A composite microbial agent, a preparation method thereof and application of the agent in remediation of heavy metal-organic compound contaminated soil

Abstract

This invention belongs to the field of environmental microbiology and soil remediation technology, specifically relating to a composite microbial agent, its preparation method, and its application in the remediation of soils contaminated with heavy metals and organic matter. The composite microbial agent provided by this invention combines sulfur-modified biochar, a composite functional microbial community, and slow-release nutrients in a specific ratio. The sulfur-modified biochar contains sulfur-containing functional groups such as S=O and C-S, enabling specific adsorption and fixation of heavy metals. The composite functional microbial community consists of organic pollutant-degrading bacteria, plant growth-promoting bacteria, and heavy metal-converting bacteria in a specific ratio of viable bacteria, complementing each other to synergistically complete the degradation of organic matter and the conversion of heavy metals. The slow-release nutrient component provides long-term nutritional support. The preparation method of this composite microbial agent is simple and suitable for the remediation of heavy metal-organic matter contaminated soils in industrial, mining, and chemical sites, offering advantages such as high efficiency, long-term effectiveness, low cost, and environmental friendliness.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology technology and soil remediation technology, specifically relating to a composite microbial agent, its preparation method, and its application in the remediation of soils contaminated with heavy metals and organic matter. Background Technology

[0002] With industrialization and urbanization, soils surrounding industrial and mining areas, gas stations, and chemical industrial parks are generally facing complex pollution from heavy metals (such as lead (Pb), cadmium (Cd), and copper (Cu)) and persistent organic pollutants (such as polycyclic aromatic hydrocarbons (PAHs) and total petroleum hydrocarbons (TPH)). This complex pollution effect is multifaceted; the persistent toxicity of heavy metals inhibits the activity of microorganisms that degrade organic matter, while the presence of organic matter can affect the form and mobility of heavy metals, making remediation far more difficult than with single pollution.

[0003] Studies have shown that the synergistic toxicity of heavy metals (such as Pb and Cd) and PAHs significantly inhibits microbial community activity, leading to a decrease in remediation efficiency (Li Wanyi, Yu Weiwei, Yu Qiongyang, et al. Research progress on environmental effects and remediation technologies of soil heavy metal-organic compound pollution [J]. Soil, 2023, 55 (03): 453-463. DOI:10.13758 / j.cnki.tr.2023.03.001.). Single chemical or physical remediation technologies are costly and may generate secondary pollution. Therefore, developing an integrated technology that can respond to compound pollution stress, ensure the survival and activity of functional microorganisms, and simultaneously remove or passivate both types of pollutants has become an urgent need in this field. Summary of the Invention

[0004] The purpose of this invention is to provide a composite microbial agent and its preparation method, as well as its application in the remediation of soil contaminated with heavy metals and organic matter, thereby improving the survival rate, colonization ability and metabolic activity of functional microorganisms in contaminated soil, and achieving simultaneous passivation of heavy metals and efficient degradation of organic matter.

[0005] This invention provides a composite microbial agent, which includes sulfur-modified biochar, a composite functional microbial community, and slow-release nutrients; The mass ratio of the sulfur-modified biochar, the composite functional microbial community, and the slow-release nutrients is (12~16):(0.5~3):1; The composite functional microbial community includes organic pollutant degrading bacteria, plant growth-promoting bacteria, and heavy metal transforming bacteria; the total viable count of the composite functional microbial community is ≥1×10⁻⁶. 10 CFU / mL, the live bacteria ratio of organic pollutant degrading bacteria, plant growth promoting bacteria and heavy metal transforming bacteria is 1:(0.5~1):(0.5~1).

[0006] Preferably, the organic pollutant degrading bacteria include *Aureobacterium tumefaciens* (…). Ochrobactrum sp.); the plant growth-promoting bacteria include Burkholderia (sp.); Burkholderia sp.); the heavy metal transforming bacteria include desulfurized spore-forming Campylobacter (sp.); Desulfosporosinus sp.); The slow-release nutrient comprises calcium alginate-diammonium hydrogen phosphate composite gel microspheres.

[0007] Preferably, the desulfurized Campylobacter includes desulfurized Campylobacter TLRB, with accession number CGMCC No. 41181; The preparation method of the calcium alginate-diammonium hydrogen phosphate composite gel microspheres includes: mixing sodium alginate solution and diammonium hydrogen phosphate and then dripping the mixture into calcium chloride solution for cross-linking and curing.

