydrolysis activity of deltamethrin was determined at 30℃ and pH 7.0, and the value was maintained at no less than 0.10 mg·L⁻¹·min. - 1·g -1 This process yields a granulated Aspergillus niger repair agent that meets the engineering application requirements.

[0144] On-site sampling and testing were conducted on soil contaminated with deltamethrin. High-performance liquid chromatography (HPLC) was used to determine the initial concentrations of deltamethrin and 3-phenoxybenzoic acid in the soil. Based on the test results, the dosage of granulated Aspergillus niger remediation agent was set in stages and mixed with the soil for 10–20 min. During the remediation period, the soil moisture content was controlled within 55–70% of the relative field capacity, the pH was stabilized at 6.0–7.5, and the ambient temperature was maintained at 20–35℃. Mixing and maintenance were carried out every 7 days. Pollutant concentrations were monitored on days 0, 7, 14, and 28. A supplementation mechanism was triggered when 3-phenoxybenzoic acid showed an increasing cumulative trend. After remediation, an external magnetic field with a surface magnetic induction intensity of 0.3T was applied, and the remediation agent was recovered within 120 s. The recovered particles were reactivated in phosphate buffer containing 0.5% glucose at 30℃ for 12 h. Once the spore survival rate and hydrolysis activity met the threshold requirements, the particles were used in the next cycle, with a stable cycle of no less than 3 cycles.

Claims

1. A *Aspergillus niger* remediation agent for deltamethrin-contaminated soil and its preparation method, characterized in that, Includes the following steps: S100, Configure a liquid culture system containing deltamethrin, set up deltamethrin concentration gradient acclimatization and continuous subculturing, perform acclimatized cell collection and dry cell quality normalization, and perform esterase activity detection and activity enhancement confirmation per unit dry cell. S200: Perform solid culture medium preparation and aseptic treatment, set the inoculation and solid culture conditions for the preparation production strain, prepare Aspergillus niger spore elution and spore suspension, set Aspergillus niger spore concentration and moisture load control, set vacuum low temperature drying and Aspergillus niger spore powder preparation. S300, configure the selection and particle size range of inorganic porous framework carrier, perform dust removal and cleaning and heat treatment to stabilize the inorganic porous framework carrier, configure iron salt mixed solution and pore wetting adsorption, configure co-precipitation reaction system, and generate Fe3O4 magnetic microparticle layer in situ, configure magnetic response inorganic porous carrier particle cleaning, neutralization and drying, and perform Fe3O4 magnetic microparticle layer loading confirmation and magnetic response performance threshold determination; S400: Prepare a sterile chitosan solution and limit the mass fraction and pH of the chitosan solution; prepare a sterile dialdehyde starch crosslinking agent solution; prepare a composite core loading mixture and limit the component ratio; prepare the composite core loading mixture into the sterile chitosan solution to form chitosan dialdehyde starch crosslinked microcapsules; and then clean and solidify them. S500, set extrusion rounding granulation molding, limit the particle size range of composite particles, configure single particle compressive strength quality control, set single particle compressive strength threshold, configure spore survival rate quality control, configure deltamethrin initial hydrolysis characterization activity quality control; S600: Conduct on-site sampling and testing of deltamethrin and 3-phenoxybenzoic acid concentrations, set the dosage of granulated Aspergillus niger remediation agent, set collaborative control of remediation environmental parameters, and conduct phased monitoring of removal rate and 3-phenoxybenzoic acid accumulation trend.

2. The Aspergillus niger remediation agent for deltamethrin-contaminated soil and its preparation method according to claim 1, characterized in that, S100 specifically includes: S100.

