Hydrothermal carbon-based microbial agent for remediation of cadmium / lead contaminated soil in coal mine area and preparation method and application thereof

Abstract

The application discloses a preparation method of a hydrothermal carbon-based microbial agent for repairing cadmium / lead contaminated soil in a coal mine area, and is characterized by the following steps: 1) using D-ribose as a biomass raw material, and using sodium oleate and PEO-PPO-PEO as a template agent to prepare regular-morphology hydrothermal carbon (DHC); 2) using a NaOH solution to modify the regular-morphology hydrothermal carbon (NDHC); 3) expanding culture of Bacillus pasteurii bacterial liquid; and 4) using an adsorption method to immobilize Bacillus pasteurii to prepare the hydrothermal carbon-based microbial agent. After 30 days of repair by the hydrothermal carbon microbial agent, the leaching amount of Cd in the soil in the coal mine area is reduced by 89.37%, and the leaching amount of Pb is reduced by 87.79%, so that the bioavailability and migration rate of heavy metals in the soil are effectively reduced. The hydrothermal carbon microbial agent provided by the application can increase soil fertility, adsorb, complex and solidify heavy metals to reduce the bioavailability of heavy metals in the soil, and can be used for ecological repair of soil in a coal mine area.

Description

Technical Field

[0001] This invention belongs to the field of environmental pollution control, specifically a hydrothermal carbon-based microbial agent for the remediation of cadmium / lead contaminated soil in coal mining areas, its preparation method, and its application. Background Technology

[0002] Soil safety is a crucial guarantee for the sustainable development of human society. Excessive cadmium and lead not only threaten plant growth and microbial diversity, but also easily enter the human body through the food chain, seriously endangering human health.

[0003] Although there are numerous remediation technologies for heavy metal contaminated soil, such as soil flushing, passivation and solidification, steam extraction, and bioreduction, these technologies suffer from problems such as high energy consumption, poor stability, and long remediation cycles. Carbon materials, with their porous, environmentally friendly, stable, and carbon-rich properties, have attracted widespread attention in the field of soil remediation. For example, CN113501518A discloses a type of biochar made from corn stalks carbonized at 680–750℃ for 1–2 hours and then dried. After three treatments with plastic film, the biochar achieved an adsorption efficiency of 65.8% for copper ions and 77.9% for mercury ions. CN104307855A discloses biochar prepared from rice straw, sawdust, and other biological waste carbonized at 700–900℃ for 20 hours. The biochar contains 6.0 wt% N, 21.0 wt% O, and 5.0 wt% P, respectively. After remediation, the proportions of mobile cadmium ions decreased to 15 wt%, while the proportions of residual cadmium ions increased to 53 wt%. The publication number CN109570208A describes soaking plant materials in a solution with a concentration of 2.5 mol·L⁻¹. -1 The plant powder was dissolved in FeCl3 solution for 12-48 hours, followed by pyrolysis under a nitrogen atmosphere (600-800℃) to obtain plant powder material. The plant powder was then thoroughly mixed with a 1 mol / L acetic acid solution and dried at 60-70℃ to obtain modified biochar material. After 60 days of remediation, it showed positive effects on Cu. 2+ The adsorption efficiency reached 87.6%. A study published in CN114410527A, using rice straw biochar as a carrier to immobilize *Geobacterium sulfide* HMP-1, showed that after 30 days of soil remediation, the available heavy metal contents of Cd, Pb, and Zn all decreased by more than 70%.

[0004] Existing research has explored the potential applications of carbon materials in the remediation of heavy metal pollution in soil. However, biochar suffers from low yield, high energy consumption, and poor adsorption stability, making it prone to secondary leaching of heavy metals. Hydrothermal char, on the other hand, is a black solid product, primarily composed of carbon and rich in oxygen-containing functional groups, obtained through a hydrothermal reaction at 150℃-375℃ and autogenous pressure. Compared to biochar, it boasts a higher yield, and its abundant carbon components can effectively improve soil fertility. However, because hydrothermal char is pyrolyzed at low temperatures, it suffers from incomplete decomposition, underdeveloped pore structure, and a small specific surface area.

