A heavy metal contaminated soil remediation preparation based on directional regulation of microbial flora and a remediation method

Abstract

This invention relates to the field of microbial-nanomaterial cross-integration technology, specifically to a remediation agent and method for heavy metal contaminated soil based on the targeted regulation of microbial communities. The agent comprises a gene-edited composite microbial community, a nanocatalytic composite system, intelligent regulatory additives, and magnetically responsive materials. The method includes soil electric field pretreatment, agent compounding, magnetoelectric synergistic remediation, dynamic regulation, and maturation monitoring. It synergistically reduces the bioavailable form of heavy metals through photocatalysis, magnetic response, and gene editing technologies, thereby improving remediation efficiency. This invention enhances the tolerance and remediation efficiency of the microbial community through gene editing, nanocatalysis, and magnetoelectric synergistic technologies, shortens the remediation cycle, achieves a significant reduction in the bioavailable form of heavy metals, and simultaneously improves soil ecology. It is suitable for complex contaminated soils, environmentally friendly, and causes no secondary pollution.

Description

Technical Field

[0001] This invention relates to the field of microbial-nanomaterial cross-integration technology, specifically to a remediation agent and method for heavy metal contaminated soil based on the directional regulation of microbial communities. Background Technology

[0002] The remediation of heavy metal-contaminated soil is a significant challenge in environmental governance. Human activities such as industrial wastewater discharge and mining lead to the deposition of large amounts of heavy metals, such as cadmium and lead, in the soil, affecting not only crop growth but also threatening human health through the food chain. While commonly used physicochemical remediation methods (such as soil washing and solidification stabilization) are highly efficient, they suffer from high costs and the potential for secondary pollution. Traditional bioremediation methods (such as phytoremediation and microbial remediation), while environmentally friendly, face bottlenecks such as insufficient tolerance of microorganisms to heavy metals, long remediation cycles, and the easy inactivation of functional microbial communities, making them particularly difficult to address the high-efficiency remediation needs of sites with complex heavy metal contamination.

[0003] Microbial community remediation technology has been widely studied due to its low cost and eco-friendliness, but its application is limited by three major technical obstacles: First, wild-type microorganisms have poor tolerance to high concentrations of heavy metals and low survival rates in contaminated soils; second, traditional remediation agents lack mechanisms for targeted enrichment and transformation of heavy metals, and remediation efficiency relies on the natural metabolism of the microbial community, making it difficult to rapidly reduce the bioavailability of heavy metals; third, the remediation process lacks environmental response regulation mechanisms, making it impossible to dynamically optimize microbial activity based on the soil microenvironment (such as pH and redox potential), resulting in significant differences in remediation effects in areas with different levels of pollution. Although some studies have attempted to modify microorganisms through genetic engineering, without combining them with nanomaterials and intelligent response systems, it is difficult to achieve a systematic improvement in remediation efficiency.

[0004] The application of nanomaterials in environmental remediation offers new insights into overcoming the aforementioned bottlenecks. For example, metal-organic frameworks (MOFs) exhibit high adsorption capacity and photocatalytic performance, while nano-zero-valent iron possesses reducing properties. However, without synergistic design with functional microbial communities, issues such as nanomaterial aggregation and weak microbial-material interface interactions can easily arise. Furthermore, photocatalytic remediation technologies are severely affected by soil organic matter, and the directional migration mechanism of magnetically responsive materials in soil remains unclear, preventing current technologies from meeting the demands for efficient and precise remediation of complex contaminated soils.

[0005] (a) Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a remediation agent and method for heavy metal contaminated soil based on the targeted regulation of microbial communities.

[0006] Technical solution A soil remediation agent for heavy metal contaminated soil, comprising the following components: Gene-edited microbial community: CRISPR-Cas9-edited Bacillus, cadmium-resistant Pseudomonas, and sulfur-reducing Desulfovibrio were mixed in a 3:2:1 mass ratio, with a total viable count ≥2×10⁻⁶. 10 CFU / g; Nanocatalytic composite system: 1-2% graphene quantum dot GQDs modified biochar supported MOF-808, 0.6-1.5% sulfurized nano-zero ferrous dithiocarbamate core-shell structure nZVI-S-DTC, 0.8-1.2% polyglutamic acid-metal-organic framework coupling γ-PGA-MOF; Intelligent control additives: 0.5-1% photoresponsive azobenzene derivative AZO, 0.3-0.7% pH-sensitive sodium alginate-chitosan microspheres SA-CS.

[0007] Preferably, it also includes magnetically responsive nano-hydroxyapatite (MNHAP), which has an Fe3O4 content of 10-15% and a particle size of 50-100 nm, and can migrate directionally to the heavy metal enrichment region under an applied magnetic field.

[0008] Preferably, it also includes a self-assembled microbial extracellular polymeric polymer (EPS)-nanoferrite composite gel, wherein the EPS content is 30-40% and the Fe3O4 nanoparticles have a particle size of 20-30 nm, which can trigger the adsorption of heavy metals through redox potential (ORP).

[0009] Preferably, the metallothionein expression level of the gene-edited Bacillus is 3-5 times that of the wild type, and the cadmium removal rate of the cadmium-resistant Pseudomonas can reach 92-98%.

[0010] Preferably, the preparation method of the GQDs-MOF-808 is as follows: graphene quantum dots and MOF-808 are mixed at a mass ratio of 1:10 and ultrasonically dispersed in N,N-dimethylformamide (DMF) for 24 hours, with a GQDs loading rate of 15-20% and an optical absorption threshold extended to 650 nm.

[0011] Preferably, the preparation method of the nZVI-S-DTC core-shell structure is as follows: nZVI is treated with sodium sulfide solution for 2 hours, and then reacted with DTC-Na at pH 8-9 for 3 hours, with a shell thickness of 5-10 nm.

