Composite microbial agent and application thereof in heavy metal pollution remediation

Abstract

The application relates to the technical field of environmental microbial remediation, and particularly discloses a composite microbial agent and application thereof in heavy metal pollution remediation. The composite microbial agent comprises a composite microbial bacteria and a composite carrier for immobilizing the composite microbial bacteria; the composite carrier is porous ceramic granules loaded with adsorption slurry, the adsorption slurry is prepared by mixing plant straw powder subjected to alkali dewaxing and a gelling agent solution; and the composite microbial bacteria are composed of Bacillus cereus and Pseudomonas fluorescens. The composite microbial agent of the application is prepared by mixing the straw subjected to alkali dewaxing and porous ceramic granules, so that the composite carrier capable of loading the microbial bacteria is obtained, and the composite microbial agent has microbial planting capacity and heavy metal remediation effect far exceeding those of a traditional simple mixed carrier.

Description

Technical Field

[0001] This application relates to the field of environmental microbial remediation technology, and more specifically, it relates to a composite microbial agent and its application in the remediation of heavy metal pollution. Background Technology

[0002] While mining has supported economic development, it has also led to severe heavy metal pollution in soil. Heavy metals such as lead (Pb), cadmium (Cd), copper (Cu), zinc (Zn), and arsenic (As) enter the environment through mining activities and persist in the soil for extended periods, not only damaging ecosystems but also posing a serious threat to human health through bioaccumulation in the food chain. Compared to costly and potentially polluting physical / chemical remediation methods, microbial remediation technology is considered a green and sustainable solution due to its environmental friendliness, lower cost, and ability to be implemented in situ.

[0003] The core of microbial remediation technology lies in functional microorganisms. Utilizing the adsorption, precipitation, and redox effects of microorganisms, soluble and highly toxic heavy metals in the soil can be transformed into less toxic or more stable forms. However, directly applying microbial inoculants to contaminated soil, especially in extremely harsh environments such as mining areas with high acidity, high heavy metal concentrations, and nutrient depletion, presents survival challenges for exogenous microorganisms. They struggle to effectively colonize and perform remediation functions, resulting in unstable remediation effects and low efficiency.

[0004] To address this issue, microbial immobilization technology has emerged. This technology immobilizes functional microorganisms on a carrier material, providing them with a "micro-sanctuary" to enhance their resistance to harsh environments. The performance of the carrier material directly determines the success or failure of the microbial remediation agent. Currently, commonly used carriers can be categorized as follows, but all have significant limitations: Inorganic carriers, such as porous ceramsite, activated carbon, and zeolite, typically possess high specific surface area and good stability. However, they are mostly inert materials and cannot provide the necessary nutritional support for the growth and metabolism of microorganisms. In nutrient-deficient mining soils, the microorganisms attached to them will rapidly decrease in activity or even die due to "starvation." For example, while using ceramsite alone can provide attachment points, the remediation effect is difficult to sustain.

[0005] Organic carriers: These are represented by various agricultural wastes (such as straw and sawdust). These materials are widely available and inexpensive, and their decomposition process gradually releases nutrients such as organic carbon, which is beneficial for the long-term survival of microorganisms. However, these materials are easily and rapidly decomposed in the environment, exhibiting poor structural stability, leading to premature carrier disintegration and an inability to provide long-term protection for microorganisms. Furthermore, their simple physical structure limits their efficiency in immobilizing microorganisms, making them prone to loss.

[0006] Composite Carriers: To balance stability and nutrition, existing technologies attempt to simply physically mix different materials. For example, straw powder is mixed with ceramsite. However, this simple physical mixing fails to achieve functional synergy and structural optimization between materials. Straw powder is still easily decomposed and lost, releasing nutrients too quickly and failing to accurately supply microorganisms; the binding force between ceramsite and microorganisms and straw is weak, and the structure is easily destroyed by soil water erosion, failing to form an effective micro-ecological environment. A Chinese patent application, publication number CN121203901A, discloses a heavy metal-resistant Bacillus belye and a microbial remediation agent and its application, which, although it treats straw with Fe... 3+ / H2O2 modification can improve its performance, but its carrier system still mainly relies on the characteristics of the single modified straw. There is still room for improvement in terms of the mechanical strength of the structure, the refined construction of microbial colonization space, and the synergistic support of multiple functional microbial communities.

