Sporosarcina, microbial inoculant thereof and application of sporosarcina in remediation and improvement of heavy metal contaminated soil

Abstract

The application discloses a spore octosphaeraceae, a microbial inoculum thereof and application of the spore octosphaeraceae in remediation and improvement of heavy metal contaminated soil, and belongs to the technical field of microorganisms and heavy metal pollution remediation. Sporosarcina The preservation number of the spore octosphaeraceae (S. sp.) is GDMCC No.68218. The application further provides a microbial inoculum containing the strain and a method for remediation of heavy metal contaminated soil and synchronous improvement of soil by using the strain or the inoculum and a mineralization liquid (containing urea and calcium salt). The strain has high urease activity and multiple heavy metal tolerance, can efficiently fix multiple heavy metals such as Cd, Cu, Mn, Ni and Zn in soil, significantly improves total porosity, water stable aggregate content, available nitrogen, phosphorus, potassium and organic matter content in soil, realizes integration of remediation, soil improvement and soil fertilization, and can guarantee safe planting of crops after remediation.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and heavy metal pollution remediation technology, specifically involving a strain of *Dystrophus occulta* with high urease activity and tolerance to multiple heavy metals. Sporosarcina (sp.), microbial agents containing this strain, and their application in remediating heavy metal contaminated soil and simultaneously improving soil. Background Technology

[0002] Microbial-induced carbonate precipitation (MICP) technology has been widely applied in recent years due to its environmental friendliness and high mineralization efficiency in the remediation of heavy metal-contaminated sites, including high-salinity wastewater from shale gas extraction, agricultural soils, sulfide mine tailings soils, gold mine tailings ponds, selenium-rich soils, and desertified areas. The core of MICP technology is to utilize the metabolic activities of microorganisms to promote carbonate precipitation, thereby effectively fixing heavy metals in the soil. Urease-producing microorganisms, also known as urease-degrading bacteria, hydrolyze urea, gradually releasing it to form HCO3-. - NH4 + and OH - and with native soil or exogenous Ca 2+ This process involves the combination of calcium and calcium ions to form carbonate mineral precipitates. Because the radius of calcium ions is similar to that of many heavy metal ions, these heavy metal ions can spontaneously replace calcium sites in the calcium carbonate crystal lattice or enter the interstitial spaces of the mineral crystal lattice to form (Ca) 1-x M x CO3 solid solution is mineralized and fixed.

[0003] Existing MIP (Microcystis Removal by Polymerization) technologies primarily focus on the immobilization of single heavy metals. For example, model strains such as *Bacillus pasteurellii* ATCC11859 and *Bacillus pasteurellii* kp-4 (publication number CN 117625453 A) only achieve the removal of single heavy metals like cadmium, without addressing soil structure improvement, fertility enhancement, or crop safety. While *Bacillus fusiformis* (publication number CN112974512 A) and *Proteus mirabilis* D2 (publication number CN 120005762 A) can remediate multiple heavy metals, they lack data on soil porosity, aggregates, and nutrient improvements, and have not conducted research on heavy metal reduction in edible parts of vegetables. This indicates that existing technologies generally neglect the synergistic improvement of soil physical structure (such as porosity and aggregates) and chemical fertility (such as available nitrogen, phosphorus, and potassium) during the remediation process. Consequently, the remediated soil may still be insufficient to support plant growth, failing to achieve the goal of safe farmland utilization through "remediation while production."

[0004] Therefore, developing an integrated MICP strain that can overcome the above-mentioned defects, possessing high urease activity, strong tolerance to multiple heavy metals, and simultaneously achieving efficient heavy metal fixation, soil structure improvement, fertility enhancement, and safe crop cultivation, is of key significance for promoting the engineering application of this technology. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a strain of *Dystrophus occulta* with high urease activity and excellent tolerance to multiple heavy metals. Sporosarcina sp.).

[0006] Another object of the present invention is to provide a microbial agent containing the said strain.

