Phosphorus-containing ore dissolving phosphorus bacterial agent, preparation method thereof, biological 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 phosphate rock dissolving phosphorus bacterial agent, a preparation method thereof, a biological bacterial fertilizer and application, the phosphate rock dissolving phosphorus bacterial agent comprising a phosphate rock dissolving bacteria, hydroxyapatite and a culture medium, and the phosphate rock dissolving bacteria is enterobacter cloacae HS-6. The hydroxyapatite is used for preparing the phosphate rock dissolving phosphorus bacterial agent, which can greatly promote the full use of phosphate rock resources and realize the dissolution of the insoluble phosphorus in the phosphate rock. The phosphate rock dissolving phosphorus bacterial agent combines the enterobacter cloacae HS-6 with the hydroxyapatite, which can utilize the phosphate rock dissolving ability of the enterobacter cloacae HS-6 and fully utilize the phosphorus resources of the hydroxyapatite. The phosphate rock dissolving phosphorus bacterial agent has a good prospect in the preparation of a biological bacterial fertilizer, application in agricultural soil improvement, application in the recovery of phosphate rock resources and application in promoting the growth of wheat.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of environment, utilization of phosphate rock resources and agricultural biotechnology, and particularly relates to a phosphate rock-containing phosphorus-dissolving bacterial agent, a preparation method thereof, a bio-bacterial fertilizer and application. BACKGROUND

[0002] Phosphorus is one of the essential elements in the process of plant growth, as a component of biological macromolecules such as nucleic acids and proteins, phosphorus participates in many metabolic processes of plants, especially plays a key role in chlorophyll synthesis, photosynthesis and organic matter transformation. The growth and yield of crops are greatly affected by the content of phosphorus in soil. Although the total content of phosphorus in soil is high, it exists in a form that is difficult for plants to absorb, resulting in a state of phosphorus deficiency despite the abundance of phosphorus in soil. Most of the phosphorus in soil exists in the form of inorganic and organic phosphorus, of which inorganic phosphorus accounts for 60%-80%, which is the main phosphorus source that can be directly absorbed and utilized by plants, but a large amount of inorganic phosphorus is still in an insoluble state and cannot be directly absorbed by plants.

[0003] Phosphate rock, as an important phosphorus-containing mineral resource, is widely used in the fields of agriculture and chemical industry. The natural form of phosphate rock is apatite, including fluorapatite, hydroxyapatite and carbon fluorapatite. However, the traditional phosphate rock processing technology has some obvious shortcomings, especially when dealing with low-grade phosphate rock, the traditional beneficiation method has high cost, low efficiency, large consumption of chemical reagents and serious environmental pollution. Therefore, there is an urgent need for a more green and environmentally friendly, low-cost phosphate rock processing technology. In recent years, biomineralization technology has been applied to the treatment of phosphate rock as a new green and environmentally friendly technology, in which phosphorus-dissolving bacteria can effectively dissolve the phosphorus in phosphate rock through its metabolic products (such as organic acids, acid phosphatase, etc.), thereby improving the utilization efficiency of phosphorus resources. As a kind of microbial resource, phosphorus-dissolving bacteria can dissolve phosphate rock through metabolic action under mild conditions, and is a potential phosphate rock processing method.

[0004] Bacterial agent is an important carrier widely used in the fields of agriculture, mining and environmental governance, and has attracted more and more attention in recent years. However, the development of phosphorus-dissolving bacterial agent and the further full utilization of phosphate rock resources need to be strengthened and expanded, and the dissolution efficiency of phosphate rock needs to be further improved. SUMMARY

[0005] In order to overcome the shortcomings of the prior art, the first object of the present application is to provide a phosphate rock-containing phosphorus-dissolving bacterial agent. The phosphate rock-containing phosphorus-dissolving bacterial agent expands the resources of phosphorus-dissolving bacterial agent by combining phosphate rock with phosphorus-dissolving bacteria, improves the dissolution efficiency of phosphate rock, and has good application effect and environmental friendliness.

[0006] In order to overcome the deficiencies of the prior art, a second object of the present application is to provide a preparation method of a phosphorus-containing ore phosphorus-dissolving bacterial agent, which is simple and easy to operate, expands the resources of the phosphorus-dissolving bacterial agent, improves the dissolution efficiency of the phosphorus ore, and has good application effect and environmental friendliness.

[0007] A third object of the present application is to provide a biological bacterial fertilizer.