[0008] Preferably, the method for preparing the sulfur-modified biochar includes: After crushing the straw, the temperature was raised to 500-600℃ at 5-15℃ / min under a nitrogen atmosphere and then pyrolyzed for 2-4 hours to obtain basic biochar. The basic biochar was modified with calcium polysulfide solution, and after washing, drying and sieving, sulfur-modified biochar was obtained.

[0009] Preferably, the mass-to-volume ratio of the basic biochar to the calcium polysulfide solution is 1 g: 10 mL. The concentration of the calcium polysulfide solution is 50~100 mg / mL; the modification temperature is 25~35℃, and the time is 6~12 h; The drying temperature is 105°C; The sieving process uses a 100-mesh sieve, and the material passing through the sieve is collected.

[0010] This invention provides a method for preparing the composite microbial agent described in the above technical solution, comprising the following steps: A premix is ​​prepared by mixing a complex functional microbial community with slow-release nutrients. Sulfur-modified biochar was placed in a vacuum environment for 20-40 minutes, and the premix was added to load the microbial community and nutrients into the carrier pores. After obtaining the premix loaded with sulfur-modified biochar, the vacuum was removed and the mixture was dried to obtain the composite microbial agent.

[0011] Preferably, the vacuum level of the vacuum environment is -0.08 to -0.1 MPa; The drying temperature is 35~40℃; The drying process also includes a granulation step to obtain a granular composite microbial agent; the particle size of the granular composite microbial agent is 1~3mm.

[0012] This invention provides the application of the composite microbial agent described in the above technical solution or the composite microbial agent obtained by the preparation method described in the above technical solution in soil remediation.

[0013] Preferably, the soil is soil contaminated with a combination of heavy metals and organic matter; the heavy metals include one or more of Pb, Cd, and Cu; The organic compounds include polycyclic aromatic hydrocarbons.

[0014] This invention provides a method for remediating soil contaminated with a combination of heavy metals and organic matter, comprising the following steps: Mix the soil contaminated with heavy metals and organic matter to be remediated and the remediation agent at a mass ratio of (50~200):1. After application, maintain the soil moisture content at 60%~80% of field capacity for one week. The repair agent is the composite microbial agent described in the above technical solution or the composite microbial agent obtained by the preparation method described in the above technical solution.

[0015] Beneficial effects: The composite microbial agent provided by this invention combines sulfur-modified biochar, a composite functional microbial community, and slow-release nutrients in a specific ratio. This significantly improves the survival rate, colonization ability, and metabolic activity of functional microorganisms in contaminated soil, achieving simultaneous passivation of heavy metals and efficient degradation of organic matter. Specifically, the sulfur-modified biochar contains sulfur-containing functional groups such as S=O and CS, enabling specific adsorption and fixation of heavy metals. The composite functional microbial community consists of organic pollutant-degrading bacteria, plant growth-promoting bacteria, and heavy metal-converting bacteria in a specific ratio of viable cells, complementing each other and synergistically completing the degradation of organic matter and the conversion of heavy metals. The slow-release nutrient component provides long-term nutritional support. The preparation method of this composite microbial agent is simple and suitable for the remediation of heavy metal-organic compound contaminated soils in industrial, mining, and chemical sites. It has advantages such as high efficiency, long-term effectiveness, low cost, and environmental friendliness.

[0016] Biological Preservation Information Desulfurized Campylobacter TLRB, classified as Desulfurized Campylobacter Desulfosporosinus sp. was deposited on January 29, 2024, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 41181. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1The image shows the appearance of the composite microbial agent obtained in Example 1; Figure 2 SEM images of the composite microbial agents obtained in Comparative Example 1 and Example 1; Figure 3 The image shows the EDS diagram of the composite microbial agent obtained in Example 1. Figure 4 The image shows the FTIR (transmittance-wavenumber) plots of the composite microbial agents obtained in Comparative Example 1 and Example 1. Detailed Implementation

[0019] This invention provides a composite microbial agent, which includes sulfur-modified biochar, a composite functional microbial community, and slow-release nutrients; The mass ratio of the sulfur-modified biochar, the composite functional microbial community, and the slow-release nutrients is (12~16):(0.5~3):1; The composite functional microbial community includes organic pollutant degrading bacteria, plant growth-promoting bacteria, and heavy metal transforming bacteria; the total viable count of the composite functional microbial community is ≥1×10⁻⁶. 10 CFU / mL, the live bacteria ratio of organic pollutant degrading bacteria, plant growth promoting bacteria and heavy metal transforming bacteria is 1:(0.5~1):(0.5~1).

