A phosphate-solubilizing agent and its preparation method, bio-fertilizer and its application

Abstract

This invention relates to the fields of environmental protection, resource utilization, and agricultural biotechnology, specifically to a phosphate-solubilizing bacterial agent with high effective phosphorus conversion, its preparation method, bio-fertilizer, and its applications. The phosphate-solubilizing bacterial agent comprises phosphate-solubilizing bacteria, bone char, and a culture medium. The phosphate-solubilizing bacteria is *Pantotheca acuminata* CT-3. By using bone char to prepare this phosphate-solubilizing bacterial agent with high effective phosphorus conversion, the full utilization of phosphorus resources in bone char can be greatly promoted, achieving efficient dissolution of insoluble phosphorus from bone char. This phosphate-solubilizing bacterial agent with high effective phosphorus conversion combines *Pantotheca acuminata* CT-3 with bone char, utilizing both the phosphorus-solubilizing ability of *Pantotheca acuminata* CT-3 and the phosphorus resources of bone char. This phosphate-solubilizing bacterial agent with high effective phosphorus conversion shows great promise in the preparation of bio-fertilizers, in agricultural soil improvement, in the recovery of phosphorus resources from bone char, and in promoting crop growth.

Description

Technical Field

[0001] This invention relates to the fields of environment, resource utilization and agricultural biotechnology, specifically to a phosphorus-solubilizing bacterial agent with high effective phosphorus conversion, its preparation method, bio-fertilizer and its application. Background Technology

[0002] Phosphorus accounts for approximately 0.2% to 10.8% of plant dry weight and is a crucial essential macronutrient for crops and global food security. However, less than 0.1% of inorganic and organic phosphorus in the soil is available for plant uptake, insufficient to meet the needs of plants and microorganisms. Due to phosphorus deficiency, it is estimated that 5.7 billion hectares of land suffer from this problem. 2 Agricultural soils are limiting crop productivity. It has been reported that at least 70% of phosphate fertilizer enters the soil and is fixed there, making it difficult for crops to absorb and utilize.

[0003] Bone char is a phosphorus-rich material with a wide range of sources, often produced from livestock and poultry waste bones through oxygen-limited pyrolysis carbonization. Compared to biochar prepared from other raw materials, bone char has a higher phosphorus content, but it mainly exists in the form of insoluble phosphates such as calcium phosphate, commonly including hydroxyapatite (Ca5(PO4)3OH) and tricalcium phosphate (Ca3(PO4)2), which are difficult for plants to absorb effectively. If the insoluble phosphorus in bone char can be fully converted into soluble phosphorus, making it available to plants, not only can phosphorus resources be utilized efficiently, but dependence on traditional phosphate fertilizers can also be reduced, thereby lowering agricultural production costs and environmental pressure.

[0004] Microorganisms play a crucial role in maintaining and regulating phosphorus properties through inorganic phosphorus dissolution and organic phosphorus mineralization, thereby enhancing phosphorus availability in plants. This has significant practical implications and application value for solving phosphorus resource utilization problems and promoting sustainable agricultural development.

[0005] Microbial agents are important carriers widely used in agriculture, mining, and environmental remediation, and have received increasing attention in recent years. However, the development of phosphate-solubilizing agents still needs to be strengthened and expanded to enrich phosphate-solubilizing agent resources and promote soil improvement and agricultural development. Summary of the Invention

[0006] To overcome the shortcomings of the prior art, the first objective of this invention is to provide a phosphate-solubilizing bacterial agent with high effective phosphorus conversion. This agent, by combining bone char with phosphate-solubilizing bacteria, expands the resources of phosphate-solubilizing bacterial agents and improves the efficiency of converting insoluble phosphorus in bone char into soluble phosphorus.

