Application of a kind of phosphorus-dissolving bacteria agent to repair cadmium-contaminated soil while increasing the content of available phosphorus in soil

Abstract

The application relates to the field of environment, resource utilization and agricultural biotechnology, and particularly relates to application of a phosphorus-dissolving bacterial agent for repairing cadmium-polluted soil and simultaneously increasing the content of available phosphorus in the soil, wherein the phosphorus-dissolving bacterial agent comprises phosphorus-dissolving bacteria, bone charcoal, a culture medium and an organic matrix; the phosphorus-dissolving bacteria are enterobacter cloacae HS-6; the phosphorus-dissolving bacterial agent is applied to Cd-polluted soil, and the bioavailability of Cd in the soil is reduced and the content of available phosphorus in the soil is increased through the cooperation of the enterobacter cloacae HS-6 and the bone charcoal. The application can effectively promote the transformation of insoluble phosphate into soluble phosphate, so that the content of available phosphorus in the soil is increased by 96.30%; on the other hand, the application can fix the heavy metal Cd in the soil, so that the content of available Cd in the soil is reduced by about 54.41%, has excellent Cd-fixing capacity, and can relieve the Cd stress of crops, and has good application value in the repair of farmland Cd pollution, the improvement of soil quality and environmental protection.

Description

Technical Field

[0001] This invention relates to the fields of environment, resource utilization, and agricultural biotechnology, specifically to the application of a phosphorus-solubilizing agent for remediating cadmium-contaminated soil while increasing the available phosphorus content in the soil. Background Technology

[0002] Many soil remediation technologies have been used to minimize the risk of heavy metal pollution, among which chemical fixation is widely used due to its low remediation cost and rapid, noticeable effects. Currently, among the various chemical amendments for Cd-contaminated soils, biochar has attracted significant attention due to its larger specific surface area and stronger Cd adsorption and fixation capacity compared to quicklime and clay minerals. Furthermore, another major advantage of biochar as a soil amendment is its ability to promote the sustainable treatment of large quantities of green waste such as straw, bagasse, and livestock bones, thereby maximizing the added value of green waste. Biochar prepared from livestock bones is simply referred to as bone char.

[0003] While chemical amendments such as biochar can fix Cd in soil to some extent, reducing its bioavailability and harm to the environment and crops, existing chemical amendments suffer from short-lived effects, short-term chemical fixation, and susceptibility to recurrence, which can also affect soil physicochemical properties. As research progresses, some drawbacks of biochar are becoming apparent; long-term use alone may lead to imbalances in certain soil nutrients.

[0004] Phosphate-solubilizing bacteria (PSBs) are a group of functional microorganisms that can convert insoluble phosphorus in the soil into available phosphorus that plants can absorb and utilize. They participate in the nutrient cycling and transformation in the soil, increasing the content of available nutrients, improving the soil microbial community structure, and enhancing soil fertility and quality. For the remediation of soil contaminated with heavy metal Cd, PSBs, as a bioremediation method, are less costly and do not produce secondary pollution compared to chemical remediation methods. However, the growth and activity of PSBs are usually limited by environmental factors, leading to a significant reduction in remediation effectiveness.

[0005] In addition, microbial agents are gradually being used for the remediation of Cd-contaminated soil. However, there are not many microbial agents that can be effectively used for the remediation of Cd-contaminated soil, and the remediation effect is not outstanding. Therefore, the development of microbial agents needs to be strengthened to enrich the resources of microbial agents for the remediation of Cd-contaminated soil and promote the remediation of Cd-contaminated soil. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, the present invention aims to provide an application of a phosphate-solubilizing agent for remediating cadmium-contaminated soil while simultaneously increasing the available phosphorus content of the soil. This phosphate-solubilizing agent is highly effective in reducing the bioavailability of cadmium, maintaining soil ecological balance, improving soil fertility, and promoting plant growth.

[0007] To achieve the objectives of the invention described above, the technical solution adopted by the present invention is as follows:

[0008] This invention provides an application of a phosphate-solubilizing agent for remediating cadmium-contaminated soil while increasing the available phosphorus content of the soil. The phosphate-solubilizing agent comprises phosphate-solubilizing bacteria, bone char, culture medium, and organic matrix; the phosphate-solubilizing bacteria is Enterobacter HS-6.

[0009] The Enterobacter hormaechei strain HS-6 was deposited on October 19, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.: 28680.

[0010] When the phosphate-solubilizing agent is applied to Cd-contaminated soil, the bioavailability of Cd in the soil is reduced by the synergistic effect of Enterobacter holmieae HS-6 and bone char, while the content of available phosphorus in the soil is increased.

[0011] Furthermore, the 16S rDNA gene sequence of the *Enterobacter holmieae* HS-6 is shown in SEQ ID No. 1.

[0012] Furthermore, the bone char has a particle size of 30 nm to 2 μm. Bone char with this particle size has a rich pore structure and a large specific surface area, which can rapidly adsorb Cd ions in the soil. In addition, Enterobacter horneri HS-6 has a good effect on converting insoluble phosphorus into soluble phosphorus in bone char with a particle size of 30 nm to 2 μm.

[0013] Furthermore, the organic matrix is ​​at least one of wheat bran, rice straw, or corn cob.