[0005] To address the problem of heavy metal pollution in soil, this invention uses hydrothermal carbon as a carrier and employs Bacillus pasteurii immobilization technology to prepare hydrothermal carbon inoculants. By combining hydrothermal carbon with microbial mineralization technology, multiple passivation mechanisms such as adsorption, complexation, and solidification are achieved to reduce the bioavailability and migration rate of heavy metals in soil. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the first objective of the present invention is to provide a hydrothermal carbon-based microbial agent for the remediation of cadmium / lead contaminated soil in coal mining areas, the second objective is to provide a method for preparing the hydrothermal carbon-based microbial agent, and the third objective is to provide its application.

[0007] To achieve the first objective mentioned above, the present invention provides the following technical solution: a method for preparing a hydrothermal carbon-based microbial agent for the remediation of cadmium / lead contaminated soil in coal mining areas, characterized by preparation according to the following steps:

[0008] 1) Using D-ribose as biomass raw material, and sodium oleate and PEO-PPO-PEO as template agents, regular hydrothermal char (DHC) was prepared.

[0009] 2) Modification of regularly morphological hydrothermal carbon (NDHC) using NaOH solution;

[0010] 3) Large-scale culture of Bacillus pasteurii bacterial suspension;

[0011] 4) A hydrothermal carbon-based bacterial agent was prepared by immobilizing Bacillus pasteurii by adsorption.

[0012] In the above scheme: Step 1) is as follows: Weigh 98% sodium oleate and PEO-PPO-PEO, dissolve them in deionized water, and stir until completely dissolved; mix the aqueous solution of D-ribose with the above solution, stir at low speed, transfer to a high-pressure reactor, and place in a 180℃ drying oven for constant temperature carbonization. After carbonization, centrifuge, wash, dry, and pass through a 0.2mm sieve to obtain DHC. 0.12 mmol of 98% sodium oleate (SO4): 0.015 mmol of PEO-PPO-PEO (P123): 3 g of D-ribose.

[0013] In the above scheme, the specific operation of step 2) is as follows: DHC is modified with 15% sodium hydroxide solution. The hydrolyzed DHC is soaked in NaOH solution at a mass ratio of 1:10. The solution is stirred for 10 minutes every 4 hours for a total of 24 hours. After soaking, the solution is centrifuged, washed with anhydrous ethanol and water to remove excess NaOH. The solution is then dried in a drying oven, ground into powder and sieved to obtain NDHC.

[0014] In the above scheme, the specific operation of step 3) is as follows: Dissolve casein peptone, urea solution, soybean peptone, sodium chloride, and agar in deionized water, adjust the pH to 7.3 with NaOH solution as the nutrient solution for the strain, add nucleating agent NaCit to the prepared nutrient solution at 0%-4%, sterilize, place in a clean bench after sterilization, turn on the ultraviolet lamp and fan for sterilization and cooling, take out the activated strain, dip a small amount of strain into the sterilized nutrient solution for inoculation, and incubate in a 30℃ biochemical incubator for 28 hours. The cultured bacterial solution should be stored in a constant temperature environment of 4℃.

[0015] In the above scheme, the operation steps of step 4) are as follows: put NDHC into an autoclave at 120°C for 20 minutes for sterilization, mix the sterilized NDHC and bacterial solution at a ratio of 98%:2% bacterial solution, adsorb and fix in a constant temperature shaker, and then centrifuge, filter, wash and dry to obtain hydrothermal carbon bacterial agent HCMA.

[0016] A method for preparing a hydrothermal carbon-based microbial agent for remediation of cadmium / lead contaminated soil in coal mining areas, wherein the hydrothermal carbon-based microbial agent is prepared.

[0017] Application of the aforementioned hydrothermal carbon-based bacterial agent in the remediation of cadmium / lead contaminated soil in coal mining areas.

[0018] In the above scheme: spray the hydrothermal carbon-based bacterial agent onto the soil to be repaired, and after the hydrothermal carbon-based bacteria are evenly mixed with the soil (after 8 hours), add the cementing liquid in the same volume ratio as the hydrothermal carbon-based bacterial agent.

