Application of phosphate-solubilizing bacterial agent to remediation of cadmium-contaminated soil and improvement of available phosphorus content in soil

Abstract

The invention relates to the field of environment, resource utilization and agricultural biotechnology, in particular to application of a phosphate-solubilizing bacterial agent to remediation of cadmium-contaminated soil and increase of available phosphorus content in the soil, in the application, the phosphate-solubilizing bacterial agent comprises phosphate-solubilizing bacteria, bone black, a culture medium and an organic matrix; the phosphate solubilizing bacteria are enterobacter hormaechei HS-6; the phosphate-solubilizing bacterial agent is applied to Cd-polluted soil, the bioavailability of Cd in the soil is reduced through the cooperation of the enterobacter hormaechei HS-6 and the bone black, and meanwhile, the content of available phosphorus in the soil is increased. On one hand, insoluble phosphate can be effectively promoted to be converted into soluble phosphate, and the content of available phosphorus in soil is increased by 96.30%; on the other hand, the soil heavy metal Cd can be fixed, so that the content of available Cd in the soil is reduced by about 54.41%, the soil heavy metal Cd-fixing agent has excellent Cd fixing capacity, can relieve Cd stress of crops, and has good application value in remediation of farmland Cd pollution, improvement of soil quality and environmental protection.

Description

Technical Field

[0001] The invention relates to the fields of environment, resource utilization and agricultural biotechnology, and in particular to the application of a phosphate-dissolving bacteria agent in repairing cadmium-contaminated soil while increasing the effective phosphorus content of the soil. Background Art

[0002] The current situation of soil cadmium (Cd) pollution is severe. Rice has a strong ability to absorb and enrich Cd, which has caused great concerns about rice safety and global food crisis, which need to be urgently addressed. Therefore, many soil remediation technologies have been used to minimize the risk of heavy metal pollution. Among them, chemical fixation has the characteristics of low remediation cost and obvious effect in a short time and is widely used. At present, among the many chemical amendments for Cd-contaminated soils, biochar has attracted much attention due to its large specific surface area and strong Cd adsorption and fixation ability compared with quicklime and clay minerals. In addition, as a soil conditioner, another major advantage of biochar is that it can promote the sustainable treatment of large amounts of green waste such as straw, bagasse, and livestock and poultry bones, thereby maximizing the value-added benefits of green waste. Among them, biochar prepared from livestock and poultry bones is referred to as bone char.

[0003] Although chemical amendments such as biochar can fix Cd in the soil to a certain extent, and can reduce the biological effectiveness of Cd in the soil to a certain extent, and reduce its harm to the ecological environment and crops, the chemical amendments in the existing technology have problems such as short-term effects, short chemical fixation time, and easy recurrence, which will also affect the physical and chemical properties of the soil. With the deepening of research, some disadvantages of biochar have gradually emerged, and long-term use alone may cause imbalance of certain nutrients in the soil.

[0004] Phosphate-solubilizing bacteria (PSB) are a group of functional microorganisms that can convert insoluble phosphorus in the soil into effective phosphorus that can be absorbed and utilized by plants. They can participate in the nutrient cycle transformation in the soil, increase the effective nutrient content in the soil, improve the soil microbial community structure, and enhance soil fertility and quality. For the remediation of soil heavy metal Cd pollution, phosphate-solubilizing bacteria, as a biological remediation method, is low-cost and does not produce secondary pollution compared to chemical remediation methods. However, the growth and activity of phosphate-solubilizing bacteria are usually restricted by environmental factors, which greatly reduces the remediation effect.

[0005] In addition, microbial agents have gradually been used to remediate 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] In order to overcome the shortcomings of the prior art, the purpose of the present invention is to provide an application of a phosphate-dissolving bacteria agent to repair cadmium-contaminated soil while increasing the effective phosphorus content of the soil. The application of the phosphate-dissolving bacteria agent to repair Cd-contaminated soil while increasing the effective phosphorus content of the soil has a significant effect in reducing the biological effectiveness of Cd, and can maintain the ecological balance of the soil, and can also improve soil fertility and promote plant growth.

[0007] In order to achieve the purpose of the above invention, the technical solution adopted by the present invention is as follows:

[0008] The present invention provides an application of a phosphate-dissolving bacteria agent for repairing cadmium-contaminated soil and increasing the effective phosphorus content of the soil. The phosphate-dissolving bacteria agent comprises phosphate-dissolving bacteria, bone charcoal, a culture medium and an organic matrix; the phosphate-dissolving bacteria is Enterobacter hallii HS-6;

[0009] The classification name of the Enterobacter hormaechei HS-6 is Enterobacter hormaechei, which was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on October 19, 2023, with a deposit number of CGMCC NO.: 28680;

[0010] The phosphate-dissolving bacteria agent is applied to Cd-contaminated soil, and the bioavailability of Cd in the soil is synergistically reduced through Enterobacter holmesii HS-6 and bone char, while the effective phosphorus content in the soil is increased.