[0008] A fourth object of the present application is to provide an application of the phosphorus-containing ore phosphorus-dissolving bacterial agent.

[0009] In order to achieve the first object of the present application, the technical solution adopted by the present application is as follows:

[0010] The present application provides a phosphorus-containing ore phosphorus-dissolving bacterial agent, which comprises phosphorus-dissolving bacteria, hydroxyapatite and culture medium; the phosphorus-dissolving bacteria is Enterobacter hormaechei HS-6, which belongs to bacteria.

[0011] The classification name of the Enterobacter hormaechei HS-6 is Enterobacter hormaechei, which has been preserved in the China General Microbiological Culture Collection Center on October 19, 2023, and the preservation number is CGMCC NO.: 28680.

[0012] Further, the 16S rDNA gene sequence of the Enterobacter hormaechei HS-6 is shown in SEQ ID No. 1.

[0013] Further, the particle size of the hydroxyapatite is 0.1-212 microns.

[0014] Preferably, the particle size of the hydroxyapatite is 0.1-1 micron. The Enterobacter hormaechei HS-6 has good dissolution effect on the hydroxyapatite with a particle size of 0.1-1 micron.

[0015] The total phosphorus content of the hydroxyapatite is 19.841%, but the effective phosphorus content in the hydroxyapatite is only 2.774%. By using the hydroxyapatite to prepare the phosphorus-containing ore phosphorus-dissolving bacterial agent, the full use of phosphorus ore resources can be greatly promoted.

[0016] The phosphorus-containing ore phosphorus-dissolving bacterial agent combines the Enterobacter hormaechei HS-6 with the hydroxyapatite, which can utilize the phosphorus-dissolving ability of the Enterobacter hormaechei HS-6 and fully utilize the phosphorus resources of the hydroxyapatite. The surface of the hydroxyapatite can also provide a carrier for the growth and reproduction of the bacterial strain. In addition, the culture medium is used to promote the growth and metabolism of the Enterobacter hormaechei HS-6.

[0017] In order to achieve the second object of the present application, the technical solution adopted by the present application is as follows:

[0018] The application provides a preparation method of a phosphorus ore phosphorus-dissolving bacterial agent, and comprises the following steps:

[0019] S1, activating culture: the Enterobacter cloacae HS-6 preserved in cold storage is streak inoculated on an LB culture medium plate, and is activated and cultured at 28 DEG C for 48 h to obtain activated culture colonies;

[0020] S2, preparing a bacterial suspension: one single colony in the activated culture colony is picked up with an inoculating loop and inoculated in LB liquid culture medium, and then is placed in a constant-temperature incubator at 28 DEG C to be oscillation cultured at 180 r / min until OD600 = 0.7, so that a bacterial suspension is obtained;

[0021] S3, preparing a bacterial agent: NBRIP culture medium is used as culture medium, and hydroxyapatite is used as the phosphorus source of the NBRIP culture medium to obtain a culture medium containing hydroxyapatite, the pH of the culture medium is controlled to be 7.0-7.2, the bacterial suspension is inoculated in the culture medium containing hydroxyapatite, and then is placed in a constant-temperature incubator at 28 DEG C to be oscillation cultured at 180 r / min for 2 days, so that the phosphorus ore phosphorus-dissolving bacterial agent is prepared.

[0022] Further, the effective viable cell count of the bacterial suspension prepared in the step S2 is 1x10 10 CFU / mL-9x10 10 CFU / mL; and / or

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

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

[0025] To achieve the third object of the application, the technical scheme adopted by the application is as follows:

[0026] The application provides a biological bacterial fertilizer prepared by the phosphorus ore phosphorus-dissolving bacterial agent.

[0027] The phosphorus ore phosphorus-dissolving bacterial agent is prepared by combining the Enterobacter cloacae HS-6 with the hydroxyapatite, so that the phosphorus-dissolving capacity of the Enterobacter cloacae HS-6 can be utilized, and the phosphorus resources of the hydroxyapatite can be fully utilized, and thus the application has a good prospect in the preparation of the biological bacterial fertilizer.

[0028] To achieve the fourth object of the application, the technical scheme adopted by the application is as follows:

[0029] The application provides the application of the above-mentioned phosphorus ore phosphorus-dissolving bacterial agent in agricultural soil improvement.