[0007] Therefore, there is an urgent need in this field for a novel microbial immobilization carrier system that can not only efficiently load and protect functional microorganisms, but also significantly improve the colonization success rate, metabolic activity and repair durability of functional bacteria in extremely polluted environments through ingenious design of materials and structures. Summary of the Invention

[0008] To address the aforementioned technical problems, this application provides a composite microbial agent and its application in the remediation of heavy metal pollution. The composite microbial agent is prepared by mixing straw after alkaline dewaxing with porous ceramsite to obtain a composite carrier capable of supporting microorganisms, exhibiting significantly higher microbial colonization capacity and heavier metal remediation effects than traditional simple mixed carriers.

[0009] In a first aspect, this application provides a compound microbial agent, which adopts the following technical solution: A composite microbial agent includes a composite microbial strain and a composite carrier for immobilizing the composite microbial strain; The composite carrier is a porous ceramic particle loaded with an adsorbent slurry, which is made by mixing plant straw powder that has undergone alkali dewaxing with a gelling agent solution. The composite microbial strain consists of Bacillus cereus and Pseudomonas fluorescens.

[0010] More preferably, the ratio of viable Bacillus cereus to Pseudomonas fluorescens is 1:(0.5-1).

[0011] More preferably, the viable count of the composite microbial agent is not less than 1.0 × 10⁻⁶.9 CFU / g.

[0012] More preferably, the porous ceramsite has a particle size of 2-5 mm and a porosity of 40-60%.

[0013] More preferably, the plant straw powder is made from crushed aquatic plant straw, which is selected from one or more of lotus pods, water hyacinths, and reeds.

[0014] More preferably, the particle size of the plant straw powder is 100-200 mesh.

[0015] The gelling agent is a natural high-molecular-weight polysaccharide that can crosslink with divalent cations or form a gel through thermal induction, specifically selected from one or more of sodium alginate, agar, κ-carrageenan, gelatin and gellan gum.

[0016] The gelling agent solution is preferably an aqueous solution of sodium alginate. After the adsorbent slurry is loaded onto porous ceramsite, it is cross-linked and cured by calcium chloride solution.

[0017] Another preferred gelling agent solution is an aqueous solution of κ-carrageenan. The adsorbent slurry is loaded onto porous ceramsite under heating conditions, and forms a gel after cooling. Optionally, it is further enhanced by potassium salt solution treatment.

[0018] Another preferred gelling agent solution is an aqueous solution of a mixture of sodium alginate and κ-carrageenan, wherein the mass ratio of sodium alginate to κ-carrageenan is 1:(0.1-1). The adsorbent slurry can be cured by first thermal induction treatment and then ionic crosslinking treatment, or it can be cured simultaneously by ionic crosslinking.

[0019] Secondly, this application provides a method for preparing a compound microbial agent, which adopts the following technical solution: A method for preparing a compound microbial agent includes the following steps: (1) Plant straw is dewaxed by alkali and then crushed to obtain plant straw powder. The plant straw powder is mixed with a gelling agent solution to make an adsorption slurry. Porous ceramsite is mixed with the adsorption slurry and then coated. After solidification, a composite carrier is obtained. (2) Add the composite carrier to the liquid culture medium, sterilize it, inoculate Bacillus cereus and Pseudomonas fluorescens for fermentation culture, dry the fermented system to obtain solid microbial agent, i.e. composite microbial agent.

[0020] More preferably, in step (1), the gelling agent solution is an aqueous solution of sodium alginate or an aqueous solution of κ-carrageenan or a mixture of sodium alginate and κ-carrageenan, preferably an aqueous solution of a mixture of sodium alginate and κ-carrageenan.

[0021] More preferably, in step (2), the liquid culture medium contains 5 g / L peptone, 3 g / L beef extract, 5 g / L NaCl, and has a pH of 7.

[0022] More preferably, in step (2), the fermentation culture temperature is 30-37℃ and the culture time is 24-48h.

[0023] More preferably, in step (2), the drying method is freeze drying.