[0007] To overcome the shortcomings of the prior art, the second objective of this invention is to provide a method for preparing a phosphate-solubilizing bacterial agent with high effective phosphorus conversion. This preparation method can achieve the efficient conversion of insoluble phosphorus in bone char into soluble phosphorus. The resulting phosphate-solubilizing bacterial agent expands the resources of phosphate-solubilizing bacterial agents and improves the efficiency of converting insoluble phosphorus in bone char into soluble phosphorus.

[0008] The third objective of this invention is to provide a bio-fertilizer.

[0009] The fourth objective of this invention is to provide an application of a phosphate-solubilizing bacterial agent with high effective phosphorus conversion.

[0010] To achieve the first objective of the invention, the technical solution adopted by the present invention is as follows:

[0011] This invention provides a phosphate-solubilizing bacterial agent with high effective phosphorus conversion, comprising phosphate-solubilizing bacteria, bone char, and culture medium; wherein the phosphate-solubilizing bacteria is Pantotheca cumulus CT-3, which belongs to the bacterial class.

[0012] The aforementioned Pantoea agglomerans CT-3 is classified as Pantoea agglomerans and was deposited on January 8, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.: 33335.

[0013] Furthermore, the 16S rDNA gene sequence of the clump-forming pantothecin CT-3 is shown in SEQ ID No. 1.

[0014] Furthermore, the particle size of the bone char is 30 nm to 2 μm. Among them, the Pantothecin CT-3 agglomerates exhibit a good effect on converting insoluble phosphorus into soluble phosphorus in bone char with a particle size of 30 nm to 2 μm.

[0015] By using bone char to prepare phosphate-solubilizing bacteria with high effective phosphorus conversion, the full utilization of bone char phosphorus resources can be greatly promoted.

[0016] This phosphate-solubilizing bacterial agent, which boasts a high effective phosphorus conversion rate, combines Pantotheca acuminata CT-3 with bone char. This allows for the utilization of both the phosphorus-solubilizing ability of Pantotheca acuminata CT-3 and the phosphorus resources of bone char. Furthermore, the surface of the bone char provides a carrier for the growth and reproduction of the bacteria. Additionally, the culture medium promotes the growth and metabolism of Pantotheca acuminata CT-3.

[0017] To achieve the second objective of the invention, the technical solution adopted by the present invention is as follows:

[0018] This invention provides a method for preparing a phosphate-solubilizing bacterial agent with high effective phosphorus conversion, comprising the following steps:

[0019] S1. Preparation of bone char: Animal bones are freeze-dried, then thermally pyrolyzed and cooled, and then ball-milled to obtain micron- and nano-scale bone char; the animal bones can be pig bones, cattle bones, or sheep bones.

[0020] S2. Activation of Pantotheca clumps: Pantotheca clumps CT-3 was inoculated into LB medium and then placed in a constant temperature shaker at 28℃ and 180r / min for 16h to allow OD to form. 600 The concentration was 0.6–0.8, resulting in an active bacterial solution;

[0021] S3. Preparation of phosphate-solubilizing bacterial agent: After sterilization by irradiation, the micron-nano multi-level bone char obtained in step S1 is added to NBRIP-P medium to obtain a reaction medium. Then, the active bacterial solution is inoculated into the reaction medium and placed in a constant temperature shaker at 28°C with shaking at 180 r / min for 3 days to obtain the phosphate-solubilizing bacterial agent with high effective phosphorus conversion.

[0022] Further, in step S1, animal bones are freeze-dried at -65℃ to -55℃ for 40 to 55 hours, then thermally pyrolyzed at 440℃ to 460℃ for 100 to 140 minutes. After cooling, large pieces of bone char are obtained. These large pieces of bone char are then ball-milled at 440 rpm to 460 rpm for 4 to 6 hours to obtain micron- to nano-scale multi-level bone char; and / or

[0023] In the ball mill, zirconium oxide particles are used to ball mill large pieces of bone char, and ethanol is added as a grinding aid; the weight ratio of zirconium oxide particles to large pieces of bone char is (15-20):1; the mass ratio of ethanol to large pieces of bone char is (1.5-2):1.