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

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

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

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

[0015] S1. Preparation of micro-nano bone charcoal: thermally crack animal bones, cool them, and then ball-mill them to obtain micron-nano multi-grade bone charcoal;

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

[0017] S3, preparation of liquid bacterial agent: adding the irradiated and sterilized micron-nano multi-grade bone charcoal obtained in step S1 and the active bacterial solution obtained in step S2 to NBRIP culture medium, and then placing in a constant temperature shaker at 28°C and shaking at 180r / min for 44h to 50h to obtain a liquid bacterial agent;

[0018] S4, organic matrix carrier treatment: after washing the organic matrix with deionized water, add deionized water that covers the organic matrix to soak the organic matrix, and sterilize at high temperature and high pressure to obtain a treated organic matrix carrier;

[0019] S5, preparation of solid bacterial agent: adding the liquid bacterial agent obtained in step S3 to the organic matrix carrier obtained in step S4, and culturing at 28° C. for 2 to 4 days to obtain the phosphate-dissolving bacterial agent;

[0020] S6. Remediation of Cd-contaminated soil: applying the phosphate-dissolving bacteria agent to Cd-contaminated soil.

[0021] Further, in step S1, the animal bone is heated to 440°C to 460°C at a heating rate of 8°C / min to 12°C / min and pyrolyzed for 100min to 140min, and after cooling, a large piece of bone char is obtained, and the large piece of bone char is ball-milled at 440rpm to 460rpm for 4h to 6h in a ball mill to obtain micron-nano multi-grade bone char; and / or

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

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

[0024] Furthermore, in step S3, the mass 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 liquid in the NBRIP culture medium is 1% of the volume percentage of the NBRIP culture medium; and / or

[0026] The radiation sterilization dosage is 3.0 kGy to 6.0 kGy.

[0027] Furthermore, in step S4, after washing the organic matrix three times with deionized water, deionized water that covers the organic matrix is ​​added to soak the organic matrix for 10 hours to 20 hours, and sterilized at high temperature and high pressure to obtain a treated organic matrix carrier;

[0028] The conditions of high temperature and high pressure sterilization are: 121° C., 104 KPa, 20 min.

[0029] Further, in step S5, the liquid bacterial agent obtained in step S3 is added to the organic matrix carrier obtained in step S4, and the mixture is placed in a polyethylene bag and sealed, and 5 to 8 air holes are pierced on the surface of the polyethylene bag with a sterile needle, and then the mixture 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-dissolving 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-dissolving bacteria agent to the Cd-contaminated soil is 3:(250-350).

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] (1) The present invention provides a method for repairing cadmium-contaminated soil and increasing the effective phosphorus content of the soil by using a phosphate-dissolving bacteria agent. The phosphate-dissolving bacteria agent includes phosphate-dissolving bacteria, bone charcoal, a culture medium and an organic matrix. The phosphate-dissolving bacteria is Enterobacter hussii HS-6. The phosphate-dissolving bacteria agent is applied to Cd-contaminated soil, and Enterobacter hussii HS-6 and bone charcoal synergistically reduce the biological effectiveness of Cd in the soil and increase the effective phosphorus content of the soil. On the one hand, the application can effectively promote the conversion of sparingly soluble phosphates into soluble phosphates, thereby increasing the effective phosphorus content of the soil by 96.30%; on the other hand, the application can fix the heavy metal Cd in the soil, thereby reducing the effective Cd content in the soil by about 54.41%. The application has excellent Cd-fixing ability and can alleviate the Cd stress of crops (such as rice). The application has great application value in remediating Cd pollution in farmland, improving soil quality and protecting the environment.