[0024] Thirdly, this application provides the application of a compound microbial agent in the remediation of soil contaminated with heavy metals, especially soil in mining areas, wherein the heavy metals include at least one of Cd, Pb, Cu, and Zn.

[0025] More preferably, the application rate of the compound microbial agent is 50-100 g / m³. 2 .

[0026] In summary, this application has the following beneficial effects: (1) This application enhances the hydrophilicity and nutrient slow-release function of straw by alkali dewaxing treatment, combines the stable skeleton of porous ceramsite, and uses a gelling agent to bond and construct a three-dimensional network carrier of straw-gel-ceramsite, thus synergistically achieving nutrient slow release and physical protection, greatly improving the colonization success rate, activity and persistence of compound bacteria in extreme soils.

[0027] (2) This application preferably combines Bacillus cereus, which has strong stress resistance and phosphate precipitation properties, with Pseudomonas fluorescens, which has EPS secretion and complexation adsorption properties. Through synergistic mechanisms such as adsorption, precipitation, and complexation, the two can efficiently convert heavy metals such as Pb, Zn, and Cd from active forms such as exchangeable and carbonate-bound states into stable residue states, thereby achieving comprehensive passivation. Detailed Implementation

[0028] The following specific examples illustrate the implementation of this application. Those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this specification. This application can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of this application.

[0029] Furthermore, it should be understood that the one or more method steps mentioned in this application do not preclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps, unless otherwise stated. Moreover, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and is not intended to limit the order of the method steps or limit the scope of implementation of this application. Changes or adjustments to their relative relationships, without substantially altering the technical content, shall also be considered as within the scope of implementation of this application.

[0030] Unless otherwise specified, the experimental conditions used in the examples are generally in accordance with conventional conditions in the art or the conditions recommended by the reagent company. Unless otherwise specified, the materials and reagents used in the examples can be purchased commercially.

[0031] In this application, Bacillus cereus was purchased from the China Industrial Microbial Culture Collection Center, accession number CICC 23828; Pseudomonas fluorescens was purchased from the China Industrial Microbial Culture Collection Center, accession number CICC 21620.

[0032] The liquid culture medium consisted of 5 g / L peptone, 3 g / L beef extract, 5 g / L NaCl, and pH 7.

[0033] Heavy metal content testing methods: The Tessier five-step extraction method was used to analyze the changes in the content and speciation of heavy metals Pb, Zn, and Cd in soil samples before and after remediation. The specific method is as follows: Exchangeable state: Weigh 1g of sample into a 50mL centrifuge tube, add 8mL of 1mol / L MgCl2, shake well, and oscillate at 250rpm for 1h. Then centrifuge at 4000rpm for 4min. Filter the supernatant through a 0.45μm filter. Add 1mL of concentrated nitric acid to the filtered supernatant and store at 4℃. Add 8mL of ultrapure water to the residue, shake well, and centrifuge at 4000rpm for 20min. Discard the supernatant.

[0034] Carbonate-bound state: Add 8 mL of 1 mol / L NaAc to the previous residue, shake well, and then shake in a constant temperature (22±5℃) shaker for 5 hours. Centrifuge at 4000 rpm for 4 minutes, filter the supernatant through a 0.45 μm filter, add 1 mL of concentrated nitric acid to the filtered supernatant, and store at 4℃. Add 8 mL of ultrapure water to the residue, shake well, and centrifuge at 4000 rpm for 20 minutes. Discard the supernatant.

[0035] Reduced state: Add 20 mL of 25% HAc solution (0.04 mol / L NH₂OH·HCl) to the previous residue and adjust the pH to 2. Incubate in a constant temperature water bath at (96±3)℃ for 6 hours, cool, centrifuge at 4000 rpm for 4 minutes, filter the supernatant through a 0.45 μm filter, add 1 mL of concentrated nitric acid to the filtered supernatant and store at 4℃. Add 8 mL of ultrapure water to the residue, shake well, centrifuge at 4000 rpm for 20 minutes, and discard the supernatant.