[0024] Furthermore, in step S2, the effective viable count of the obtained active bacterial solution is 1×10⁻⁶. 10 CFU / mL ~9×10 10 CFU / mL; and / or

[0025] In step S3, the mass-to-volume ratio of the bone char to the NBRIP-P culture medium is 0.5% to 2.5%; and / or

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

[0027] The inoculation amount of the active bacterial solution in the reaction medium is 1% of the volume of the reaction medium.

[0028] To achieve the third objective of the invention, the technical solution adopted by the present invention is as follows:

[0029] This invention provides a bio-fertilizer, which is prepared using a phosphorus-solubilizing bacterial agent with high effective phosphorus conversion rate as described above.

[0030] Among them, the phosphorus-solubilizing bacterial agent with high effective phosphorus conversion capacity combines Pantotheca granulata CT-3 with bone char, which can utilize both the phosphorus-solubilizing ability of Pantotheca granulata CT-3 and the phosphorus resources of bone char. Therefore, it has a good prospect for application in the preparation of bio-fertilizer.

[0031] To achieve the fourth objective of the invention, the technical solution adopted by the present invention is as follows:

[0032] This invention provides the application of the above-mentioned phosphorus-solubilizing bacterial agent with high effective phosphorus conversion in agricultural soil improvement.

[0033] Specifically, the phosphorus-soluble bacteria agent with high effective phosphorus conversion is applied to the soil, and the action of the clump-forming pantothecin CT-3 promotes the conversion and release of insoluble phosphorus in the soil, thereby improving the effective phosphorus content in the soil and promoting plant growth.

[0034] This invention provides the application of the above-mentioned phosphorus-solubilizing bacterial agent with high effective phosphorus conversion in the recovery of bone char phosphorus resources.

[0035] Specifically, applying the phosphorus-soluble bacterial agent with high effective phosphorus conversion rate to bone char to convert insoluble phosphorus into soluble phosphorus can improve the recovery rate of phosphorus resources in bone char.

[0036] This invention provides the application of the above-mentioned phosphorus-soluble bacterial agent with high effective phosphorus conversion in promoting crop growth.

[0037] This phosphorus-solubilizing bacterial agent, which boasts a high effective phosphorus conversion rate, combines phosphorus-solubilizing bacteria with bone char. This not only utilizes the phosphorus-solubilizing ability of the bacteria but also provides a growth and reproduction carrier for the bacteria on the surface of the bone char, thereby improving the efficiency of converting insoluble phosphorus in bone char into soluble phosphorus. This not only helps improve the recovery and utilization rate of phosphorus resources but also reduces environmental pollution from traditional chemical treatment methods, improves agricultural soil, and promotes crop growth when used as a bio-fertilizer. Therefore, it has excellent application prospects in all these areas.

[0038] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0039] (1) The present invention provides a phosphate-solubilizing agent with high effective phosphorus conversion, comprising phosphate-solubilizing bacteria, bone char, and culture medium; the phosphate-solubilizing bacteria is Pantotheca cumingii CT-3. By combining bone char with phosphate-solubilizing bacteria to prepare this phosphate-solubilizing agent with high effective phosphorus conversion, not only is the resource of phosphate-solubilizing agents expanded, but the full utilization of phosphorus resources in bone char is also greatly promoted, realizing the dissolution of insoluble phosphorus in bone char and greatly improving the efficiency of converting insoluble phosphorus in bone char into soluble phosphorus. This phosphate-solubilizing agent with high effective phosphorus conversion, by combining Pantotheca cumingii CT-3 with bone char, can utilize the phosphate-solubilizing ability of Pantotheca cumingii CT-3 and fully utilize the phosphorus and calcium resources of bone char. The surface of bone char can also provide a carrier for the growth and reproduction of bacteria. Therefore, this phosphate-solubilizing agent with high effective phosphorus conversion not only expands the resource of phosphate-solubilizing agents but also improves the utilization efficiency of phosphorus resources in bone char, reduces the waste of phosphorus resources in bone char during use, and thus promotes plant growth.