[0019] In the above scheme: the cementing solution consists of 0.6 mol·L⁻¹-1 Urea and 0.6 mol·L -1 Composition of a mixed solution of calcium chloride.

[0020] Beneficial effects

[0021] 1. The hydrothermal char microbial agent of this invention can remediate soil heavy metal pollution through multiple passivation mechanisms such as adsorption, complexation, and solidification, which is beneficial to reducing the bioavailability and migration rate of heavy metals in the soil and is less prone to secondary leaching. After HCMA remediation, the leaching amount of Cd decreased by 89.37%, and the leaching amount of Pb decreased by 87.79%.

[0022] 2. This invention uses D-ribose as a biomass raw material to prepare hydrothermal char. Hydrothermal char not only contains abundant oxygen-containing functional groups and has a high yield, but also has low cost and is conducive to carbon sequestration and emission reduction. Furthermore, D-ribose is a natural component in plants and is easily obtained, so it will not cause secondary pollution to the environment.

[0023] 3. This invention selects 15% NaOH solution as a modifier, which increases the specific surface area of ​​hydrothermal carbon by 70%, providing more attachment sites for Bacillus pasteurii while also providing a suitable weakly alkaline environment.

[0024] 4. This invention selects Bacillus pasteurii for immobilization due to its easily controllable mineralization process, rapid reaction rate, and high chemical conversion rate. Its precipitate has a very stable calcite structure, preventing secondary leaching of cadmium / lead. After 30 days of remediation of the tested soil samples with Bacillus pasteurii, the content of carbonate-form Cd increased by 131.42%, and the content of carbonate-form Pb increased by 187.97%. Attached Figure Description

[0025] Figure 1 Growth curves of Bacillus pasteurii under different heavy metal gradients.

[0026] Figure 2 The effect of NaCit on the quality of precipitates.

[0027] Figure 3 SEM images of hydrothermal charcoal and hydrothermal charcoal inoculants (a: HC; b: DHC; c: HCMA)

[0028] Figure 4 XPS spectra of mineralized products.

[0029] Figure 5 Heavy metal leaching experiment.

[0030] Figure 6 Distribution map of heavy metal speciation in soil. Detailed Implementation

[0031] The present invention will be further described below with reference to the accompanying drawings and embodiments:

[0032] Example 1

[0033] The preparation method of hydrothermal carbon-based microbial agent for cadmium / lead contaminated soil remediation in coal mining areas is as follows:

[0034] (1) Weigh 0.12 mmol of 98% sodium oleate (SO4) and 0.015 mmol of PEO-PPO-PEO (P123) and dissolve them in 20 mL of deionized water. Stir slowly in a water bath with a magnetic stirrer at room temperature until completely dissolved. Mix 3 g of D-ribose in 40 mL of aqueous solution with the above solution. Stir the mixture at low speed in a water bath with a magnetic stirrer at room temperature for 30 min, then transfer it to a 100 mL reaction vessel. Place it in a drying oven at 180 °C for constant temperature carbonization for 10 h. After carbonization, centrifuge, wash, dry, and pass through a 0.2 mm sieve to obtain DHC.

[0035] (2) HC was modified using 15% sodium hydroxide solution. The hydrolyzed HC was soaked in NaOH solution at a mass ratio of 1:10, and stirred for 10 minutes every 4 hours for a total soaking time of 24 hours. After soaking, the HC was centrifuged, washed three times with water, and then washed once with anhydrous ethanol to remove excess NaOH. The HC was dried in an 80℃ drying oven, ground into powder, and sieved. The product is alkaline D-ribose hydrothermal carbon (NDHC).