[0034] (2) The application of a phosphate-dissolving bacteria agent of the present invention to repair cadmium-contaminated soil while increasing the effective phosphorus content in the soil, compared with the Cd pollution remediation technology in the prior art that only focuses on a single mechanism, the application technology of the present invention is a combined remediation technology that uses synergistic effects of Enterobacter holmii HS-6 and micron-nano multi-level bone charcoal. The micron-nano multi-level bone charcoal in the phosphate-dissolving bacteria agent has rich pore structure, huge specific surface area and strong ion exchange capacity, which can efficiently and quickly adsorb Cd ions in the soil, has excellent Cd fixation ability in the soil, and can also provide a stable habitat and rich nutrients for Enterobacter holmii HS-6, which can protect Enterobacter holmii HS-6 from the influence of adverse environmental factors, enhance the survival ability and activity of Enterobacter holmii HS-6 in a polluted environment, and thereby improve the repair ability of Enterobacter holmii HS-6 for heavy metals. After the combination of Enterobacter holmii HS-6 and micron-nano multi-grade bone charcoal, the biochemical regulation effect of Enterobacter holmii HS-6 is prominent, thereby further strengthening the fixation of Cd, and the coexistence of Enterobacter holmii HS-6 and micron-nano multi-grade bone charcoal is beneficial to the growth and reproduction of Enterobacter holmii HS-6 itself and may continue to dissolve phosphorus, thereby continuously activating soil nutrients and optimizing Cd forms. Therefore, the synergy of Enterobacter holmii HS-6 and micron-nano multi-grade bone charcoal greatly improves the removal and fixation efficiency of Cd, greatly reduces the bioavailability of Cd in the soil, and reduces its harm to the ecological environment and crops. In addition, the metabolites produced by Enterobacter holmii HS-6 in the process of dissolving phosphorus can also form stable complexes or precipitations with Cd ions, further reducing the bioavailability of Cd. Therefore, compared with the method of using physical or chemical adsorption alone, the present invention has a more significant repair effect in reducing the bioavailability of Cd in the soil.

[0035] (3) The application of a phosphate-dissolving bacteria agent of the present invention to repair cadmium-contaminated soil and increase the effective phosphorus content of the soil is different from the traditional chemical remediation method, which uses a large amount of chemical agents and is prone to cause agent residues and secondary pollution. The repair technology of the present invention uses Enterobacter holmii HS-6 and micron-nano multi-grade bone charcoal, which are both of natural origin or biodegradable materials, and will not introduce harmful chemicals into the soil, which is more friendly to the soil and the surrounding ecological environment. In addition, Enterobacter holmii HS-6 can also improve the structure of soil microbial communities and promote the growth and reproduction of beneficial microorganisms, while micron-nano multi-grade bone charcoal can provide a good habitat for soil microorganisms and jointly maintain the ecological balance of the soil, which is difficult to achieve with many traditional repair technologies. Therefore, the application of the present invention has the advantage of being green and environmentally friendly.

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

[0037] (5) The application of a phosphate-dissolving bacteria agent of the present invention to repair cadmium-contaminated soil and increase the effective phosphorus content in the soil, through the synergistic effect of Enterobacter holliformis HS-6 and micron-nano multi-grade bone charcoal to reduce the toxic effect of Cd in the soil on crops, and enhance the stress resistance of crops, which is a function that simple physical or chemical repair technology does not have. On the other hand, micron-nano multi-grade bone charcoal is rich in various nutrients such as calcium, nitrogen, phosphorus and magnesium, which are exactly the nutrients necessary for plant growth. Therefore, the micron-nano multi-grade bone charcoal of the present invention can also serve as a fertilizer to supplement the fertility of the soil, promote the healthy growth of crops, and thus improve agricultural productivity. Therefore, the application of the present invention has the advantage of promoting plant growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

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

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

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

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

[0043] Figure 5 This is a graph showing the results of determining the available phosphorus content in soil from samples collected at different times from different experimental groups.

[0044] Figure 6 This is a graph showing the results of determining the effective Cd content in soil of samples collected from different experimental groups at different times.

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

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

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

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

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

[0050] Fig.12 This is a graph showing the results of measuring the root length of rice cultivated using soil samples from different experimental groups.

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

[0052] Fig.14 This is a graph showing the results of measuring the fresh weight of the underground part of rice cultivated with soil samples from different experimental groups. DETAILED DESCRIPTION

[0053] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0054] The terms used in the embodiments of the present invention are only for the purpose of describing specific embodiments, and are not intended to limit the present invention. In the present invention, the singular forms "a", "said" and "the" used in the embodiments and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

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

[0056] The culture medium formula described in the following examples is as follows:

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

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

[0059] NBRIP medium (g / L): glucose 10g, MgCl 2 6H 2 O 5g, MgSO 4 7H 2 O 0.25g, FeSO 4 7H 2 O0.01g, KCl 0.2g, (NH 4 ) 2 SO 4 0.1g, Ca 3 (PO 4 ) 2 5.0g, pH 7.0~7.4.

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

[0061] Example 1

[0062] A use of a phosphate-dissolving bacteria agent to repair cadmium-contaminated soil while increasing the effective phosphorus content of the soil, the phosphate-dissolving bacteria agent comprising phosphate-dissolving bacteria, bone charcoal, culture medium and wheat bran; the phosphate-dissolving bacteria is Enterobacter hormaechei HS-6. The classification name of Enterobacter hormaechei HS-6 is Enterobacter hormaechei, and it has been deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on October 19, 2023, with a deposit number of CGMCC NO.: 28680, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. Among them, the scanning electron microscope image of Enterobacter hormaechei HS-6 is as follows Figure 1 shown.

