Phosphorus-dissolving bacterial agent for degrading hydroxyapatite, preparation method thereof, bio-bacterial fertilizer and application

Abstract

The present application relates to the field of environment, the utilization of phosphate rock resources and the field of agricultural biotechnology, and in particular to a kind of hydroxyapatite-degrading phosphate-solubilizing bacterial agent and its preparation method, biological bacterial fertilizer and application, the hydroxyapatite-degrading phosphate-solubilizing bacterial agent includes phosphate-solubilizing bacteria, hydroxyapatite and culture medium, the phosphate-solubilizing bacteria is Pantoea agglomerate CT3.By using hydroxyapatite to prepare the hydroxyapatite-degrading phosphate-solubilizing bacterial agent, the full use of phosphate rock resources can be greatly promoted, and the dissolution of insoluble phosphorus in phosphate rock is realized.The hydroxyapatite-degrading phosphate-solubilizing bacterial agent combines Pantoea agglomerate CT3 with hydroxyapatite, which can utilize the phosphate-solubilizing ability of Pantoea agglomerate CT3 and fully utilize the phosphorus resources of hydroxyapatite.The hydroxyapatite-degrading phosphate-solubilizing bacterial agent has good prospects in the preparation of biological bacterial fertilizer, application in agricultural soil improvement, application in phosphate rock resource recovery and application in promoting wheat growth.

Description

Technical Field

[0001] This invention relates to the fields of environmental protection, phosphate rock resource utilization, and agricultural biotechnology, specifically to a phosphorus-soluble bacterial agent for degrading hydroxyapatite, its preparation method, and bio-fertilizer and its application. Background Technology

[0002] Phosphate rock, as an important phosphorus-containing mineral resource, is widely used in agriculture and chemical industries. Phosphate minerals exist naturally in the form of apatite, including fluorapatite, hydroxyapatite, and carbofluorapatite.

[0003] Many countries directly apply phosphate rock as phosphate fertilizer. The size, surface area, and dissolution rate of phosphate rock particles all have different effects on soil properties and plant growth. Studies have shown that the specific surface area of ​​phosphate rock has a significant impact on the chemical reactions used to extract phosphorus using chemical extractants. The particle size of phosphate rock directly determines the specific surface area of ​​the material; fine-particle phosphate rock has a larger surface area, while coarse-particle phosphate rock has a smaller surface area. The dissolution rate of phosphate rock increases with increasing specific surface area. Small-particle phosphate rock has a larger specific surface area, allowing for closer contact with soil microorganisms and thus a greater impact on the soil microbial community. Although coarse-particle ore has a slower dissolution rate, it can continuously release phosphorus over a longer period, providing a more sustained effect on soil phosphorus supply.

[0004] Furthermore, the interaction between phosphate rock and microorganisms also affects the application efficiency of phosphate rock. Different strains exhibit varying dissolution abilities for different types and particle sizes of phosphate rock; smaller particle sizes do not necessarily lead to higher phosphate dissolution efficiency. Therefore, further development of phosphate-dissolving bacterial agents is needed to promote the full utilization of phosphate rock resources, and the dissolution efficiency of phosphate rock needs further improvement. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, the first objective of this invention is to provide a phosphate-solubilizing agent for degrading hydroxyapatite. This phosphate-solubilizing agent, by combining phosphate rock with phosphate-solubilizing bacteria, expands the resources of phosphate-solubilizing agents, improves the utilization efficiency of phosphate rock resources, reduces waste of phosphate rock resources during use, and promotes plant growth, exhibiting good application effects and environmental friendliness.

[0006] To overcome the shortcomings of the prior art, the second objective of this invention is to provide a method for preparing a phosphorus-solubilizing agent that degrades hydroxyapatite. This preparation method is simple and easy to operate, and the resulting phosphorus-solubilizing agent expands the phosphorus-solubilizing agent resources, improves the utilization efficiency of phosphate rock resources, and has good application effects and environmental friendliness.

[0007] The third objective of this invention is to provide a bio-fertilizer.

[0008] The fourth objective of this invention is to provide an application of a phosphate-solubilizing agent for degrading hydroxyapatite.