[0007] Another objective of this invention is to provide the application of the strain or agent in the remediation of heavy metal contaminated soil, particularly to achieve an integrated application of heavy metal fixation, soil structure improvement, fertility enhancement, and safe crop cultivation.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a spore-forming *Dystrophus* ( Sporosarcina sp.), the taxonomic name of this strain is: Sporosarcina sp. was deposited on May 7, 2026 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, with accession number GDMCC No. 68218.

[0009] The strain was obtained from heavy metal-contaminated soil in a coal mining area through a gradual increase in heavy metal concentration during acclimatization and screening using an adaptive laboratory evolution method. This strain possesses the following excellent characteristics: High urease activity: It can efficiently decompose urea, providing sufficient carbonate ions for the MIP process.

[0010] Multiple heavy metal tolerance: It has excellent tolerance to multiple heavy metals such as Cd, As, and Mn, and can grow normally under high concentration of heavy metal stress.

[0011] Highly efficient mineralization capacity: In aqueous phase, it can mineralize Cd² within 24 hours. + Its fixation efficiency is as high as 97.4%.

[0012] The mineralized products exhibit high stability: under extremely acidic conditions (pH=2), the Cd-containing calcite precipitate induced by the product shows a Cd re-release rate of only 0.13% after 28 days, demonstrating excellent long-term stability.

[0013] Soil structure improvement capability: It can significantly increase the total porosity and water-stable aggregate content of soil, and improve the physical structure of soil.

[0014] Soil fertility enhancement capability: It can simultaneously increase the content of available nitrogen, phosphorus, potassium and organic matter in the soil.

[0015] Ensuring safe crop cultivation: Restored soil can significantly reduce the heavy metal content in the edible parts of crops such as lettuce.

[0016] Secondly, the present invention provides a microbial agent comprising live cells, spores or cultures of the aforementioned *Dystrophus spores*.

[0017] Thirdly, the present invention provides the application of the above-mentioned Bacillus spores or the above-mentioned microbial agents in the preparation of products for remediating heavy metal contaminated soil.

[0018] Preferably, the repair includes at least one of the following functions: (a) Fixing heavy metals in soil; (b) Improve the physical structure of the soil; (c) Improve soil fertility; (d) Reduce the heavy metal content in crops grown in the soil.

[0019] Preferably, the heavy metal is one or more of Cd, Cu, Mn, Ni, and Zn.

[0020] Fourthly, the present invention provides a method for remediating heavy metal-contaminated soil and simultaneously improving the soil using the above-mentioned Bacillus subtilis or the above-mentioned microbial inoculant, comprising the following steps: (1) Apply the above-mentioned Bacillus spores culture medium or the above-mentioned microbial agent and mineralizing solution to the heavy metal contaminated soil to be remediated; wherein the mineralizing solution contains urea and calcium salt; (2) Cultivate the strain to induce calcium carbonate precipitation, so as to fix heavy metals and improve the soil.

[0021] Preferably, the ratio of the applied volume of the strain culture solution or microbial agent to the soil mass in step (1) is 5-15:100, where the unit is mL / g; the ratio of the applied volume of the mineralizing solution to the soil mass is 30-50:100, where the unit is mL / g, and more preferably 40:100.

[0022] Preferably, the mineralization solution in step (1) further comprises microbial culture medium components. These microbial culture medium components can provide the necessary nutrients for microbial growth, such as, but not limited to, tryptone, beef meal, and yeast powder.

[0023] Preferably, the mineralizing solution is an NBU mineralizing solution, which comprises tryptone, beef meal, yeast extract, urea, and calcium chloride. In one specific embodiment, the NBU mineralizing solution has the following composition: 5 g / L tryptone, 1.5 g / L beef meal, 1.5 g / L yeast extract, 20 g / L urea, and 50 mM anhydrous calcium chloride.

[0024] Fifthly, the present invention provides a product for remediating soil in heavy metal-contaminated mining areas and simultaneously improving the soil, characterized in that it comprises the above-mentioned Bacillus cereus or the above-mentioned microbial agent, and a mineralizing solution containing urea and calcium salts.