1. Configure a liquid culture system containing deltamethrin, and set up a stepwise acclimatization and continuous subculturing of deltamethrin concentration gradient; A liquid culture system containing deltamethrin was prepared under aseptic conditions. The liquid culture system containing deltamethrin included a carbon source, a nitrogen source, inorganic salts, and deltamethrin stock solution. The carbon source was glucose with a mass concentration of 20 g / L. The nitrogen source was peptone with a mass concentration of 5 g / L. The inorganic salts included potassium dihydrogen phosphate and magnesium sulfate heptahydrate with a mass concentration of 1 g / L and a mass concentration of 0.5 g / L. The initial pH of the liquid culture system containing deltamethrin was set to 6.0 ± 0.

2. Aspergillus niger was inoculated into a liquid culture system containing deltamethrin. The inoculation amount of Aspergillus niger was measured as a spore suspension, and the spore concentration was set at 1×10⁻⁶. 7 The inoculum volume fraction was set at 5% (CFU / mL). The liquid culture system containing deltamethrin was cultured under constant temperature and shaking conditions. The culture temperature was set at 30℃±1℃, the shaking speed was set at 180 rpm, and the single culture time was set at 48h. Each grade of deltamethrin concentration gradient was continuously passaged 3 times. The continuous passage method was set as follows: at the end of 48h of culture, Aspergillus niger cells were collected, washed twice with sterile physiological saline, and then cell homogenate was prepared. The cell homogenate was inoculated into a liquid culture system containing deltamethrin of the same grade of deltamethrin concentration at a volume fraction of 10% to form the second and third generation cultures. The entry conditions for increasing the deltamethrin concentration gradient from 5 mg / L to 10 mg / L, from 10 mg / L to 20 mg / L, and from 20 mg / L to 40 mg / L were set as follows: at the end of the third generation of culture with the same deltamethrin concentration, the dry mycelial mass concentration of Aspergillus niger was not less than 1.0 g / L, and the mycelial morphology of Aspergillus niger maintained a continuous filamentous aggregate morphology without completely fragmented flocculent sedimentation morphology. At the end of the third generation of culture with a 40 mg / L deltamethrin concentration gradient, the acclimatized bacterial cells were collected. The acclimatized bacterial cells were separated from the culture medium by vacuum filtration with a filter membrane pore size of 0.45 μm. The acclimatized bacterial cells were washed twice with sterile phosphate buffer with a pH of 7.0 ± 0.

1. S100.2, Perform the collection of domesticated bacterial cells and the normalization of dry bacterial cell quality, and perform esterase activity detection and confirmation of activity improvement in unit dry bacterial cells; The dry cell mass of the acclimatized bacteria is used for subsequent calculation of esterase activity per unit dry cell. The method for determining the dry cell mass of the acclimatized bacteria is set as follows: the acclimatized bacteria are dried at a constant temperature of 60℃ until the mass is constant. The criterion for constant mass is set as the difference between two consecutive weighings not exceeding 1mg. An esterase activity detection reaction system was constructed, which included crude enzyme extract, phosphate buffer, and p-nitrophenol acetate substrate solution. The pH of the phosphate buffer was set to 7.0±0.1, the final concentration of p-nitrophenol acetate substrate was set to 1 mmol / L, the reaction temperature of the esterase activity detection reaction system was set to 30℃±1℃, and the reaction time was set to 10 min. The activity enhancement was confirmed using an unacclimated Aspergillus niger baseline microbial population as a control. After culturing the unacclimated Aspergillus niger baseline microbial population in a liquid culture system without deltamethrin for 48 hours, the esterase activity per unit dry cell was obtained through the following methods: collection of acclimated microbial cells, normalization of dry cell mass, detection of esterase activity per unit dry cell, and confirmation of activity enhancement. The activity enhancement was defined as the increase in esterase activity per unit dry cell of acclimated microbial population relative to that of unacclimated Aspergillus niger baseline microbial population. The threshold for determining the formulation production strain was set at an activity enhancement of not less than 30%. Acclimated microbial populations that met the activity enhancement threshold were defined as formulation production strains.

3. The Aspergillus niger remediation agent for deltamethrin-contaminated soil and its preparation method according to claim 1, characterized in that, S200 specifically includes: S200.