[0009] To achieve the first objective of the invention, the technical solution adopted by the present invention is as follows:

[0010] This invention provides a phosphate-solubilizing agent for degrading hydroxyapatite, comprising phosphate-solubilizing bacteria, hydroxyapatite, and culture medium; wherein the phosphate-solubilizing bacteria is Pantotheca clumps CT3, which belongs to the bacterial class.

[0011] The aforementioned Pantoea agglomerans CT3 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.

[0012] Furthermore, the 16S rDNA gene sequence of the clump-forming pantothecin CT3 is shown in SEQ ID No. 1.

[0013] Furthermore, the particle size of the hydroxyapatite is 0.1 μm to 212 μm.

[0014] Preferably, the hydroxyapatite has a particle size of 0.1 μm to 1 μm. Specifically, the agglomerating pantothecin CT3 exhibits good dissolution effects on hydroxyapatite with a particle size of 0.1 μm to 1 μm.

[0015] The total phosphorus content of hydroxyapatite is 19.841%, but the available phosphorus content is only 2.774%. By using hydroxyapatite to prepare phosphorus-soluble bacteria that degrade hydroxyapatite, the full utilization of phosphate rock resources can be greatly promoted.

[0016] This phosphate-solubilizing agent for degrading hydroxyapatite combines *Pantotheca acuminata* CT3 with hydroxyapatite, utilizing both the phosphate-solubilizing ability of *Pantotheca acuminata* CT3 and the phosphorus resources of hydroxyapatite. The surface of hydroxyapatite also provides a carrier for the growth and reproduction of the bacteria. Additionally, the culture medium promotes the growth and metabolism of *Pantotheca acuminata* CT3.

[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 agent for degrading hydroxyapatite, comprising the following steps:

[0019] S1. Activation culture: Inoculate the clustered pantothecin CT3 onto LB medium plates and activate the culture at 28℃ for 24h to 48h to obtain activated colonies;

[0020] S2. Preparation of bacterial suspension: Using an inoculation loop, pick one single colony from the activated culture and inoculate it into LB liquid medium. Then, place it in a constant temperature shaker at 28°C and shake at 180 r / min until OD reaches the target concentration. 600 =0.7, thus obtaining a bacterial suspension;

[0021] S3. Preparation of bacterial agent: Hydroxyapatite is used to replace the phosphorus source in NBRIP medium to obtain a hydroxyapatite-containing medium. The pH of the medium is controlled at 7.0-7.2. The bacterial suspension is inoculated into the hydroxyapatite-containing medium and then placed in a constant temperature shaker at 28°C with shaking at 180 r / min for 2 days to obtain the phosphorus-soluble bacterial agent containing phosphate rock.

[0022] Furthermore, the effective viable bacteria count of the bacterial suspension obtained in step S2 is 1 × 10⁻⁶. 10 CFU / mL ~9×10 10 CFU / mL; and / or

[0023] In step S3, the concentration of hydroxyapatite in the culture medium is 5 g / L;

[0024] The inoculation amount of the bacterial suspension in the hydroxyapatite-containing culture medium is 1% of the culture medium volume.

[0025] To achieve the third objective of the invention, the technical solution adopted by the present invention is as follows:

[0026] This invention provides a bio-fertilizer, which is prepared using the above-mentioned phosphorus-solubilizing agent for degrading hydroxyapatite.

[0027] Among them, the phosphorus-solubilizing agent for degrading hydroxyapatite combines Pantotheca granulata CT3 with hydroxyapatite, which can utilize both the phosphorus-solubilizing ability of Pantotheca granulata CT3 and the phosphorus resources of hydroxyapatite. Therefore, it has a promising application in the preparation of bio-fertilizers.

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

[0029] This invention provides the application of the above-described phosphorus-soluble bacterial agent for degrading hydroxyapatite in agricultural soil improvement.

[0030] Specifically, the phosphorus-soluble bacteria that degrade hydroxyapatite are applied to the soil, and the action of the clump-forming pantothenic acid CT3 promotes the dissolution and release of phosphorus in the soil, thereby improving the available phosphorus content in the soil and promoting plant growth.

[0031] This invention provides the application of the above-described phosphate-solubilizing agent for degrading hydroxyapatite in the recovery of phosphate rock resources.

[0032] Specifically, applying the phosphorus-dissolving agent that degrades hydroxyapatite to the biological dissolution of waste phosphate rock or low-grade phosphate rock (hydroxyapatite) can improve the recovery rate of phosphate rock resources.