[0020] In one embodiment, the mass ratio of sulfur-modified biochar, composite functional microbial community and slow-release nutrients in this invention is 7:0.7:0.5.

[0021] As one embodiment, the preparation method of the sulfur-modified biochar of the present invention includes: crushing straw and heating it to 500-600°C at 5-15°C / min under a nitrogen atmosphere, and then pyrolyzing it for 2-4 hours to obtain basic biochar; modifying the basic biochar with calcium polysulfide solution, and then washing, drying and sieving to obtain the sulfur-modified biochar.

[0022] In one embodiment, the straw mentioned in this invention includes one or more of soybean straw, corn straw, wheat straw, and rice straw. In one embodiment, the particle size of the crushed straw is 2-5 cm; in another embodiment, the particle size of the crushed straw is 3-4 cm. In one embodiment, the straw is crushed and then pyrolyzed at 550°C under a nitrogen atmosphere at a rate of 10°C / min for 3 hours.

[0023] In one embodiment, the mass-to-volume ratio of the basic biochar and the calcium polysulfide solution is 1 g:10 mL, and the concentration of the calcium polysulfide solution is 50-100 mg / mL; in another embodiment, the concentration of the calcium polysulfide solution is 60-90 mg / mL; in yet another embodiment, the concentration of the calcium polysulfide solution is 70-80 mg / mL. In one embodiment, the modification temperature is 25-35°C; in another embodiment, the modification temperature is 26-30°C; in yet another embodiment, the modification temperature is 28°C. In one embodiment, the modification time is 6-12 h; in another embodiment, the modification time is 7-10 h; in yet another embodiment, the modification time is 8 h. This invention utilizes calcium polysulfide solution for modification, which not only retains the huge specific surface area and rich pore structure of basic biochar, but also successfully introduces sulfur-containing functional groups such as S=O and CS into its surface. These functional groups can specifically coordinate with heavy metal ions or form metal sulfide precipitates, thereby achieving efficient passivation.

[0024] In one embodiment, the washing process of the present invention uses deionized water to wash the modified basic biochar until the filtrate is neutral. In another embodiment, the drying temperature of the present invention is 105°C. In yet another embodiment, the sieving process of the present invention uses a 100-mesh sieve, and the material passing through the sieve is collected.

[0025] In one embodiment, the viable cell ratio of the organic pollutant-degrading bacteria, plant growth-promoting bacteria, and heavy metal-converting bacteria in this invention is 1:1:1. In another embodiment, the organic pollutant-degrading bacteria, plant growth-promoting bacteria, and heavy metal-converting bacteria are cultured to the logarithmic growth phase, the cells are collected by centrifugation, resuspended in sterile physiological saline, and mixed to obtain the aforementioned composite functional microbial community. This invention combines organic pollutant-degrading bacteria, plant growth-promoting bacteria, and heavy metal-converting bacteria in a certain proportion, resulting in complementary functions and a synergistic effect.

[0026] As one embodiment, the organic pollutant degrading bacteria of the present invention include *Ailuropoda spp.* The *Ailuropoda spp.* described in this invention exhibits multiple tolerances to Pb, Cd, and Cu, and can degrade polycyclic aromatic hydrocarbons. The *Ailuropoda spp.* used in the specific embodiments of this invention was purchased from Shanghai Guyan Industrial Co., Ltd., product number GOY-5144.

[0027] As one embodiment, the plant growth-promoting bacteria of this invention include Burkholderia. The Burkholderia of this invention possesses the ability to solubilize phosphorus, fix nitrogen, and secrete indoleacetic acid, thus improving soil microecology and indirectly promoting the activity of functional bacterial communities. The Burkholderia used in the specific embodiments of this invention was purchased from the China Industrial Microbial Culture Collection Center (CICC), with the catalog number CICC 10877.