[0040] (2) The present invention provides a method for preparing a phosphate-solubilizing agent with high effective phosphorus conversion, thereby achieving resource recycling. Since bone char is usually a product of animal bones and other waste, utilizing Pantothecin CT-3 to convert phosphorus in bone char is a secondary utilization of waste resources, which helps to achieve resource recycling, reduces dependence on primary phosphate rock resources, and meets the requirements of sustainable development. The method is simple and easy to operate, and the resulting phosphate-solubilizing agent expands the resources of phosphate-solubilizing agents, improves the utilization efficiency of phosphorus resources in bone char, and has good application effects and environmental friendliness.

[0041] (3) The preparation method of the phosphate-solubilizing bacterial agent with high effective phosphorus conversion of the present invention is as follows: Since Pantotheca cumulus CT-3 has a strong phosphorus-solubilizing ability and bone char is rich in insoluble phosphorus, after Pantotheca cumulus CT-3 reacts with bone char, it can convert a large amount of insoluble phosphorus in bone char into soluble phosphorus in a short time, and has the advantage of high soluble phosphorus conversion efficiency. The phosphorus solubility reaches 4907.65 mg / L in 72 h, and the effect of converting insoluble phosphorus into soluble phosphorus is stable. The phosphorus solubility can also reach 2702.02 mg / L in 168 h, which improves the effectiveness and persistence of phosphorus, and thus has the advantage of high effective phosphorus conversion. Therefore, the prepared phosphate-solubilizing bacterial agent has a good application prospect.

[0042] (4) The bio-fertilizer of the present invention is prepared using a phosphorus-solubilizing agent with high effective phosphorus conversion rate, as described above. Therefore, it can promote the conversion of insoluble phosphorus into soluble phosphorus and has a good application effect. In particular, the phosphorus-solubilizing agent with high effective phosphorus conversion rate combines Pantotheca cumulus CT-3 with bone char, which can utilize both the phosphorus-solubilizing ability of Pantotheca cumulus CT-3 and the phosphorus and calcium resources of bone char. Therefore, it has a good prospect for application in the preparation of bio-fertilizer.

[0043] (4) The application of the phosphorus-solubilizing agent with high effective phosphorus conversion capacity of the present invention is as follows: By combining phosphorus-solubilizing bacteria with bone char, this agent can utilize the phosphorus-solubilizing ability of the bacteria and provide a growth and reproduction carrier for the bacteria through the surface of the bone char, thereby improving the efficiency of converting insoluble phosphorus in bone char into soluble phosphorus. This not only helps to improve the recovery and utilization rate of phosphorus resources, but also reduces environmental pollution from traditional chemical treatment methods, improves agricultural soil, and promotes crop growth when used as a bio-fertilizer. Therefore, it has excellent application prospects in agricultural soil improvement, phosphorus resource recovery from bone char, and promotion of crop growth. Attached Figure Description

[0044] 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.

[0045] Figure 1 This is a scanning electron microscope image of the CT-3 cluster of pantothecin bacteria according to the present invention.

[0046] Figure 2 This is a scanning electron microscope image of the clump-forming pantothecin CT-3 of the present invention after interaction with bone char.

[0047] Figure 3 This is a graph showing the results of testing the phosphorus-dissolving ability of the Pantotheca CT-3 of the present invention with different amounts of bone char applied.

[0048] Figure 4 This is a graph showing the dehydrogenase activity detection results of the CT-3 clumps of the present invention under different amounts of bone char application. Detailed Implementation

[0049] 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.

[0050] 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.

[0051] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0052] The culture medium formulations described in the following examples are as follows:

[0053] LB medium (g / L): 10g peptone, 5g yeast extract, 10g NaCl, pH 7.0-7.2.

[0054] LB solid medium: Add 18g to 20g of agar powder to every 1L of LB medium.