[0033] This invention provides the application of the above-described phosphate-solubilizing agent for degrading hydroxyapatite in promoting wheat growth.

[0034] This phosphate-solubilizing agent for degrading hydroxyapatite combines phosphate-solubilizing bacteria with phosphate rock. It utilizes the phosphate-solubilizing ability of the bacteria while providing a growth and reproduction carrier for the bacteria on the surface of the phosphate rock, thereby improving the dissolution efficiency of the phosphate rock. This not only helps improve the recovery and utilization rate of phosphorus resources but also reduces environmental pollution from traditional chemical treatment methods. Furthermore, it can improve agricultural soil, increasing the available phosphorus content in agricultural soil by up to 10.90 times. When used as a bio-fertilizer, it can also promote wheat growth. Therefore, it has excellent application prospects in all these applications.

[0035] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0036] (1) The present invention provides a phosphorus-solubilizing agent for degrading hydroxyapatite, comprising phosphorus-solubilizing bacteria, hydroxyapatite, and a culture medium; wherein the phosphorus-solubilizing bacteria is *Panthera philoxeroides* CT3. By using hydroxyapatite to prepare this phosphorus-solubilizing agent for degrading hydroxyapatite, the full utilization of phosphate rock resources can be greatly promoted, and the leaching of insoluble phosphorus in phosphate rock can be achieved. This phosphorus-solubilizing agent for degrading hydroxyapatite combines *Panthera philoxeroides* CT3 with hydroxyapatite, utilizing both the phosphorus-solubilizing ability of *Panthera philoxeroides* CT3 and the phosphorus resources of hydroxyapatite. The surface of hydroxyapatite can also provide a carrier for the growth and reproduction of the bacteria. Therefore, this phosphorus-solubilizing agent for degrading hydroxyapatite not only expands the resources of phosphorus-solubilizing agents and improves the utilization efficiency of phosphate rock resources, reduces the waste of phosphate rock resources during use, and promotes plant growth, but also has good application effects and environmental friendliness.

[0037] (2) The method for preparing a phosphorus-soluble agent for degrading hydroxyapatite of the present invention has the characteristics of simple preparation method and easy operation, and the prepared phosphorus-soluble agent expands the phosphorus-soluble agent resources, improves the utilization efficiency of phosphate rock resources, and has good application effect and environmental friendliness.

[0038] (3) The bio-fertilizer of the present invention is prepared using the above-mentioned phosphorus-solubilizing agent for degrading hydroxyapatite, thus promoting the dissolution of insoluble phosphorus and having a good application effect. Specifically, this phosphorus-solubilizing agent for degrading hydroxyapatite combines Pantothenia glutinosa CT3 with hydroxyapatite, utilizing both the phosphorus-solubilizing ability of Pantothenia glutinosa CT3 and the phosphorus resources of hydroxyapatite, thus showing great promise for its application in the preparation of bio-fertilizers.

[0039] (4) The application of the phosphate-solubilizing agent for degrading hydroxyapatite of the present invention: Because this phosphate-solubilizing agent for degrading hydroxyapatite combines phosphate-solubilizing bacteria with phosphate rock, it can utilize the phosphate-solubilizing ability of the bacteria and provide a carrier for the growth and reproduction of the bacteria through the surface of the phosphate rock, thereby improving the dissolution efficiency of the phosphate rock. This not only helps to improve the recovery and utilization rate of phosphorus resources, but also reduces the environmental pollution of traditional chemical treatment methods, and can improve agricultural soil, increasing the effective phosphorus content in agricultural soil by up to 10.90 times. When used as a bio-fertilizer, it can also promote wheat growth. Therefore, it has excellent application prospects in agricultural soil improvement, phosphate rock resource recovery, and wheat growth promotion. Attached Figure Description

[0040] 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.

[0041] Figure 1 This is an observational image of the formation of phosphate-solubilizing rings by the clump-forming pantothecin CT3 bacteria of the present invention on a culture medium containing hydroxyapatite.

[0042] Figure 2 This is a scanning electron microscope image of the CT3 cluster of pantothecin bacteria according to the present invention.

[0043] Figure 3 This is a graph showing the results of the detection of the phosphorus-dissolving ability of the CT3 clump-forming bacteria of the present invention on hydroxyapatite of different sizes at different time periods.