[0028] In one embodiment, the heavy metal transforming bacteria of the present invention include *Campylobacter desulfurization*. In another embodiment, the *Campylobacter desulfurization* of the present invention includes *Campylobacter desulfurization* TLRB, with accession number CGMCC No. 41181. The *Campylobacter desulfurization* TLRB of the present invention can reduce sulfate to sulfide (H₂S / S) under anaerobic or microaerobic conditions using organic matter as an electron donor. 2- This leads to the combination of free heavy metal ions to form extremely insoluble sulfide crystals (such as PbS and CdS), fundamentally reducing the bioavailability and mobility of heavy metals.

[0029] In one embodiment, the slow-release nutrient of the present invention comprises calcium alginate-diammonium hydrogen phosphate composite gel microspheres. In another embodiment, the preparation method of the calcium alginate-diammonium hydrogen phosphate composite gel microspheres of the present invention includes: mixing sodium alginate solution and diammonium hydrogen phosphate, and then dripping the mixture into a calcium chloride solution for cross-linking and solidification. In one embodiment, the concentration of the sodium alginate solution of the present invention is 20-80 mg / mL; in another embodiment, the concentration of the sodium alginate solution of the present invention is 50 mg / mL. In one embodiment, the volume-to-mass ratio of the sodium alginate solution to diammonium hydrogen phosphate of the present invention is 5-15 mL:0.5-1.5 g; in another embodiment, the volume-to-mass ratio of the sodium alginate solution to diammonium hydrogen phosphate of the present invention is 10 mL:1 g. In one embodiment, the concentration of the calcium chloride solution of the present invention is 10-30 mg / mL; in another embodiment, the concentration of the calcium chloride solution of the present invention is 20 mg / mL. In another embodiment, the present invention mixes sodium alginate solution and diammonium hydrogen phosphate and then adds it dropwise to calcium chloride solution to form gel microspheres encapsulating nitrogen and phosphorus nutrients.

[0030] This invention provides a method for preparing the composite microbial agent described in the above technical solution, comprising the following steps: A premix is ​​prepared by mixing a complex functional microbial community with slow-release nutrients. Sulfur-modified biochar was placed in a vacuum environment for 20-40 minutes, and the premix was added to load the microbial community and nutrients into the carrier pores. After obtaining the premix loaded with sulfur-modified biochar, the vacuum was removed and the mixture was dried to obtain the composite microbial agent.

[0031] In one embodiment, the vacuum degree of the vacuum environment described in this invention is -0.08 to -0.1 MPa. In one embodiment, the drying temperature described in this invention is 35 to 40°C; in another embodiment, the drying temperature described in this invention is 36 to 38°C. By adding the premix to sulfur-modified biochar under vacuum conditions, this invention allows the bacterial community and slow-release nutrients to be loaded to the maximum extent within the carrier pores.

[0032] In one embodiment, the present invention further includes a granulation step after drying to obtain a granular composite microbial agent; the particle size of the granular composite microbial agent is 1~3mm.

[0033] In specific applications, the composite microbial agent of the present invention has the following advantages: (1) Targeted passivation and microenvironment creation: Sulfur-modified biochar takes the lead in exerting its effect, and its surface sulfur-containing functional groups are effective against heavy metal ions (such as Pb). 2+ Cd 2+ (1) Specific adsorption and chemical fixation, transforming it from a highly active free state into a highly inert sulfide-bound state, rapidly reducing the concentration of heavy metals in the soil solution, creating a "low-toxicity" or "non-toxic" safe microenvironment for subsequent microbial implantation. (2) Synergistic degradation and micro-enrichment effect: The huge specific surface area of ​​sulfur-modified biochar simultaneously produces strong adsorption of organic pollutants (such as PAHs), causing them to be relatively enriched around the carrier, greatly improving the local contact concentration and reaction efficiency of pollutants and degrading bacteria, thereby accelerating the degradation process. (3) Long-term remediation mechanism: Slow-release nutrients can continuously and stably provide microorganisms with essential nutrients such as nitrogen and phosphorus for several weeks or even months, avoiding the waste and later depletion caused by pulsed nutrient supply, ensuring the long-term growth stability and remediation efficiency of the microbial community. At the same time, growth-promoting bacteria indirectly enhance the function of the entire microbial community by secreting substances such as plant hormones. (4) Waste resource utilization and green sustainability: Using straw as the main raw material, high-value-added resource utilization of agricultural waste is realized. The cost is low and the entire restoration process is free of secondary pollution, which perfectly fits the development concept of green circular economy.