[0036] Oxidized state: Add 3 mL of 0.02 mol / L HNO3 solution and 5 mL of 30% H2O2 solution to the previous residue, and adjust the pH to 2. Nitrify at (85±2)℃ for 2 hours with intermittent stirring; add 3 mL of 30% H2O2 solution and continue nitrification for 3 hours with intermittent stirring; add 5 mL of 20% HNO3 solution of 3.2 mol / L NH4Ac, dilute to 20 mL, and maintain constant temperature and shaking at (22±5)℃ for 30 minutes. Centrifuge at 4000 rpm for 4 minutes, filter the supernatant through a 0.45 μm filter, add 1 mL of concentrated nitric acid to the filtered supernatant and store at 4℃. Add 8 mL of ultrapure water to the residue, shake well, centrifuge at 4000 rpm for 20 minutes, and discard the supernatant.

[0037] Residual state: Take out the previous stage residue, dry it at 105℃ and digest it. Refer to the total determination method for the determination procedure to extract the heavy metals in the residual state.

[0038] The concentration of heavy metal ions in the supernatant after filtration in each of the above steps was determined by flame atomic absorption spectrometry (F-AAS). The F-AAS test conditions were: wavelength 283.3 nm; slit width 1.3 mm; lamp current 7.5 mA; flame type Air-C2H2; fuel gas flow rate 2 L / min; oxidizing gas flow rate 15 L / min. Standard solutions with concentrations of 0, 1, 5, 10, 15, and 20 mg / L were measured, and standard curves were automatically generated by the flame atomic absorption spectrometer.

[0039] Examples of preparation of compound microbial agents Example 1

[0040] A compound microbial agent is prepared through the following steps: (1) Crush the dried lotus pod straw, pass it through a 100-mesh sieve, soak it in a 2% sodium hydroxide solution, heat it to 70℃, and heat-treat it for 2 hours. After treatment, wash it repeatedly with clean water until the pH of the wash solution is 7. Weigh 100g of straw powder after alkali dewaxing treatment, mix it with 1L of 3wt% sodium alginate solution, and stir at high speed to make a uniform adsorption slurry.

[0041] (2) Weigh 100g of porous ceramsite with a particle size of 2-5mm and mix it with the adsorption slurry for 30min to ensure that the ceramsite surface and pores are fully coated with slurry. Pour the coated ceramsite into a 5% calcium chloride solution and crosslink and solidify for 30min. After removing it, rinse it slightly with deionized water and drain it to obtain the composite carrier.

[0042] (3) Weigh 100g of the composite carrier and add it to 1L of liquid culture medium. Sterilize at 121℃ for 15min. After cooling, inoculate with activated Bacillus cereus and Pseudomonas fluorescens, respectively, at an inoculation volume ratio of 1:0.5. Incubate at 32℃ and 180r / min with shaking for 48h. After incubation, freeze-dry for 36h to obtain the composite microbial agent, with a total viable count of 1.0×10⁻⁶. 10 CFU / g level.

[0043] Example 2

[0044] A compound microbial agent is prepared through the following steps: (1) Crush the dried lotus pod straw, pass it through a 100-mesh sieve, soak it in a 2% sodium hydroxide solution, heat it to 70℃, and heat-treat it for 2 hours. After treatment, wash it repeatedly with clean water until the pH of the wash solution is 7. Weigh 100g of straw powder after alkali dewaxing treatment, mix it with 1L of 2% (w / v) κ-carrageenan solution, and stir it continuously in a 65℃ water bath for 1 hour to prepare a uniform adsorption slurry.

[0045] (2) Weigh 100g of porous ceramsite with a particle size of 2-5mm and mix it with the adsorption slurry for 30min to ensure that the ceramsite surface and pores are fully coated with slurry. Take out the coated ceramsite, let it stand and cool at room temperature for 1h, then immerse it in 0.3mol / L KCl solution and treat it at 4℃ for 30min. After rinsing with deionized water, the composite carrier is obtained.

[0046] (3) Weigh 100g of the composite carrier and add it to 1L of liquid culture medium. Sterilize at 121℃ for 15min. After cooling, inoculate with activated Bacillus cereus and Pseudomonas fluorescens, respectively, at an inoculation volume ratio of 1:0.5. Incubate at 32℃ and 180r / min with shaking for 48h. After incubation, freeze-dry for 36h to obtain the composite microbial agent, with a total viable count of 1.0×10⁻⁶. 10 CFU / g level.