[0063] This application applies phosphate-dissolving bacteria to Cd-contaminated soil, synergistically reducing the bioavailability of Cd in the soil through Enterobacter HS-6 and bone char, while increasing the effective phosphorus content in the soil.

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

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

[0066] The application of the phosphate-dissolving bacteria agent to repair cadmium-contaminated soil and increase the effective phosphorus content in the soil comprises the following steps:

[0067] S1. Preparation of micro-nano bone char: animal bones are heated to 450°C at a heating rate of 10°C / min, thermally cracked at this temperature for 120min, and cooled to obtain large pieces of bone char. The large pieces of bone char are ball-milled at 450rpm for 5h in a ball mill to obtain micron-nano multi-grade bone char. In this embodiment, the animal bones are selected from pig bones.

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

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

[0070] S3, preparation of liquid bacterial agent: adding the irradiated and sterilized micron-nano multi-grade bone char prepared in step S1 and the active bacterial solution prepared in step S2 to NBRIP culture medium, and then placing in a constant temperature shaker at 28°C and shaking at 180r / min for 48h to obtain a liquid bacterial agent;

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

[0072] S4. Treatment of organic matrix carrier: After rinsing the organic matrix with deionized water for three times, add deionized water that covers the organic matrix to soak the organic matrix for 15 hours, and sterilize it at high temperature and high pressure to obtain a treated organic matrix carrier; wherein, the conditions of high temperature and high pressure sterilization are: 121°C, 104KPa, 20min.

[0073] S5. Preparation of solid bacterial agent: adding the liquid bacterial agent obtained in step S3 to the organic matrix carrier obtained in step S4, putting the mixture into a polyethylene bag and sealing it, piercing 5 air holes on the surface of the polyethylene bag with a sterile needle, and then placing the mixture into a constant temperature shaker at 28° C. and shaking and culturing at 180 r / min for 3 days to obtain a phosphate-dissolving bacterial agent; in this embodiment, the mass ratio of the liquid bacterial agent to the organic matrix carrier is 15:85;

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

[0075] Example 2

[0076] The invention discloses an application of a phosphate-dissolving bacteria agent to repair cadmium-contaminated soil while increasing the effective phosphorus content of the soil. The difference between this embodiment and embodiment 1 is that in step S3, the mass volume ratio of bone char to NBRIP culture medium is 1%, the dose of irradiation sterilization is 3.0 kGy, and the shaking culture is carried out for 44 hours. The remaining conditions and methods of this embodiment are the same as those of embodiment 1.

[0077] Example 3

[0078] The invention discloses an application of a phosphate-dissolving bacteria agent to repair cadmium-contaminated soil while increasing the effective phosphorus content of the soil. The difference between this embodiment and embodiment 1 is that in this embodiment, the organic matrix is ​​rice straw; in step S3, the mass volume ratio of bone char to NBRIP culture medium is 2%, the dose of irradiation sterilization is 4.0 kGy, and the shaking culture is carried out for 46 hours. The remaining conditions and methods of this embodiment are the same as those of embodiment 1.

[0079] Example 4

[0080] The invention discloses an application of a phosphate-dissolving bacteria agent to repair cadmium-contaminated soil while increasing the effective phosphorus content of the soil. The difference between this embodiment and embodiment 1 is that in this embodiment, the organic matrix is ​​corn cobs; in step S3, the mass volume ratio of bone char to NBRIP culture medium is 2.5%, the dose of irradiation sterilization is 6.0 kGy, and the shaking culture is carried out for 50 hours. The remaining conditions and methods of this embodiment are the same as those of embodiment 1.

[0081] Example 5

[0082] The invention discloses an application of a phosphate-dissolving bacteria agent to repair cadmium-contaminated soil while increasing the effective phosphorus content of the soil. The difference between this embodiment and embodiment 1 is that, in this embodiment, step S1, preparation of micro-nano bone charcoal: heating animal bones to 440°C at a heating rate of 8°C / min, thermally cracking at this temperature for 140 minutes, cooling to obtain large pieces of bone charcoal, ball milling the large pieces of bone charcoal at 440 rpm for 6 hours in a ball mill to obtain micron-nano multi-grade bone charcoal.