[0025] Compared with the prior art, the present invention has the following significant advantages: Highly efficient immobilization of multiple heavy metals: The strain can simultaneously and efficiently immobilize multiple heavy metals such as Cd, Cu, Mn, Ni, and Zn, with the exchangeable content decreasing by more than 60%. The immobilization effect on Cd is particularly significant, with a 24-hour aqueous phase removal rate of 97.4%.

[0026] Long-term stability of mineralized products: Under extremely acidic conditions (pH=2), the re-release rate of Cd in mineralized products is extremely low (only 0.13% after 28 days), effectively solving the problem of secondary release of heavy metals caused by acid rain leaching.

[0027] Synergistic improvement of soil structure and fertility: While repairing heavy metals, it can significantly increase the total porosity of the soil (+37.44%), water-stable aggregates (+93%), and simultaneously increase the content of available nitrogen, phosphorus, potassium and organic matter in the soil, achieving the integration of "remediation-soil improvement-fertilization".

[0028] Ensuring safe crop cultivation: Remediated soil can significantly reduce the heavy metal content in edible parts of crops (such as lettuce) by 36%-75%, providing a feasible solution for the simultaneous remediation and safe utilization of polluted farmland.

[0029] Environmentally friendly and low-cost: Based on indigenous microorganisms, it requires no large amount of chemical reagents, causes no secondary pollution, is easy to operate, and is suitable for large-scale promotion in mining areas.

[0030] The strain involved in this invention was deposited on May 7, 2026, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, with accession number GDMCC No. 68218. Its taxonomic name is: Sporosarcina sp. Attached Figure Description

[0031] Figure 1 The image shows the morphology of Ureaplasma urealyticum on urea phenol red agar plates in Example 1. Figure 2 This is a graph showing the comparison results of different urease activities in Example 1; Figure 3 This is a graph showing the heavy metal tolerance test results of the top 9 urease-producing bacteria strains in Example 1; Figure 4 Different urealytic bacteria in Example 2 affect Cd² + Figure (b) shows the fixation efficiency (a) and the stability test results of the mineralized products of strain DF-C31. Figure 5 The images show scanning electron microscope (SEM) images (a) and X-ray diffraction (XRD) patterns (b) of the mineralized Cd precipitate from strain DF-C31 in Example 2. Figure 6 This is a comparison diagram of the distribution of heavy metal speciation before and after soil remediation in Example 3; Figure 7 This is a comparison diagram of the distribution of heavy metal speciation before and after soil remediation in Example 4; Figure 8 This is a comparison diagram of the distribution of heavy metal speciation before and after soil remediation in Example 5; Figure 9 This is a comparison chart of heavy metal content in lettuce leaves in Example 6; Figure 10 The graph shows the changes in available nitrogen (a), available phosphorus (b), available potassium (c), and organic matter (d) in the soil before and after mineralization in Example 6. Detailed Implementation

[0032] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0033] The processes, conditions, reagents, and experimental methods used in implementing this invention, except as specifically mentioned below, are all common knowledge and general knowledge in the field, and this invention does not have any particular limitations. Experimental methods in the embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0034] Unless otherwise stated, all technical terms and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. However, in the event of any conflict, the specification containing the definitions shall prevail.

[0035] The culture medium formulations used in the following examples are as follows: NBU liquid culture medium: 5 g / L tryptone, 1.5 g / L beef meal, 1.5 g / L yeast extract, 5 g / L sodium chloride, 20 g / L urea (added after filtration and sterilization).

[0036] Urea-phenol red solid medium: Add 20 g / L agar and 8 mL / L 0.2% phenol red solution to NBU liquid medium.