[0014] Furthermore, the application includes the following steps:

[0015] S1. Preparation of micro / nano bone char: Animal bone was thermally pyrolyzed and cooled, then ball-milled to obtain micro / nano multi-level bone char;

[0016] S2. Activation of Enterobacter holmieae: Enterobacter holmieae HS-6 was inoculated into LB medium and then placed in a constant temperature shaker at 28°C with shaking at 180 r / min until OD. 600 =0.6~0.8, to obtain an active bacterial solution;

[0017] S3. Preparation of liquid bacterial agent: After sterilization by irradiation, the micron-nano multi-level bone char obtained in step S1 and the active bacterial solution obtained in step S2 are added to NBRIP medium and then placed in a constant temperature shaker at 28℃ and shaken at 180r / min for 44h-50h to obtain liquid bacterial agent.

[0018] S4. Organic matrix carrier treatment: After rinsing the organic matrix with deionized water, add deionized water to cover the organic matrix and soak it. Then, sterilize it under high temperature and high pressure to obtain the treated organic matrix carrier.

[0019] S5. Preparation of solid bacterial agent: The liquid bacterial agent obtained in step S3 is added to the organic matrix carrier obtained in step S4, and cultured at 28°C for 2 to 4 days to obtain the phosphate-solubilizing bacterial agent.

[0020] S6. Remediation of Cd-contaminated soil: Apply the phosphate-solubilizing agent to Cd-contaminated soil.

[0021] Further, in step S1, the animal bone is heated to 440℃~460℃ at a heating rate of 8℃ / min~12℃ / min for 100min~140min for thermal decomposition. After cooling, large pieces of bone char are obtained. The large pieces of bone char are then ball-milled at 440rpm~460rpm for 4h~6h to obtain micron-nano-level multi-grade bone char; and / or

[0022] In the ball mill, zirconium oxide particles are used to ball mill large bone char pieces, and ethanol is added as a grinding aid; the weight ratio of zirconium oxide particles to large bone char pieces is (15-20):1; the mass ratio of ethanol to large bone char pieces is (1.5-2):1.

[0023] Furthermore, in step S2, the effective viable count of the prepared active bacterial solution is 1×10⁻⁶. 10 CFU / mL ~9×10 10 CFU / mL.

[0024] Further, in step S3, the mass-to-volume ratio of the bone char to the NBRIP culture medium is 0.5% to 2.5%; and / or

[0025] The inoculation amount of the active bacterial solution in the NBRIP medium is 1% of the volume percentage of the NBRIP medium; and / or

[0026] The irradiation sterilization dose is 3.0 kGy to 6.0 kGy.

[0027] Furthermore, in step S4, after rinsing the organic matrix three times with deionized water, the organic matrix is ​​soaked in deionized water that covers it for 10 to 20 hours, and then sterilized under high temperature and high pressure to obtain the treated organic matrix carrier.

[0028] The conditions for high-temperature and high-pressure sterilization are: 121℃, 104KPa, 20min.

[0029] Further, in step S5, the liquid bacterial agent obtained in step S3 is added to the organic matrix carrier obtained in step S4, packed into a polyethylene bag and sealed. Five to eight air holes are punched on the surface of the polyethylene bag with a sterile needle. Then, it is placed in a constant temperature shaker at 28°C and shaken at 180 r / min for 2 to 4 days to obtain the phosphate-solubilizing bacterial agent.

[0030] In step S5, the mass ratio of the liquid bacterial agent to the organic matrix carrier is 15:(70-100);

[0031] In step S6, the mass ratio of the phosphate-solubilizing agent to the Cd-contaminated soil is 3:(250-350).

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0033] (1) An application of a phosphate-solubilizing agent of the present invention for remediating cadmium-contaminated soil while increasing the available phosphorus content of the soil. The phosphate-solubilizing agent includes phosphate-solubilizing bacteria, bone char, culture medium, and organic matrix; the phosphate-solubilizing bacteria is Enterobacter horneri HS-6; when the phosphate-solubilizing agent is applied to Cd-contaminated soil, Enterobacter horneri HS-6 and bone char synergistically reduce the bioavailability of Cd in the soil, while increasing the available phosphorus content of the soil. This application can effectively promote the conversion of insoluble phosphates into soluble phosphates, thereby increasing the available phosphorus content of the soil by 96.30%; on the other hand, it can fix the heavy metal Cd in the soil, thereby reducing the available Cd content of the soil by about 54.41%, showing excellent Cd fixation ability and alleviating Cd stress in crops (e.g., rice). It has great application value in the remediation of Cd pollution in farmland, improvement of soil quality, and environmental protection.

[0034] (2) The application of the phosphate-solubilizing agent of the present invention to remediate cadmium-contaminated soil and increase the available phosphorus content of the soil is different from the existing Cd pollution remediation technology that only focuses on a single mechanism. The application technology of the present invention adopts the combined remediation technology of Enterobacter holmieae HS-6 and micron-nano multi-level bone char. Through the rich pore structure, huge specific surface area and strong ion exchange capacity of the micron-nano multi-level bone char in the phosphate-solubilizing agent, it can efficiently and quickly adsorb Cd ions in the soil, and has an excellent ability to fix Cd in the soil. It can also provide a stable habitat and rich nutrients for Enterobacter holmieae HS-6, protect Enterobacter holmieae HS-6 from adverse environmental factors, enhance the survival ability and activity of Enterobacter holmieae HS-6 in the polluted environment, and thus improve the remediation ability of Enterobacter holmieae HS-6 for heavy metals. The combination of *Enterobacter holmieae* HS-6 and micron- or nano-scale bone char significantly enhances the biochemical regulatory effect of *Enterobacter holmieae* HS-6, thereby further strengthening Cd fixation. Furthermore, the coexistence of *Enterobacter holmieae* HS-6 and micron- or nano-scale bone char not only facilitates the growth and reproduction of *Enterobacter holmieae* HS-6 but also allows for continuous phosphorus dissolution, thus continuously activating soil nutrients and optimizing Cd speciation. Therefore, the synergistic effect of *Enterobacter holmieae* HS-6 and micron- or nano-scale bone char greatly improves the removal and fixation efficiency of Cd, significantly reducing the bioavailability of Cd in the soil and mitigating its harm to the ecological environment and crops. In addition, the metabolites produced by *Enterobacter holmieae* HS-6 during phosphorus dissolution can form stable complexes or precipitates with Cd ions, further reducing Cd bioavailability. Therefore, compared with methods using physical or chemical adsorption alone, this invention has a more significant remediation effect in reducing the bioavailability of Cd in soil.