[0034] Based on the effects of the composite microbial agent provided by the present invention, the present invention provides the application of the composite microbial agent described in the above technical solution or the composite microbial agent obtained by the preparation method described in the above technical solution in soil remediation.

[0035] In one embodiment, the soil described in this invention is soil contaminated with a combination of heavy metals and organic matter. In another embodiment, the heavy metals described in this invention include one or more of Pb, Cd, and Cu.

[0036] In one embodiment, the concentration of Pb in the heavy metal-organic compound contaminated soil of the present invention is 450-800 mg / kg; in another embodiment, the concentration of Pb in the heavy metal-organic compound contaminated soil of the present invention is 500-700 mg / kg; in yet another embodiment, the concentration of Pb in the heavy metal-organic compound contaminated soil of the present invention is 600 mg / kg. In one embodiment, the source of Pb in the heavy metal-organic compound contaminated soil of the present invention is lead nitrate.

[0037] In one embodiment, the concentration of Cd in the heavy metal-organic compound contaminated soil of the present invention is 120-200 mg / kg; in another embodiment, the concentration of Cd in the heavy metal-organic compound contaminated soil of the present invention is 150-180 mg / kg; in yet another embodiment, the concentration of Cd in the heavy metal-organic compound contaminated soil of the present invention is 160 mg / kg. In one embodiment, the source of Cd in the heavy metal-organic compound contaminated soil of the present invention is cadmium chloride.

[0038] In one embodiment, the concentration of Cu in the heavy metal-organic compound contaminated soil of the present invention is 360~1000 mg / kg; in another embodiment, the concentration of Cu in the heavy metal-organic compound contaminated soil of the present invention is 400~800 mg / kg; in yet another embodiment, the concentration of Cu in the heavy metal-organic compound contaminated soil of the present invention is 500~700 mg / kg; in yet another embodiment, the concentration of Cu in the heavy metal-organic compound contaminated soil of the present invention is 600 mg / kg. In one embodiment, the source of Cu in the heavy metal-organic compound contaminated soil of the present invention is copper sulfate.

[0039] In one embodiment, the organic matter described in this invention includes polycyclic aromatic hydrocarbons (PAHs). In one embodiment, the concentration of PAHs in the heavy metal-organic compound contaminated soil is 8-300 mg / kg; in another embodiment, the concentration is 10-250 mg / kg; in yet another embodiment, the concentration is 50-200 mg / kg; in yet another embodiment, the concentration is 60-180 mg / kg; in yet another embodiment, the concentration is 80-160 mg / kg; in yet another embodiment, the concentration is 100-150 mg / kg. In one embodiment, the PAH is benzo[a]pyrene.

[0040] This invention provides a method for remediating soil contaminated with a combination of heavy metals and organic matter, comprising the following steps: Mix the soil contaminated with heavy metals and organic matter to be remediated and the remediation agent at a mass ratio of (50~200):1. Maintain the soil moisture content at 60%~80% of field capacity for one week after application. The remediation agent is the compound microbial agent described in the above technical solution or the compound microbial agent prepared by the preparation method described in the above technical solution.

[0041] In one embodiment, the mass ratio of the soil to be remediated due to heavy metal-organic composite pollution to the remediation agent is (60~180):1; in another embodiment, the mass ratio is (80~150):1; and in yet another embodiment, the mass ratio is (100~120):1. In one embodiment, the remediation agent is added to the surface of the heavy metal-organic composite pollution to be remediated and then mixed with the top 0-20cm of soil by rotary tillage.

[0042] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, provides a composite microbial agent, its preparation method, and its application in the remediation of soil contaminated with heavy metals and organic matter. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0043] Example 1 Preparation of compound microbial agents 1. Carrier Preparation: Dry and pulverized corn stalks were pyrolyzed at 550℃ for 3 hours under a N2 atmosphere at a rate of 10℃ / min to obtain basic biochar. The basic biochar was then soaked in an 8% calcium polysulfide solution and shaken at 30℃ for 8 hours. The mass-to-volume ratio of basic biochar to 8% calcium polysulfide solution was 1 g:10 mL. The biochar was washed, dried, and passed through a 100-mesh sieve. The material passing through the sieve was collected to obtain sulfur-modified biochar (S-BC), which was then set aside.