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

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

[0085] Example 6

[0086] The invention discloses an application of a phosphate-dissolving bacteria agent to repair cadmium-contaminated soil while increasing the effective phosphorus content of the soil. The difference between this embodiment and embodiment 1 is that, in this embodiment, step S1, preparation of micro-nano bone charcoal: heating animal bones to 460°C at a heating rate of 12°C / min, thermally cracking at this temperature for 100 minutes, and obtaining large pieces of bone charcoal after cooling. The large pieces of bone are ball-milled at 460 rpm for 4 hours using a ball mill to obtain micron-nano multi-grade bone charcoal.

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

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

[0089] Example 7

[0090] A use of a phosphate-dissolving bacteria agent to repair cadmium-contaminated soil while increasing the effective phosphorus content of the soil. The difference between this embodiment and embodiment 1 is that in this embodiment, in step S4, deionized water that covers the organic matrix is ​​added to soak the organic matrix for 10 hours. In step S5, 6 air holes are pierced on the surface of the polyethylene bag with a sterile needle, and then placed in a constant temperature shaker at 28°C and shaken at 180r / min for 2 days to obtain a phosphate-dissolving bacteria agent; in this embodiment, the mass ratio of the liquid bacteria agent to the organic matrix carrier is 15:100. In step S6, the mass ratio of the phosphate-dissolving bacteria agent to the Cd-contaminated soil is 3:250.

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

[0092] Example 8

[0093] The invention discloses an application of a phosphate-dissolving bacteria agent to repair cadmium-contaminated soil and simultaneously increase the effective phosphorus content of the soil. The difference between this embodiment and embodiment 1 is that in this embodiment, in step S4, deionized water that covers the organic matrix is ​​added to soak the organic matrix for 20 hours. In step S5, 8 air holes are pierced on the surface of the polyethylene bag with a sterile needle, and then the bag is placed in a constant temperature shaker at 28°C and cultured at 180r / min for 4 days to obtain a phosphate-dissolving bacteria agent. In this embodiment, the mass ratio of the liquid bacteria agent to the organic matrix carrier is 15:70. In step S6, the mass ratio of the phosphate-dissolving bacteria agent to the Cd-contaminated soil is 3:350.

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

[0095] Experimental testing:

[0096] 1. Scanning electron microscope detection

[0097] Among them, the scanning electron micrograph of the strain Enterobacter holmesii HS-6 of the present invention is shown in Figure 1 .in, Figure 1 (b) Yes Figure 1 (a) The enlarged view of the part in the dotted box. Figure 1 As shown, Enterobacter hallii HS-6 is a rod-shaped bacterium 1 μm in length.

[0098] In addition, the scanning electron microscopy of Enterobacter HS-6 after reacting with bone charcoal can be seen in Figure 2 ,in Figure 2 (b) Figure 2 (a) Enlarged view of the dotted box. Figure 2 It can be seen that Enterobacter holmii HS-6 is adsorbed on the surface of bone charcoal, and the morphology of Enterobacter holmii HS-6 is complete and plump, indicating that bone charcoal provides favorable conditions for the growth and physiological state of Enterobacter holmii HS-6.

[0099] 2. Remediation of Cd-contaminated soil

[0100] 1. Soil collection

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

[0102] 2. Experimental sample design plan:

[0103] The design of specific experimental samples is shown in Table 1 below.

[0104] Table 1 Design plan of experimental samples

[0105]

[0106] There are four experimental groups in Table 1, and each experimental group has four parallels. The total weight of each experimental group sample is 300g, and it is placed in a brown wide-mouth bottle for culture. The bottle is placed in a constant temperature environment of 25℃, and water is added daily to keep the moisture content constant at 60%. Soil samples were collected on the 0th, 3rd, 10th, 18th and 30th day, respectively, ground after cold drying, and passed through a 100-mesh sieve for subsequent testing.

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

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

[0109] The soil pH values ​​of the samples designed according to Table 1 and collected at different times (four replicates for each sample) were measured according to the Potentiometric Method for Determination of Soil pH Value (HJ 962-2018). The results can be found in Figure 3 . Figure 3 In the figure, the mark “Cd” represents the Cd soil group, the mark “Cd MNBC” represents the Cd MNBC soil group, and the mark “Cd MNBC HS-6” represents the Cd MNBC HS-6 soil group.

[0110] Depend on Figure 3 The measurement results show that the pH values ​​of the samples in the blank control group and the Cd soil group at each sampling period were about 4.5, and the pH values ​​of the samples in the Cd MNBC soil group and the Cd MNBC HS-6 soil group at each sampling period were about 6, indicating that the pH value of the soil with the addition of bone char or bone char and Enterobacter holmesii HS-6 was higher than that of the blank control group, which can improve the acidic soil.

[0111] in addition, Figure 3 The soil pH basically showed a trend of first increasing and then decreasing, reaching the highest on the 10th day, indicating that the soil pH value increased significantly after applying bone char or applying bone char and Enterobacter holmesii HS-6.