[0012] Preferably, the method for remediating heavy metal contaminated soil using the formulation includes the following steps: S1. Soil electric field pretreatment: Apply a DC electric field of 0.5-1V / cm for 72 hours to promote the migration and activation of heavy metal ions to the anode; S2. Microbial community-nano system compound: Gene-edited microbial community and nanocatalytic composite system are mixed at a mass ratio of 1:5, intelligent regulatory additives are added, and SA-CS microspheres are used to form microcapsules with a diameter of 50-100μm. S3. Magnetoelectric Synergistic Remediation: After the microcapsules are applied to the soil, an alternating magnetic field of 0.1-0.3T is applied, and visible light is irradiated at the same time; S4. Dynamic regulation: Soil pH, ORP and heavy metal concentration are monitored in real time through IoT sensors. When pH < 6, SA-CS microspheres release CaCO3; when ORP > -50mV, AZO derivatives trigger the photocatalytic reaction of GQDs-MOF-808. S5. Bio-enhancement: Starting from day 5 of the repair process, an inducer containing 1 mmol / L IPTG is injected to induce the gene-edited bacterial community to express metallothionein for 15 days.

[0013] Preferably, the frequency of the alternating magnetic field in S3 is 5-10Hz, which causes MNHAP to aggregate in a directional manner to form a "nano-repair grid". At the same time, the ·OH free radicals generated by GQDs-MOF-808 under light have a degradation rate of 60-80% on soil organic matter.

[0014] Preferably, in step S4, the IoT sensor monitors once per hour, and the data is transmitted to the cloud AI system to automatically adjust the injection of regulator into the drip irrigation system.

[0015] Preferably, after the remediation cycle, the available content of Cd, Pb and Cu in the soil decreased by 75-90%, and third-generation sequencing confirmed that the relative abundance of functional genes in the microbial community increased by 4-6 times, and the soil enzyme activity increased by 1.8-2.5 times.

[0016] (III) Beneficial Effects Compared with existing technologies, the beneficial effects of this invention are: 1. The heavy metal contaminated soil remediation technology based on targeted regulation of microbial communities provided by this invention achieves a dual improvement in remediation efficiency and stability through multi-dimensional innovation. Compared with wild-type strains, the gene-edited composite microbial community exhibits enhanced tolerance to cadmium and lead, and can continuously express heavy metal-binding proteins in high-concentration contaminated soil, directly reducing the content of available heavy metals. The nanocatalytic composite system utilizes photocatalysis to reduce heavy metal ions to inert elements, while the generated •OH free radicals can degrade soil organic matter, removing its complexation protection against heavy metals and synergistically improving remediation efficiency.

[0017] 2. Magnetic responsive material MNHAP migrates directionally to areas of heavy metal accumulation under an applied magnetic field, forming a "nano-remediation grid." This increases the concentration of remediation agents in hotspot areas, enabling precise remediation of contaminated sites. Intelligent regulatory additives dynamically trigger photocatalytic reactions and alkali release based on soil ORP and pH, maintaining an optimal growth environment for microbial communities and shortening the remediation cycle compared to traditional methods. This technology can reduce the available cadmium and lead content in the soil, increase microbial community diversity, simultaneously improve soil enzyme activity, and rebuild soil ecological functions.

[0018] 3. The formulation utilizes microencapsulation technology to encapsulate gene-edited bacteria and nanomaterials within sodium alginate-chitosan microspheres. After six months of storage, the viable bacterial count remains extremely high, overcoming the problem of easy inactivation in traditional bacterial agents. The photocatalytic-magnetic-electric synergistic remediation mode is adaptable to soils with varying degrees of pollution, showing significant advantages, especially for sites with complex heavy metal contamination. The risk of secondary soil pollution after remediation approaches zero, providing an efficient and green solution for the safe utilization of heavy metal-contaminated farmland and industrial sites. Attached Figure Description

[0019] Figure 1 This is a flowchart of a remediation agent for heavy metal contaminated soil based on the targeted regulation of microbial communities; Figure 2 This is a bar chart comparing the Cd removal rate and Pb removal rate of the examples and comparative examples; Figure 3 This is a line graph comparing soil pH and the percentage of available Cd in the examples and comparative examples; Figure 4 This is a bar and line graph comparing the effective Pb percentage and duration of action in the examples and comparative examples. Detailed Implementation

[0020] according to Figures 1 to 4 The specific embodiments of the present invention are as follows: Example 1: Basic Heavy Metal Contaminated Soil Remediation Agents and Methods Repair formulation components (by weight percentage): Gene-edited microbial community: 35% CRISPR-Cas9-edited Bacillus (mtf-1 gene expression level 2.3 pg / cell), and cadmium-resistant Pseudomonas (czcABC gene copy number 1.2 × 10⁻⁶). 6 25% (copies / μL), 20% sulfur-reducing bacteria, total viable count 2.1×10⁻⁶ 10 CFU / g; Nanocatalytic composite system: GQDs-modified biochar-supported MOF-808 (GQDs loading rate 18%) 1.5%, nZVI-S-DTC (shell thickness 8nm) 1.2%, γ-PGA-MOF (coupling rate 86%) 1.0%; Intelligent regulatory additives: 0.8% AZO derivative and 0.5% SA-CS microspheres (80μm particle size).

[0021] Preparation steps S1: Culture of gene-edited microbiota Bacillus gene editing and culture: Strain activation: Wild-type Bacillus ATCC 6633 lyophilized powder was inoculated into 50 mL LB liquid medium (containing 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0), and cultured at 37℃ with shaking at 200 rpm for 12 hours until the logarithmic growth phase (OD200). 600 =0.6).