[0044] 2. Microbial community expansion and compounding: *Bacillus pallida*, *Burkholderia*, and *Campylobacter desulfosporidum* (CGMCC No. 41181) were cultured separately in LB medium to OD. 600 The value was 1.0. The bacterial cells were collected by centrifugation and mixed with live bacteria at a ratio of 1:1:1 to obtain a bacterial suspension.

[0045] 3. Preparation of nutrient microspheres: Prepare a 5% sodium alginate solution, add diammonium hydrogen phosphate powder to dissolve it to a final concentration of 1 g / mL, and obtain a mixture. Use a syringe to dropwise add the mixture into a 2% CaCl2 solution to form spherical gel particles; the volume ratio of the mixture to the 2% CaCl2 solution is 1:5.

[0046] 4. Vacuum Loading and Granulation: Place S-BC in a vacuum dryer and evacuate to -0.09 MPa. Inject the mixture of bacterial suspension and nutrient microspheres and let it stand for 30 minutes. After releasing the vacuum, remove the material and dry it at 37℃. Then, granulate it using a granulator (particle size approximately 2 mm), seal and package it to obtain the composite microbial agent, which looks like... Figure 1 As shown; the mass ratio (dry weight ratio) of S-BC, bacterial suspension and nutrient microspheres is 70g:7g:5g. The dry weight of the bacterial suspension is based on the dry weight of the complex functional microbial community in the bacterial suspension. The dry weight of the nutrient microspheres is 10% of the wet weight. That is, the ratio of the dry weight of S-BC, the dry weight of the bacterial suspension and the wet weight of the nutrient microspheres is 70g:7g:50g.

[0047] Comparative Example 1 The composite microbial agent was prepared according to the method in Example 1, with the only difference being that the biochar was not subjected to sulfur modification treatment, and sulfur-modified biochar (S-BC) was replaced with basic biochar.

[0048] Test Example 1 1. The composite microbial inoculant obtained in Comparative Example 1 (denoted as unmodified biochar-SEM) and the composite microbial inoculant obtained in Example 1 (denoted as sulfur-modified biochar-SEM) were observed by SEM. The results are as follows: Figure 2 As shown, the composite microbial agent prepared based on sulfur-modified biochar (S-BC) has a multi-microporous structure of about 2~4μm, a smooth surface, and a large specific surface area, thus exhibiting good adsorption effect.

[0049] 2. The composite microbial inoculant obtained in Example 1 (denoted as sulfur-modified biochar-EDS) was subjected to EDS observation. The results are as follows: Figure 3 As shown, the composite microbial agent prepared based on sulfur-modified biochar (S-BC) was successfully loaded with sulfur, and the S content was uniform and sufficient. This structure is conducive to the full exposure of sulfur-containing functional groups, and can exhibit excellent performance in the adsorption of heavy metals and organic matter.

[0050] 3. The FTIR (transmittance-wavenumber) values ​​of the composite microbial agent obtained in Comparative Example 1 (denoted as BC) and the composite microbial agent obtained in Example 1 (denoted as SBC) were measured respectively. The results are as follows: Figure 4 As shown, the FTIR spectra of the composite microbial agent obtained in Comparative Example 1 show that wavenumber 3055 corresponds to the alkyl CH stretching vibration, and 1537 corresponds to the C=C / C=S stretching vibration. The FTIR spectra of the composite microbial agent obtained in Example 1 show that wavenumber 3055 corresponds to the alkyl CH stretching vibration, 1537 / 1423 corresponds to the C=S stretching vibration, 1211 corresponds to the S=O bending vibration, and 632 corresponds to the CS stretching vibration. Both the composite microbial agents obtained in Comparative Example 1 and Example 1 contain hydroxyl groups, which indicates good adsorption performance. The composite microbial agent obtained in Example 1, with the addition of sulfur, is richer in S=O and CS bonds than the composite microbial agent in Comparative Example 1, resulting in even better adsorption performance.

[0051] Comparative Example 2 The compound microbial agent was prepared according to the method in Example 1, with the only difference being that no *Bacillus annuus* was added.

[0052] Comparative Example 3 The compound microbial agent was prepared according to the method of Example 1, with the only difference being that Burkholderia was not added.

[0053] Comparative Example 4 The compound microbial agent was prepared according to the method in Example 1, the only difference being that desulfurized Campylobacter spores were not added.