[0112] A large number of studies have shown that in acidic soils (pH <6), the activity of some phosphate-dissolving bacteria may be inhibited, but in neutral (pH 6-7) or slightly alkaline (pH>7) soils, many phosphate-dissolving bacteria are highly active, which is conducive to the release of phosphorus. Therefore, the application of a phosphate-dissolving bacteria agent of the present invention to repair cadmium-contaminated soil while increasing the effective phosphorus content of the soil can make the soil close to neutral, thereby increasing the activity of Enterobacter holmesii HS-6 and promoting the increase of the effective phosphorus content of the soil. Therefore, the application of the phosphate-dissolving bacteria agent of the present invention in the soil, combined with the effects of bone charcoal and Enterobacter holmesii HS-6, is conducive to the survival of Enterobacter holmesii HS-6 in paddy soil and better play of the phosphate-dissolving effect.

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

[0114] The soil cation exchange capacity (CEC) was determined by sampling samples designed according to Table 1 and collected at different times (four replicates were set for each sample). The results can be found in Figure 4 Among them, the determination of soil cation exchange capacity is carried out in accordance with the "Determination of soil cation exchange capacity - Hexaamminecobalt trichloride extraction-spectrophotometric method" (HJ 889-2017). Figure 4 In the figure, the mark “Cd” represents the Cd soil group, the mark “Cd MNBC” represents the Cd MNBC soil group, and the mark “Cd MNBC HS-6” represents the Cd MNBC HS-6 soil group.

[0115] Depend on Figure 4 The test results show that the soil cation exchange capacity of the samples in the Cd MNBC soil group and the Cd MNBC HS-6 soil group at each sampling period was significantly improved compared with the blank control group and the Cd soil group. The soil cation exchange capacity basically showed a trend of first increasing and then decreasing, reaching the highest on the 10th day, indicating that the soil cation exchange capacity of the application of bone char or the application of bone char and E. hallii HS-6 was significantly improved.

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

[0117] The samples designed and collected at different times according to Table 1 (four replicates for each sample) were used to determine the soil available phosphorus content. The results can be found in Figure 5 . Figure 5 In the figure, the mark “Cd” represents the Cd soil group, the mark “Cd MNBC” represents the Cd MNBC soil group, and the mark “Cd MNBC HS-6” represents the Cd MNBC HS-6 soil group.

[0118] Depend on Figure 5 The measurement results show that the effective phosphorus content of the Cd MNBC soil group and the Cd MNBC HS-6 soil group is significantly increased compared with the blank control group and the Cd soil group. It shows that the application of micron-nano multi-grade bone char and Escherichia coli HS-6 in the soil can significantly increase the effective phosphorus content of the soil. Interestingly, compared with the Cd MNBC soil group, the effective phosphorus content of the soil in the Cd MNBC HS-6 soil group under the action of micron-nano multi-grade bone char combined with Escherichia coli HS-6 increased by 20.27% on the 3rd day, indicating that under Cd pollution, the application of the present invention, i.e., the coordinated use of bone char and Escherichia coli HS-6, can further increase the effective phosphorus content of the soil. After 30 days, the effective phosphorus content of the soil in the Cd MNBC HS-6 soil group increased by about 96.30%.

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

[0120] The samples designed and collected at different times according to Table 1 (four replicates for each sample) were used to determine the soil available Cd content. The results are shown in Figure 6 . Figure 6 In the figure, the mark “Cd” represents the Cd soil group, the mark “Cd MNBC” represents the Cd MNBC soil group, and the mark “Cd MNBC HS-6” represents the Cd MNBC HS-6 soil group.

[0121] The determination of soil available Cd content was carried out according to the "Determination of 8 Available Elements in Soil - Diethylenetriaminepentaacetic Acid Extraction-Inductively Coupled Plasma Optical 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 (CaCl 2 )-triethanolamine (TEA) extractant (pH 7.3), reciprocating at 160 r / min at room temperature for 2 h, centrifuged, and the supernatant was filtered through a 0.22 μm filter membrane for testing.

[0122] Depend on Figure 6 The results of the determination show that the effective Cd content of the Cd MNBC soil group and the Cd MNBC HS-6 soil group was significantly reduced compared with the Cd soil group. In particular, the Cd MNBC HS-6 soil group had a greater reduction in the effective Cd content than the Cd MNBC soil group, indicating that the micron-nano multi-grade bone char combined with Escherichia coli HS-6 had a better effect in reducing the effective Cd content in the soil. The analysis of the Cd soil group on the 3rd day found that the effective Cd content in the Cd MNBC soil group and the Cd MNBC HS-6 soil group was reduced by 39.88% and 53.09%, respectively. After 30 days of the action of micron-nano multi-grade bone char combined with Escherichia coli HS-6, the effective Cd content of the Cd MNBC HS-6 soil group was reduced by 54.41%. It shows that the application of a phosphate-dissolving bacteria agent of the present invention to repair cadmium-contaminated soil while increasing the effective phosphorus content in the soil can greatly reduce the effective Cd content and has a more significant effect in reducing the biological effectiveness of Cd in the soil.