[0030] Specifically, the phosphorus-soluble bacteria agent containing phosphorus ore is applied to the soil, and the action of Enterobacter holmieae HS-6 is used to promote 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-mentioned phosphorus-soluble bacteria agent in the recovery of phosphate rock resources.

[0032] Specifically, applying the phosphorus-soluble bacteria agent 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-mentioned phosphorus-containing rock phosphate-solubilizing agent in promoting wheat growth.

[0034] This phosphorus-solubilizing bacterial agent for phosphate rock combines phosphorus-solubilizing bacteria with phosphate rock. It utilizes the phosphorus-solubilizing ability of the bacteria while providing a growth and reproduction carrier for the bacteria on the surface of the ore, 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 13.38 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 phosphate-containing rocks, comprising phosphorus-solubilizing bacteria, hydroxyapatite, and a culture medium, wherein the phosphorus-solubilizing bacteria is Enterobacter holmium HS-6. By using hydroxyapatite to prepare this phosphorus-solubilizing agent for phosphate-containing rocks, 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 phosphate-containing rocks combines Enterobacter holmium HS-6 with hydroxyapatite, which can utilize both the phosphorus-solubilizing ability of Enterobacter holmium HS-6 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 phosphate-containing rocks not only expands the resources of phosphorus-solubilizing agents and improves the dissolution efficiency of phosphate rock, but also has good application effects and environmental friendliness.

[0037] (2) The method for preparing a phosphorus-solubilizing agent for phosphorus-containing ore of the present invention has the characteristics of simple preparation method and easy operation, and the obtained phosphorus-solubilizing agent expands the phosphorus-solubilizing agent resources, improves the dissolution efficiency of phosphorus ore, and has good application effect and environmental friendliness.

[0038] (3) The bio-fertilizer of the present invention is prepared using the above-mentioned phosphorus-soluble bacteria agent containing phosphorus ore, thus promoting the dissolution of insoluble phosphorus and having a good application effect. Among them, the phosphorus-soluble bacteria agent containing phosphorus ore combines Enterobacter holmieae HS-6 with hydroxyapatite, which can utilize both the phosphorus-soluble ability of Enterobacter holmieae HS-6 and the phosphorus resources of hydroxyapatite, thus showing great promise for the preparation of bio-fertilizer.

[0039] (4) The application of the phosphorus-solubilizing bacterial agent for phosphorus-containing ore of the present invention, because the phosphorus-solubilizing bacterial agent combines phosphorus-solubilizing bacteria with phosphate rock, can utilize the phosphorus-solubilizing ability of the bacteria and provide a carrier for the growth and reproduction of the bacteria through the surface of the ore, thereby improving the dissolution efficiency of 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 13.38 times. When used as a bio-fertilizer, it can also promote wheat growth. Therefore, it has excellent application prospects in agricultural soil improvement, phosphorus 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 Enterobacter HS-6 of the present invention on a culture medium containing hydroxyapatite.

[0042] Figure 2 This is a scanning electron microscope image of Enterobacter HS-6 of the present invention.

[0043] Figure 3 This is a graph showing the results of the detection of the phosphorus-dissolving ability of Enterobacter holometabolum HS-6 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 h of phosphorus dissolution treatment by Enterobacter holometabolum HS-6 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 bacteria agent and bacterial suspension prepared in Examples 1, 2 and 3 of this invention.

[0046] Figure 6The graph shows the test results of the phosphorus-soluble bacteria and bacterial suspension prepared by applying the phosphorus-containing rock phosphate-solubilizing agent and bacterial suspension obtained by applying the present invention in Examples 1, 2 and 3, and the root and stem length of wheat without any treatment.

[0047] Figure 7 The graph shows the test results of the phosphorus-soluble bacteria agent and bacterial suspension prepared by applying the phosphorus-containing ore to the soil in Examples 1, 2 and 3 of this invention, and the fresh weight of the aboveground and underground parts of wheat without any treatment.

[0048] Figure 8 The graph shows the detection results of chlorophyll a and chlorophyll b content in wheat prepared by applying phosphorus-containing ore phosphate-solubilizing bacteria and bacterial suspensions obtained by applying Examples 1, 2 and 3 of this invention, and wheat without any treatment. 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 phosphate-containing rocks includes phosphate-solubilizing bacteria, hydroxyapatite, and a culture medium; the phosphate-solubilizing bacteria is *Enterobacter hormaechei* HS-6; *Enterobacter hormaechei* HS-6, taxonomically named *Enterobacter hormaechei*, was deposited on October 19, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.: 28680, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. A scanning electron microscope image of *Enterobacter hormaechei* HS-6 is shown below. Figure 2 As shown.