[0035] (3) The application of the phosphate-solubilizing agent of this invention to remediate cadmium-contaminated soil while increasing the available phosphorus content of the soil offers advantages over traditional chemical remediation methods. Traditional chemical remediation methods rely heavily on chemical agents, which can lead to residues and secondary pollution. In contrast, the remediation technology of this invention utilizes *Enterobacter holmieae* HS-6 and micron- and nano-scaled bone char, both of which are naturally derived or biodegradable materials. These materials do not introduce harmful chemicals into the soil, making them more environmentally friendly to the soil and surrounding ecosystem. Furthermore, *Enterobacter holmieae* HS-6 can improve the soil microbial community structure and promote the growth and reproduction of beneficial microorganisms, while micron- and nano-scaled bone char provides a favorable habitat for soil microorganisms, jointly maintaining the soil ecological balance—something many traditional remediation technologies struggle to achieve. Therefore, the application of this invention has the advantage of being green and environmentally friendly.

[0036] (4) The application of the phosphate-solubilizing agent of the present invention to remediate cadmium-contaminated soil and increase the effective phosphorus content of the soil. Since the Enterobacter HS-6 in the phosphate-solubilizing agent can convert the insoluble phosphorus in the soil into effective phosphorus, thereby increasing the phosphorus nutrition of the soil, while the micron-nano multi-level bone char can increase the pH value and cation exchange capacity of the soil and enhance the soil's water and fertilizer retention capacity, the combination of the two can comprehensively improve soil fertility and create good nutritional conditions for crop growth.

[0037] (5) The application of the phosphorus-solubilizing agent of this invention to remediate cadmium-contaminated soil while increasing the available phosphorus content of the soil, through the synergistic effect of Enterobacter holmieae HS-6 and micron-nano-level bone char, reduces the toxic effects of Cd on crops in the soil and enhances the crop's resistance to stress. This is a function that simple physical or chemical remediation technologies do not possess. On the other hand, micron-nano-level bone char is rich in various nutrients such as calcium, nitrogen, phosphorus, and magnesium, which are precisely the nutrients necessary for plant growth. Therefore, the micron-nano-level bone char of this invention can also act as fertilizer, replenishing soil fertility, promoting the vigorous growth of crops, and thus improving agricultural productivity. Therefore, the application of this invention has advantages in promoting plant growth. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1 This is a scanning electron microscope image of Enterobacter HS-6 of the present invention.

[0040] Figure 2 This is a scanning electron microscope image of Enterobacter HS-6 of the present invention after reaction with bone char.

[0041] Figure 3 This is a graph showing the soil pH measurement results of samples collected from different experimental groups at different times.

[0042] Figure 4 This is a graph showing the results of soil cation exchange capacity measurements from samples collected at different times in different experimental groups.

[0043] Figure 5 This is a graph showing the results of soil available phosphorus content determination for samples collected from different experimental groups at different time periods.

[0044] Figure 6 This is a graph showing the results of soil available Cd content determination for samples collected from different experimental groups at different time periods.

[0045] Figure 7This is a graph showing the results of soil catalase activity assays for samples collected from different experimental groups at different time periods.

[0046] Figure 8 This is a graph showing the results of soil sucrase activity assays on samples collected from different experimental groups at different time periods.

[0047] Figure 9 This is a graph showing the results of soil urease activity measurements from samples collected at different times in different experimental groups.

[0048] Figure 10 This is a graph showing the results of soil acid phosphatase activity measurements from samples collected at different times in different experimental groups.

[0049] Figure 11 This is a graph showing the results of rice plant height measurements using soil samples from different experimental groups.

[0050] Figure 12 This is a graph showing the results of root length measurement of rice cultivated from soil samples in different experimental groups.

[0051] Figure 13 This is a graph showing the results of measuring the fresh weight of the aboveground parts of rice grown from soil samples in different experimental groups.

[0052] Figure 14 This is a graph showing the results of measuring the fresh weight of the underground parts of rice grown from soil samples in different experimental groups. Detailed Implementation

[0053] To make the technical problem to be solved, the technical solution, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0054] The terminology used in the embodiments of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. In this invention, the singular forms “a,” “the,” and “the” as used in the embodiments and appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0055] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0056] The culture medium formulations described in the following examples are as follows:

[0057] LB medium (g / L): 10g peptone, 5g yeast extract, 10g NaCl, pH 7.0-7.2.

[0058] LB solid medium: Add 18g to 20g of agar powder to every 1L of LB medium.

[0059] NBRIP medium (g / L): glucose 10g, MgCl2·6H2O 5g, MgSO4·7H2O 0.25g, FeSO4·7H2O 0.01g, KCl 0.2g, (NH4)2SO4 0.1g, Ca3(PO4)2 5.0g, pH 7.0~7.4.

[0060] Solid medium for NBRIP: Add 18g to 20g of agar powder to every 1L of NBRIP-P medium.