[0054] Comparative Example 5 The composite microbial agent was prepared according to the method of Example 1, with the only difference being that the nutrient microspheres were replaced with blank microspheres, that is, diammonium hydrogen phosphate was not added, only the physical structure was provided.

[0055] Example 2 Validation of the remediation effect of compound microbial agents on simulated heavy metal-PAHs co-contaminated soil 1. Preparation of contaminated soil: Take clean garden soil and artificially add lead nitrate, cadmium chloride, copper sulfate and benzo[a]pyrene (BaP) (as PAHs model) to make the initial concentrations of Pb, Cd, Cu and naphthalene in the soil 800 mg / kg, 200 mg / kg, 1000 mg / kg and 300 mg / kg, respectively.

[0056] 2. Experimental setup: Different treatment groups were set up, with 3 replicates for each group; Group A (blank): Blank control (no formulation added); Group B (S-BC only): The contaminated soil and the S-BC obtained in Example 1 were mixed evenly, with a mass ratio of contaminated soil to S-BC of 100:1. Group C (complete components): The contaminated soil and the composite microbial agent obtained in Example 1 are mixed evenly, with a mass ratio of contaminated soil to composite microbial agent of 100:1. Group D (lacking Bacillus anthocyanin): The contaminated soil and the compound microbial agent obtained in Comparative Example 2 were mixed evenly, with a mass ratio of contaminated soil to compound microbial agent of 100:1. Group E (Burkholderia septembrittlement): The contaminated soil and the compound microbial agent obtained in Comparative Example 3 were mixed evenly, with a mass ratio of contaminated soil to compound microbial agent of 100:1. Group F (deficient desulfurization bacteria): The contaminated soil and the compound microbial agent obtained in Comparative Example 4 were mixed evenly, with a mass ratio of contaminated soil to compound microbial agent of 100:1. Group G (Nutrient Deficiency): The contaminated soil and the compound microbial agent obtained in Comparative Example 5 were mixed evenly, with a mass ratio of contaminated soil to compound microbial agent of 100:1.

[0057] 3. Cultivation and Monitoring: Soil samples from each group were placed in a 25℃ constant temperature incubator, and water was added regularly to maintain humidity. Samples were taken on day 0 and day 30 for testing, and the results are shown in Table 1.

[0058] Table 1. Remediation effects of different treatment groups on contaminated soil

[0059] As shown in Table 1, after 30 days, the proportion of acid-extractable Pb (the most bioavailable form) in the soil of group C decreased from 45% to 12%, the proportion of acid-extractable Cd (the most bioavailable form) decreased from 64% to 22%, and the proportion of acid-extractable Cu (the most bioavailable form) decreased from 51% to 17%, while the proportions of residual and sulfide Pb, Cd, and Cu increased significantly. After 30 days, the degradation rate of naphthalene in the soil of group C reached 92%, significantly higher than that of group B (35%) and group A (<5%). The enzyme activity of group C remained at a high level throughout the experimental period, approximately four times that of group A. The composite microbial agent obtained in Example 1 can effectively passivate heavy metals and efficiently degrade organic matter. When *Bacillus pachymansi* was missing (Group D), the degradation rate of benzo[a]pyrene decreased by 37%, proving that it dominated the degradation of organic matter; when *Campylobacter desulfurization* was missing (Group F), the reduction in acid-extractable Pb was insufficient and no sulfide form was generated, highlighting its core role in heavy metal transformation; the absence of nutrient microspheres (Group G) led to the overall inhibition of microbial activity, confirming the necessity of slow-release nutrients for long-term repair.

[0060] Example 3 Experiment on compound microbial agents in contaminated soil at a former industrial and mining site 1. Test site: A decommissioned coking plant site in North China, with severe historical soil pollution. Typical pollutants are Pb: 450 mg / kg, Cd: 120 mg / kg, Cu: 360 mg / kg, and benzo[a]pyrene (BaP): 8 mg / kg.

[0061] 2. The compound microbial agent obtained in Example 1 was added to the surface of the test site at a mass ratio of 1:150 and mixed with the top 0-20cm soil by rotary tillage. After 6 months of remediation, the passivation rate of heavy metals was: Pb>91%, Cd>98%, Cu>88%; and the degradation rate of benzo[a]pyrene (BaP) was >95%.