[0047] Example 3

[0048] A compound microbial agent is prepared through the following steps: (1) Crush the dried lotus pod straw, pass it through a 100-mesh sieve, soak it in a 2% sodium hydroxide solution, heat it to 70°C, heat it for 2 hours, and after the treatment is completed, wash it repeatedly with water until the pH of the washing liquid is 7, and obtain the straw powder treated by alkali dewaxing.

[0049] (2) Weigh 30g of sodium alginate and dissolve it in 500mL of deionized water at 50℃. Stir until completely dissolved to obtain a 6% (w / v) sodium alginate solution. Weigh 20g of κ-carrageenan and dissolve it in 500mL of deionized water at 85℃. Stir continuously in an 80℃ water bath until completely dissolved to obtain a 4% (w / v) κ-carrageenan solution. Under the condition of continuous stirring and maintaining the temperature of the κ-carrageenan solution at 70-75℃, slowly pour the sodium alginate solution into the κ-carrageenan solution. After mixing, continue stirring in a 70℃ water bath for 30min to obtain a uniform sodium alginate and κ-carrageenan composite gelling agent solution. Weigh 100g of straw powder after alkali dewaxing treatment and mix it with the composite gelling agent solution. Stir at high speed to prepare a uniform adsorption slurry.

[0050] (3) Weigh 100g of porous ceramsite with a particle size of 2-5mm and mix it with the adsorption slurry for 30min to ensure that the ceramsite surface and pores are fully coated with slurry. Take out the coated ceramsite and let it stand and cool at 4℃ for 2h. Then immerse it in a mixed solution containing 0.1mol / L calcium chloride and 0.05mol / L potassium chloride and crosslink and solidify it at 4℃ for 4h. After taking it out, rinse it slightly with deionized water and drain it to obtain the composite carrier.

[0051] (4) Weigh 100g of the composite carrier and add it to 1L of liquid culture medium. Sterilize at 121℃ for 15min. After cooling, inoculate with activated Bacillus cereus and Pseudomonas fluorescens, respectively, at an inoculation volume ratio of 1:0.5. Incubate at 32℃ and 180r / min with shaking for 48h. After incubation, freeze-dry for 36h to obtain the composite microbial agent, with a total viable count of 1.0×10⁻⁶. 10 CFU / g level.

[0052] Comparative Example 1 The microbial inoculum used for comparison was obtained through the following preparation steps: (1) Crush the dried lotus pod straw, pass it through a 100-mesh sieve, soak it in a 2% sodium hydroxide solution, heat it to 70°C, heat it for 2 hours, and after the treatment is completed, wash it repeatedly with water until the pH of the washing liquid is 7, and obtain the straw powder treated by alkali dewaxing.

[0053] (2) Weigh 100g of straw powder after alkali dewaxing treatment and 100g of porous ceramsite with a particle size of 2-5mm, mix them using a simple physical dry method, add the mixture to 1L of liquid culture medium, sterilize at 121℃ for 15min, and after cooling, inoculate with activated Bacillus cereus and Pseudomonas fluorescens respectively, with an inoculation volume ratio of 1:0.5. Incubate at 32℃ and 180r / min with shaking for 48h. After the incubation is completed, freeze-dry for 36h to obtain the compound microbial agent, with a total viable count of 1.0×10⁻⁶. 10 CFU / g level.

[0054] Comparative Example 2 The microbial inoculum used for comparison was obtained through the following preparation steps: (1) Take liquid culture medium, sterilize at 121℃ for 15 min, cool, and then inoculate with activated Bacillus cereus and Pseudomonas fluorescens at a volume ratio of 1:0.5. Incubate at 32℃ and 180 r / min for 48 h with shaking. After the culture is completed, centrifuge to collect the bacterial cells, resuspend them in sterile physiological saline, and prepare a high-concentration compound bacterial suspension.

[0055] (2) Weigh 30g of sodium alginate, dissolve it in 1L of deionized water at 50℃, stir until completely dissolved to obtain a 3% (w / v) sodium alginate solution, sterilize the solution and cool it to room temperature.

[0056] (3) Under sterile conditions, the composite bacterial suspension prepared in step (1) and the sodium alginate solution in step (2) are mixed evenly at a volume ratio of 1:4 to form a bacterial-gel mixture.