[0061] Example 1

[0062] An application of a phosphate-solubilizing agent for remediating cadmium-contaminated soil while simultaneously increasing the available phosphorus content in the soil is disclosed. This agent comprises phosphate-solubilizing bacteria, bone char, culture medium, and wheat bran; the phosphate-solubilizing bacteria is *Enterobacter hormaechei* HS-6. *Enterobacter hormaechei* HS-6 was deposited on October 19, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.: 28680, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. A scanning electron microscope image of *Enterobacter hormaechei* HS-6 is shown below. Figure 1 As shown.

[0063] This application involves applying phosphate-solubilizing agents to Cd-contaminated soil, using Enterobacter holmieae HS-6 and bone char to synergistically reduce the bioavailability of Cd in the soil while increasing the available phosphorus content.

[0064] The 16S rDNA gene sequence of Enterobacter holmieae HS-6 is shown in SEQ ID No. 1. The gene sequence length is 1476.

[0065] In this embodiment, the particle size of bone char is 30 nm to 2 μm.

[0066] The application of this phosphate-solubilizing agent to remediate cadmium-contaminated soil while increasing the available phosphorus content in the soil includes the following steps:

[0067] S1. Preparation of micro / nano bone char: Animal bone was heated to 450℃ at a heating rate of 10℃ / min and thermally decomposed at this temperature for 120 min. After cooling, large pieces of bone char were obtained. The large pieces of bone char were ball-milled at 450 rpm for 5 h to obtain micro / nano multi-level bone char. In this embodiment, pig bone was selected as the animal bone.

[0068] In the ball mill, zirconium oxide particles are used to ball mill large bone char pieces, and ethanol is added as a grinding aid; the weight ratio of zirconium oxide particles to large bone char pieces is 18:1; the mass ratio of ethanol to large bone char pieces is 1.5:1.

[0069] S2. Activation of Enterobacter holmieae: Enterobacter holmieae HS-6 was inoculated into LB medium and then placed in a constant temperature shaker at 28°C with shaking at 180 r / min until OD. 600 =0.6~0.8, to obtain an active bacterial solution; wherein, the effective viable count of the active bacterial solution is 1×10 10 CFU / mL ~9×10 10 CFU / mL.

[0070] S3. Preparation of liquid bacterial agent: After sterilization by irradiation, the micron-nano multi-level bone char obtained in step S1 and the active bacterial solution obtained in step S2 are added to NBRIP medium and then placed in a constant temperature shaker at 28℃ and shaken at 180r / min for 48h to obtain liquid bacterial agent.

[0071] In this embodiment, the mass-to-volume ratio of bone char to NBRIP medium is 0.5%, and the irradiation sterilization dose is 5.0 kGy; wherein, the inoculum amount of active bacterial solution in the NBRIP medium is 1% of the volume percentage of the NBRIP medium.

[0072] S4. Organic matrix carrier treatment: After rinsing the organic matrix three times with deionized water, soak the organic matrix in deionized water that covers it for 15 hours, and then sterilize it under high temperature and high pressure to obtain the treated organic matrix carrier; the conditions for high temperature and high pressure sterilization are: 121℃, 104KPa, 20min.

[0073] S5. Preparation of solid bacterial agent: The liquid bacterial agent obtained in step S3 is added to the organic matrix carrier obtained in step S4, packed into a polyethylene bag and sealed. Five air holes are punched on the surface of the polyethylene bag with a sterile needle. Then, it is placed in a constant temperature shaker at 28°C and shaken at 180 r / min for 3 days to obtain the phosphate-solubilizing bacterial agent. In this embodiment, the mass ratio of liquid bacterial agent to organic matrix carrier is 15:85.

[0074] S6. Remediation of Cd-contaminated soil: Applying phosphate-solubilizing bacteria to Cd-contaminated soil. In this embodiment, the mass ratio of phosphate-solubilizing bacteria to Cd-contaminated soil is 3:300.

[0075] Example 2

[0076] An application of a phosphate-solubilizing bacterial agent to remediate cadmium-contaminated soil while increasing the available phosphorus content of the soil is described. The difference between this embodiment and Example 1 is that in step S3, the mass-to-volume ratio of bone char to NBRIP medium is 1%, the irradiation sterilization dose is 3.0 kGy, and the culture is shaken for 44 h. All other conditions and methods in this embodiment are the same as in Example 1.

[0077] Example 3

[0078] An application of a phosphate-solubilizing bacterial agent to remediate cadmium-contaminated soil while increasing the available phosphorus content of the soil is described. The difference between this embodiment and Example 1 is that in this embodiment, the organic substrate is rice straw; in step S3, the mass-to-volume ratio of bone char to NBRIP medium is 2%, the irradiation sterilization dose is 4.0 kGy, and the culture is shaken for 46 h. All other conditions and methods in this embodiment are the same as in Example 1.

[0079] Example 4

[0080] An application of a phosphate-solubilizing bacterial agent to remediate cadmium-contaminated soil while increasing the available phosphorus content of the soil is described. The difference between this embodiment and Example 1 is that in this embodiment, the organic substrate is corn cob; in step S3, the mass-to-volume ratio of bone char to NBRIP medium is 2.5%, the irradiation sterilization dose is 6.0 kGy, and the culture is shaken for 50 h. All other conditions and methods in this embodiment are the same as in Example 1.

[0081] Example 5

[0082] An application of a phosphorus-solubilizing bacterial agent to remediate cadmium-contaminated soil while increasing the available phosphorus content in the soil. The difference between this embodiment and Embodiment 1 is that in this embodiment, step S1, preparation of micro-nano bone char: animal bone is heated to 440°C at a heating rate of 8°C / min, and thermally decomposed at this temperature for 140 min. After cooling, large pieces of bone char are obtained. The large pieces of bone char are ball-milled at 440 rpm for 6 h to obtain micron-nano multi-level bone char.