[0022] Electroporation preparation: Take 5 mL of bacterial culture, centrifuge at 8000 r / min for 10 minutes at 4℃, discard the supernatant, wash the bacterial cells 3 times with pre-cooled 10% glycerol solution, and finally resuspend in 100 μL of 10% glycerol to prepare competent cells.

[0023] Plasmid preparation: The pHT01 plasmid carrying the mtf-1 gene expression cassette (promoter P43, selection marker erm gene) was extracted and the concentration was determined to be 500 ng / μL using NanoDrop.

[0024] Electroporation: Add 10 μL of plasmid (500 ng) to competent cells and transfer to a 2 mm electroporation cuvette. Set the electroporation parameters to 2.5 kV, 25 μF, and 200 Ω. Immediately after electroporation, add 1 mL of LB medium and incubate at 37°C for 1 hour.

[0025] Screening and validation: The resuscitation solution was spread on LB agar plates containing erythromycin (5 μg / mL) and incubated at 37°C for 16 hours. Single colonies were picked and amplified by PCR using primers F (5'-ATGAAGCTTTATCAGCTG-3') and R (5'-TTATTTGTTTATGCCTTCTT-3'). The products were validated by 1% agarose gel electrophoresis. Positive clones were inoculated on LB medium containing 1 mmol / L IPTG and incubated at 37°C with shaking at 200 rpm for 12 hours.

[0026] Cell collection: The culture was centrifuged at 8000 r / min for 10 minutes at 4℃, washed twice with pre-cooled PBS (pH 7.4), and the metallothionein content was determined by BCA method to be 1.8 mg / g of cells. The cells were then lyophilized for later use.

[0027] Screening and culture of cadmium-resistant Pseudomonas aeruginosa: Sample collection and enrichment: 50g of cadmium-contaminated soil from a smelter was collected and added to 100mL of M9 liquid culture medium containing 50mg / L CdCl2 (formula: Na2HPO4・7H2O 12.8g, KH2PO4 3g, NaCl 0.5g, NH4Cl 1g, glucose 2g, MgSO4 0.24g, CaCl2 0.011g, and distilled water to a final volume of 1L). The medium was then incubated at 30℃ and 180r / min for 48 hours with shaking.

[0028] Separation and purification: The enrichment solution was serially diluted to 10. -6 Spread the culture onto M9 solid plates containing 50 mg / L CdCl2 and incubate at 30°C for 24 hours. Select well-grown single colonies and repeat streak purification three times.

[0029] Genetic detection: Genomic DNA was extracted from single colonies, and the czcABC gene was amplified using primers F (5'-GCGTATGATGGTGTTGGTG-3') and R (5'-CGTCATGTCCTTGTAGCGG-3'). After sequencing verification, high-expression clones were screened by flow cytometry.

[0030] Optimized culture: The high-expression clones were inoculated into M9 medium containing 50 mg / L CdCl2 and cultured at 30℃ and 180 r / min for 16 hours. The cells were collected by centrifugation and the cadmium removal rate was measured to be 95% (initial concentration 50 mg / L). The cells were then lyophilized for later use.

[0031] Sulfur-reducing bacteria culture: Culture medium preparation: Prepare Postgate C medium (formula: KH2PO4 0.5g, NH4Cl 1g, CaSO4·2H2O 1.5g, MgSO4·7H2O 2g, yeast extract 1g, sodium lactate 3.5g, FeSO4·7H2O 0.5g, ascorbic acid 0.1g, distilled water to a final volume of 1L, pH 7.2), sterilize at 121℃ for 20 minutes, cool, and then purge with a N2:CO2:H2 (80:10:10, v / v) mixed gas for 30 minutes to remove oxygen.

[0032] Inoculation and culture: Inoculate Desulfovibrio vulgaris DSM 644 lyophilized powder into anaerobic medium, incubate at 35°C for 24 hours, collect the cells by centrifugation at 4°C and 6000 r / min for 15 minutes, wash twice with anaerobic PBS, and lyophilize for later use.

[0033] Microbial community mixing: Bacillus, Pseudomonas, and sulfur-reducing bacteria were mixed in a mass ratio of 3:2:1, and 10% glycerol was added as a preservative. The mixture was then freeze-dried at -80°C for 48 hours to obtain a total viable count of 2.1 × 10⁻⁶. 10 Compound bacterial powder with CFU / g.

[0034] S2: Preparation of GQDs-MOF-808 Synthesis of graphene quantum dots (GQDs): Preparation of graphite oxide: Place 5g of flake graphite powder in a 250mL beaker, add 100mL of concentrated sulfuric acid (98%) and 2.5g of sodium nitrate, stir in an ice bath for 30 minutes, slowly add 15g of potassium permanganate (in 3 portions, 10 minutes apart each time), control the temperature to <20℃, and stir at room temperature for 2 hours.

[0035] Graphite oxide exfoliation: Heat to 35℃ and stir for 1 hour. Slowly add 200mL of deionized water (exothermic reaction, drop rate needs to be controlled). Heat to 98℃ and stir for 15 minutes. Add 30% hydrogen peroxide dropwise until the solution turns bright yellow (about 10mL). Filter while hot using a sintered glass funnel.

[0036] GQDs preparation: The filter cake was washed three times with 5% hydrochloric acid solution, then washed with deionized water until neutral, dispersed in 200 mL of deionized water, sonicated at 400 W for 4 hours, centrifuged at 12000 r / min for 30 minutes, and the supernatant was dialyzed in a 3500 Da dialysis bag for 48 hours to obtain a GQDs solution with a particle size of 5 nm (concentration of about 1 mg / mL).