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

[0124] The samples designed according to Table 1 and collected at different times (four replicates for each sample) were tested for soil catalase activity, sucrase activity, urease activity and acid phosphatase activity. The test results are shown in Table 1. Figure 7 , Figure 8 , Fig. 9 and Fig.10 .

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

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

[0127] Among them, urease, sucrase and catalase are important soil enzymes widely used to evaluate the effects of soil conditioners on soil biological functions. Urease and sucrase participate in the decomposition and mineralization of carbohydrates in the soil, providing nutrition for the growth of soil microorganisms and crops, while catalase can degrade H in the soil. 2 O 2 , reducing its toxic effects on organisms. Figure 7 It can be seen that compared with the Cd soil group, the micron-nano multi-grade bone char combined with E. hallii HS-6 can significantly increase the catalase activity in Cd-contaminated soil by 13.84%. Figures 8 to 9 The measurement results showed that the micron-nano multi-grade bone char combined with Enterobacter holmesii HS-6 significantly promoted the activities of sucrase and urease in Cd-contaminated soil, which were increased by about 366.09% and 470.52% respectively compared with the Cd soil group.

[0128] In addition, acid phosphatase is a type of enzyme that catalyzes the mineralization of organic phosphorus in the soil. Its activity directly affects the decomposition and transformation of organic phosphorus in the soil and its biological effectiveness. It is an indicator for evaluating the direction and intensity of soil phosphorus biological transformation. Acid phosphatase is significantly affected by soil carbon and nitrogen content, available phosphorus content and pH. Fig.10 The results of the test showed that compared with the Cd soil group, in the Cd MNBC HS-6 soil group, the micron-nano multi-grade bone char combined with E. coli HS-6 could significantly increase the acid phosphatase activity in the Cd contaminated soil by 82.46%. Therefore, the micron-nano multi-grade bone char combined with E. coli HS-6 enhanced the activity of acid phosphatase in Cd contaminated soil, and this enhancement may be closely related to the unique properties of micron-nano multi-grade bone char. Bone char is an additional source of C and P elements, providing more suitable habitat conditions for E. coli HS-6 in the soil and promoting their growth.

[0129] 3. Experiments to promote crop growth

[0130] The rice variety selected was Xiuzhan 15. The rice was first cultivated in a plant incubator. After 14 days of cultivation, the rice seedlings of the same size were transplanted into the four experimental group samples designed according to Table 1 for pot cultivation. Six parallel samples were set for each sample and placed at room temperature for a 28-day exposure experiment. The rice plants were harvested and the plant height, root length, aboveground fresh weight and underground fresh weight of the rice were measured. The results of the measurements are shown in Fig.11 , Fig.12 , Fig.13 and Fig.14 .

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

[0132] Depend on Figures 11 to 14 The results of the test 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, by 22.55%, 25.87%, 25.23% and 32.00% respectively, indicating that Cd-contaminated soil seriously affects the growth of crops. Compared with the Cd soil group, 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. Among them, the plant height, root length, aboveground fresh weight and underground fresh weight of rice in the CdMNBC HS-6 soil group increased by 18.30%, 34.89%, 61.87% and 18.18% respectively compared with the Cd soil group. It shows that the application of a phosphate-dissolving bacteria agent to repair cadmium-contaminated soil and increase the effective phosphorus content in the soil can promote the healthy growth of crops and thus improve agricultural productivity.

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

[0134]

Claims

1. An application of a phosphate-dissolving bacteria agent to repair cadmium-contaminated soil and increase the effective phosphorus content of the soil, characterized in that: The phosphate-dissolving bacteria agent comprises phosphate-dissolving bacteria, bone charcoal, culture medium and organic matrix; the phosphate-dissolving bacteria is Enterobacter holmesii HS-6; The classification name of the Enterobacter hormaechei HS-6 is Enterobacter hormaechei, which was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on October 19, 2023, with a deposit number of CGMCC NO.: 28680; The phosphate-dissolving bacteria agent is applied to Cd-contaminated soil, and the bioavailability of Cd in the soil is synergistically reduced through Enterobacter holmesii HS-6 and bone char, while the effective phosphorus content in the soil is increased.