[0036] 3) Bacillus pasteurii was a commercial strain (CGMCC 1.803) purchased from the Guangdong Microbial Culture Collection Center. 3g of casein peptone and 20g·L⁻¹ were added... -1 Urea solution, 5g soybean peptone, 5g sodium chloride, 20g·L -1 Dissolve the agar in 200 mL of deionized water, and use 1 mol·L⁻¹ water. -1 The pH of the culture medium was adjusted to 7.3 using NaOH solution as the nutrient solution for the bacterial strain. To increase the nucleation sites of Bacillus pasteurii, nucleating agent (NaCit) was added to the prepared nutrient solution at concentrations of 0%, 1%, 2%, 3%, and 4% to investigate the effect of the nucleating agent on the amount of calcium carbonate precipitation. Before inoculation, the nutrient solution containing the nucleating agent, petri dishes, Erlenmeyer flasks, and inoculation instruments were all sterilized in a high-temperature autoclave. After sterilization, they were placed in a clean bench and sterilized and cooled using ultraviolet lamps and fans. The activated bacterial strain was removed, and a small amount of the strain was dipped into the sterilized nutrient solution using a sterilized toothpick for inoculation. The culture was then placed in a 30℃ biochemical incubator for 28 hours. The cultured bacterial solution was stored in a constant temperature environment at 4℃.

[0037] 4) Sterilize NDHC in an autoclave at 120℃ for 20 minutes. Mix the sterilized NDHC with 2% of the culture solution containing nucleating agent (Bacillus pasteurii) that has been cultured for 28 hours. Sterilize at 30℃ and 180 rpm. -1 The hydrothermal char agent (HCMA) was obtained by adsorption and fixation in a constant temperature shaker for 18 hours, followed by centrifugation, filtration, washing and drying.

[0038] CO(NH2)2+H2O→2NH3+NH2COOH2 (1)

[0039] NH₂COOH₂ + 2H₂O → H₂CO₃ + NH₃ (2)

[0040] H2CO3→H + +CO3 2- (3)

[0041] NH3 + H2O → NH4 + +OH - (4)

[0042] CO2 + OH - →HCO3 - (5)

[0043] Ca 2+ +HCO3 - +OH - →H2O+CaCO3↓ (6)

[0044] Heavy metal tolerance tests were conducted on Bacillus pasteurii, and a 1 g·L⁻¹ solution was prepared. -1 The mother liquor of cadmium nitrate and lead nitrate was used to adjust the Cd content in the liquid culture medium. 2+ Pb 2+ The concentrations were in gradients of 0, 50, 100, 150, and 200 mg·L⁻¹. -1 After inoculation, the samples were kept at 30℃ and 180 rpm. -1 Bacillus pasteurii was cultured in a constant-temperature shaker for 60 hours, and samples were taken every 4 hours to measure the OD600 value using a UV spectrophotometer to investigate the effects of different concentrations of heavy metals (Cd). 2+ Pb 2+ Tolerance to heavy metals was assessed using the bacterial growth inhibition rate as an indicator, as shown in formula (1). The results are as follows: Figure 1 As shown.

[0045] Bacterial growth inhibition rate = (OD of control group) 600 -Experimental group OD 600) / Control group OD 600 (7)

[0046] Figure 1 As can be seen, the growth curve of Bacillus pasteurii is S-shaped, which can be divided into a lag phase (0-10h), a logarithmic growth phase (10-35h), and a stationary phase (35-52h). Cd 2+ Pb 2+ Concentrations range from 0 to 150 mg / L -1 Within the range, Cd had no significant effect on bacterial growth. 2+ At 200 mg·L -1 It showed significant inhibition of bacterial cells, with an inhibition rate of 27.8% after 52 hours of culture. Pb 2+ At 200 mg·L -1 It showed significant inhibition of bacterial cells, with an inhibition rate of 18.2% after 52 hours of culture. *Bacillus pasteurellii* showed resistance to Cd... 2+ Pb 2+ The highest tolerated concentration is 200 mg·L. -1 Cd 2+ The inhibition rate against Bacillus pasteurellosis is stronger than that against Pb. 2+ .

[0047] BET tests were performed on DHC and NDHC to investigate the changes in specific surface area before and after modification. The results are shown in Table 1.

[0048] Table 1 BET Analysis of DHC and NDHC

[0049]

[0050] As shown in Table 1, the specific surface area of ​​DHC is 104.34 m². 2 ·g -1 The pore volume is 4.967 cm³. 3 ·g -1 The average particle size is 5.143 nm. The specific surface area of ​​NDHC is 178.459 m². 2 ·g -1 The pore volume is 6.171 cm³. 3 ·g -1 The average particle size was 3.797 nm. After modification with NaOH, the specific surface area of ​​NDHC increased significantly, and the average particle size decreased, making it more suitable for adsorbing heavy metal pollutants and immobilizing microorganisms. This is because the D-ribose was corroded by NaOH, causing changes in its carbon skeleton, making it easier to form pores and increase the number of micropores during the hydrothermal carbonization process of D-ribose.