1. Perform solid culture medium preparation and aseptic treatment, set the inoculation and solid culture conditions for the preparation production strain, and prepare Aspergillus niger spore elution and spore suspension; Solid culture medium was prepared, consisting of wheat bran and corn cob, with a mass ratio of wheat bran to corn cob of 7:

3. After adding sterile water, the moisture content of the solid culture medium was adjusted to 55% ± 2%. The solid culture medium was then loaded into solid culture containers, with a filling thickness of 2 cm to 4 cm. The solid culture containers were then subjected to high-temperature sterilization, with a sterilization temperature of 121°C and a sterilization time of 20 min. After the solid culture containers were cooled to 25°C to 30°C, they were ready for inoculation. A suspension of spores from the microbial strains used in the formulation production was prepared using sterile physiological saline, and the spore concentration was set at 1×10⁻⁶. 7 CFU / mL up to 5×10 7 CFU / mL, the spore suspension of the formulation production strain was inoculated into the solid culture medium. The inoculation volume fraction of the spore suspension of the formulation production strain was set at 5% ± 1%. After inoculation, the solid culture medium was stirred and mixed. The stirring was set to be 3 to 5 times. The stirring goal was to make the spore suspension of the formulation production strain evenly distributed in the solid culture medium. The solid culture container was placed in a constant temperature and humidity culture environment. The culture temperature was set to 28℃ to 32℃, the relative humidity was set to 70% to 85%, and the culture time was set to 72h. During the culture process, the oxygen supply method was set to open the lid for ventilation for 2 to 5 minutes every 12 hours to maintain the aerobic state on the surface of the solid culture medium and promote the formation of Aspergillus niger spores. A sterile preservative solution was prepared, which contained trehalose and skim milk powder. The mass fraction of trehalose was set at 5%, and the mass fraction of skim milk powder was set at 10%. The sterile preservative solution was prepared with sterile water and filtered through a 0.22 μm filter for sterilization. After solid-state culture, the sterile preservative solution was added to the solid-state culture container. The volume added was sufficient to completely wet the surface of the solid culture medium. The wetting time was set at 10 min. After wetting, Aspergillus niger spores were released by aseptic scraping. The scraping time was set at 3 min to 8 min to obtain an Aspergillus niger spore suspension. The Aspergillus niger spore suspension was filtered through two layers of sterile gauze. The filtration target was to remove bran particles and corn cob fibers while retaining Aspergillus niger spores and a small amount of mycelial fragments. After the Aspergillus niger spore suspension was allowed to stand for 5 minutes, the supernatant was taken and entered into the concentration step. S200.2, Set the concentration and moisture load control of Aspergillus niger spores, and set the vacuum low-temperature drying and Aspergillus niger spore powder preparation; Aspergillus niger spore suspension was concentrated by centrifugation to obtain Aspergillus niger spore concentrate. The centrifugation conditions were set at 4000g to 6000g and the centrifugation time was set at 8min to 12min. After centrifugation, the supernatant was discarded, and the precipitate was defined as Aspergillus niger spore concentrate. The water load of the Aspergillus niger spore concentrate was used to control the efficiency of subsequent vacuum low-temperature drying. The solid content of the Aspergillus niger spore concentrate was set at 15% to 25%. When the solid content of the Aspergillus niger spore concentrate was lower than 15%, the centrifugation concentration step was repeated once. When the solid content of the Aspergillus niger spore concentrate was higher than 25%, a sterile protective agent solution was added to dilute it to below 25%. Spread the concentrated Aspergillus niger spore slurry evenly onto the drying tray, with a spreading thickness of 2mm to 5mm. Place the drying tray into the vacuum low-temperature drying equipment, with the drying temperature set to 25℃ to 30℃ and the vacuum pressure set to 10kPa to 20kPa. After drying, the dried flakes are aseptically pulverized. The pulverization method is set to low-speed shear pulverization, the pulverization speed is set to 200 rpm to 600 rpm, and the pulverization time is set to 30 s to 120 s. After pulverization, the flakes are sieved through a 60-mesh sieve. The material that passes through the sieve is defined as Aspergillus niger spore powder. Spore counts were determined from Aspergillus niger spore powder using a serial dilution plating method. The incubation temperature was set at 30℃±1℃, the incubation time at 48 h, and the spore count threshold was set at at least 1×10⁻⁶ spores. 9 CFU / g; The moisture content of Aspergillus niger spore powder is checked, and the threshold for moisture content determination is set at no more than 8%. The spore powder is also subjected to contamination limit confirmation, which is performed by incubating on nutrient agar plates at 30°C for 48 hours without the appearance of bacterial colonies. Aspergillus niger spore powder that meets the thresholds for spore count, moisture content, and contamination limit is defined as raw material powder for pharmaceutical preparation. This raw material powder is packaged in aluminum foil composite bags, and the relative humidity control threshold inside the aluminum foil composite bags is set at no more than 30%. A batch number for the raw material powder is established, and the batch number is archived in correspondence with the strain number used in pharmaceutical preparation.