[0083] In the ball mill, zirconium oxide particles are used to ball mill large bone char pieces, and ethanol is added as a grinding aid; the weight ratio of zirconium oxide particles to large bone char pieces is 15:1; the mass ratio of ethanol to large bone char pieces is 1.7:1.

[0084] The remaining conditions and methods in this embodiment are the same as in Embodiment 1.

[0085] Example 6

[0086] An application of a phosphorus-solubilizing bacterial agent to remediate cadmium-contaminated soil while increasing the available phosphorus content in the soil. The difference between this embodiment and Embodiment 1 is that in this embodiment, step S1, preparation of micro-nano bone char: animal bone is heated to 460°C at a heating rate of 12°C / min, and thermally decomposed at this temperature for 100 min. After cooling, large pieces of bone char are obtained. The large pieces of bone are ball-milled at 460 rpm for 4 h to obtain micron-nano multi-level bone char.

[0087] In the ball mill, zirconium oxide particles are used to ball mill large bone char pieces, and ethanol is added as a grinding aid; the weight ratio of zirconium oxide particles to large bone char pieces is 20:1; the mass ratio of ethanol to large bone char pieces is 2:1; and the irradiation sterilization dose is 6.0 kGy.

[0088] The remaining conditions and methods in this embodiment are the same as in Embodiment 1.

[0089] Example 7

[0090] An application of a phosphate-solubilizing bacterial agent to remediate cadmium-contaminated soil while simultaneously increasing the available phosphorus content of the soil is disclosed. The difference between this embodiment and Embodiment 1 is that in this embodiment, in step S4, the organic matrix is ​​soaked in deionized water covering it for 10 hours. In step S5, six air holes are punched in the surface of a polyethylene bag using a sterile needle, and the bag is then placed in a constant-temperature shaker at 28°C and cultured at 180 r / min for 2 days to obtain the phosphate-solubilizing bacterial agent. In this embodiment, the mass ratio of the liquid bacterial agent to the organic matrix carrier is 15:100. In step S6, the mass ratio of the phosphate-solubilizing bacterial agent to the Cd-contaminated soil is 3:250.

[0091] The remaining conditions and methods in this embodiment are the same as in Embodiment 1.

[0092] Example 8

[0093] An application of a phosphate-solubilizing bacterial agent to remediate cadmium-contaminated soil while increasing the available phosphorus content of the soil is disclosed. The difference between this embodiment and Embodiment 1 is that in this embodiment, in step S4, the organic matrix is ​​soaked in deionized water covering it for 20 hours. In step S5, eight air holes are punched in the surface of a polyethylene bag using a sterile needle, and then the bag is placed in a constant temperature shaker at 28°C and shaken at 180 r / min for 4 days to obtain the phosphate-solubilizing bacterial agent. In this embodiment, the mass ratio of the liquid bacterial agent to the organic matrix carrier is 15:70. In step S6, the mass ratio of the phosphate-solubilizing bacterial agent to the Cd-contaminated soil is 3:350.

[0094] The remaining conditions and methods in this embodiment are the same as in Embodiment 1.

[0095] Experimental testing:

[0096] I. Scanning electron microscopy examination

[0097] For a scanning electron microscope image of the *Enterobacter holmieae* strain HS-6 of this invention, please refer to [link to image]. Figure 1 .in, Figure 1 (b) is Figure 1 (a) A magnified view of the area within the dashed box. For example... Figure 1 As shown, Enterobacter holmieae HS-6 is a rod-shaped bacterium 1 μm long.

[0098] In addition, scanning electron microscopy was performed on *Enterobacter holmieae* HS-6 after reaction with bone char. Please refer to [link to relevant documentation]. Figure 2 ,in Figure 2 (b) is Figure 2 (a) Enlarged view of the area within the dashed box. (From...) Figure 2As can be seen, Enterobacter holmieae HS-6 is adsorbed on the surface of bone char, and the morphology of Enterobacter holmieae HS-6 is intact and full, indicating that bone char provides favorable conditions for the growth and physiological state of Enterobacter holmieae HS-6.

[0099] II. Remediation of Cd-contaminated soil

[0100] 1. Soil collection

[0101] The soil used in the experiment was collected from the topsoil layer (0cm–20cm) of paddy fields in Huadu District, Guangzhou (23.415°N, 113.047°E). Before use, the soil was air-dried, ground, and passed through a 2mm mesh sieve.

[0102] 2. Experimental Sample Design Scheme:

[0103] The specific design scheme for the experimental samples is shown in Table 1 below.

[0104] Table 1. Experimental Sample Design Scheme

[0105]

[0106] The four experimental groups in Table 1 were each set up in quadruplicates. The total weight of the samples in each experimental group was 300g, and they were placed in brown wide-mouth bottles for incubation. The bottles were placed in a constant temperature environment of 25℃, and water was added daily to maintain a constant moisture content of 60%. Soil samples were collected on days 0, 3, 10, 18, and 30, cooled, ground, and passed through a 100-mesh sieve for subsequent testing.

[0107] The soil pH, soil cation exchange capacity (CEC), soil available phosphorus content, soil available Cd content, and soil enzyme activity of the samples from the above experimental groups were measured respectively.

[0108] (I) Determination of soil pH in different experimental groups

[0109] The soil pH values ​​of the samples collected at different time periods according to the design in Table 1 (four parallel samples for each sample) were determined according to the "Polypotential Method for Determination of Soil pH" (HJ 962-2018). The results can be found in [link to relevant documentation]. Figure 3 . Figure 3 In the diagram, “Cd” represents the Cd soil group, “Cd MNBC” represents the Cd MNBC soil group, and “Cd MNBC HS-6” represents the Cd MNBC HS-6 soil group.