[0051] This experiment investigated the effect of NaCit addition on mineralization experiments, discussing five cases with NaCit addition amounts of 0%, 1%, 2%, 3%, and 4%. The results are as follows: Figure 2 As shown. This experiment also prepared hydrothermal char (HC) using D-ribose as a biomass feedstock without the addition of sodium oleate (SO) and PEO-PPO-PEO (P123), and compared the effects of HC and DHC on morphology and structure using SME. Figure 3 ), and XPS spectroscopy analysis of the mineralized products ( Figure 4 Adjusting Cd in liquid culture medium using cadmium nitrate and lead nitrate stock solutions. 2+ Pb 2+ The concentrations of the inoculants were set at gradients of 0, 50, 100, 150, and 200 mg / L. After inoculation, the inoculants were cultured in a constant-temperature shaker at 30℃ and 180 r / min for 24 h before mineralization experiments. A cementing solution was prepared using a mixture of 0.6 mol / L urea and 0.6 mol / L calcium chloride. The cementing solution and hydrothermal charcoal inoculant were mixed at a volume ratio of 1:1 for mineralization experiments. After 7 days of mineralization, the mineralized products were washed, dried, and subjected to XPS spectroscopy.

[0052] Depend on Figure 2 As shown, without the addition of NaCl, bacterial solution, and cementing solution in a 1:1 ratio, calcium carbonate precipitated to a concentration of 12.28 g·L⁻¹. -1 The addition of NaCit to the bacterial culture significantly increased the amount of calcium carbonate precipitation. At a NaCit addition of 2%, the precipitation reached a maximum of 32.915 g, which is 3.26 times that without NaCit. When the NaCit addition was 3%, the amount of calcium carbonate precipitation began to decrease. Therefore, a NaCit addition of 2% is more conducive to the mineralization of heavy metals.

[0053] The carrier structure directly affects the life activities and adsorption strength of immobilized microorganisms. The most ideal living environment for immobilized microorganisms is porous, with regular morphology and high specific surface area. For example... Figure 3 As shown in figure a, hydrothermal carbon (HC) has a small amount of spherical structure and an irregular morphology. Figure 3 b. It was found that the hydrothermal char prepared by the template method exhibits a regular spherical structure with a large number of pores. The orderly spherical structure and well-developed pore structure of the hydrothermal char facilitate the entry and exit of nutrients and cell metabolites, making it an excellent carrier material for the preparation of hydrothermal char inoculants. Figure 3 c shows that Bacillus pasteurii was densely attached to the surface of the hydrothermal charcoal, indicating that Bacillus pasteurii was successfully colonized on the hydrothermal charcoal.

[0054] Figure 4The XPS spectroscopy results show that the main elements of the mineralization products are C, O, and Ca, with heavy metals fixed in a solidified form. The bacterial solution contains Cd. 2+ The precipitate mainly contained C, O, Ca, and Cd, with Cd accounting for 6.94%. The bacterial culture contained Pb. 2+ The precipitate mainly contained C, O, Ca, and Pb, with Pb accounting for 9.01%. This indicates that MICP technology can effectively remove Cd. 2+ Pb 2+ Solidification is then carried out. The mineralized products contain oxygen-containing functional groups such as OC=C, OC=O, C=O, and CO. These functional groups facilitate the complexation of Cd and Pb, fixing the heavy metals in calcium carbonate. The Cd3d spectral diffraction peaks show that CdCO3 is a fixative for Cd. 2+ The main form. From the Pb4f spectral diffraction peaks, it can be seen that Pb... 2+ The main fixation forms of Cd are not only PbCO3, but also Pb(OH)2. In short, Cd... 2+ and Pb 2+ The removal is attributed to the co-precipitation of Cd, Pb and CaCO3 by hydrothermal carbon and Bacillus pasteurella during treatment.