4. The Aspergillus niger remediation agent for deltamethrin-contaminated soil and its preparation method according to claim 1, characterized in that, The S300 specifically includes: S300.1, Configure the inorganic porous framework carrier selection and particle size range limitation, perform dust removal and cleaning and heat treatment to stabilize the inorganic porous framework carrier, configure iron salt mixed solution and pore wetting adsorption, configure co-precipitation reaction system, and generate Fe3O4 magnetic microparticle layer in situ. An inorganic porous framework carrier is obtained, which is set as either diatomaceous earth or palygorskite. The diatomaceous earth or palygorskite carrier is subjected to sieving and classification to obtain inorganic porous framework carrier particles. The particle size range of the inorganic porous framework carrier particles is set to 0.20 mm to 1.00 mm. Powder particles with a particle size smaller than 0.20 mm are rejected, and particles with a particle size greater than 1.00 mm are crushed and then re-sieved. The inorganic porous framework carrier particles undergo dust removal and cleaning treatment. The dust removal and cleaning treatment uses sterile deionized water with stirring and cleaning. The stirring speed is set to 200 rpm to 400 rpm, the single cleaning time is set to 5 min, and the number of cleaning times is set to 3. After the dust removal and cleaning treatment is completed, the particles undergo filtration and dehydration treatment using a 0.45 μm pore size filter membrane. Inorganic porous framework carrier particles were subjected to heat treatment to form pores and stabilize them. The heat treatment temperature was set to 200℃ to 300℃, the heat treatment time was set to 2h, and the heat treatment heating rate was set to 5℃ / min to 10℃ / min. After the heat treatment, the particles were naturally cooled to 25℃ to 30℃ to obtain heat-treated inorganic porous framework carrier particles. A mixed iron salt solution was prepared, consisting of ferric chloride hexahydrate and ferrous sulfate heptahydrate, with a molar ratio of ferric chloride hexahydrate to ferrous sulfate heptahydrate of 2:

1. The concentration of ferric chloride hexahydrate in the mixed iron salt solution was set to 0.40 mol / L, and the concentration of ferrous sulfate heptahydrate was set to 0.20 mol / L. The solvent for the mixed iron salt solution was set to deionized water, and the dissolution temperature of the mixed iron salt solution was set to 25℃ to 35℃. Heat-treated inorganic porous framework carrier particles are added to an iron salt mixed solution to form an impregnation and adsorption system. The solid-liquid ratio in the impregnation and adsorption system is set to 1:

10. The impregnation and adsorption system is subjected to ultrasonic impregnation treatment with an ultrasonic power of 150W to 250W and an ultrasonic time of 5min to 10min. After the ultrasonic impregnation treatment is completed, a stirring adsorption treatment is performed with a stirring speed of 300rpm to 600rpm and a stirring adsorption time of 20min to 40min. The adsorption system is heated to the coprecipitation reaction temperature, which is set to 70°C to 85°C. A precipitant solution is prepared, which is either an ammonia solution or a sodium hydroxide solution. The mass fraction of the ammonia solution is set to 10% to 15%, and the mass fraction of the sodium hydroxide solution is set to 2% to 4%. Fe3O4 magnetic microparticle layers are generated in situ on the surface of inorganic porous framework carrier particles and at the pore inlet position to obtain magnetically responsive inorganic porous carrier particle slurry. S300.2, Cleaning, neutralizing and drying the magnetic response inorganic porous carrier particles, and confirming the loading amount of Fe3O4 magnetic microparticle layer and determining the threshold of magnetic response performance; The magnetically responsive inorganic porous carrier particle slurry underwent solid-liquid separation, with the separation method set to magnetic separation or filtration separation. The magnetically responsive inorganic porous carrier particles underwent deionized water washing and neutralization treatment, with the number of washing cycles set to 4 to 6. The washing termination criterion was set to the pH of the washing supernatant reaching 7.0±0.

2. After washing, ethanol replacement dehydration treatment was performed, with the ethanol volume fraction set to 70% and the ethanol replacement time set to 10 min. The magnetically responsive inorganic porous carrier particles were subjected to low-temperature drying and shaping. The drying temperature was set to 45℃ to 55℃ and the drying time was set to 6h to 10h to obtain dried magnetically responsive inorganic porous carrier particles. The loading of Fe3O4 magnetic microparticle layer was confirmed for the dry magnetic response inorganic porous carrier particles. The loading of Fe3O4 magnetic microparticle layer was determined by the mass difference method. The mass difference method is defined as the mass of dry magnetic response inorganic porous carrier particles minus the mass of heat-treated inorganic porous framework carrier particles. The loading threshold of Fe3O4 magnetic microparticle layer is set to 5% to 15%. When the loading of Fe3O4 magnetic microparticle layer is less than 5%, a supplementary deposition cycle of coprecipitation reaction system construction and in-situ generation of Fe3O4 magnetic microparticle layer is performed once. When the loading of Fe3O4 magnetic microparticle layer is greater than 15%, a deionized water strong stirring and peeling treatment is performed for 5 min and then retested.

5. The Aspergillus niger remediation agent for deltamethrin-contaminated soil and its preparation method according to claim 1, characterized in that, The S400 specifically includes: S400.1 Prepare a sterile chitosan solution and limit the mass fraction and pH of the chitosan solution; prepare a sterile dialdehyde starch crosslinking agent solution; prepare a composite core loading mixture and limit the component ratio. Chitosan powder was obtained, with a degree of deacetylation of not less than 80%. Chitosan powder was added to an acidifying solvent to form a chitosan solution. The acidifying solvent was set to an aqueous solution of glacial acetic acid with a volume fraction of 1.0%. The mass fraction of chitosan in the chitosan solution was set to 1.0% to 2.0%. The chitosan solution was stirred and dissolved at 25°C to 30°C for 2 to 4 hours. The pH of the chitosan solution was adjusted to 5.0 ± 0.

2. The chitosan solution was then filtered through a 0.22 μm filter to obtain a sterile chitosan solution. Dialdehyde starch crosslinking agent powder was obtained. The dialdehyde starch crosslinking agent powder was added to sterile deionized water to form a dialdehyde starch crosslinking agent solution. The mass fraction of dialdehyde starch crosslinking agent in the dialdehyde starch crosslinking agent solution was set to 0.3% to 0.8%. The aldehyde content of the dialdehyde starch crosslinking agent solution was confirmed by hydroxylamine titration. The aldehyde content threshold was set to be not less than 2.5 mmol / g. The dialdehyde starch crosslinking agent solution that met the aldehyde content threshold was sterilized by 0.22 μm filtration to obtain a sterile dialdehyde starch crosslinking agent solution. A magnetically responsive structural carrier was obtained. The magnetically responsive structural carrier was then contacted with sterile phosphate buffer to form a wetting magnetically responsive structural carrier. The pH of the sterile phosphate buffer was set to 7.0±0.1, and the wetting time was set to 10 min to 20 min. The raw material powder for the formulation was obtained and the wetting magnetically responsive structural carrier was added. The mass ratio of the wetting magnetically responsive structural carrier to the raw material powder for the formulation was set to 10:1 to 20:

1. The mixing method was set to low-shear stirring, and the stirring time was set to 3 min to 8 min to obtain the spore-loaded magnetically responsive structural carrier. Obtain induced enzyme-producing solidified material, defined as solidified particles containing esterase-inducing substrate, with particle size range set from 0.5 mm to 2.0 mm, and the amount of induced enzyme-producing solidified material added set from 10% to 20% based on the mass of the spore-loaded magnetic response structure carrier, to obtain an enzyme-inducing composite carrier. Buffer oxygen-supplying microparticles were obtained. Buffer oxygen-supplying microparticles were defined as a mixture of calcium carbonate microparticles and magnesium peroxide microparticles. The mass ratio of calcium carbonate microparticles to magnesium peroxide microparticles was set to 4:

1. The amount of buffer oxygen-supplying microparticles added was set to 3% to 8% based on the mass of the enzyme-inducing composite carrier, and a composite core-loaded mixture was obtained. S400.

2. The composite core loading mixture is introduced into a sterile chitosan solution to form chitosan dialdehyde starch cross-linked microcapsules, which are then cleaned and solidified. The composite core loading mixture was added to a sterile chitosan solution to form a coating dispersion system. The solid-liquid ratio of the coating dispersion system was set to 1:8 to 1:15, the temperature of the coating dispersion system was set to 25℃ to 30℃, the stirring speed of the coating dispersion system was set to 200rpm to 400rpm, and the pre-coating time was set to 5min to 12min to obtain chitosan pre-coated composite particles. A sterile dialdehyde starch crosslinking agent solution is added to chitosan pre-coated composite particles to form a crosslinking reaction system. The amount of sterile dialdehyde starch crosslinking agent solution added is set to 5% to 15% based on the volume of sterile chitosan solution. The reaction time of the crosslinking reaction system is set to 20 min to 40 min. The pH of the crosslinking reaction system is maintained at 5.0 ± 0.2 to form chitosan dialdehyde starch crosslinked microcapsules. Chitosan-dialdehyde starch cross-linked microcapsules were separated from the liquid phase using magnetic separation. The microcapsules were then washed with sterile phosphate buffer, and the washing termination criterion was set at a pH of 7.0±0.2 for the supernatant. The microcapsules were then cured at 25℃ to 30℃ for 30 to 60 minutes. The spore encapsulation efficiency of the microcapsules was then confirmed.

6. The Aspergillus niger remediation agent for deltamethrin-contaminated soil and its preparation method according to claim 1, characterized in that, The S500 specifically includes: S500.1 Set the extrusion rounding granulation molding, limit the particle size range of composite particles, configure the single particle compressive strength quality control, and set the single particle compressive strength threshold. A microencapsulated repair agent intermediate is obtained, and an extrusion spheroidization granulation device is acquired. The extrusion spheroidization granulation device includes a screw extrusion unit and a spheroidization shaping unit. The microencapsulated repair agent intermediate enters a mixing granulation system, which includes the microencapsulated repair agent intermediate, microcrystalline cellulose powder, and starch powder. The mass ratio of the microencapsulated repair agent intermediate to microcrystalline cellulose powder to starch powder is set at 5:3:

2. Sterile deionized water is added to the mixing granulation system for moisture content adjustment, and the moisture content of the mixing granulation system is set to 25% to 35%. The mixing granulation system then enters the screw extrusion unit to form extruded strips. The die diameter of the rod extrusion unit is set to 1.0 mm to 1.5 mm. The extruded strip enters the rounding and shaping unit to form composite particles. The rotation speed of the rounding and shaping unit is set to 600 rpm to 1200 rpm, and the rounding and shaping time is set to 3 min to 8 min. The composite particles are screened and graded to obtain target composite particles. The particle size range of the target composite particles is set to 1 mm to 3 mm. The target composite particles enter low-temperature drying and curing. The low-temperature drying and curing temperature is set to 35℃ to 45℃, and the low-temperature drying and curing time is set to 4 h to 8 h. The moisture content verification threshold of the target composite particles is set to not exceed 10%. A single-particle compressive strength tester was obtained. The loading mode of the single-particle compressive strength tester was set to constant-speed compression loading, and the loading speed was set to 1 mm / min. Thirty target composite particles were extracted as strength sampling samples. Each sample was subjected to a crush test and the crushing force was recorded. The crushing force was defined as the peak load when the target composite particle underwent structural rupture. The single-particle compressive strength was defined as the crushing force value. The threshold for single-particle compressive strength was set to be no less than 10 N. When the single-particle compressive strength was less than 10 N, the moisture content of the mixed granulation system was increased by 5 percentage points and the extrusion and rounding granulation process was repeated once. S500.2, Configure spore survival rate quality control and configure deltamethrin initial hydrolysis characterization activity quality control; A spore counting detection system was obtained using the serial dilution plating method. The incubation temperature was set at 30℃±1℃, and the incubation time was set at 48h. The intermediate of the microencapsulated repair agent was obtained, and the spore count of the intermediate was determined. The spore count of the intermediate was defined as the number of colony-forming units per unit mass of the intermediate, denoted as N. mid The target composite particles were obtained and their spore count was determined. The spore count of the target composite particles was defined as the number of colony-forming units per unit mass of the target composite particles, denoted as N. pel ; A hydrolysis characterization reaction system for deltamethrin was obtained. The hydrolysis characterization reaction system for deltamethrin included sterile phosphate buffer and deltamethrin standard solution. The pH of the sterile phosphate buffer was set to 7.0±0.1, the initial mass concentration of the deltamethrin standard solution was set to 20 mg / L, the temperature of the hydrolysis characterization reaction system was set to 30℃±1℃, and the shaking speed was set to 180 rpm. The target composite particles were added to the deltamethrin hydrolysis characterization reaction system, and the mass of the target composite particles added was denoted as m. pel (g), m pel The target concentration was set to 1.00g. The deltamethrin concentration detection module used high-performance liquid chromatography (HPLC) to obtain the deltamethrin mass concentration. The deltamethrin mass concentration at 0 min after adding the target composite particles was recorded as C0, and the deltamethrin mass concentration at 10 min after adding the target composite particles was recorded as C10. 10 ; The initial hydrolysis activity threshold for deltamethrin was set as A0 ≥ 0.10 mg·L⁻¹. -1 ·min -1 ·g -1 When the initial hydrolysis characterization activity of deltamethrin is lower than the threshold, the mass ratio of the microencapsulated repair agent intermediate in the mixed granulation system is increased by 5 percentage points and the extrusion and spheroidization granulation process is repeated once. The target composite particles that meet the single-particle compressive strength threshold, spore survival rate threshold and initial hydrolysis characterization activity threshold of deltamethrin are defined as granulated Aspergillus niger repair agent finished products.

7. The Aspergillus niger remediation agent for deltamethrin-contaminated soil and its preparation method according to claim 1, characterized in that, The S600 specifically includes: S600.