[0037] MOF-808 Synthesis: Ligand preparation: Weigh 1.5g of pyromellitic acid (H3BTC) and dissolve it in 100mL of N,N-dimethylformamide (DMF), and sonicate for 30 minutes.

[0038] Preparation of metal salt solution: Weigh 2.0g ZrOCl2・8H2O and dissolve it in 100mL DMF. Add 10mL concentrated hydrochloric acid (catalytic reaction) and stir until completely dissolved.

[0039] Crystallization reaction: H3BTC solution was slowly added to ZrOCl2 solution, stirred for 30 minutes, and then transferred to a 250mL polytetrafluoroethylene-lined reactor. The reaction was carried out in an oven at 130℃ for 24 hours. After cooling to room temperature, the solid was collected by centrifugation, washed 3 times with DMF and 3 times with anhydrous ethanol, and dried under vacuum at 60℃ for 12 hours to obtain white MOF-808 powder.

[0040] GQDs load: Mixing and dispersing: Mix GQDs solution with MOF-808 at a mass ratio of 1:10, add an appropriate amount of DMF (total volume 100mL), and sonicate at 400W for 24 hours (frequency 40kHz).

[0041] Concentration and purification: DMF was removed by rotary evaporation at 80℃. The residue was washed three times with anhydrous ethanol and dried under vacuum at 60℃ for 12 hours. XPS analysis showed that the GQD loading rate was 18%, and UV-Vis DRS showed that the light absorption threshold was extended to 650nm.

[0042] S3: nZVI-S-DTC Synthesis Preparation of nano-zero valent iron (nZVI): Precursor solution preparation: Weigh 20g FeSO4・7H2O and dissolve it in 200mL deionized water, then pass high-purity nitrogen gas through for 30 minutes to remove dissolved oxygen.

[0043] Reduction reaction: Quickly add 100 mL of 0.5 mol / L NaBH4 solution (freshly prepared, 20% excess), stir vigorously for 30 minutes. Reaction equation: Fe²⁺ + 2BH₄⁻ + 6H₂O → Fe₂O + 2B(OH)₃ + 7H₂O Purification: Black nZVI was collected by magnetic separation, washed three times with oxygen-free deionized water, washed three times with anhydrous ethanol, and dried under vacuum at 60°C for 12 hours to obtain nZVI powder with a particle size of 60 nm.

[0044] Vulcanization treatment: Preparation of sulfidation solution: Prepare 100 mL of 0.2 mol / L Na2S solution and remove oxygen with nitrogen for 10 minutes.

[0045] Sulfidation reaction: nZVI was added to Na2S solution at a solid-liquid ratio of 1:100, stirred in a constant temperature water bath at 30℃ for 2 hours, the product was collected by magnetic separation, and washed 3 times with oxygen-free deionized water to obtain nZVI-S.

[0046] DTC coating: DTC solution preparation: Weigh 0.94g sodium dimethyl dithiocarbamate (DTC-Na) and dissolve it in 100mL of deionized water. Adjust the pH to 8.5 with NaOH and remove oxygen with nitrogen for 10 minutes.

[0047] Coating reaction: nZVI-S was added to DTC-Na solution at a solid-liquid ratio of 1:50, stirred at 30°C for 3 hours, washed three times with ethanol, and vacuum dried at 60°C for 12 hours. TEM observation showed that the shell thickness was 8 nm.

[0048] S4: Soil Remediation Implementation Electric field pretreatment: Soil sampling and pretreatment: Soil samples were collected from farmland surrounding a smelter (Cd 20 mg / kg, Pb 500 mg / kg), air-dried, passed through a 2 mm sieve, and the pH was measured to be 5.8. The moisture content was then adjusted to 25%.

[0049] Electric field device setup: Take 5 kg of soil and put it into an organic glass column with an inner diameter of 10 cm and a height of 60 cm. Insert a graphite anode plate of 10 cm × 10 cm × 0.5 cm and an iron cathode plate at both ends respectively. Connect a DC power supply and apply an electric field strength of 0.8 V / cm. Continue to energize for 72 hours. During this period, deionized water is added to the anode chamber through a peristaltic pump (flow rate of 5 mL / h) to maintain the soil moisture content.

[0050] Effect test: After the power was turned on, soil samples were collected, and the available forms of heavy metals were determined by the 0.1 mol / L HCl extraction method. The proportion of available forms of Cd increased from 35% to 65%, and the proportion of available forms of Pb increased from 12% to 42%.

[0051] Formulation compounding: Raw material mixing: The freeze-dried bacterial powder was mixed with GQDs-MOF-808, nZVI-S-DTC and γ-PGA-MOF at a mass ratio of 1:5, and AZO derivative and SA-CS microspheres (particle size 80μm) were added.

[0052] Microcapsule preparation: Prepare a 3% sodium alginate solution (containing 0.5% Tween 80), add the mixed powder at a mass ratio of 1:10, stir magnetically to form a uniform suspension, add 5% CaCl2 solution (containing 0.1% Span 80) dropwise with a 10mL syringe, crosslink and solidify for 30 minutes to form microcapsules with a particle size of 80μm.

[0053] Purification and preservation: Wash the microcapsules twice with deionized water and store at 4°C for later use.

[0054] Magnetoelectric Synergistic Repair: On-site construction: Apply the microcapsules at a rate of 1% (w / w) to the surface of the pretreated soil, and then till the soil to a depth of 25cm with a shovel to mix them evenly.

[0055] Magnetic field and illumination setup: An electromagnetic coil (1000 turns) is arranged around the soil column and connected to an alternating magnetic field generator to apply a 0.2T, 8Hz alternating magnetic field; an LED light source (wavelength 400-700nm, light intensity 800μmol / m²) is installed on top. 2 •s), 12 hours of sunlight per day.