[0055] A mixed culture experiment was conducted between the remediation agent and contaminated soil, followed by a heavy metal leaching experiment. 150g of air-dried coal mine soil sample was added to a plastic cup, and the soil was moistened with deionized water to 60% of the mine soil's water holding capacity. HC, DHC, NDHC, MA, and HCMA (NaCit added at 2%) were thoroughly mixed with cadmium and lead-contaminated soil at a ratio of 3%. (The hydrothermal carbon-based bacterial agent was sprayed onto the soil to be remediated. After the hydrothermal carbon-based bacteria and soil were evenly mixed (8 hours later), a cementing solution was added at the same volume ratio as the hydrothermal carbon-based bacterial agent. The cementing solution consisted of 0.6 mol·L⁻¹...) -1 Urea and 0.6 mol·L -1 The soil was prepared using a mixed solution of calcium chloride (calcium chloride). A control group without added remediation agents was also included, with three replicates in each group. The soil was then pre-cultured in the dark at 25±2℃ for 30 days. All plastic cups were covered with plastic wrap, leaving small holes to ensure gas exchange and minimize moisture loss. Weighing was performed every 5 days, and appropriate amounts of water were added to maintain a constant moisture content throughout the incubation period. Destructive sampling of the soil was conducted at days 15 and 30 to determine changes in the speciation and content of heavy metals. The soil samples were then digested using a microwave digester, and the leaching content of Pb and Cd was analyzed using inductively coupled plasma mass spectrometry (ICP-OES). Simultaneously, the Tessier method was used to investigate the component content of Cd and Pb in the soil environment. Results are as follows: Figure 5 , Figure 6 As shown.

[0056] Figure 5 The results show that the initial TCLP-Cd content in the unremediated soil sample was 14.31 mg·kg⁻¹. -1 The initial TCLP-Pb content was 21.96 mg·kg⁻¹. -1 This indicates that Cd and Pb are highly toxic in soil. After 30 days of remediation with HC, DHC, NDHC, bacteria (MA), and hydrothermal charcoal (HCMA), the TCLP-Cd content in the soil samples was 11.63 mg·kg⁻¹. -1 7.18 3 mg·kg -1 5.313 mg·kg -1 4.73 3 mg·kg -1 1.12 mg·kg -1 This effectively reduced the migration rate and toxicity of Cd. Specifically, after HCMA remediation, the leaching of Cd decreased by 89.37%, demonstrating a good passivation and consolidation effect on Cd. After 30 days of adsorption and passivation of soil Pb by HC, DHC, NDHC, MA, and HCMA, the TCLP-Pb content was 17.37 mg·kg⁻¹. -1 12.71 mg·kg -1 10.16 mg·kg -1 8.68 mg·kg -1 and 2.68 mg·kg -1 HCMA effectively reduced the TCLP-Pb content in the soil, with a reduction of 87.79% after remediation. In summary, HCMA effectively reduced the bioavailability of Cd and Pb in the soil.

[0057] Figure 6It can be seen that Cd exists in the tested soil samples in the following forms: exchangeable (EXC, 44.76%), carbonate-bound (CARB, 18.24%), iron-manganese oxide-bound (MnOX, 15.68%), organic matter-bound (OMI, 11.51%), and residual (RES, 9.81%). Compared with the control (CK), the EXC-Cd content decreased by 43.43% after DHC treatment, while the OMI-Cd and RES-Cd contents increased by 92.87% and 63.09%, respectively. After 30 days of MA remediation of the tested soil samples, EXC-Cd mainly transformed into CARB-Cd and RES-Cd, with the EXC-Cd content decreasing by 54.53% and the CARB-Cd and RES-Cd contents increasing by 86.41% and 75.12%, respectively. After 30 days of HCMA treatment of the tested soil samples, the EXC-Cd content decreased by 76.12%. The tested soil samples contained 49.14% EXC-Pb, 12.31% CARB-Pb, 13.02% MnOX-Pb, 18.34% OMI-Pb, and 7.19% RES-Pb. Compared with the control (CK), soil samples treated with HC, MA, and HCMA for 30 days showed a decrease in EXC-Pb content of 57.08–80.97%, while the MnOX-Pb content was similar to that of the CK components. After DHC treatment, the OMI-Pb and RES-Pb contents increased by 55.61% and 118.77%, respectively. After 30 days of MA remediation, the CARB-Pb content increased by 187.97%. Compared with the CK, HCMA remediation significantly reduced the EXC-Pb content. However, soil samples cultured with MA for 30 days showed a significant increase in CARB-Cd and CARB-Pb contents, which is attributed to CO3 produced by Bacillus pasteurellis. 2- Cd and Pb combine to form CdCO3 and PbCO3. Therefore, HCMA can effectively reduce the migration rate and toxicity of Cd and Pb in mining soils.