[0037] Example 1: Isolation, screening and identification of strain DF-C31 1. Strains domestication and screening Heavy metal contaminated soil samples were collected from the coal mining area of ​​Dafang County, Bijie City, Guizhou Province. 1 g of soil sample was placed in an initial culture medium containing 5 mg / L Cd, Cu, Mn, Ni, and Zn, and acclimatized at 30 ℃ using an automated microbial adaptive evolution instrument. The culture medium was changed every week, and the heavy metal concentration was increased by 5 mg / L. After two months, the acclimatized samples were serially diluted and plated on urea phenol red agar, and incubated at 30 ℃ for 1-2 days. Single colonies that turned the agar deep red color fastest were selected, and after multiple streak purifications, 25 candidate strains were obtained. Morphological photographs of some strains on urea phenol red agar plates are shown below. Figure 1 .

[0038] 2. Urease activity assay The urease activity of the candidate strains was determined using the phenol-nitroglycerin-hypochlorite method. The results are as follows: Figure 2 As shown, the top 9 bacteria with the highest urease activity (including DF-C31) were selected, and their enzyme activities were all greater than 85 ΔU.

[0039] 3. Heavy metal tolerance test The above nine candidate strains were inoculated into NBU liquid medium containing 50 mg / L and 100 mg / L Cd, As, and Mn, respectively, and cultured at 30 ℃ and 150 rpm. Their growth curves were then measured. The results are as follows: Figure 3 As shown, strain DF-C31 exhibited the highest growth rate and strongest Cd tolerance under Cd stress of 50 mg / L and 100 mg / L. Based on the combined results of enzyme activity and heavy metal tolerance, DF-C31 was selected as the preferred strain for subsequent experiments.

[0040] 4. Strain identification Genomic DNA was extracted from strain DF-C31, and 16S rDNA gene amplification and sequencing were performed. The sequencing results were then compared with the NCBI database using BLAST, and the results showed that it was similar to... Sporosarcina It showed the highest similarity to strains of the genus, and was therefore identified as *Bacillus occulta*. Sporosarcina This strain (sp.) was named DF-C31. It was deposited at the Guangdong Provincial Microbial Culture Collection Center on May 7, 2026, with accession number GDMCC No. 68218.

[0041] Example 2: Verification of the aqueous mineralization ability of strain DF-C31 1.Cd² + Fixed efficiency test Strain strain DF-C31 was cultured in NBU liquid medium at 30 °C and 150 rpm for 24 hours to prepare a seed culture. A solution containing 50 mg / L Cd²⁻ was prepared. + NBU liquid medium was used to inoculate the seed culture at a 2% inoculum, and the culture was mineralized at 30 °C and 150 rpm for 5 days. The supernatant was sampled periodically for Cd²⁻ determination. + Concentration, calculate fixation rate.

[0042] The results are as follows Figure 4 As shown in (a), strain DF-C31 showed an effect on Cd² within 24 hours. + The fixation efficiency reached 97.4%, which was significantly higher than that of other candidate strains.

[0043] 2. Stability testing of mineralized products Collect the Cd-containing calcium carbonate precipitate after the above mineralization reaction, wash it, and freeze-dry it. Take 0.3 g of the precipitate and add it to deionized water at pH = 2.0, 4.0, 7.0, and 10.0, respectively. Take the supernatant every week to determine Cd². + Adjust the concentration and replenish with an equal amount of freshly prepared acid-base solution, continuing for 4 weeks.

[0044] The results are as follows Figure 4 As shown in (b), even under extremely acidic conditions (pH=2), Cd² + The re-release rate also showed a downward trend, reaching only 0.13% after 28 days, indicating that the mineralized products have extremely high long-term stability.

[0045] 3. Characterization of mineralized products The precipitates after DF-C31 mineralization were analyzed by scanning electron microscopy (SEM) and X-ray diffraction (XRD). The results are as follows: Figure 5 As shown, SEM reveals that the mineral crystals exhibit typical rhombic hexagonal unit cells (calcite characteristics), and XRD patterns confirm that Cd is successfully encapsulated within a stable calcite lattice.

[0046] Example 3: Remediation and Improvement of Heavy Metal Contaminated Soil in Mining Areas The soil used in this embodiment comes from the manganese mining pollution area in Zhenlong Village, Honghuagang District, Zunyi City. It is a soil contaminated with multiple heavy metals, and its basic properties are: cadmium 12 mg / kg, copper 400 mg / kg, manganese 3500 mg / kg, nickel 200 mg / kg, and zinc 2500 mg / kg.