[0044] Figure 4 The images are scanning electron microscope (SEM) images of hydroxyapatite of different sizes before and after 168 hours of phosphorus dissolution treatment using the CT3 CT3 of the present invention.

[0045] Figure 5 The graph shows the results of testing the effective phosphorus content of the soil before and after applying the phosphorus-soluble bacterial agent and bacterial suspension prepared according to Examples 1, 2 and 3 of this invention to degrade hydroxyapatite.

[0046] Figure 6 The graph shows the results of testing the root and stem lengths of wheat grown in the original soil after applying the phosphorus-soluble bacterial agent and bacterial suspension prepared according to Examples 1, 2 and 3 of this invention.

[0047] Figure 7The graph shows the test results of the aboveground and underground fresh weights of wheat grown in the original soil, obtained by applying the phosphorus-soluble bacterial agent and bacterial suspension of the hydroxyapatite-degrading bacteria prepared in Examples 1, 2 and 3 of this invention.

[0048] Figure 8 The graph shows the detection results of chlorophyll a and chlorophyll b content in wheat grown in the original soil after applying the phosphate-solubilizing agent and bacterial suspension prepared according to Examples 1, 2 and 3 of this invention. 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] PVK medium (g / L): NaCl 0.2g, Ca3(PO4)2 5g, MgSO4 0.1g, (NH4)2SO4 0.5g, glucose 15g, chloramphenicol 50mg, streptomycin 50mg, pH 6.8~7.0.

[0054] Solid medium for PVK: Add 18g to 20g of agar powder to every 1L of PVK medium.

[0055] LB medium (g / L): 10g peptone, 5g yeast extract, 10g NaCl, pH 7.0-7.2.

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

[0057] NBRIP medium (g / L): glucose 10g, (NH4)2SO4 0.5g, NaCl 0.3g, MgSO4·7H2O 0.3g, FeSO4·7H2O 0.03g, MnSO4·2H2O 0.03g, Ca3(PO4)2 5g, KCl 0.3g, lecithin 0.2g, pH 7.2~7.4.

[0058] Solid medium for NBRIP: Add 18g to 20g of agar powder to every 1L of NBRIP medium.

[0059] Hydroxyapatite, also known as basic calcium phosphate, is a naturally occurring mineralization of calcium apatite, and its molecular formula is usually written as (Ca...). 10 In the form of (PO4)6(OH)2).

[0060] Example 1

[0061] A phosphate-solubilizing agent for degrading hydroxyapatite includes phosphate-solubilizing bacteria, hydroxyapatite, and a culture medium; the phosphate-solubilizing bacteria is *Pantoea agglomerans* CT3; wherein *Pantoea agglomerans* CT3 is taxonomically named *Pantoea agglomerans*, and 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* CT3 is shown below. Figure 2 As shown.

[0062] The 16S rDNA gene sequence of *Pantotheca cumulus* CT3 is shown in SEQ ID No. 1. The length of the gene sequence is 1411.

[0063] In this embodiment, the particle size of hydroxyapatite is 0.1 μm to 1 μm.

[0064] The preparation method of this phosphate-solubilizing agent for degrading hydroxyapatite includes the following steps:

[0065] S1. Activation culture: Inoculate the clustered pantothecin CT3 onto LB medium plates and activate the culture at 28℃ for 48h to obtain activated colonies;

[0066] S2. Preparation of bacterial suspension: Using an inoculation loop, pick one single colony from the activated culture and inoculate it into LB liquid medium. Then, place it in a constant temperature shaker at 28°C and shake at 180 r / min until OD reaches the target concentration. 600 =0.7, thus obtaining a bacterial suspension;

[0067] S3. Preparation of bacterial agent: Hydroxyapatite is used to replace the phosphorus source in NBRIP medium to obtain a hydroxyapatite-containing medium. The pH of the medium is controlled at 7.1. The bacterial suspension is inoculated into the hydroxyapatite-containing medium and then placed in a constant temperature shaker at 28°C and shaken at 180 r / min for 2 days to obtain a phosphorus-soluble bacterial agent containing phosphate rock.

[0068] In this study, hydroxyapatite was used to replace the phosphorus source in the NBRIP medium (i.e., hydroxyapatite was used to replace Ca3(PO4)2), and the concentration of hydroxyapatite in the medium was 5 g / L.