[0058] As shown in Table 2, after 30 days of remediation with hydrothermal charcoal inoculant, the soil pH increased to 7.93. This is because urease decomposed urea, producing some OH-. - The soil organic matter content also increased significantly, from the original 16.46 g·kg⁻¹. -1 Increased to 27.34 g·kg -1 The soil cation exchange capacity and organic carbon content also increased by 66%. Specifically, the organic carbon content increased by 86%. This demonstrates that the hydrothermal charcoal inoculant effectively improved the soil's physical and chemical properties and increased soil fertility.

[0059] Table 2 Changes in soil physicochemical properties after 30 days of remediation

[0060]

[0061] 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 equivalents.

Claims

1. A method for preparing a hydrothermal carbon-based microbial agent for the remediation of cadmium / lead contaminated soil in coal mining areas, characterized in that, Prepare according to the following steps: 1) Using D-ribose as a biomass raw material, weigh 98% sodium oleate and PEO-PPO-PEO and dissolve them in deionized water, stirring until completely dissolved; mix the aqueous solution of D-ribose with the above solution, stir at low speed, transfer to a high-pressure reactor, and place in a 180℃ drying oven for constant temperature carbonization. After carbonization, centrifuge, wash, dry, and pass through a 0.2mm sieve to obtain DHC; 2) Modification of NDHC with regular-morphology hydrothermal carbon using NaOH solution; 3) Bacillus pasteurii Large-scale culture of bacterial culture: Casein peptone, urea solution, soybean peptone, sodium chloride, and agar were dissolved in deionized water, and the pH was adjusted to 7.3 with NaOH solution as the nutrient solution for the bacterial strain. The prepared nutrient solution was then inoculated with 2% nucleating agent NaCit and sterilized. After sterilization, the culture was placed in a clean bench and sterilized and cooled by turning on the ultraviolet lamp and fan. The activated bacterial culture was then removed, and a small amount of bacterial culture was inoculated into the sterilized nutrient solution using a sterilized toothpick. The culture was then placed in a 30℃ biochemical incubator for 28 h. The cultured bacterial culture should be stored in a constant temperature environment at 4℃. Bacillus pasteurii The bacterial strain is identified as CGMCC 1.803; 4) Immobilization by adsorption method Bacillus pasteurii Hydrothermal carbon-based bacterial agent was prepared. NDHC was sterilized in an autoclave at 120°C for 20 minutes. The bacterial solution and sterilized NDHC were mixed at a ratio of 98%:2%, and the mixture was adsorbed and fixed in a constant temperature shaker. After centrifugation, filtration, washing and drying, hydrothermal carbon-based bacterial agent HCMA was obtained.

2. The preparation method of the hydrothermal carbon-based microbial agent for cadmium / lead contaminated soil remediation in coal mining areas according to claim 1, characterized in that, Step 2) is as follows: DHC is modified with 15% sodium hydroxide solution. The hydrolyzed DHC is soaked in NaOH solution at a mass ratio of 1:

10. The solution is stirred for 10 minutes every 4 hours for a total of 24 hours. After soaking, the solution is centrifuged, washed with anhydrous ethanol and water to remove excess NaOH. The solution is then dried in a drying oven, ground into powder and sieved to obtain NDHC.

3. A hydrothermal carbon-based microbial agent prepared by the method described in any one of claims 1-2 for remediation of cadmium / lead contaminated soil in coal mining areas.

4. The application of the hydrothermal carbon-based microbial agent according to claim 3 in the remediation of cadmium / lead contaminated soil in coal mining areas.

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