[0055] NBRIP-P medium (g / L): glucose 10g, MgCl2·6H2O 5g, MgSO4·7H2O 0.25g, FeSO4·7H2O 0.01g, KCl 0.2g, (NH4)2SO4 0.1g, pH 7.0~7.4.

[0056] NBRIP-P solid medium: Add 18g to 20g of agar powder to every 1L of NBRIP-P medium.

[0057] Example 1

[0058] A phosphate-solubilizing bacterial agent with high effective phosphorus conversion includes phosphate-solubilizing bacteria, bone char, and culture medium; the phosphate-solubilizing bacteria is *Pantoea agglomerans* CT-3; *Pantoea agglomerans* CT-3 was deposited on January 8, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.: 33335, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. A scanning electron microscope image of *Pantoea agglomerans* CT-3 is shown below. Figure 1 As shown.

[0059] The 16S rDNA gene sequence of *Pantotheca cumulus* CT-3 is shown in SEQ ID No. 1. The length of the gene sequence is 1411.

[0060] In this embodiment, the particle size of bone char is 30 nm to 2 μm.

[0061] The preparation method of this phosphate-solubilizing bacterial agent with high effective phosphorus conversion includes the following steps:

[0062] S1. Preparation of bone char: Pig bones were freeze-dried at -60℃ for 48h, then thermally pyrolyzed at 450℃ for 120min. After cooling, large pieces of bone char were obtained. The large pieces of bone char were then ball-milled at 450rpm for 5h to obtain micron-nano multi-level bone char.

[0063] 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.

[0064] S2. Activation of Pantotheca clumps: Pantotheca clumps CT-3 was inoculated into LB medium and then placed in a constant temperature shaker at 28℃ and 180r / min for 16h to allow OD to form. 600 The concentration was 0.6–0.8, resulting in an active bacterial solution; wherein the effective viable count of the active bacterial solution was 1 × 10⁻⁶. 10 CFU / mL ~9×10 10 CFU / mL;

[0065] S3. Preparation of phosphate-solubilizing bacterial agent: After sterilization by irradiation, the micron-nano multi-level bone char obtained in step S1 is added to NBRIP-P medium to obtain a reaction medium. Then, the active bacterial solution is inoculated into the reaction medium and placed in a constant temperature shaker at 28°C with shaking at 180 r / min for 3 days to obtain the phosphate-solubilizing bacterial agent with high effective phosphorus conversion.

[0066] In this embodiment, the irradiation sterilization dose was 5.0 kGy; the mass-to-volume ratio of bone char to NBRIP-P medium was 0.5%. The inoculum amount of active bacteria in the reaction medium was 1% of the reaction medium volume.

[0067] Example 2

[0068] A phosphate-solubilizing bacterial agent with high effective phosphorus conversion is described in this embodiment. The difference between this embodiment and Example 1 is that the mass-to-volume ratio of bone char to NBRIP-P culture medium is 1% in this embodiment. All other conditions and methods in this embodiment are the same as in Example 1.

[0069] Example 3

[0070] A phosphate-solubilizing bacterial agent with high effective phosphorus conversion is described in this embodiment. The difference between this embodiment and Example 1 is that the mass-to-volume ratio of bone char to NBRIP-P culture medium is 2% in this embodiment. All other conditions and methods in this embodiment are the same as in Example 1.

[0071] Example 4

[0072] A phosphate-solubilizing bacterial agent with high effective phosphorus conversion is described in this embodiment. The difference between this embodiment and Example 1 is that the mass-to-volume ratio of bone char to NBRIP-P culture medium is 2.5% in this embodiment. All other conditions and methods are the same as in Example 1.

[0073] Example 5

[0074] A phosphorus-solubilizing bacterial agent with high effective phosphorus conversion. The difference between this embodiment and Example 1 is that in this embodiment, step S1, preparation of bone char: pig bones are freeze-dried at -65℃ for 40h, then thermally pyrolyzed at 440℃ for 140min, and after cooling, large pieces of bone char are obtained. Then, the large pieces of bone char are ball-milled at 440rpm for 6h to obtain micron-nano multi-level bone char.