[0069] The inoculation amount of the bacterial suspension in the hydroxyapatite-containing culture medium was 1% of the culture medium volume.

[0070] Example 2

[0071] A phosphate-solubilizing agent for degrading hydroxyapatite. The difference between this embodiment and Example 1 is that the particle size of the hydroxyapatite in this embodiment is 75 μm to 90 μm. All other conditions and methods in this embodiment are the same as in Example 1.

[0072] Example 3

[0073] A phosphate-solubilizing agent for degrading hydroxyapatite. The difference between this embodiment and Example 1 is that the particle size of the hydroxyapatite in this embodiment is 200 μm to 212 μm. All other conditions and methods in this embodiment are the same as in Example 1.

[0074] Example 4

[0075] A phosphate-solubilizing agent for degrading hydroxyapatite. The difference between this embodiment and Example 1 is that in this embodiment, in step S1, the agent is activated and cultured at 28°C for 24 hours; and in step S3, the pH of the culture medium is controlled to be 7.0 during the preparation of the agent. The remaining conditions and methods of this embodiment are the same as those of Example 1.

[0076] Example 5

[0077] A phosphate-solubilizing agent for degrading hydroxyapatite. The difference between this embodiment and Example 1 is that in this embodiment, in step S1, the agent is activated and cultured at 28°C for 36 hours; and in step S3, the pH of the culture medium is controlled to be 7.2 during the preparation of the agent. The remaining conditions and methods of this embodiment are the same as those of Example 1.

[0078] Example 6

[0079] A bio-fertilizer is prepared using a phosphorus-soluble bacterial agent that degrades hydroxyapatite, obtained from any one of Examples 1 to 5.

[0080] Experimental testing:

[0081] Experiment Example 1: Testing the ability of CT3, a type of pantothenic acid bacteria, to dissolve hydroxyapatite.

[0082] Hydroxyapatite was used to replace the phosphorus source in PVK solid culture medium (i.e., replacing Ca3(PO4)2) to form a hydroxyapatite-containing culture medium. Aggregates of *Pantotheca cumulus* CT3 were spotted onto the surface of the hydroxyapatite-containing medium and then incubated at 28°C for 3–5 days. Observation revealed that the aggregated *Pantotheca cumulus* CT3 produced distinct phosphorus-solubilizing zones on the hydroxyapatite-containing medium, such as… Figure 1 As shown, this demonstrates that the clustered pantothecin CT3 has the ability to dissolve hydroxyapatite.

[0083] Experiment 2: Testing the phosphorus-solubilizing ability of clustered pantothenic acid CT3 on hydroxyapatite of different sizes.

[0084] Hydroxyapatite of 0.1 μm–1 μm, 75 μm–90 μm, and 200 μm–212 μm respectively was used to replace the phosphorus source (i.e., replace Ca3(PO4)2) in NBRIP medium, respectively, to form hydroxyapatite-containing culture media. The bacterial suspension prepared in Example 1 was inoculated into the hydroxyapatite-containing culture media at an inoculation rate of 1% (v / v) and then cultured in a constant temperature shaker at 28°C with shaking at 180 rpm for 7 days, with the available phosphorus content measured daily. For the results of the available phosphorus content measurement, please refer to [link to relevant documentation]. Figure 3 .

[0085] Depend on Figure 3 It can be seen that when using 0.1μm to 1μm hydroxyapatite, the phosphorus solubility reaches 69.284 mg / L in 24 hours and peaks at 94.205 mg / L in 168 hours. When using 75μm to 90μm hydroxyapatite, the phosphorus solubility is only 1.059 mg / L in 24 hours and peaks at 87.304 mg / L in 108 hours. When using 200μm to 212μm hydroxyapatite, the phosphorus solubility is only 6.572 mg / L in 24 hours and peaks at 69.011 mg / L in 144 hours.

[0086] Therefore, the CT3 clump-forming bacteria of the present invention has better phosphorus solubility for 0.1 μm to 1 μm hydroxyapatite.

[0087] In addition, the hydroxyapatite (0.1μm–1μm, 75μm–90μm, and 200μm–212μm) treated with CT3 by *Plasmodium styracifolium* for 168 hours, as well as the hydroxyapatite before treatment, were examined using scanning electron microscopy. The results are as follows: Figure 3 As shown.