[0057] (4) The above mixture was injected dropwise into a sterile 0.1 mol / L calcium chloride solution that was constantly stirred using a syringe. The mixture was allowed to stand at 4°C for 24 h for cross-linking and then freeze-dried under vacuum for 24 h to obtain sodium alginate-encapsulated granular bacterial agent.

[0058] Examples of using compound microbial agents to remediate heavy metal contaminated soil Example 4

[0059] A lead-zinc tailings mine in Hunan Province was used as a soil sample contaminated with heavy metals. The sample was spread out to a thickness of 5 cm, and the composite microbial agent prepared in Example 1 was added to the soil sample at a concentration of 50 g / m³. 2 Mix thoroughly and then place in an environment with a temperature of 25±5℃ and a relative humidity of 75±5% for 2 months.

[0060] After two months of cultivation, the changes in heavy metal content and speciation in the soil before and after remediation were measured. The results are as follows: Table 1. Changes in different forms of Pb before and after soil remediation Results analysis: After applying the bacterial agent from Example 1, the bioavailable form of Pb (exchangeable form + carbonate-bound form) decreased from 58.6% to 35.0%, with a significant reduction in the proportion of exchangeable form. The proportion of residual form increased from 20.5% to 50.9%, indicating that the carrier of sodium alginate gel can effectively support microorganisms in converting active Pb into a stable form.

[0061] Table 2 Changes in different forms of Zn before and after soil remediation Results analysis: The bioavailable form of Zn decreased from 42.5% to 28.1%, while the residual form increased significantly from 40.1% to 60.8%, showing a good stabilization effect.

[0062] Table 3. Changes in different forms of Cd before and after soil remediation Results analysis: The bioavailable form of Cd decreased from 18.4% to 14.0%, while the residual form increased from 67.5% to 75.4%, indicating a clear remediation trend.

[0063] Example 5

[0064] A lead-zinc tailings mine in Hunan Province was used as a soil sample contaminated with heavy metals. The sample was spread out to a thickness of 5 cm, and the composite microbial agent prepared in Example 2 was added to the soil sample at a concentration of 50 g / m³. 2 Mix thoroughly and then place in an environment with a temperature of 25±5℃ and a relative humidity of 75±5% for 2 months.

[0065] After two months of cultivation, the changes in heavy metal content and speciation in the soil before and after remediation were measured. The results are as follows: Table 4. Changes in different forms of Pb before and after soil remediation Results analysis: The repair effect of the bacterial agent in Example 2 on Pb was comparable to that in Example 1, with the effective state decreasing to 33.6% and the residual state increasing to 53.6%, indicating that the κ-carrageenan system can also construct an effective microbial colonization environment.

[0066] Table 5. Changes in different forms of Zn before and after soil remediation Results analysis: The repair effect of Zn was slightly better than that of the compound microbial agent in Example 1. The effective state decreased to 25.9% and the residual state reached 64.0%. This may be related to the thermal reversibility of κ-carrageenan gel and the more suitable microenvironment provided by potassium ion enhancement, which is more conducive to microbial metabolic activities.

[0067] Table 6. Changes in different forms of Cd before and after soil remediation Results analysis: The bioavailable form of Cd decreased from 18.4% to 13.4%, while the residual form increased from 67.5% to 76.6%. The remediation effect was clear and similar to that of the bacterial agent in Example 1, further confirming the universal remediation capability of this carrier system for various heavy metals.

[0068] Example 6

[0069] A lead-zinc tailings mine in Hunan Province was used as a soil sample contaminated with heavy metals. The sample was spread out to a thickness of 5 cm, and the composite microbial agent prepared in Example 3 was added to the soil sample at a concentration of 50 g / m³. 2 Mix thoroughly and then place in an environment with a temperature of 25±5℃ and a relative humidity of 75±5% for 2 months.

[0070] After two months of cultivation, the changes in heavy metal content and speciation in the soil before and after remediation were measured. The results are as follows: Table 7. Changes in different forms of Pb before and after soil remediation Results Analysis: After application of the compound microbial agent of this application, the content of bioavailable Pb (exchangeable + carbonate-bound) in the soil decreased significantly, from 58.6% to 29.7%, a reduction of 49.3%. Among them, the proportion of exchangeable Pb, which has the highest activity and the strongest toxicity, decreased from 31.7% to 15.6%. At the same time, the proportion of stable residual Pb increased significantly, from 20.5% to 57.9%, an increase of 182%. This indicates that this agent can efficiently drive the transformation of Pb from its active form to its inert form, greatly reducing the environmental risk and bioavailability of Pb.