[0062] The 16S rDNA gene sequence of Enterobacter holmieae HS-6 is shown in SEQ ID No. 1. The gene sequence length is 1476.

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

[0064] The preparation method of this phosphorus-soluble ore spore-dissolving bacterial agent includes the following steps:

[0065] S1. Activation culture: Refrigerated Enterobacter HS-6 was streaked onto LB agar plates and activated at 28°C for 48 h 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 OD600 = 0.7 to obtain a bacterial suspension; wherein, the effective viable count of the bacterial suspension is 1 × 10⁻⁶. 10 CFU / mL ~9×10 10 CFU / mL;

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

[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 phosphorus-soluble bacterial agent for phosphorus-containing ore is disclosed. The difference between this embodiment and Example 1 is that the particle size of 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 phosphorus-soluble bacterial agent for phosphorus-containing ore is disclosed. The difference between this embodiment and Example 1 is that the particle size of 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 phosphorus-solubilizing bacterial agent for phosphorus-containing rocks is disclosed. The difference between this embodiment and Example 1 is that in this embodiment, the pH of the culture medium is controlled at 7.0 during step S3 of the bacterial agent preparation. All other conditions and methods in this embodiment are the same as in Example 1.

[0076] Example 5

[0077] A phosphorus-solubilizing bacterial agent for phosphorus-containing rocks is disclosed in this embodiment. The difference between this embodiment and Example 1 is that, in step S3 of preparing the bacterial agent, the pH of the culture medium is controlled at 7.2. All other conditions and methods in this embodiment are the same as in Example 1.

[0078] Example 6

[0079] A bio-fertilizer is prepared using a phosphorus-soluble bacterial agent from phosphorus-containing ore obtained in any one of Examples 1 to 5.

[0080] Experimental testing:

[0081] Experiment Example 1: Detection of whether Enterobacter holometabolum HS-6 has the ability 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. The strain *Enterobacter holmieae* HS-6 was spotted onto the surface of the hydroxyapatite-containing medium and then incubated at 28°C for 3–5 days. Observation revealed that *Enterobacter holmieae* HS-6 produced distinct phosphorus-solubilizing zones on the hydroxyapatite-containing medium, such as… Figure 1 As shown, this demonstrates that Enterobacter HS-6 has the ability to dissolve hydroxyapatite.

[0083] Experiment Example 2: Detection of the phosphorus-solubilizing ability of Enterobacter holometabolum HS-6 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 is evident that when using 0.1μm–1μm hydroxyapatite, the phosphorus solubility reaches 89.00 mg / L after 48 hours, and can reach a maximum of 105.16 mg / L after 168 hours. When using 75μm–90μm hydroxyapatite, the phosphorus solubility reaches 48.96 mg / L after 48 hours, and can reach a maximum of 71.75 mg / L after 168 hours. When using 200μm–212μm hydroxyapatite, the phosphorus solubility reaches 51.47 mg / L after 48 hours, and can reach a maximum of 62.97 mg / L after 168 hours.

[0086] Therefore, the Enterobacter HS-6 of the present invention has better phosphorus solubility for 0.1 μm to 1 μm hydroxyapatite.

[0087] In addition, hydroxyapatite of 0.1 μm–1 μm, 75 μm–90 μm, and 200 μm–212 μm sizes treated with Enterobacter holometabolum HS-6 for 168 h, as well as hydroxyapatite before treatment, were analyzed using scanning electron microscopy. The results are as follows: Figure 4 As shown.

[0088] Specifically, after reacting Enterobacter HS-6 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 hydroxyapatite of 0.1 μm to 1 μm size treated by Enterobacter holometabolum HS-6 for 168 h; c) shows SEM images of hydroxyapatite of 75 μm to 90 μm size treated by Enterobacter holometabolum HS-6 for 168 h; and d) shows SEM images of hydroxyapatite of 200 μm to 212 μm size treated by Enterobacter holometabolum HS-6 for 168 h.