[0056] Inducer addition: Inject 1 mmol / L IPTG solution (5% of soil volume) every 10 days through the drip irrigation system to induce mtf-1 gene expression.

[0057] Monitoring frequency: Soil samples were collected every 5 days to determine pH, ORP, available heavy metals (ICP-MS), and microbial count (plate count method).

[0058] Example 2: Photocatalytically Enhanced Remediation Agent and Method for Heavy Metal Contaminated Soil Repair formulation components (by weight percentage): Gene-edited microbial community: CRISPR-Cas9-edited Bacillus (mtf-1 gene expression level 2.5 pg / cell) 38%, cadmium-resistant Pseudomonas (czcABC gene copy number 1.3 × 10⁻⁶) 6 The bacterial count was 22% (copies / μL), 18% sulfur-reducing bacteria, and the total viable count was 2.3 × 10⁻⁶. 10 CFU / g; Nanocatalytic composite system: GQDs-modified biochar-supported MOF-808 (GQDs loading rate 20%) 2.0%, nZVI-S-DTC (shell thickness 8nm) 1.5%, γ-PGA-MOF (coupling rate 88%) 1.2%; Intelligent regulatory additives: 1.0% AZO derivative and 0.7% SA-CS microspheres (80μm particle size).

[0059] Preparation steps S1: Culture of gene-edited microbiota Bacillus gene editing and culture: Strain activation: Wild-type Bacillus ATCC 6633 lyophilized powder was inoculated into 50 mL LB liquid medium (containing 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0) and cultured at 37℃ with shaking at 200 rpm for 12 h until the logarithmic growth phase (OD200). 600 =0.6).

[0060] Electroporation preparation: Centrifuge 5 mL of bacterial culture at 4℃ and 8000 r / min for 10 min, wash 3 times with 10% glycerol before pre-cooling, and resuspend in 100 μL of 10% glycerol to prepare competent cells.

[0061] Plasmid preparation: Extract the pHT01 plasmid (promoter P43, selection marker erm gene) carrying the mtf-1 gene expression cassette at a concentration of 500 ng / μL.

[0062] Electroporation: 10 μL of plasmid was added to competent cells, and the parameters of the 2 mm electroporation cuvette were set to 2.5 kV, 25 μF, and 200 Ω. After electroporation, 1 mL of LB medium was added and the cells were incubated at 37°C for 1 h.

[0063] Screening and validation: The resuscitation solution was spread on LB plates containing erythromycin (5 μg / mL) and incubated at 37°C for 16 h. Single clones were picked and amplified by PCR using primers F (5'-ATGAAGCTTTATCAGCTG-3') and R (5'-TTATTTGTTTATGCCTTCTT-3'). Positive clones were verified by agarose gel electrophoresis and inoculated into LB medium containing 1 mmol / L IPTG and incubated at 37°C and 200 r / min for 12 h.

[0064] Cell collection: The culture was centrifuged at 8000 r / min for 10 min at 4℃, washed twice with PBS (pH 7.4), and the metallothionein content was determined by BCA method to be 2.0 mg / g of cells. The cells were then lyophilized for later use.

[0065] Screening and culture of cadmium-resistant Pseudomonas aeruginosa: Sample collection and enrichment: 50g of cadmium-contaminated soil from the smelter was added to 100mL of M9 liquid culture medium containing 50mg / L CdCl2 (formula: Na2HPO4・7H2O 12.8g, KH2PO4 3g, NaCl 0.5g, NH4Cl 1g, glucose 2g, MgSO4 0.24g, CaCl2 0.011g, and distilled water to a final volume of 1L), and cultured at 30℃ and 180r / min for 48h with shaking.

[0066] Separation and purification: The enrichment solution was serially diluted to 10. -6 Spread the culture onto M9 solid plates containing 50 mg / L CdCl2, incubate at 30°C for 24 h, and then streak a single colony three times for purification.

[0067] Genetic detection: Genomic DNA was extracted, and the czcABC gene was amplified using primers F (5'-GCGTATGATGGTGTTGGTG-3') and R (5'-CGTCATGTCCTTGTAGCGG-3'). After sequencing, high-expression clones were screened by flow cytometry.

[0068] Optimized culture: High-expression clones were inoculated into M9 medium containing 50 mg / L CdCl2 and cultured at 30℃ and 180 r / min for 16 h. The cells were collected by centrifugation, and the cadmium removal rate reached 96% (initial concentration 50 mg / L). The cells were then lyophilized for later use.

[0069] Sulfur-reducing bacteria culture: Culture medium preparation: Postgate C medium (formulation: KH2PO4 0.5g, NH4Cl 1g, CaSO4・2H2O 1.5g, MgSO4・7H2O 2g, yeast extract 1g, sodium lactate 3.5g, FeSO4・7H2O 0.5g, ascorbic acid 0.1g, distilled water to a final volume of 1L, pH 7.2), sterilized at 121℃ for 20min, cooled, and then purged with a N2:CO2:H2 (80:10:10, v / v) mixed gas for 30min to remove oxygen.

[0070] Inoculation and culture: Desulfovibrio vulgaris DSM 644 lyophilized powder was inoculated into anaerobic medium, incubated at 35°C for 24 h, centrifuged at 4°C and 6000 r / min for 15 min to collect the cells, washed twice with anaerobic PBS, and lyophilized for later use.

[0071] Microbial conjugation: Bacillus, Pseudomonas, and sulfur-reducing bacteria were mixed at a mass ratio of 3.8:2.2:1.8, with 10% glycerol as a preservative added, and freeze-dried at -80℃ for 48 hours to obtain a total viable count of 2.3 × 10⁻⁶. 10 Compound bacterial powder with CFU / g.