[0047] Take 600 g of the above-mentioned contaminated soil and remediate it according to the following steps: (1) The strain DF-C31 was cultured in NBU liquid medium until OD600≥1.0 to obtain bacterial solution.

[0048] (2) Mix 60 mL of bacterial solution with 240 mL of NBU mineralization solution, apply the mixture to the soil, and mix thoroughly. The NBU mineralization solution consists of: 5 g / L tryptone, 1.5 g / L beef meal, 1.5 g / L yeast powder, 20 g / L urea, and 50 mM anhydrous calcium chloride.

[0049] Let it stand at room temperature for mineralization for 1 month, and add 100 mL of water every 5 days to maintain humidity.

[0050] After mineralization, the speciation of five heavy metals, Cd, Cu, Mn, Ni and Zn, in the soil was analyzed using the Tessier sequential extraction method.

[0051] Soil physical structure was tested in accordance with NYT 1121.19-2008 Soil Testing Part 19: Determination of Soil Water-Stable Macroaggregate Composition, to detect changes in soil particle size and aggregates after remediation, and porosity changes were detected by X-ray tomography.

[0052] Heavy metal remediation: Results as follows Figure 6 As shown, the exchangeable contents of Cd, Cu, Mn, Ni, and Zn in the remediated soil all decreased significantly, while the residual contents increased significantly. Specifically, exchangeable Cd decreased from 4.68 mg / kg to 0.93 mg / kg, a reduction of 80.08%, while residual Cd increased from 0.56 mg / kg to 3.32 mg / kg. Exchangeable Cu decreased from 12.69 mg / kg to 8.11 mg / kg, a reduction of 36.12%, while residual Cu increased from 104.45 mg / kg to 161.40 mg / kg. Exchangeable Mn decreased from 34.38 mg / kg to 11.78 mg / kg, a reduction of 65.75%, while residual Mn increased from 122.62 mg / kg to 499.10 mg / kg, a three-fold increase. The exchangeable Ni decreased from 14.35 mg / kg to 3.12 mg / kg, a reduction of 78.26%, while the residual Ni increased from 17.01 mg / kg to 38.35 mg / kg. The exchangeable Zn decreased from 25.71 mg / kg to 8.85 mg / kg, a reduction of 65.58%, while the residual Zn increased from 834.28 mg / kg to 1513.93 mg / kg.

[0053] Improvement of soil physical structure: As shown in Table 1, the proportion of large particles larger than 3 mm in the soil increased significantly after remediation (from 45.31% to 63.11%), while the proportion of fine particles smaller than 0.25 mm decreased significantly (from 8.20% to 3.73%).

[0054] Table 1 Soil particle size after MICP remediation

[0055] The mean weight diameter (MWD) of soil aggregates is an important indicator of soil structural stability, reflecting the soil's resistance to erosion and its ability to retain organic matter. Water-stable aggregates (WSA) are an important indicator of soil structure, referring to aggregates of soil particles that remain stable and undamaged in water. Table 2 shows that after MIP mineralization remediation, the soil MWD increased from 3.65 mm to 4.86 mm, an increase of 33%, and the proportion of WSA increased from 27.96% to 54.05%, an increase of 93% compared to the control, indicating that the soil aggregates are more stable and can effectively prevent soil erosion.

[0056] Table 2 Changes in soil aggregates after MICP remediation

[0057] Further analysis of the soil pore structure, as shown in Table 3, revealed a significant increase in total porosity, pore number, and fractal dimension after remediation. The fractal dimension increased from 2.34 to 2.56, indicating an increase in surface roughness and spatial irregularity. Combined with the substantial increase in pore number (from approximately 30,000 to nearly 130,000), the remediation process did not simply "block" the pores but rather generated a large number of complex-shaped mesopores. The interconnected porosity was also improved, and the effective permeability coefficient increased, demonstrating that this MIP technology effectively enhances soil aeration, water retention, and erosion resistance, which is crucial for improving soil fertility and sustainable agriculture.