[0075] 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; and the irradiation sterilization dose is 3.0 kGy.

[0076] The remaining conditions and methods in this embodiment are the same as in Embodiment 1.

[0077] Example 6

[0078] A phosphorus-solubilizing bacterial agent with high effective phosphorus conversion. The difference between this embodiment and Example 1 is that in this embodiment, step S1, preparation of bone char: pig bones are freeze-dried at -55℃ for 55h, then thermally pyrolyzed at 460℃ for 100min, and after cooling, large pieces of bone char are obtained. Then, the large pieces of bone char are ball-milled at 460rpm for 4h to obtain micron-nano multi-level bone char.

[0079] 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.

[0080] The remaining conditions and methods in this embodiment are the same as in Embodiment 1.

[0081] Example 7

[0082] A bio-fertilizer is prepared using a phosphorus-solubilizing bacterial agent with high effective phosphorus conversion obtained from any one of Examples 1 to 6.

[0083] Example 8

[0084] This invention relates to the application of a phosphate-solubilizing bacterial agent with high effective phosphorus conversion. Specifically, the phosphate-solubilizing bacterial agent prepared by this invention can be used for agricultural soil improvement. When applied to the soil, the agent utilizes the action of the clump-forming Pantothecin CT-3 to promote the conversion and release of insoluble phosphorus in the soil, thereby improving the effective phosphorus content and promoting plant growth. Furthermore, the phosphate-solubilizing bacterial agent prepared by this invention can be used for bone char phosphorus resource recovery. By applying this agent to bone char, insoluble phosphorus in bone char is converted into soluble phosphorus, improving the recovery rate of bone char phosphorus resources. Additionally, the phosphate-solubilizing bacterial agent prepared by this invention can be used to promote crop growth. When applied to the soil where crops grow, it provides sufficient soluble phosphorus and soluble calcium to the crops, thus effectively promoting crop growth.

[0085] Experimental testing:

[0086] (I) Scanning electron microscopy detection

[0087] For a scanning electron microscope image of the pantothenic acid CT-3 strain of the present 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, the CT-3 cluster of pantothecin bacteria consists of rod-shaped bacteria 1 μm long.

[0088] In addition, scanning electron microscopy was performed on the clumps of pantothenic acid CT-3 after interaction 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 2 As can be seen, clustered pantothenic acid CT-3 is adsorbed on the surface of bone char, and the morphology of the clustered pantothenic acid CT-3 is intact and full, indicating that bone char provides favorable conditions for the growth and physiological state of clustered pantothenic acid CT-3.

[0089] (II) Testing the phosphorus-solubilizing ability of Pantotheca agglomerata CT-3 with different amounts of bone char applied.

[0090] Sample Preparation: The micron- and nano-scale multi-level bone char prepared in Example 1 was added to NBRIP-P medium to obtain a reaction medium. Then, the active bacterial solution prepared in Example 1 was inoculated into the reaction medium to prepare samples. The inoculation amount of the active bacterial solution in the reaction medium was 1% of the reaction medium volume. The mass-to-volume ratio of bone char in the NBRIP-P medium was set to 0.5%, 1%, 2%, and 2.5%, respectively, resulting in four samples. Additionally, a blank control sample was prepared by inoculating 1% of *Pantotheca acuminata* CT-3 (1% of the NBRIP-P medium volume) into the NBRIP-P medium without adding bone char. Three replicates were prepared for each sample.

[0091] The above samples were placed in a constant temperature shaker at 28℃ and shaken at 180 r / min for 168 h. Samples were taken every 24 h to detect the available phosphorus content. For the results of the available phosphorus content detection, please refer to [link to relevant documentation]. Figure 3 .