[0090] Depend on Figure 4 visible, Figure 3 (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 *Enterobacter holmieae* HS-6 could fully contact 0.1 μm to 1 μm hydroxyapatite, promoting its dissolution and release of phosphorus. Additionally, observations... Figure 4 (c) It was found that the surface of the 75μm–90μm hydroxyapatite after treatment became slightly smoother, but the platy structure was still visible, indicating that the metabolites of Enterobacter horneri HS-6 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 particles still accumulated in large quantities, with an uneven surface and slightly smoother edges, indicating that the metabolites of *Enterobacter holometabolum* HS-6 were less effective at dissolving them compared to 75μm–90μm hydroxyapatite. Overall, *Enterobacter holometabolum* HS-6 showed little difference in its dissolution effect on 75μm–90μm and 200μm–212μm hydroxyapatite, but 0.1μm–1μm hydroxyapatite showed a stronger affinity for dissolving *Enterobacter holometabolum* HS-6. This indicates that *Enterobacter holometabolum* HS-6 has a better phosphorus-dissolving ability for smaller particle sizes of hydroxyapatite.

[0091] Experimental Example 3: Application of Phosphate-Soluble Bacterial Agents from Phosphate-Containing Rocks in Improving Soil Physicochemical Properties

[0092] The application of a phosphorus-soluble mineral agent containing phosphorus rock in agricultural soil improvement involved applying the phosphorus-soluble mineral agents prepared in Examples 1, 2, and 3, as well as the bacterial suspension prepared in Example 1, to the soil for 14 days. The available phosphorus content of the soil in the four samples after application was then measured, and the available phosphorus content of the soil without any treatment was also measured for comparison. The test results are as follows: Figure 5 As shown.

[0093] Figure 5 In the text, CK represents soil without any treatment, Enterobacter holmierei represents the bacterial suspension prepared in Example 1, "Enterobacter holmierei +0.1-1μm" represents the phosphorus-soluble bacterial agent for phosphorus-containing rocks prepared in Example 1, "Enterobacter holmierei +75-90μm" represents the phosphorus-soluble bacterial agent for phosphorus-containing rocks prepared in Example 2, and "Enterobacter holmierei +200-212μm" represents the phosphorus-soluble bacterial agent for phosphorus-containing rocks prepared in Example 3.

[0094] Depend on Figure 5 The test results show that the phosphorus-soluble bacteria agents prepared in Examples 1, 2, and 3, as well as the bacterial suspension prepared in Example 1, all showed a significant increase in available phosphorus content compared to untreated soil after 14 days of application. Specifically, the phosphorus-soluble bacteria agent prepared in Example 1 (hydroxyapatite particle size of 0.1 μm–1 μm) increased the available phosphorus content by 13.38 times. The phosphorus-soluble bacteria agent prepared in Example 2 (hydroxyapatite particle size of 75 μm–90 μm) increased the available phosphorus content by 3.03 times. The phosphorus-soluble bacteria agent prepared in Example 3 (hydroxyapatite particle size of 200 μm–212 μm) increased the available phosphorus content by 3.68 times. The bacterial suspension prepared in Example 1 increased the available phosphorus content by 2.74 times.

[0095] When the phosphorus-soluble bacterial agent prepared by this invention is applied to the soil, on the one hand, *Enterobacter holmieae* HS-6 can further convert the insoluble phosphorus in the hydroxyapatite of the bacterial agent into available phosphorus; on the other hand, *Enterobacter holmieae* HS-6 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 by this invention, due to the presence of *Enterobacter holmieae* HS-6, 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 phosphorus-soluble bacterial agent containing phosphate rock prepared in Example 1 (with a particle size of 0.1 μm to 1 μm of hydroxyapatite) can significantly increase the available phosphorus content in the soil. This indicates that the agent contains 0.1 μm to 1 μm 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 phosphorus-soluble bacterial agent containing phosphate rock 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, Enterobacter horneri HS-6 further converts the insoluble phosphorus in the original soil into available phosphorus. Therefore, the phosphorus-soluble bacterial agent containing phosphate rock prepared in this invention can be applied to bio-fertilizers and has a promising application prospect.

[0097] Experiment Example 4: Application of Phosphate-Soluble Bacterial Agents Containing Phosphate Rocks in Promoting Wheat Growth

[0098] The application of a phosphorus-containing rock phosphate-solubilizing bacterial agent in promoting wheat growth: The phosphorus-containing rock phosphate-solubilizing bacterial agents 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] Figure 6 to Figure 8 In the text, CK represents wheat without any treatment, Enterobacter holmierei represents the bacterial suspension prepared in Example 1, "Enterobacter holmierei +0.1-1μm" represents the phosphorus-soluble bacterial agent for phosphorus-containing rocks prepared in Example 1, "Enterobacter holmierei +75-90μm" represents the phosphorus-soluble bacterial agent for phosphorus-containing rocks prepared in Example 2, and "Enterobacter holmierei +200-212μm" represents the phosphorus-soluble bacterial agent for phosphorus-containing rocks prepared in Example 3.