[0071] Table 8. Changes in different forms of Zn before and after soil remediation Results analysis: After remediation, the total bioavailable form of Zn decreased significantly, from 42.5% to 21.4%, a reduction of nearly half. In particular, exchangeable Zn decreased from 28.9% to 12.5%. Conversely, residual Zn became the dominant form, with its proportion increasing dramatically from 40.1% to 70.3%, an increase of 75%. This confirms that the compound microbial agent of this application has a strong stabilizing ability for Zn and can effectively alleviate Zn toxicity and migration.

[0072] Table 9. Changes in different forms of Cd before and after soil remediation Results Analysis: Although the initial residual Cd content in the original soil was relatively high, this microbial agent further optimized its speciation. After remediation, the bioavailable Cd content decreased from 18.4% to 12.0%, a reduction of 34.8%. Exchangeable Cd decreased from 11.1% to 6.9%. The proportion of residual Cd was consolidated and improved, increasing from 67.5% to 82.1%, an increase of 21.6%. This indicates that the microbial agent also has a clear passivation effect on Cd, further reducing its ecological risk.

[0073] Comparing the test results of Examples 4-6, all three composite microbial agents of this application using different gelling agent systems showed significant heavy metal remediation effects, but the effects exhibited a gradient, demonstrating the crucial impact of optimizing the carrier construction strategy on the final performance. Example 3, using a composite gelling agent of sodium alginate and κ-carrageenan, achieved improvements in the residual state of Pb, Zn, and Cd of 182%, 75%, and 21.6%, respectively, all higher than Examples 1 and 2. This is attributed to the synergistic effect of sodium alginate and κ-carrageenan: sodium alginate, through Ca... 2+ Crosslinking forms a stable structure, providing initial strength and embedding effect; the thermally reversible gelation properties of κ-carrageenan and K + The resulting dense network further enhances the water-holding capacity and mechanical stability of the carrier. The combination of these two elements forms a more stable, water-retaining, and biocompatible three-dimensional network, which not only provides excellent physical protection for functional microorganisms, but its optimized pore structure also facilitates the transport of nutrients and metabolites, thereby maximizing the synergistic repair function of the microbial community and achieving the most efficient and stable passivation of various heavy metals.

[0074] Comparative Example 3 A lead-zinc tailings mine in Hunan Province was used as a soil sample contaminated with heavy metals. The sample was spread out to a thickness of 5 cm. The composite microbial agent prepared in Comparative Example 1 was added to the soil sample at a concentration of 50 g / m³. 2 Mix thoroughly and then place in an environment with a temperature of 25±5℃ and a relative humidity of 75±5% for 2 months.

[0075] After two months of cultivation, the changes in heavy metal content and speciation in the soil before and after remediation were measured. The results are as follows: Table 10 Changes in different forms of Pb before and after soil remediation Results Analysis: The remediation effect of the microbial agent using a simple physical mixing carrier was limited. The bioavailable Pb content decreased only from 58.6% to 46.2%, while the residual content increased to 37.3%, significantly worse than in Examples 4-6. This indicates that the carrier structure lacking a gelling agent is loose, making it easy for microorganisms to be lost and difficult to colonize and function in harsh soils for a long time.

[0076] Table 11 Changes in different forms of Zn before and after soil remediation Results analysis: The effective state of Zn decreased to 37.7%, while the residual state increased to 49.2%, which is far less effective than the embodiments of this application.

[0077] Table 12 Changes in different forms of Cd before and after soil remediation Results analysis: The repair effect on Cd was weak, with the residual state only slightly increasing to 71.5%, further confirming the key influence of the carrier structure on the repair effect.

[0078] Comparative Example 4 A lead-zinc tailings mine in Hunan Province was used as a soil sample for heavy metal contamination. The sample was spread out to a thickness of 5 cm. The composite microbial agent prepared in Comparative Example 2 was added to the soil sample at a concentration of 50 g / m³. 2 Mix thoroughly and then place in an environment with a temperature of 25±5℃ and a relative humidity of 75±5% for 2 months.