[0110] Depend on Figure 3The results showed that the pH values ​​of the samples from the blank control group and the Cd soil group were approximately 4.5 at each sampling time, while the pH values ​​of the samples from the Cd MNBC soil group and the Cd MNBC HS-6 soil group were approximately 6 at each sampling time. This indicates that the soil with added bone char or with added bone char and Enterobacter horneri HS-6 had a higher pH value compared to the blank control group, which can improve acidic soil.

[0111] in addition, Figure 3 During the study, the soil pH generally showed a trend of first increasing and then decreasing, reaching its highest point on day 10, indicating that the application of bone char or the application of bone char and Enterobacter horneri HS-6 significantly increased the soil pH.

[0112] Numerous studies have shown that the activity of some phosphate-solubilizing bacteria may be inhibited in acidic soils (pH < 6), but many phosphate-solubilizing bacteria exhibit high activity in neutral (pH 6-7) or slightly alkaline (pH > 7) soils, which is conducive to phosphorus release. Therefore, the application of this invention's phosphate-solubilizing agent to remediate cadmium-contaminated soil while simultaneously increasing the available phosphorus content can bring the soil closer to neutral, thereby increasing the activity of *Enterobacter holmieae* HS-6 and promoting an increase in the available phosphorus content of the soil. Therefore, applying this invention's phosphate-solubilizing agent to the soil, in combination with the effects of bone char and *Enterobacter holmieae* HS-6, is beneficial for the survival of *Enterobacter holmieae* HS-6 in paddy soil and for better exertion of its phosphate-solubilizing effect.

[0113] (II) Determination of soil cation exchange capacity of samples from different experimental groups

[0114] Soil cation exchange capacity (CEC) was determined using samples collected at different time periods according to the design in Table 1 (four parallel samples were set up for each sample). The results can be found in [link to table]. Figure 4 The determination of soil cation exchange capacity was carried out in accordance with the "Determination of Soil Cation Exchange Capacity by Hexaamminecobalt Trichloride Extraction-Spectrophotometric Method" (HJ 889-2017). Figure 4 In the diagram, “Cd” represents the Cd soil group, “Cd MNBC” represents the Cd MNBC soil group, and “Cd MNBC HS-6” represents the Cd MNBC HS-6 soil group.

[0115] Depend on Figure 4 The results showed that, compared with the blank control group and the Cd soil group, the soil cation exchange capacity of samples from the Cd MNBC soil group and the Cd MNBC HS-6 soil group at each sampling time point was significantly increased. The soil cation exchange capacity generally showed a trend of first increasing and then decreasing, reaching its maximum on day 10, indicating that the application of bone char or the application of bone char and Enterobacter horneri HS-6 significantly improved the soil cation exchange capacity.

[0116] (III) Determination of available phosphorus content in soil samples from different experimental groups

[0117] The available phosphorus content in the soil was determined using samples collected at different time periods according to the design in Table 1 (four parallel samples were set up for each sample). The determination results can be found in [link to table]. Figure 5 . Figure 5 In the diagram, “Cd” represents the Cd soil group, “Cd MNBC” represents the Cd MNBC soil group, and “Cd MNBC HS-6” represents the Cd MNBC HS-6 soil group.

[0118] Depend on Figure 5 The results showed that the available phosphorus content in the Cd MNBC soil group and the Cd MNBC HS-6 soil group was significantly increased compared to the blank control group and the Cd soil group. This indicates that the application of micron- and nano-level bone char and Enterobacter horneri HS-6 to the soil can significantly increase the available phosphorus content. Interestingly, compared to the Cd MNBC soil group, the available phosphorus content in the Cd MNBC HS-6 soil group increased by 20.27% on day 3 under the combined action of micron- and nano-level bone char and Enterobacter horneri HS-6, indicating that under Cd pollution, the application of this invention, namely the synergistic effect of bone char and Enterobacter horneri HS-6, can further increase the available phosphorus content in the soil. After 30 days, the available phosphorus content in the Cd MNBC HS-6 soil group increased by approximately 96.30%.

[0119] (iv) Determination of available Cd content in soil samples from different experimental groups

[0120] The available Cd content in the soil was determined using samples collected at different time periods according to the design in Table 1 (four parallel samples were set up for each sample). The determination results can be found in [link to table]. Figure 6 . Figure 6 In the diagram, “Cd” represents the Cd soil group, “Cd MNBC” represents the Cd MNBC soil group, and “Cd MNBC HS-6” represents the Cd MNBC HS-6 soil group.

[0121] The determination of available Cd content in soil was carried out according to the "Determination of 8 Available Elements in Soil: Diethylenetriaminepentaacetic Acid Extraction-Inductively Coupled Plasma Atomic Emission Spectrometry" (HJ 804-2016). 5.00 g of freeze-dried and sieved soil sample was weighed and placed in a 50 mL centrifuge tube. 20 mL of diethylenetriaminepentaacetic acid (DTPA)-calcium chloride (CaCl2)-triethanolamine (TEA) extractant (pH 7.3) was added. The sample was extracted at room temperature with reciprocating shaking at 160 r / min for 2 h. After centrifugation, the supernatant was filtered through a 0.22 μm filter membrane and then tested.