[0092] Depend on Figure 3 In the sample, “1%PSB3+0.5%BC” represents a sample with bone char comprising 0.5% of the NBRIP-P medium by mass volume, “1%PSB3+1%BC” represents a sample with bone char comprising 1% of the NBRIP-P medium by mass volume, “1%PSB3+2%BC” represents a sample with bone char comprising 2% of the NBRIP-P medium by mass volume, and “1%PSB3+2.5%BC” represents a sample with bone char comprising 2.5% of the NBRIP-P medium by mass volume.

[0093] Depend on Figure 3As can be seen, after the reaction of Pantothenic Acid CT-3 with bone char, the phosphorus solubility of the "1% PSB3 + 2% BC" sample reached a peak of 1071.35 mg / L at 48 h, the "1% PSB3 + 2.5% BC" sample reached 1898.41 mg / L at 72 h, the "1% PSB3 + 1% BC" sample reached 4907.65 mg / L at 72 h, and the "1% PSB3 + 0.5% BC" sample reached 3456.80 mg / L at 72 h. As time progressed, the "1% PSB3 + 0.5% BC" sample showed a higher phosphorus solubility than other samples, reaching 2702.02 mg / L even after 168 hours. This indicates that in the "1% PSB3 + 0.5% BC" sample, the 1% inoculum of Pantotheca cumulosa CT-3 and 0.5% bone char interacted more fully, more efficiently converting insoluble phosphorus in bone char into soluble phosphorus, and exhibiting excellent conversion stability, continuously and efficiently converting insoluble phosphorus into soluble phosphorus. Therefore, the high effective phosphorus conversion phosphorus-solubilizing agent of the present invention, through Pantotheca cumulosa CT-3, can efficiently and stably convert phosphorus in bone char into soluble phosphorus, demonstrating excellent phosphorus-solubilizing ability and providing sufficient soluble phosphorus for crop growth. This high effective phosphorus conversion phosphorus-solubilizing agent or the bio-fertilizer made from the phosphorus-solubilizing agent of the present invention will be a new agricultural input for green and sustainable agricultural development.

[0094] (III) Detection of dehydrogenase activity of Pantotheca agglomerata CT-3 under different bone char application amounts

[0095] Sample Preparation: The micron- and nano-scale multi-level bone char prepared in Example 1 was added to NBRIP-P medium to obtain a reaction medium. Then, the active bacterial solution prepared in Example 1 was inoculated into the reaction medium to prepare samples. The inoculation amount of the active bacterial solution in the reaction medium was 1% of the reaction medium volume. The mass-to-volume ratio of bone char in the NBRIP-P medium was set to 0.5%, 1%, 2%, and 2.5%, respectively, resulting in four samples. Additionally, a blank control sample was prepared by inoculating 1% of *Pantotheca acuminata* CT-3 (1% of the NBRIP-P medium volume) into the NBRIP-P medium without adding bone char. Three replicates were prepared for each sample.

[0096] The above samples were incubated in a constant temperature shaker at 28℃ with shaking at 180 rpm for 168 h, and samples were taken every 24 h to detect dehydrogenase activity. For the results of the dehydrogenase activity detection, please refer to [link to relevant documentation]. Figure 4 .

[0097] Depend on Figure 4In the sample, “1%PSB3+0.5%BC” represents a sample with bone char comprising 0.5% of the NBRIP-P medium by mass volume, “1%PSB3+1%BC” represents a sample with bone char comprising 1% of the NBRIP-P medium by mass volume, “1%PSB3+2%BC” represents a sample with bone char comprising 2% of the NBRIP-P medium by mass volume, and “1%PSB3+2.5%BC” represents a sample with bone char comprising 2.5% of the NBRIP-P medium by mass volume.