[0088] Specifically, after the clumps of pantothenic acid CT3 were reacted with hydroxyapatite of different sizes for 168 hours, the supernatant was removed, and the remaining hydroxyapatite at the bottom was dried and observed using a scanning electron microscope.

[0089] Figure 4 In the figure, a shows hydroxyapatite before treatment; b shows SEM images of 0.1μm to 1μm hydroxyapatite treated by *Plasmodium spp.* CT3 for 168 h; c shows SEM images of 75μm to 90μm hydroxyapatite treated by *Plasmodium spp.* CT3 for 168 h; and d shows SEM images of 200μm to 212μm hydroxyapatite treated by *Plasmodium spp.* CT3 for 168 h.

[0090] Depend on Figure 4 visible, Figure 4 (b) Hydroxyapatite compared to untreated Figure 4 (a) Hydroxyapatite was more dispersed, and the edges of the treated hydroxyapatite tended to be smoother with increased porosity, indicating that the clustered pantothenic acid CT3 could fully contact 0.1 μm to 1 μm hydroxyapatite, promoting the dissolution of hydroxyapatite and the release of phosphorus. Additionally, observations... Figure 4 (c) It was found that the surface of the 75μm–90μm hydroxyapatite was slightly smoother after treatment, but the platy structure was still visible, indicating that the metabolites of clump-forming pantothenic acid CT3 were less soluble in it than in the 0.1μm–1μm hydroxyapatite. Observation Figure 4 (d) It was found that the treated 200μm–212μm hydroxyapatite still accumulated in large quantities, with an uneven surface and slightly smoother edges, indicating that the metabolites of *Pantotheca acuminata* CT3 were less effective at dissolving it than the 75μm–90μm hydroxyapatite. Overall, the dissolution effect of *Pantotheca acuminata* CT3 on 75μm–90μm and 200μm–212μm hydroxyapatite was not significantly different, but the 0.1μm–1μm hydroxyapatite showed a stronger affinity for *Pantotheca acuminata* CT3. This indicates that *Pantotheca acuminata* CT3 has a better phosphorus-dissolving ability for 0.1μm–1μm hydroxyapatite.

[0091] Experimental Example 3: Application of the phosphorus-soluble bacterial agent for degrading hydroxyapatite in this invention in improving soil physical and chemical properties.

[0092] The application of a phosphate-solubilizing agent for degrading hydroxyapatite in agricultural soil improvement involved applying the phosphate-solubilizing agents for degrading hydroxyapatite prepared in Examples 1, 2, and 3, as well as the bacterial suspension prepared in Example 1, to the soil. After 14 days, the available phosphorus content of the soil in the four samples was measured, and the available phosphorus content of the original soil was also measured for comparison. The test results are as follows: Figure 5 As shown.

[0093] Figure 5 In the text, CK represents the original soil, "Clustered Pantothenic Acid" represents the bacterial suspension prepared in Example 1, "Clustered Pantothenic Acid + 0.1-1μm" represents the phosphate-solubilizing agent for degrading hydroxyapatite prepared in Example 1, "Clustered Pantothenic Acid + 75-90μm" represents the phosphate-solubilizing agent for degrading hydroxyapatite prepared in Example 2, and "Clustered Pantothenic Acid + 200-212μm" represents the phosphate-solubilizing agent for degrading hydroxyapatite prepared in Example 3.

[0094] Depend on Figure 5 The test results show that the phosphorus-soluble bacteria for degrading hydroxyapatite prepared in Examples 1, 2, and 3, as well as the bacterial suspension prepared in Example 1, all showed an increase in available phosphorus content relative to the original soil after 14 days of application. Specifically, the phosphorus-soluble bacteria for degrading hydroxyapatite prepared in Example 1 (hydroxyapatite particle size of 0.1 μm to 1 μm) increased the available phosphorus content by 10.90 times. The phosphorus-soluble bacteria for degrading hydroxyapatite prepared in Example 2 (hydroxyapatite particle size of 75 μm to 90 μm) increased the available phosphorus content by 1.64 times. The phosphorus-soluble bacteria for degrading hydroxyapatite prepared in Example 3 (hydroxyapatite particle size of 200 μm to 212 μm) increased the available phosphorus content by 7.16 times. The bacterial suspension prepared in Example 1 increased the available phosphorus content by 3.53 times.