[0122] Depend on Figure 6The results showed that, compared to the Cd soil group, the available Cd content in both the Cd MNBC soil group and the Cd MNBC HS-6 soil group was significantly reduced. In particular, the reduction in available Cd content in the Cd MNBC HS-6 soil group was greater than that in the Cd MNBC soil group, indicating that the combined treatment with micron- and nano-level bone char and *Enterobacter horneri* HS-6 resulted in a better reduction in available Cd content. Analysis on day 3 compared to the Cd soil group revealed that the available Cd content in the Cd MNBC soil group and the Cd MNBC HS-6 soil group decreased by 39.88% and 53.09%, respectively. After 30 days of treatment with micron- and nano-level bone char combined with *Enterobacter horneri* HS-6, the available Cd content in the Cd MNBC HS-6 soil group decreased by 54.41%. This demonstrates that the application of this invention's phosphate-solubilizing agent for remediating cadmium-contaminated soil while simultaneously increasing available phosphorus content can significantly reduce available Cd content, exhibiting a more significant effect in reducing the bioavailability of Cd in soil.

[0123] (v) Determination of soil enzyme activity in samples from different experimental groups

[0124] Soil catalase, sucrase, urease, and acid phosphatase activities were measured using samples collected at different time periods according to the design in Table 1 (four parallel samples were set up for each sample). The measurement results can be found in the respective tables. Figure 7 , Figure 8 , Figure 9 and Figure 10 .

[0125] The method for determining soil enzyme activity is as follows: Using the kit from Iseku Biotechnology Co., Ltd. (China) according to the manufacturer's instructions, and using a multifunctional enzyme marker (Synergy HTX, BioTek, USA), the activities of four typical soil enzymes—catalase, sucrase, urease, and acid phosphatase—were determined.

[0126] Figures 7 to 10 In the diagram, “Cd” represents the Cd soil group, “Cd MNBC” represents the Cd MNBC soil group, and “Cd MNBC HS-6” represents the Cd MNBC HS-6 soil group.

[0127] Urease, sucrase, and catalase are important soil enzymes widely used to evaluate the impact of soil conditioners on soil biological functions. Urease and sucrase participate in the decomposition and mineralization of carbohydrates in the soil, providing nutrients for soil microorganisms and crop growth, while catalase can degrade H2O2 in the soil, reducing its toxic effects on organisms. Figure 7It is evident that, compared to the Cd-contaminated soil group, micron- and nano-level bone char combined with *Enterobacter holmium* HS-6 significantly enhanced catalase activity in Cd-contaminated soil, increasing it by 13.84%. Figures 8 to 9 The results showed that micron- and nano-level bone char combined with Enterobacter holmium HS-6 significantly promoted the activities of sucrase and urease in Cd-contaminated soil, increasing them by approximately 366.09% and 470.52% respectively compared with the Cd soil group.

[0128] In addition, acid phosphatase is a class of enzymes that catalyze the mineralization of soil organic phosphorus. Its activity directly affects the decomposition, transformation, and bioavailability of organic phosphorus in the soil, and serves as an indicator for evaluating the direction and intensity of soil phosphorus biotransformation. Acid phosphatase activity is significantly influenced by soil carbon and nitrogen content, available phosphorus content, and pH. Figure 10 The results showed that, compared with the Cd soil group, the micron- and nano-level bone char combined with *Enterobacter holmierei* HS-6 significantly enhanced acid phosphatase activity in Cd-contaminated soil by 82.46% in the Cd MNBC HS-6 soil group. Therefore, the combination of micron- and nano-level bone char and *Enterobacter holmierei* HS-6 enhanced acid phosphatase activity in Cd-contaminated soil. This enhancement may be closely related to the unique properties of micron- and nano-level bone char, which is an additional source of C and P elements, providing more suitable habitat conditions for *Enterobacter holmierei* HS-6 in the soil and promoting its growth.

[0129] III. Experiments to Promote Crop Growth

[0130] The selected rice variety was Xiuzhan 15. The rice seedlings were first cultivated in a plant culture box for 14 days. Then, uniformly sized seedlings were transplanted into four experimental groups (as designed in Table 1) for pot cultivation. Six replicates were prepared for each group. The plants were placed at room temperature for a 28-day exposure experiment. The rice plants were harvested, and plant height, root length, aboveground fresh weight, and underground fresh weight were measured. The results are shown in the respective tables. Figure 11 , Figure 12 , Figure 13 and Figure 14 .

[0131] Figures 11 to 14 In the diagram, “Cd” represents the Cd soil group, “Cd MNBC” represents the Cd MNBC soil group, and “Cd MNBC HS-6” represents the Cd MNBC HS-6 soil group.

[0132] Depend on Figures 11 to 14The results showed that, compared with the blank control group, the plant height, root length, aboveground fresh weight, and underground fresh weight of rice cultivated in the Cd soil group were significantly reduced, decreasing by 22.55%, 25.87%, 25.23%, and 32.00%, respectively, indicating that Cd-contaminated soil severely affects crop growth. Compared with the Cd soil group, both the Cd MNBC soil group and the CdMNBC HS-6 soil group significantly increased the plant height, root length, aboveground fresh weight, and underground fresh weight of rice. Specifically, the CdMNBC HS-6 soil group, compared with the Cd soil group, increased the plant height, root length, aboveground fresh weight, and underground fresh weight of rice by 18.30%, 34.89%, 61.87%, and 18.18%, respectively. This demonstrates that the application of a phosphorus-solubilizing agent to remediate cadmium-contaminated soil while increasing the available phosphorus content in the soil can promote robust crop growth and thus improve agricultural productivity.