2. The use of a phosphate-dissolving bacteria agent as claimed in claim 1 for repairing cadmium-contaminated soil while increasing the effective phosphorus content of the soil, characterized in that: The 16S rDNA gene sequence of the Enterobacter holmesii HS-6 is shown in SEQ ID No.

1.

3. The use of a phosphate-dissolving bacteria agent for repairing cadmium-contaminated soil and increasing the effective phosphorus content of the soil as claimed in claim 1, characterized in that: The particle size of the bone char is 30nm-2μm.

4. The use of a phosphate-dissolving bacteria agent for repairing cadmium-contaminated soil and increasing the effective phosphorus content of the soil as claimed in claim 1, characterized in that: The organic matrix is ​​at least one of wheat bran, rice straw or corn cob.

5. The use of a phosphate-dissolving bacteria agent for repairing cadmium-contaminated soil and increasing the effective phosphorus content of the soil as claimed in claim 1, characterized in that: The application comprises the following steps: S1. Preparation of micro-nano bone charcoal: thermally crack animal bones, cool them, and then ball-mill them to obtain micron-nano multi-grade bone charcoal; S2. Activation of Enterobacter holmesii: Enterobacter holmesii HS-6 was inoculated into LB medium and then placed in a constant temperature shaker at 28°C and shaken at 180 r / min until OD 600 =0.6-0.8, to obtain active bacterial solution; S3, preparation of liquid bacterial agent: adding the irradiated and sterilized micron-nano multi-grade bone charcoal obtained in step S1 and the active bacterial solution obtained in step S2 to NBRIP culture medium, and then placing in a constant temperature shaker at 28°C and shaking at 180r / min for 44h to 50h to obtain a liquid bacterial agent; S4, organic matrix carrier treatment: after washing the organic matrix with deionized water, add deionized water that covers the organic matrix to soak the organic matrix, and sterilize at high temperature and high pressure to obtain a treated organic matrix carrier; S5, preparation of solid bacterial agent: adding the liquid bacterial agent obtained in step S3 to the organic matrix carrier obtained in step S4, and culturing at 28° C. for 2 to 4 days to obtain the phosphate-dissolving bacterial agent; S6. Remediation of Cd-contaminated soil: applying the phosphate-dissolving bacteria agent to Cd-contaminated soil.

6. The use of a phosphate-dissolving bacteria agent for repairing cadmium-contaminated soil and increasing the effective phosphorus content of the soil as claimed in claim 5, characterized in that: In step S1, the animal bone is heated to 440°C to 460°C at a heating rate of 8°C / min to 12°C / min and thermally cracked for 100min to 140min, and then cooled to obtain a large piece of bone char, which is ball-milled in a ball mill at 440rpm to 460rpm for 4h to 6h to obtain micron-nano multi-grade bone char; and / or In the ball mill, zirconium oxide particles are used to ball-mill the bulk bone char, and ethanol is added as a grinding aid; the weight ratio of the zirconium oxide particles to the bulk bone char is (15-20):1; the mass ratio of the ethanol to the bulk bone char is (1.5-2):

1.

7. The use of a phosphate-dissolving bacteria agent for repairing cadmium-contaminated soil and increasing the effective phosphorus content of the soil as claimed in claim 5, characterized in that: In step S2, the effective viable bacteria count of the prepared active bacterial solution is 1×10 10 CFU / mL~9×10 10 CFU / mL.

8. The use of a phosphate-dissolving bacteria agent for repairing cadmium-contaminated soil and increasing the effective phosphorus content in the soil as claimed in claim 5, characterized in that: In step S3, the mass 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 liquid in the NBRIP culture medium is 1% of the volume percentage of the NBRIP culture medium; and / or The radiation sterilization dosage is 3.0 kGy to 6.0 kGy.

9. The use of a phosphate-dissolving bacteria agent for repairing cadmium-contaminated soil and increasing the effective phosphorus content of the soil as claimed in claim 5, characterized in that: In step S4, after washing the organic matrix three times with deionized water, adding deionized water that covers the organic matrix to soak the organic matrix for 10 hours to 20 hours, and sterilizing the organic matrix carrier with high temperature and high pressure; The conditions of high temperature and high pressure sterilization are: 121° C., 104 KPa, 20 min.

10. The use of a phosphate-dissolving bacteria agent for repairing cadmium-contaminated soil and increasing the effective phosphorus content of the soil as claimed in claim 5, 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 the mixture is placed in a polyethylene bag and sealed, and 5 to 8 air holes are pierced on the surface of the polyethylene bag with a sterile needle, and then the mixture 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-dissolving 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-dissolving bacteria agent to the Cd-contaminated soil is 3:(250-350).

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