[0095] When the phosphorus-soluble bacterial agent for degrading hydroxyapatite prepared in this invention is applied to the soil, on the one hand, the clustered pantothenic acid CT3 can further convert the insoluble phosphorus in the hydroxyapatite in the bacterial agent into available phosphorus; on the other hand, the clustered pantothenic acid CT3 can convert the insoluble phosphorus in the soil into available phosphorus, thereby increasing the phosphorus content in the soil that can be absorbed by plants. In addition, the bacterial suspension prepared in this invention, due to the presence of clustered pantothenic acid CT3, can convert the insoluble phosphorus in the soil into available phosphorus; however, since the content of insoluble phosphorus in the soil is limited, the increase in available phosphorus content is not significant.

[0096] Depend on Figure 5 The test results show that applying the hydroxyapatite-degrading phosphate-solubilizing bacterial agent prepared in Example 1 (hydroxyapatite particle size of 0.1μm to 1μm) can significantly increase the available phosphorus content in the soil, indicating that the bacterial agent contains 0.1μm to 1μm hydroxyapatite, which can greatly increase the available phosphorus content in the soil. The available phosphorus content in the soil is closely related to plant growth; the higher the available phosphorus content, the stronger the plant growth and the higher the yield. The hydroxyapatite-degrading phosphate-solubilizing bacterial agent prepared in Example 1 can effectively convert the insoluble phosphorus in hydroxyapatite into soluble phosphorus that can be directly absorbed and utilized by plants. At the same time, the clump-forming Pantothecin CT3 further converts the insoluble phosphorus in the original soil into available phosphorus. Therefore, the hydroxyapatite-degrading phosphate-solubilizing bacterial agent prepared in this invention can be applied to bio-fertilizers and has a good application prospect.

[0097] Experimental Example 4: Application of the phosphate-solubilizing agent for degrading hydroxyapatite of the present invention in promoting wheat growth.

[0098] The application of a phosphate-solubilizing agent for degrading hydroxyapatite in promoting wheat growth: The phosphate-solubilizing agents for degrading hydroxyapatite prepared in Examples 1, 2 and 3, as well as the bacterial suspension prepared in Example 1, were used as fertilizers for wheat growth and applied to the soil. After 14 days, the root length, stem length, aboveground fresh weight and underground fresh weight of the wheat were measured.

[0099] Among them, the test results for the root length and stem length of wheat are as follows: Figure 6 As shown in the figure. The test results of the aboveground and underground fresh weights of wheat are as follows. Figure 7 As shown in the figure. The detection results of chlorophyll a and chlorophyll b content in wheat are as follows. Figure 8 As shown.

[0100] Figures 6 to 8 In the text, CK represents the original soil without any added materials, "Clustered Pantothenic Acid" represents the bacterial suspension prepared in Example 1, "Clustered Pantothenic Acid + 0.1-1μm" represents the phosphate-solubilizing agent for degrading hydroxyapatite prepared in Example 1, "Clustered Pantothenic Acid + 75-90μm" represents the phosphate-solubilizing agent for degrading hydroxyapatite prepared in Example 2, and "Clustered Pantothenic Acid + 200-212μm" represents the phosphate-solubilizing agent for degrading hydroxyapatite prepared in Example 3.

[0101] Depend on Figure 6 The test results show that, compared with wheat grown in the original soil, the root length and stem length of the phosphate-solubilizing agents for degrading hydroxyapatite prepared in Examples 1, 2 and 3, as well as the bacterial suspension prepared in Example 1, increased.

[0102] Figure 7 middle, Figure 7 (A) shows the fresh weight of the aboveground parts. Figure 7 (B) shows the fresh weight of the underground parts. Figure 7 The test results show that, compared with wheat grown in the original soil, the application of the hydroxyapatite-degrading phosphate-solubilizing agents prepared in Examples 1, 2, and 3, as well as the bacterial suspension prepared in Example 1, significantly increased the aboveground and underground fresh weights of wheat. Furthermore, the increase in aboveground fresh weight of wheat caused by the hydroxyapatite-degrading phosphate-solubilizing agents prepared in Examples 1, 2, and 3 was significantly higher than that caused by the bacterial suspension prepared in Example 1, especially the application of the hydroxyapatite-degrading phosphate-solubilizing agent prepared in Example 1 (hydroxyapatite particle size of 0.1 μm to 1 μm), which resulted in the most significant increase in aboveground fresh weight. This demonstrates that the application of the hydroxyapatite-degrading phosphate-solubilizing agent prepared in this invention has a highly efficient effect on promoting wheat growth.