[0133] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

[0134]

Claims

1. An application of a phosphate-solubilizing bacterial agent for remediating cadmium-contaminated soil while simultaneously increasing the available phosphorus content of the soil, characterized in that, The phosphate-solubilizing agent comprises phosphate-solubilizing bacteria, bone char, culture medium, and organic matrix; the phosphate-solubilizing bacteria is Enterobacter HS-6. The classification name of the Enterobacter HS-6 is... Enterobacter hormaechei It was deposited at the China General Microbiological Culture Collection Center on October 19, 2023, with accession number CGMCC NO.: 28680; When the phosphate-solubilizing agent is applied to Cd-contaminated soil, the bioavailability of Cd in the soil is reduced by the synergistic effect of Enterobacter holmieae HS-6 and bone char, while the content of available phosphorus in the soil is increased. The particle size of the bone char is 30 nm to 2 μm.

2. The application of the phosphorus-solubilizing agent as described in claim 1 for remediating cadmium-contaminated soil while simultaneously increasing the available phosphorus content of the soil, characterized in that, The 16S rDNA gene sequence of *Enterobacter holmieae* HS-6 is shown in SEQ ID No.

1.

3. The application of the phosphorus-solubilizing bacterial agent as described in claim 1 for remediating cadmium-contaminated soil while simultaneously increasing the available phosphorus content of the soil, characterized in that, The organic matrix is ​​at least one of wheat bran, rice straw, or corn cob.

4. The application of the phosphorus-solubilizing bacterial agent as described in claim 1 for remediating cadmium-contaminated soil while simultaneously increasing the available phosphorus content of the soil, characterized in that, The application includes the following steps: S1. Preparation of micro / nano bone char: Animal bone was thermally pyrolyzed and cooled, then ball-milled to obtain micro / nano multi-level bone char; S2. Activation of Enterobacter holmieae: Enterobacter holmieae HS-6 was inoculated into LB medium and then placed in a constant temperature shaker at 28°C with shaking at 180 r / min until OD. 600 =0.6~0.8, to obtain active bacterial solution; S3. Preparation of liquid bacterial agent: After sterilization by irradiation, the micron-nano multi-level bone char obtained in step S1 and the active bacterial solution obtained in step S2 are added to NBRIP medium and then placed in a constant temperature shaker at 28℃ and shaken at 180r / min for 44h~50h to obtain liquid bacterial agent. S4. Organic matrix carrier treatment: After rinsing the organic matrix with deionized water, add deionized water to cover the organic matrix and soak it. Then, sterilize it under high temperature and high pressure to obtain the treated organic matrix carrier. S5. Preparation of solid bacterial agent: The liquid bacterial agent obtained in step S3 is added to the organic matrix carrier obtained in step S4, and cultured at 28°C for 2-4 days to obtain the phosphate-solubilizing bacterial agent; S6. Remediation of Cd-contaminated soil: Apply the phosphate-solubilizing agent to Cd-contaminated soil.

5. The application of the phosphorus-solubilizing agent as described in claim 4 for remediating cadmium-contaminated soil while simultaneously increasing the available phosphorus content of the soil, characterized in that... In step S1, animal bones are heated to 440℃~460℃ at a heating rate of 8℃ / min~12℃ / min for 100min~140min for thermal decomposition. After cooling, large pieces of bone char are obtained. The large pieces of bone char are then ball-milled at 440rpm~460rpm for 4h~6h to obtain micron-nano-level multi-grade bone char; and / or In the ball mill, zirconium oxide particles are used to ball mill large bone char, and ethanol is added as a grinding aid; the weight ratio of zirconium oxide particles to large bone char is (15~20):1; the mass ratio of ethanol to large bone char is (1.5~2):

1.

6. The application of the phosphorus-solubilizing bacterial agent as described in claim 4 for remediating cadmium-contaminated soil while simultaneously increasing the available phosphorus content of the soil, characterized in that, In step S2, the effective viable count of the prepared active bacterial solution is 1×10⁻⁶. 10 CFU / mL ~9×10 10 CFU / mL.

7. The application of the phosphorus-solubilizing bacterial agent as described in claim 4 for remediating cadmium-contaminated soil while simultaneously increasing the available phosphorus content of the soil, characterized in that, In step S3, the mass-to-volume ratio of the bone char to the NBRIP culture medium is 0.5% to 2.5%; and / or The inoculation amount of the active bacterial solution in the NBRIP medium is 1% of the volume percentage of the NBRIP medium; and / or The irradiation sterilization dose is 3.0 kGy to 6.0 kGy.

8. The application of the phosphorus-solubilizing bacterial agent as described in claim 4 for remediating cadmium-contaminated soil while simultaneously increasing the available phosphorus content of the soil, characterized in that, In step S4, after rinsing the organic matrix three times with deionized water, the organic matrix is ​​soaked in deionized water that covers it for 10-20 hours, and then sterilized under high temperature and high pressure to obtain the treated organic matrix carrier. The conditions for high-temperature and high-pressure sterilization are: 121℃, 104KPa, 20min.

9. The application of a phosphorus-solubilizing agent as described in any one of claims 4 to 5 for remediating cadmium-contaminated soil while simultaneously increasing the available phosphorus content of the soil, characterized in that, In step S5, the liquid bacterial agent obtained in step S3 is added to the organic matrix carrier obtained in step S4, and then placed in a polyethylene bag and sealed. Five to eight air holes are punched on the surface of the polyethylene bag with a sterile needle. The bag is then placed in a constant temperature shaker at 28°C and shaken at 180 r / min for 2 to 4 days to obtain the phosphate-solubilizing bacterial agent. In step S5, the mass ratio of the liquid bacterial agent to the organic matrix carrier is 15:(70~100). In step S6, the mass ratio of the phosphate-solubilizing agent to the Cd-contaminated soil is 3:(250~350).

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