[0101] Depend on Figure 6 The test results show that, compared with wheat without any treatment, although there was no significant difference in stem and root length among wheat treated with the phosphorus-soluble bacteria inoculants from Examples 1, 2, and 3, and the bacterial suspension prepared in Example 1, the stem length of wheat treated with Examples 1 and 3 increased by 3.97% and 3.31%, respectively, and the root length of wheat treated with the phosphorus-soluble bacteria inoculants from Examples 1, 2, and 3 increased by 25.14%, 13.33%, and 12.38%, respectively.

[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 7The test results show that, compared with untreated wheat, the application of the phosphate-containing rock-soluble bacteria agents prepared in Examples 1, 2, and 3, as well as the bacterial suspension prepared in Example 1, significantly increased the fresh weight of both the aboveground and underground parts. Furthermore, the increase in aboveground fresh weight of wheat from the phosphate-containing rock-soluble bacteria agents prepared in Examples 1, 2, and 3 was significantly higher than that from the bacterial suspension prepared in Example 1, especially the application of the phosphate-containing rock-soluble bacteria 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 phosphate-containing rock-soluble bacteria agent prepared in this invention has a highly efficient effect on promoting wheat growth.

[0103] Figure 8 In this context, chla represents chlorophyll a, and chlb represents chlorophyll b. Figure 8 The test results showed that, compared with untreated wheat, the chlorophyll content of wheat treated with the phosphate rock-containing phosphate-solubilizing bacteria prepared in Examples 1 and 3 increased significantly by 27.87% and 8.73%, respectively, and the chlorophyll content of wheat treated with the phosphate rock-containing phosphate-solubilizing bacteria prepared in Examples 1 and 3 increased significantly by 64.94% and 13.64%, respectively. There was no significant difference in chlorophyll content between wheat treated with the phosphate rock-containing phosphate-solubilizing bacteria prepared in Example 2 and the bacterial suspension prepared in Example 1. Among the phosphate rock-containing phosphate-solubilizing bacteria prepared in this invention, the phosphate rock-containing phosphate-solubilizing bacteria prepared in Example 1 (with a 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 phosphorus-containing rock phosphate-solubilizing bacteria agent 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] sequence list

[0107] SEQ ID No.1

Claims

1. A phosphorus-soluble bacterial agent for phosphorus-containing ore, characterized in that, It includes phosphate-solubilizing bacteria, hydroxyapatite, and culture medium; the phosphate-solubilizing bacteria is Enterobacter holmieae HS-6; the hydroxyapatite serves as the phosphorus source for the culture medium; The classification name of the Enterobacter HS-6 is... Enterobacter hormaechei It was deposited at the China General Microbiological Culture Collection Center on October 19, 2023, with accession number CGMCC NO.28680; 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 phosphorus-soluble bacterial agent for phosphorus-containing ore 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 phosphorus-soluble bacterial agent for phosphorus-containing ore according to any one of claims 1 to 2, characterized in that, Includes the following steps: S1. Activation culture: Refrigerated Enterobacter HS-6 was streaked onto LB agar plates and activated at 28°C for 48 h to obtain activated colonies. 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 OD600 = 0.7 to obtain bacterial suspension. S3. Preparation of bacterial agent: Using NBRIP medium as the culture medium and hydroxyapatite as the phosphorus source of NBRIP medium, a hydroxyapatite-containing culture medium is obtained. The pH of the culture medium is controlled at 7.0-7.

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

4. The method for preparing a phosphorus-soluble bacterial agent for phosphorus-containing ore as described in claim 3, characterized in that, The effective viable 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 phosphorus-soluble bacteria agent for phosphorus-containing ore as described in any one of claims 1 to 2.

6. The application of the phosphorus-soluble phosphate-containing mineral agent according to any one of claims 1 to 2 in agricultural soil improvement.

7. The application of the phosphorus-soluble bacteria agent for phosphorus-containing ore as described in any one of claims 1 to 2 in the recovery of phosphorus resources.

8. The application of the phosphorus-soluble phosphate-containing rock agent according to any one of claims 1 to 2 in promoting wheat growth.

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