[0072] S2: Preparation of GQDs-MOF-808 (Optimized Process) Synthesis of graphene quantum dots (GQDs): Preparation of graphite oxide: Add 5g of flake graphite powder to 100mL of concentrated sulfuric acid (98%) and 2.5g of sodium nitrate, stir in an ice bath for 30min, add 15g of potassium permanganate in 3 portions (10min interval), control the temperature to <20℃, and stir at room temperature for 2h.

[0073] Graphite oxide exfoliation: Heat to 35℃ and stir for 1 hour, slowly add 200 mL of deionized water, heat to 98℃ and stir for 15 minutes, add 30% hydrogen peroxide dropwise until bright yellow (about 10 mL), and filter while hot.

[0074] GQDs preparation: The filter cake was washed three times with 5% hydrochloric acid, washed with deionized water until neutral, dispersed in 200 mL of deionized water, sonicated at 400 W for 4 h, centrifuged at 12000 r / min for 30 min, and the supernatant was dialyzed in a 3500 Da dialysis bag for 48 h to obtain a GQDs solution with a particle size of 5 nm (concentration of about 1 mg / mL).

[0075] MOF-808 Synthesis: Ligand preparation: 1.5 g of pyromellitic acid (H3BTC) was dissolved in 100 mL of DMF and sonicated for 30 min.

[0076] Preparation of metal salt solution: Dissolve 2.0 g ZrOCl2・8H2O in 100 mL DMF, add 10 mL concentrated hydrochloric acid, and stir until dissolved.

[0077] Crystallization reaction: H3BTC solution was slowly added to ZrOCl2 solution, stirred for 30 min and then transferred to a polytetrafluoroethylene reactor. The reaction was carried out at 130℃ for 24 h. After cooling, the solid was collected by centrifugation, washed 3 times with DMF and 3 times with anhydrous ethanol, and dried under vacuum at 60℃ for 12 h to obtain MOF-808 powder.

[0078] GQDs load optimization: Mixing and dispersion: GQDs solution and MOF-808 were mixed at a mass ratio of 1:8, DMF was added to 100 mL, and the mixture was ultrasonically dispersed at 400 W for 36 h (frequency 40 kHz), followed by hydrothermal treatment at 120 °C for 24 h.

[0079] Concentration and purification: DMF was removed by rotary evaporation at 80℃, washed three times with anhydrous ethanol, and dried under vacuum at 60℃ for 12 h. XPS analysis showed that the GQD loading rate was 20%, and PL spectroscopy showed that the separation efficiency of photogenerated electron-hole pairs was improved by 20%.

[0080] S3: Synthesis of nZVI-S-DTC (same as Example 1) Preparation of nano-zero valent iron (nZVI): 20g FeSO4・7H2O was dissolved in 200mL deionized water, nitrogen gas was purged for 30min, 100mL 0.5mol / L NaBH4 solution was added for reduction, nZVI was collected by magnetic separation, washed and dried to obtain nZVI powder with a particle size of 60nm.

[0081] Sulfurization treatment: nZVI was added to 0.2 mol / L Na2S solution at a solid-liquid ratio of 1:100, stirred at 30℃ for 2 h, and magnetically separated to obtain nZVI-S.

[0082] DTC coating: 0.94g DTC-Na was dissolved in 100mL deionized water, the pH was adjusted to 8.5, nZVI-S was added at a solid-liquid ratio of 1:50, stirred at 30℃ for 3h, washed and dried, and TEM showed a shell thickness of 8nm.

[0083] S4: Soil Remediation Implementation Electric field pretreatment (same as in Example 1): Soil sampling and pretreatment: Soil from farmland surrounding the smelter (Cd 20 mg / kg, Pb 500 mg / kg) was air-dried, sieved, and the moisture content was adjusted to 25%.

[0084] Electric field setup: 5 kg of soil was placed in an plexiglass column, and a graphite anode and an iron cathode were inserted. An electric field strength of 0.8 V / cm was applied for 72 hours. Deionized water was added to the anode chamber to maintain the moisture content.

[0085] Effect test: The proportion of bioavailable Cd increased from 35% to 65%, and the proportion of bioavailable Pb increased from 12% to 42%.

[0086] Formulation compounding: Raw material mixing: freeze-dried bacterial powder is mixed with GQDs-MOF-808, nZVI-S-DTC and γ-PGA-MOF at a mass ratio of 1:5, and AZO derivative and SA-CS microspheres are added.

[0087] Microcapsule preparation: 3% sodium alginate solution (containing 0.5% Tween 80) was mixed with the mixed powder at a mass ratio of 1:10. 5% CaCl2 solution (containing 0.1% Span 80) was added dropwise and cross-linked and cured for 30 min to form microcapsules with a particle size of 80 μm. After washing, the microcapsules were refrigerated at 4℃ for later use.

[0088] Photocatalytic synergistic repair: On-site construction: Microcapsules are applied to the pretreated soil at a rate of 1% (w / w) and then tilled to a depth of 25cm.

[0089] Lighting system upgrade: Top-mounted LED array light source (wavelength 400-700nm, light intensity 1000μmol / m²) 2 •s), with 16 hours of sunlight per day, and a reflector to improve light utilization.

[0090] Intelligent sensing and control: The WSEN-ISB02 type ORP sensor is embedded to transmit data to the PLC controller in real time. When ORP > -50mV, it triggers the isomerization of AZO derivatives (cis → trans) and starts the MOF-808 photocatalytic reaction.

[0091] Inducer addition: Drip irrigation with 1 mmol / L IPTG solution (5% of soil volume) every 10 days to induce mtf-1 gene expression.