1. Conduct on-site sampling and testing of deltamethrin and 3-phenoxybenzoic acid concentrations, and set the dosage of granulated Aspergillus niger repair agent. Soil contaminated with deltamethrin to be remediated was obtained. A deltamethrin concentration detection module and a 3-phenoxybenzoic acid concentration detection module were also acquired. The deltamethrin concentration detection module used high-performance liquid chromatography (HPLC) to determine the deltamethrin concentration in the soil. The soil deltamethrin concentration was defined as C0. DM The 3-phenoxybenzoic acid concentration detection module uses high-performance liquid chromatography (HPLC) to obtain the soil 3-phenoxybenzoic acid concentration in the soil contaminated with deltamethrin to be remediated. The soil 3-phenoxybenzoic acid concentration is defined as C3. 3PBA ; To obtain the granulated Aspergillus niger remediation agent, the dosage of the granulated Aspergillus niger remediation agent is defined as D based on the unit soil mass, and the dosage segmentation rule is set as: C. DM When ≤1mg / kg, D=2g / kg, 1 <C DM When ≤10mg / kg, D=5g / kg, C DM When the concentration is >10mg / kg, D = 8g / kg. The granulated Aspergillus niger remediation agent is mixed with the soil contaminated with deltamethrin to be remediated. The mixing time is set to 10min to 20min. S600.2, Set up collaborative control of remediation environment parameters, and perform phased monitoring of removal rate and 3-phenoxybenzoic acid accumulation trend; Obtain the field water holding capacity (FBC) measurement value; the soil moisture content corresponding to the FBC is defined as θ. FC The soil moisture content contaminated with deltamethrin to be remediated is defined as θ, and the relative field capacity is defined as W. FC (%)=θ / θ FC ×100%, the relative field water holding capacity control threshold is set at 55% to 70%, the pH control threshold for the soil contaminated with deltamethrin to be remediated is set at 6.0 to 7.5, the remediation environment temperature control threshold is set at 20℃ to 35℃, the turning and maintenance cycle is set at 7 days, and the turning and maintenance is used to maintain the aerobic state. Monitoring time points were set at days 0, 7, 14, and 28. Soil deltamethrin concentration C was recorded on day 0. DM,0 Record the soil deltamethrin concentration C on day td. DM,t The removal rate of deltamethrin is defined as η. t ; The soil 3-phenoxybenzoic acid concentration C was recorded on days 0, 7, and 14. 3PBA,0 C 3PBA,7 and C 3PBA,14 The criterion for the increasing trend of 3-phenoxybenzoic acid accumulation in soil was set as C. 3PBA,14 >C 3PBA,7 And C 3PBA,7 >C 3PBA,0 The additional trigger condition is determined on day 14, and the additional trigger condition is set to η. 14 <70% or meeting the criterion of an increasing trend in the accumulation of 3-phenoxybenzoic acid in the soil, the supplementary dosage is defined as D add ; The granulated Aspergillus niger remediation agent corresponding to the dosage was added into the soil contaminated with deltamethrin to be remediated and then mixed thoroughly. After day 28, an external magnetic field recovery component was obtained. The external magnetic field recovery component was set to a permanent magnet with a surface magnetic induction intensity of 0.3T, the distance between the permanent magnet and the outer wall of the soil container was set to 2cm, and the magnetic response aggregation time was set to no more than 120s. The granulated Aspergillus niger remediation agent product after magnetic response aggregation was separated to obtain recovered particles. A reactivation solution was prepared, comprising sterile phosphate buffer and glucose aqueous solution. The pH of the sterile phosphate buffer was set to 7.0 ± 0.1, and the glucose mass fraction was set to 0.5%. The recovered particles were then placed in the reactivation solution for reactivation culture. The reactivation culture temperature was set to 30℃ ± 1℃, the shaking speed was set to 180 rpm, and the reactivation culture time was set to 12 h. After reactivation, the recovered particles underwent spore survival rate verification and deltamethrin initial hydrolysis activity verification. The spore survival rate threshold was set to be no less than 80%, and the deltamethrin initial hydrolysis activity threshold was set to be no less than 0.10 mg·L⁻¹·min⁻¹. -1 ·g -1 Particles that are reactivated and meet the spore survival rate threshold and the initial hydrolysis characterization activity threshold of deltamethrin are defined as reusable particles. Reusable particles are returned and the next round of addition is performed. The threshold for the number of reuse cycles is set to be no less than 3 rounds.

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