[0098] Depend on Figure 4 It is evident that the metabolic activity of *Pantospira clumps* CT-3 is dynamic. Initially, when a certain amount of insoluble phosphorus is present in the environment, the low available phosphorus content stimulates *Pantospira clumps* CT-3 to increase dehydrogenase activity, enhancing its metabolic capacity to secrete more phosphate-solubilizing substances to convert insoluble phosphorus into soluble phosphorus, thereby increasing the available phosphorus content. This increase in available phosphorus content provides more energy and material basis for phosphate-solubilizing bacteria, further promoting the growth of *Pantospira clumps* CT-3 and increasing dehydrogenase activity, forming a positive feedback loop. At 72 hours, the phosphate solubility reaches its peak. Excessively high available phosphorus content in the bacterial culture may alter intracellular osmotic pressure, affecting normal cellular physiological functions, and thus triggering negative feedback regulation, reducing the phosphate-solubilizing capacity and metabolic activity of *Pantospira clumps* CT-3, thereby decreasing dehydrogenase activity. Therefore, in summary... Figure 3 and Figure 4 The "1% PSB3+0.5% BC" sample has a superior phosphorus-solubilizing ability. That is, the phosphorus-solubilizing agent with high effective phosphorus conversion obtained in Example 1 can better realize the efficient conversion of insoluble phosphorus in bone char into soluble phosphorus, better improve the utilization rate of phosphorus resources in bone char, and provide an efficient and stable phosphorus source for crop growth, thereby promoting crop growth.

[0099] 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.

[0100] sequence list

[0101] SEQ ID No.1

[0102]

Claims

1. A phosphate-solubilizing agent, characterized in that, Includes phosphate-solubilizing bacteria, bone char, and culture medium; the phosphate-solubilizing bacteria is Pantotheca clumps CT-3; The CT-3 clump of Pantoea agglomerans was deposited on January 8, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.33335. The particle size of the bone char is 30 nm to 2 μm.

2. The method for preparing a phosphate-solubilizing agent according to claim 1, characterized in that, Includes the following steps: S1. Preparation of bone char: Animal bones are freeze-dried, then thermally pyrolyzed and cooled, and then ball-milled to obtain micron- and nano-scale bone char. S2. Activation of Pantotheca clumps: Pantotheca clumps CT-3 was inoculated into LB medium and then placed in a constant temperature shaker at 28℃ and 180r / min for 16h to allow OD to form. 600 The concentration was 0.6–0.8, resulting in an active bacterial solution; S3. Preparation of phosphate-solubilizing bacterial agent: After sterilization by irradiation, the micron-nano multi-level bone char obtained in step S1 is added to NBRIP-P medium to obtain a reaction medium. Then, the active bacterial solution is inoculated into the reaction medium and placed in a constant temperature shaker at 28°C with shaking at 180 r / min for 3 days to obtain the phosphate-solubilizing bacterial agent.

3. The method for preparing a phosphate-solubilizing agent as described in claim 2, characterized in that, In step S1, animal bones are freeze-dried at -65℃ to -55℃ for 40h to 55h, and then thermally pyrolyzed at 440℃ to 460℃ for 100min to 140min. After cooling, large pieces of bone char are obtained. The large pieces of bone char are then ball-milled at 440rpm to 460rpm for 4h to 6h to obtain micron-nano multi-level bone char. In the ball mill, zirconium oxide particles are used to ball mill large pieces of bone char, and ethanol is added as a grinding aid; the weight ratio of zirconium oxide particles to large pieces of bone char is (15-20):1; the mass ratio of ethanol to large pieces of bone char is (1.5-2):

1.

4. The method for preparing a phosphate-solubilizing agent as described in claim 2, 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; and / or In step S3, the mass-to-volume ratio of the bone char to the NBRIP-P culture medium is 0.5% to 2.5%; and / or The irradiation sterilization dose is 3.0 kGy to 6.0 kGy; The inoculation amount of the active bacterial solution in the reaction medium is 1% of the volume of the reaction medium.

5. A bio-fertilizer, characterized in that, It is prepared using the phosphate-solubilizing agent described in claim 1.

6. The application of the phosphate-solubilizing agent according to claim 1 in agricultural soil improvement.

7. The application of the phosphate-solubilizing agent according to claim 1 in the recovery of bone char phosphorus resources.

8. The application of the phosphate-solubilizing agent according to claim 1 in promoting crop growth.

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