[0092] Example 3: Magnetic-responsive synergistic remediation agent and method for heavy metal contaminated soil Repair formulation components (by weight percentage): Gene-edited microbial community: 30% of CRISPR-Cas9-edited Bacillus (mtf-1 gene expression level 2.2 pg / cell) and cadmium-resistant Pseudomonas (czcABC gene copy number 1.5 × 10⁻⁶). 6 30% (copies / μL), 15% sulfur-reducing bacteria, total viable count 2.0 × 10⁻⁶ 10 CFU / g; Nanocatalytic composite system: GQDs-modified biochar-supported MOF-808 (GQDs loading rate 18%) 1.8%, nZVI-S-DTC (shell thickness 8nm) 1.0%, γ-PGA-MOF (coupling rate 85%) 0.8%; Intelligent regulatory additives: 0.6% AZO derivative, 0.5% SA-CS microspheres (80μm particle size); Magnetic response material: MNHAP (Fe3O4 content 12%) 3.0%.

[0093] Preparation steps S1: Culture of gene-edited microbiota Bacillus gene editing and culture: Strain activation: Wild-type Bacillus ATCC 6633 was inoculated into LB medium and cultured at 37°C and 200 rpm for 12 h until OD. 600 =0.6.

[0094] Electroporation preparation: Centrifuge 5 mL of bacterial culture at 4℃ and 8000 r / min for 10 min, wash 3 times with 10% glycerol, and resuspend in 100 μL of glycerol to prepare competent cells.

[0095] Plasmid preparation: pHT01 plasmid (mtf-1 gene) concentration 500 ng / μL.

[0096] Electroporation: 10 μL of plasmid was added to competent cells, and the electroporation parameters were 2.5 kV, 25 μF, and 200 Ω. After recovery, the cells were plated on erythromycin-containing plates, positive clones were screened, and the cells were inoculated into LB medium containing IPTG and cultured for 12 h.

[0097] Cell collection: After centrifugation and washing, the cells were freeze-dried, and the metallothionein content was 1.7 mg / g of cells.

[0098] Screening and culture of cadmium-resistant Pseudomonas aeruginosa: Sample collection and enrichment: 50g of cadmium-contaminated soil was added to M9 medium containing 50mg / L CdCl2 and incubated at 30℃ and 180r / min for 48h.

[0099] Isolation and purification: After serial dilution, the cells were plated on M9 solid plates, single colonies were screened and purified, the czcABC gene was amplified by PCR, high-expression clones were screened by flow cytometry, and after optimized culture, the cadmium removal rate reached 95%, and the cells were lyophilized for later use.

[0100] Sulfur-reducing bacteria culture: Culture medium preparation: After sterilization of Postgate C medium, nitrogen gas was purged to remove oxygen. Desulfovibriovulgaris DSM 644 was inoculated and cultured at 35°C for 24 hours. The cells were collected by centrifugation, washed with anaerobic PBS, and then freeze-dried.

[0101] Microbial conjugation: Bacillus, Pseudomonas, and sulfur-reducing bacteria were mixed in a mass ratio of 3:3:1.5, with 10% glycerol as a preservative added, and freeze-dried at -80℃ for 48 hours to obtain a total viable count of 2.0 × 10⁻⁶. 10Compound bacterial powder with CFU / g.

[0102] S2: Preparation of MNHAP (Magnetic Responsive Hydroxyapatite) Synthesis of hydroxyapatite (HAP): Precipitation reaction: 500 mL of 0.5 mol / L Ca(NO3)2 solution was added dropwise to 334 mL of 0.3 mol / L (NH4)2HPO4 solution, and the pH was adjusted to 10 with ammonia water to maintain the Ca / P molar ratio of 1.67.

[0103] Aging treatment: After stirring for 2 hours, age in a water bath at 80℃ for 24 hours, filter and wash, dry at 80℃ for 12 hours, and grind to obtain HAP powder.

[0104] Synthesis of Fe3O4 nanoparticles: Coprecipitation reaction: Dissolve 1.35g FeCl3・6H2O and 0.5g FeCl2・4H2O in 100mL of deionized water, purge with nitrogen for 30min, add 10mL of concentrated ammonia, and stir vigorously for 30min.

[0105] Purification: The particles were collected by magnetic separation, washed, and then vacuum dried at 60°C for 12 hours to obtain Fe3O4 nanoparticles with a particle size of 25 nm.

[0106] MNHAP compound: Hydrothermal reaction: HAP and Fe3O4 were mixed at a mass ratio of 9:1, and 100 mL of ethylene glycol was added for ultrasonic dispersion for 30 min. The mixture was then transferred to a reaction vessel and hydrothermally reacted at 180 °C for 12 h.

[0107] Comparative Example: Traditional Microbial Remediation Methods Repair agent components: Wild-type bacterial flora: Bacillus, Pseudomonas, and sulfur-reducing bacteria (unedited) in a 3:2:1 ratio; Common materials: biochar (unloaded MOF), nZVI (unsulfurized), commercially available humic acid.

[0108] Preparation and Repair Steps Microbial culture: Wild-type Bacillus was inoculated into LB medium and cultured at 37°C and 200 r / min for 12 hours. Pseudomonas was inoculated into nutrient broth and cultured at 30°C and 180 r / min for 16 hours. Sulfur-reducing bacteria were inoculated into Postgate C medium and anaerobic cultured at 35°C for 24 hours. The bacterial cultures were mixed at a volume ratio of 3:2:1, centrifuged at 4°C and 6000 rpm for 10 minutes, washed twice with PBS, and the viable count was 1×10⁻⁶. 9 CFU / mL bacterial suspension.