[0058] Table 3 Changes in soil porosity after MICP remediation

[0059] Example 4 The difference from Example 3 is that the amounts of bacterial solution and NBU mineralization solution used are different; in Example 3, the amounts are 30 mL of bacterial solution and 300 mL of NBU mineralization solution. Everything else is the same as in Example 3.

[0060] Heavy metal remediation results as follows Figure 7As shown, compared with the control (CK), the exchangeable Cd in the remediated soil decreased from 4.68 mg / kg to 2.47 mg / kg, a reduction of 47.13%, while the residual Cd increased from 0.56 mg / kg to 2.22 mg / kg. Exchangeable Cu decreased from 12.69 mg / kg to 10.94 mg / kg, a reduction of 13.81%, while the residual Cu increased from 104.45 mg / kg to 132.11 mg / kg. Exchangeable Mn decreased from 34.38 mg / kg to 20.37 mg / kg, a reduction of 40.75%, while the residual Mn increased from 122.62 mg / kg to 452.95 mg / kg, an increase of 2.7 times. Exchangeable Ni decreased from 14.35 mg / kg to 9.28 mg / kg, a reduction of 35.34%, while the residual Ni increased from 17.01 mg / kg to 25.75 mg / kg. The exchangeable Zn decreased from 25.71 mg / kg to 11.89 mg / kg, a decrease of 53.75%, while the residual Zn increased from 834.28 mg / kg to 1004.28 mg / kg.

[0061] Example 5 The difference from Example 3 is that the amounts of bacterial solution and NBU mineralization solution are different; in Example 3, the amounts are 90 mL of bacterial solution and 180 mL of NBU mineralization solution. Everything else is the same as in Example 3.

[0062] Heavy metal remediation results as follows Figure 8 As shown, compared with the control (CK), the exchangeable Cd in the remediated soil decreased from 4.68 mg / kg to 1.06 mg / kg, a reduction of 77.29%, while the residual Cd increased from 0.56 mg / kg to 4.56 mg / kg. Exchangeable Cu decreased from 12.69 mg / kg to 7.88 mg / kg, a reduction of 37.88%, while the residual Cu increased from 104.45 mg / kg to 172.84 mg / kg. Exchangeable Mn decreased from 34.38 mg / kg to 9.35 mg / kg, a reduction of 72.81%, while the residual Mn increased from 122.62 mg / kg to 523.62 mg / kg, an increase of 3.3 times. Exchangeable Ni decreased from 14.35 mg / kg to 3.64 mg / kg, a reduction of 74.66%, while the residual Ni increased from 17.01 mg / kg to 43.21 mg / kg. The exchangeable Zn content decreased from 25.71 mg / kg to 7.26 mg / kg, a decrease of 71.76%, while the residual Zn content increased from 834.28 mg / kg to 1564.28 mg / kg.

[0063] Example 6: Verification of safe crop planting in remediated soil The soil restored in Example 3 and the control (CK) soil were placed in cultivation pots, and Italian lettuce seedlings were transplanted and managed with conventional water and fertilizer. The lettuce was harvested after 35 days, and the biomass and heavy metal content of the leaves were measured.

[0064] Lettuce biomass: After MIP treatment, the fresh weight of the aboveground parts of lettuce was 3.36 g and the fresh weight of the underground parts was 1.34 g, which were not significantly different from the control (3.42 g and 2.18 g, respectively).

[0065] Results of heavy metal content in leaves are shown below Figure 9 The leaf Cd content was 1.41 mg / kg, a decrease of 75% compared to the control; Cu content was 9.95 mg / kg, a decrease of 56% compared to the control; Mn content was 111.90 mg / kg, a decrease of 63% compared to the control; Ni content was 11.91 mg / kg, a decrease of 36% compared to the control; and Zn content was 35.01 mg / kg, a decrease of 58% compared to the control. As trace elements essential for the human body, the internationally accepted tolerable upper intake levels (UL) for Cu and Zn are 8 mg / day and 40 mg / day, respectively, so the lettuce contained these two elements and met the standards. However, because the tested soil was collected from the center of the mining area, the heavy metal pollution level was extremely high and involved multiple elements, so the total heavy metal content of the vegetables grown did not meet the edible standards. However, most non-mining arable land is in low-pollution areas with fewer types of pollutants than mining areas. After MIP mineralization remediation, the heavy metal content in the edible parts of crops grown there is expected to be below the national safety limits.