[0103] Figure 8In this context, chla represents chlorophyll a, and chlb represents chlorophyll b. Figure 8 The test results show that the application of the hydroxyapatite-degrading phosphate-solubilizing agents prepared in Examples 1, 2, and 3 increased the chlorophyll content in wheat. The application of the bacterial suspension prepared in Example 1 did not significantly increase the chlorophyll a content compared to the original soil, but significantly increased the chlorophyll b content. Among the hydroxyapatite-degrading phosphate-solubilizing agents prepared in this invention, the hydroxyapatite-degrading phosphate-solubilizing agent of Example 1 (hydroxyapatite particle size of 0.1 μm to 1 μm) showed the most significant increase in chlorophyll content.

[0104] Therefore, by Figure 6 , Figure 7 and Figure 8 The test results show that the phosphate-solubilizing agent for degrading hydroxyapatite of the present invention can effectively promote the growth of wheat.

[0105] 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.

[0106]

Claims

1. A phosphate-solubilizing agent for degrading hydroxyapatite, characterized in that, It includes phosphate-solubilizing bacteria, hydroxyapatite, and culture medium; the phosphate-solubilizing bacteria is Pantotheca clumps CT3; the hydroxyapatite serves as the phosphorus source for the culture medium; The classification name of the pantothecin CT3 cluster is... Pantoea agglomerans It was deposited at the China General Microbiological Culture Collection Center on January 8, 2025, with accession number CGMCC NO.33335; The hydroxyapatite has a particle size of 0.1 μm to 212 μm; the concentration of the hydroxyapatite in the culture medium is 5 g / L.

2. The phosphate-solubilizing agent for degrading hydroxyapatite as described in claim 1, characterized in that, The hydroxyapatite has a particle size of 0.1 μm to 1 μm.

3. A method for preparing a phosphate-solubilizing agent for degrading hydroxyapatite according to any one of claims 1 to 2, characterized in that, Includes the following steps: S1. Activation culture: Inoculate the clustered pantothecin CT3 onto LB medium plates and activate the culture at 28℃ for 24 h to 48 h to obtain activated colonies; S2, preparation of bacterial suspension: pick up one single colony from the activated culture with a loop and inoculate into LB liquid medium, then put into a constant temperature shaker at 28°C and shake at 180 r / min until OD 600 = 0.7, to obtain bacterial suspension; S3. Preparation of bacterial agent: Hydroxyapatite is used to replace the phosphorus source in NBRIP medium to obtain a hydroxyapatite-containing medium. The pH of the medium is controlled at 7.0-7.

2. The bacterial suspension is inoculated into the hydroxyapatite-containing medium and then placed in a constant temperature shaker at 28°C with shaking at 180 r / min for 2 days to obtain the phosphorus-soluble bacterial agent containing phosphate rock.

4. The method for preparing a phosphate-solubilizing agent for degrading hydroxyapatite as described in claim 3, characterized in that, The effective viable bacteria count of the bacterial suspension obtained in step S2 is 1×10⁻⁶. 10 CFU / mL ~9×10 10 CFU / mL; and / or In step S3, the concentration of hydroxyapatite in the culture medium is 5 g / L; The inoculation amount of the bacterial suspension in the hydroxyapatite-containing culture medium is 1% of the culture medium volume.

5. A bio-fertilizer, characterized in that, It is prepared using a phosphate-solubilizing agent for degrading hydroxyapatite as described in any one of claims 1 to 2.

6. The application of the phosphorus-soluble bacterial agent for degrading hydroxyapatite as described in any one of claims 1 to 2 in agricultural soil improvement.

7. The application of the phosphorus-soluble bacteria agent for degrading hydroxyapatite as described in any one of claims 1 to 2 in the recovery of phosphate rock resources.

8. The application of the phosphate-solubilizing agent for degrading hydroxyapatite as described in any one of claims 1 to 2 in promoting wheat growth.

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