[0079] After two months of cultivation, the changes in heavy metal content and speciation in the soil before and after remediation were measured. The results are as follows: Table 13 Changes in different forms of Pb before and after soil remediation Results analysis: The alginate-encapsulated microbial agent showed some effect in the early stages of remediation, reducing the bioavailable form of Pb to 39.1% and increasing the residual form to 44.2%, which was better than Comparative Example 3. However, due to the lack of a long-lasting organic carbon source provided by alkali-dewaxed straw and a stable physical shelter provided by porous ceramsite, the microorganisms could not maintain high activity in the nutrient-poor mining soil for a long time, resulting in limited remediation durability and efficiency, and the effect was still significantly inferior to the composite carrier of this application.

[0080] Table 14 Changes in different forms of Zn before and after soil remediation Results analysis: The trend of the effect is similar to that of Pb, with the effective state of Zn decreasing to 30.9% and the residual state increasing to 56.9%. This further demonstrates that single embedding materials have limitations in providing a comprehensive micro-ecological environment that combines "nutritional support" and "structural protection".

[0081] Table 15 Changes in different forms of Cd before and after soil remediation Results analysis: The repair of Cd was between that of Comparative Example 3 and Example 4 of this application, with the effective state decreasing to 15.3% and the residual state increasing to 73.0%. The results further demonstrate the superiority of the design of this application, which combines nutrient slow release (straw), structural support (ceramsite), and fixation protection (gel).

[0082] The above description is merely a preferred embodiment of this application and does not constitute any limitation on this application in any form or substance. It should be noted that those skilled in the art can make several improvements and additions without departing from the method of this application, and these improvements and additions should also be considered within the scope of protection of this application. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of this application are equivalent embodiments of this application; furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of this application still fall within the scope of the technical solution of this application.

Claims

1. A compound microbial agent, characterized in that, It includes a composite microbial strain and a composite carrier for immobilizing the composite microbial strain; The composite carrier is a porous ceramic particle loaded with an adsorbent slurry, which is made by mixing plant straw powder that has undergone alkali dewaxing with a gelling agent solution. The composite microbial strain consists of Bacillus cereus and Pseudomonas fluorescens; The gelling agent solution is an aqueous solution of a mixture of sodium alginate and κ-carrageenan.

2. The compound microbial agent according to claim 1, characterized in that, The ratio of viable Bacillus cereus to viable Pseudomonas fluorescens is 1:(0.5-1), and the total viable count is not less than 1.0 × 10⁻⁶. 9 CFU / g.

3. The compound microbial agent according to claim 1, characterized in that, The porous ceramsite has a particle size of 2-5 mm and a porosity of 40-60%.

4. The compound microbial agent according to claim 1, characterized in that, The plant straw powder is made by crushing aquatic plant straw, which is selected from one or more of lotus pods, water hyacinths, and reeds, and the particle size of the plant straw powder is 100-200 mesh.

5. A method for preparing the composite microbial agent according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Plant straw is dewaxed by alkali and then crushed to obtain plant straw powder. The plant straw powder is mixed with a gelling agent solution to make an adsorption slurry. Porous ceramsite is mixed with the adsorption slurry and then coated. After solidification, a composite carrier is obtained. (2) Add the composite carrier to the liquid culture medium, sterilize it, inoculate it with Bacillus cereus and Pseudomonas fluorescens for fermentation culture, dry the fermented system to obtain solid microbial agent, i.e. composite microbial agent.

6. The method for preparing the composite microbial agent according to claim 5, characterized in that, In step (2), the liquid culture medium contains 5 g / L peptone, 3 g / L beef extract powder, 5 g / L NaCl, and pH 7.

7. The method for preparing the composite microbial agent according to claim 5, characterized in that, In step (2), the fermentation culture temperature is 30-37℃, the culture time is 24-48h, and the drying method is freeze drying.

8. The application of the compound microbial agent according to any one of claims 1-4 in the remediation of heavy metal contaminated soil, wherein the application rate of the compound microbial agent is 50-100 g / m³. 2 .

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