[0066] Referring to "NYT1848-2010 Determination of Ammonium Nitrogen, Available Phosphorus, and Available Potassium in Neutral and Calcareous Soils by Combined Extraction-Colorimetric Method" and "NYT 525-2021 Organic Fertilizers", the available nitrogen, phosphorus, potassium, and organic matter content of the soil after MIP remediation were tested immediately after mineralization, after one round of lettuce planting, and after two rounds of lettuce planting.

[0067] See results Figure 10 The results showed that the available nitrogen, phosphorus, potassium, and organic matter content in the soil after MIP remediation was higher than that in the control group immediately after mineralization, after one round of lettuce planting, and after two rounds of lettuce planting. This indicates that in addition to improving soil physical structure and increasing aeration, this technology is also beneficial for improving the soil's nutrient-poor state. After MIP mineralization, the contents of available nitrogen (0.22 g / kg), available phosphorus (20.43 mg / kg), available potassium (134.98 mg / kg), and organic matter (11.25 g / kg) increased by 130%, 53%, 33%, and 17%, respectively, compared with the control group. After two rounds of crop planting, the available nitrogen, phosphorus, potassium, and organic matter content were still 694%, 421%, 320%, and 60% higher than those in the control soil, respectively. This indicates that MIP treatment not only reduces crop absorption of heavy metals but also improves soil fertility.

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A type of spore-forming Micrococcus ( Sporosarcina sp.) DF-C31, characterized in that, It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 7, 2026, with accession number GDMCC No. 68218.

2. A microbial inoculant, characterized in that, The microbial agent comprises live cells of Bacillus spores or a culture thereof as described in claim 1.

3. The application of the *Bacillus sporeans* as described in claim 1 or the microbial agent as described in claim 2 in the preparation of products for remediating heavy metal contaminated soil, characterized in that... The repair includes at least one of the following functions: (a) Fixing heavy metals in soil; (b) Improve the physical structure of the soil; (c) Improve soil fertility; (d) Reduce the heavy metal content in crops grown in the soil; The heavy metal is one or more of Cd, Cu, Mn, Ni, and Zn.

4. A method for remediating heavy metal-contaminated soil and simultaneously improving the soil using the Bacillus subtilis of claim 1 or the microbial agent of claim 2, characterized in that, Includes the following steps: (1) Apply the Bacillus spores culture medium of claim 1 or the microbial agent of claim 2 and the mineralizing solution to the heavy metal contaminated soil to be remediated; wherein the mineralizing solution contains urea and calcium salt; (2) Cultivate the strain to induce calcium carbonate precipitation, so as to fix heavy metals and improve the soil; The heavy metal is one or more of Cd, Cu, Mn, Ni, and Zn.

5. The method according to claim 4, characterized in that, In step (1), the ratio of the applied volume of Bacillus spores culture medium or microbial agent to the soil mass is 5-15:100, with units of mL / g; the ratio of the applied volume of mineralizing solution to the soil mass is 30-50:100, with units of mL / g.

6. The method according to claim 4, characterized in that, The mineralizing solution described in step (1) also contains microbial culture medium components.

7. The method according to claim 6, characterized in that, The mineralizing solution is an NBU mineralizing solution, which contains tryptone, beef meal, yeast powder, urea and calcium chloride.

8. A product for remediating soil in heavy metal-contaminated mining areas and simultaneously improving the soil, characterized in that, It comprises the spore-forming Micrococcus of claim 1 or the microbial agent of claim 2 and a mineralizing solution containing urea and calcium salts.

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