[0062] 3. Ecological Restoration: A mung bean germination experiment was conducted on the restored soil. The results showed that the germination rate recovered from 40% before restoration to 85%.

[0063] After the contaminated soil was remediated using the compound microbial agent obtained in Example 1, all indicators were better than the relevant risk control standards.

[0064] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A compound microbial agent, characterized in that, The composite microbial agent includes sulfur-modified biochar, a composite functional microbial community, and slow-release nutrients; The mass ratio of the sulfur-modified biochar, the composite functional microbial community, and the slow-release nutrients is (12~16):(0.5~3):1; The composite functional microbial community includes organic pollutant degrading bacteria, plant growth-promoting bacteria, and heavy metal transforming bacteria; the total viable count of the composite functional microbial community is ≥1×10⁻⁶. 10 CFU / mL, the live bacteria ratio of organic pollutant degrading bacteria, plant growth promoting bacteria and heavy metal transforming bacteria is 1:(0.5~1):(0.5~1).

2. The compound microbial agent according to claim 1, characterized in that, The organic pollutant degrading bacteria include *Aureobacterium acnes* (… Ochrobactrum sp.); the plant growth-promoting bacteria include Burkholderia (sp.); Burkholderia sp.); the heavy metal transforming bacteria include desulfurized spore-forming Campylobacter (sp.); Desulfosporosinus sp.); The slow-release nutrient comprises calcium alginate-diammonium hydrogen phosphate composite gel microspheres.

3. The compound microbial agent according to claim 2, characterized in that, The desulfurized Campylobacter includes Desulfurized Campylobacter TLRB, with accession number CGMCC No. 41181; The preparation method of the calcium alginate-diammonium hydrogen phosphate composite gel microspheres includes: mixing sodium alginate solution and diammonium hydrogen phosphate and then dripping the mixture into calcium chloride solution for cross-linking and curing.

4. The compound microbial agent according to claim 1, characterized in that, The method for preparing the sulfur-modified biochar includes: After crushing the straw, the temperature was raised to 500-600℃ at 5-15℃ / min under a nitrogen atmosphere and then pyrolyzed for 2-4 hours to obtain basic biochar. The basic biochar was modified with calcium polysulfide solution, and after washing, drying and sieving, sulfur-modified biochar was obtained.

5. The compound microbial agent according to claim 4, characterized in that, The mass-to-volume ratio of the basic biochar to the calcium polysulfide solution is 1 g: 10 mL; The concentration of the calcium polysulfide solution is 50~100 mg / mL; the modification temperature is 25~35℃, and the time is 6~12h; The drying temperature is 105°C; The sieving process uses a 100-mesh sieve, and the material passing through the sieve is collected.

6. The method for preparing the composite microbial agent according to any one of claims 1 to 5, characterized in that, Includes the following steps: A premix is ​​prepared by mixing a complex functional microbial community with slow-release nutrients. Sulfur-modified biochar was placed in a vacuum environment for 20-40 minutes, and the premix was added to load the microbial community and nutrients into the carrier pores. After obtaining the premix loaded with sulfur-modified biochar, the vacuum was removed and the mixture was dried to obtain the composite microbial agent.

7. The preparation method according to claim 6, characterized in that, The vacuum level of the vacuum environment is -0.08 to -0.1 MPa; The drying temperature is 35~40℃; The drying process also includes a granulation step to obtain a granular composite microbial agent; the particle size of the granular composite microbial agent is 1~3mm.

8. The application of the composite microbial agent according to any one of claims 1 to 5 or the composite microbial agent obtained by the preparation method according to claim 6 or 7 in soil remediation.

9. The application according to claim 8, characterized in that, The soil is a complex polluted soil containing heavy metals and organic matter; the heavy metals include one or more of Pb, Cd, and Cu. The organic compounds include polycyclic aromatic hydrocarbons.

10. A method for remediating soil contaminated with a combination of heavy metals and organic matter, characterized in that, Includes the following steps: Mix the soil contaminated with heavy metals and organic matter to be remediated and the remediation agent at a mass ratio of (50~200):

1. After application, maintain the soil moisture content at 60%~80% of field capacity for one week. The repair agent is the composite microbial agent according to any one of claims 1 to 5 or the composite microbial agent obtained by the preparation method according to claim 6 or 7.

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