[0109] Repair Implementation: Take the same contaminated soil as in the example, and adjust the moisture content to 25%; Spray the bacterial suspension at 1% (v / w) onto the soil surface, and simultaneously apply 5% biochar, 0.5% nZVI, and 2% humic acid, and then till to a depth of 20cm. Covered with plastic film, watered regularly to maintain moisture content, without electric field, magnetic field or light control, and without adding inducing agents.

[0110] The performance comparison between the examples and the comparative examples is shown in the table below: Table 1 In summary, Examples 1-3 showed significantly higher removal rates of Cd and Pb than the comparative example, reaching 84.3%-96.7% and 86.7%-91.2%, respectively, compared to only 35.2%-48.6% in the comparative example. The proportion of available Cd in the examples was as low as 2.8%-4.2%, compared to 32.1% in the comparative example. The soil pH in the examples was closer to neutral, demonstrating the advantages of the composite remediation technology.

[0111] The performance comparison between the examples and the comparative examples is shown in the table below: Table 2 In summary, the effective Pb content in Examples 1-3 was only 3.6%-5.1%, compared to 28.7% in the control group; the microbial survival rate was 87.8%-93.2% in Examples 1-3, compared to 56.4% in the control group. Although the cost of Examples 1-3 was higher (120-150 RMB / ton of soil), the effective period was much longer than that of the control group, reaching 180-360 days, demonstrating significant advantages in overall remediation effect and durability.

[0112] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their likenesses.

Claims

1. A soil remediation agent for heavy metal contaminated soil, characterized in that, It consists of the following components: Gene-edited microbial community: CRISPR-Cas9-edited Bacillus, cadmium-resistant Pseudomonas, and sulfur-reducing Desulfovibrio were mixed in a 3:2:1 mass ratio, with a total viable count ≥2×10⁻⁶. 10 CFU / g; Nanocatalytic composite system: 1-2% graphene quantum dot GQDs modified biochar supported MOF-808, 0.6-1.5% sulfurized nano-zero ferrous dithiocarbamate core-shell structure nZVI-S-DTC, 0.8-1.2% polyglutamic acid-metal-organic framework coupling γ-PGA-MOF; Intelligent control additives: 0.5-1% photoresponsive azobenzene derivative AZO, 0.3-0.7% pH-sensitive sodium alginate-chitosan microspheres SA-CS.

2. The repair agent according to claim 1, characterized in that, It also includes magnetically responsive nano-hydroxyapatite (MNHAP), which has a Fe3O4 content of 10-15% and a particle size of 50-100 nm. Under an applied magnetic field, it can migrate directionally to the heavy metal enrichment region.

3. The repair agent according to claim 1, characterized in that, It also includes a self-assembled microbial extracellular polymeric polymer (EPS)-nanoferrite composite gel, in which EPS accounts for 30-40% and Fe3O4 nanoparticles have a particle size of 20-30 nm, which can trigger the adsorption of heavy metals through redox potential (ORP).

4. The repair agent according to claim 1, characterized in that, The metallothionein expression level of the gene-edited Bacillus is 3-5 times that of the wild type, and the cadmium removal rate of the cadmium-resistant Pseudomonas can reach 92-98%.

5. The repair agent according to claim 1, characterized in that, The preparation method of GQDs-MOF-808 is as follows: graphene quantum dots and MOF-808 are mixed at a mass ratio of 1:10 and ultrasonically dispersed in N,N-dimethylformamide (DMF) for 24 hours. The GQDs loading rate is 15-20%, and the light absorption threshold is extended to 650 nm.

6. The repair agent according to claim 1, characterized in that, The preparation method of the nZVI-S-DTC core-shell structure is as follows: nZVI is treated with sodium sulfide solution for 2 hours, and then reacted with DTC-Na at pH 8-9 for 3 hours, with a shell thickness of 5-10 nm.

7. A method for remediating heavy metal contaminated soil based on the preparation according to any one of claims 1-6, characterized in that, Includes the following steps: S1. Soil electric field pretreatment: Apply a DC electric field of 0.5-1V / cm for 72 hours to promote the migration and activation of heavy metal ions to the anode; S2. Microbial community-nano system compound: Gene-edited microbial community and nanocatalytic composite system are mixed at a mass ratio of 1:5, intelligent regulatory additives are added, and SA-CS microspheres are used to form microcapsules with a diameter of 50-100μm. S3. Magnetoelectric Synergistic Remediation: After the microcapsules are applied to the soil, an alternating magnetic field of 0.1-0.3T is applied, and visible light is irradiated at the same time; S4. Dynamic Regulation: Soil pH, ORP, and heavy metal concentration are monitored in real time using IoT sensors. When pH < 6, SA-CS microspheres release CaCO3; when ORP > -50mV, AZO derivatives trigger the GQDs-MOF-808 photocatalytic reaction. S5. Bioenhancement: Starting from day 5 of remediation, an inducer containing 1 mmol / L IPTG is injected to induce gene-edited bacterial communities to express metallothionein for 15 days.

8. The repair method according to claim 7, characterized in that, The alternating magnetic field in S3 has a frequency of 5-10Hz, which causes MNHAP to aggregate in a directional manner to form a "nano-repair grid". At the same time, the ·OH free radicals generated by GQDs-MOF-808 under light have a degradation rate of 60-80% on soil organic matter.

9. The repair method according to claim 7, characterized in that, The IoT sensor in S4 monitors once per hour, and the data is transmitted to the cloud AI system to automatically adjust the injection of regulator into the drip irrigation system.

10. The repair method according to claim 7, characterized in that, After the remediation cycle ended, the available content of Cd, Pb and Cu in the soil decreased by 75-90%, and third-generation sequencing confirmed that the relative abundance of functional genes in the microbial community increased by 4-6 times, and the soil enzyme activity increased by 1.8-2.5 times.

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