Phosphorus-solubilizing bacterial agent for degrading hydroxyapatite, preparation method of phosphate-solubilizing bacterial agent, biological bacterial fertilizer and application

Abstract

The invention relates to the field of environment, phosphorite resource utilization and agricultural biotechnology, in particular to a phosphate-solubilizing bacterial agent for degrading hydroxyapatite, a preparation method of the phosphate-solubilizing bacterial agent, a biological bacterial fertilizer and application of the phosphate-solubilizing bacterial agent for degrading hydroxyapatite, the phosphate-solubilizing bacterial agent for degrading hydroxyapatite comprises phosphate-solubilizing bacteria, hydroxyapatite and a culture medium, and the phosphate-solubilizing bacteria are pantoea agglomerans CT3. The hydroxyapatite is used for preparing the phosphate-solubilizing bacterial agent for degrading hydroxyapatite, so that full utilization of phosphate ore resources can be greatly promoted, and dissolution of insoluble phosphorus in phosphate ore is realized. According to the phosphate-solubilizing bacterial agent for degrading hydroxyapatite, the pantoea agglomerans CT3 is combined with the hydroxyapatite, so that the phosphate-solubilizing capacity of the pantoea agglomerans CT3 can be utilized, and the phosphorus resource of the hydroxyapatite can be fully utilized. The phosphate-solubilizing bacterial agent capable of degrading hydroxyapatite has good prospects in application to preparation of biological bacterial fertilizer, application to agricultural soil improvement, application to phosphorite resource recovery and application to promotion of wheat growth.

Description

Technical Field

[0001] The invention relates to the fields of environment, utilization of phosphate rock resources and agricultural biotechnology, and in particular to a phosphate-dissolving bacterial agent for degrading hydroxyapatite, a preparation method thereof, a biological bacterial fertilizer and application thereof. Background Art

[0002] Phosphate ore is an important phosphorus-containing mineral resource and is widely used in agriculture and chemical industry. The natural form of phosphate mineral is apatite, including fluoroapatite, hydroxyapatite and carbon fluoroapatite.

[0003] Many countries directly apply phosphate rock as phosphate fertilizer. The size, surface area and dissolution rate of phosphate rock powder particles will have different effects on soil properties and plant growth. Studies have shown that the specific surface area of ​​phosphate rock has a greater impact on the chemical reaction of extracting phosphorus using chemical extractants. The particle size of phosphate rock directly determines the specific surface area of ​​the material. Fine-grained phosphate rock has a larger surface area, while coarse-grained phosphate rock has a smaller surface area. The dissolution rate of phosphate rock increases with the increase of specific surface area. Small-grained phosphate rock has a larger specific surface area, so it can have closer contact with microorganisms in the soil, and thus have more impact on the soil microbial community; although the dissolution rate of coarse-grained ore is slow, it can continuously release phosphorus for a long time, which has a more lasting effect on the soil phosphorus supply.

[0004] In addition, the interaction between phosphate rock and microorganisms will also affect the application effect of phosphate rock. Different strains have different solubility abilities for different types and particle sizes of phosphate rock. It is not the case that the smaller the particle size of phosphate rock, the higher the efficiency of phosphate dissolution. Therefore, further development of phosphate-dissolving bacteria is needed to promote the further full utilization of phosphate rock resources, and the dissolution efficiency of phosphate rock needs to be further improved. Summary of the invention

[0005] In order to overcome the shortcomings of the prior art, the first purpose of the present invention is to provide a phosphate-dissolving bacteria agent for degrading hydroxyapatite. The phosphate-dissolving bacteria agent for degrading hydroxyapatite expands the resources of phosphate-dissolving bacteria agent by combining phosphate ore with phosphate-dissolving bacteria, improves the utilization efficiency of phosphate ore resources, reduces the waste of phosphate ore resources during use, and can promote plant growth, and has good application effect and environmental friendliness.

[0006] In order to overcome the shortcomings of the prior art, the second purpose of the present invention is to provide a method for preparing a phosphate-dissolving bacteria agent for degrading hydroxyapatite. The preparation method is simple and easy to operate. The prepared phosphate-dissolving bacteria agent expands the resources of phosphate-dissolving bacteria agents, improves the utilization efficiency of phosphate rock resources, and has good application effect and environmental friendliness.

[0007] The third object of the present invention is to provide a biological fertilizer.

[0008] The fourth object of the present invention is to provide an application of a phosphate-dissolving bacteria agent for degrading hydroxyapatite.

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

[0010] The present invention provides a phosphate-dissolving bacteria agent for degrading hydroxyapatite, comprising phosphate-dissolving bacteria, hydroxyapatite and a culture medium; the phosphate-dissolving bacteria is Pantoea agglomerans CT3, which belongs to bacteria;

[0011] The classification name of the Pantoea agglomerans CT3 is Pantoea agglomerans, which was deposited in the General Microbiological Center of the China Culture Collection Administration on January 8, 2025, with the deposit number CGMCCNO.: 33335.

[0012] Furthermore, the 16S rDNA gene sequence of Pantoea agglomerans 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 particle size of the hydroxyapatite is 0.1 μm to 1 μm. Among them, Pantoea agglomerans CT3 has a good dissolving effect 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 effective phosphorus content in hydroxyapatite is only 2.774%. By using hydroxyapatite to prepare the phosphate-dissolving bacteria agent for degrading hydroxyapatite, the full utilization of phosphate rock resources can be greatly promoted.

[0016] The phosphate-dissolving bacteria agent for degrading hydroxyapatite combines Pantoea agglomerans CT3 with hydroxyapatite, which can not only utilize the phosphate-dissolving ability of Pantoea agglomerans CT3, but also make full use of the phosphorus resources of hydroxyapatite. The surface of hydroxyapatite can also provide a carrier for the growth and reproduction of bacteria. In addition, the culture medium is used to promote the growth and metabolism of Pantoea agglomerans CT3.

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

[0018] The present invention provides a method for preparing a phosphate-dissolving bacterial agent for degrading hydroxyapatite, comprising the following steps:

[0019] S1. Activation culture: Pantoea agglomerans CT3 was inoculated on an LB medium plate and activated and cultured at 28°C for 24 h to 48 h to obtain activated culture colonies;

[0020] S2. Prepare bacterial suspension: Use an inoculation loop to pick a single colony from the activated cultured colonies and inoculate it into LB liquid culture medium, then place it in a 28°C constant temperature shaker at 180 r / min and culture until OD 600 =0.7, and obtain bacterial suspension;

[0021] S3. Preparation of bacterial agent: using hydroxyapatite to replace the phosphorus source of NBRIP culture medium to obtain a culture medium containing hydroxyapatite, controlling the pH of the culture medium to 7.0-7.2, inoculating the bacterial suspension into the culture medium containing hydroxyapatite, and then placing the culture medium in a constant temperature shaker at 28°C and shaking and culturing at 180r / min for 2 days to obtain the phosphate-containing phosphate-dissolving bacterial agent.

[0022] Furthermore, the effective viable count of the bacterial suspension obtained in step S2 is 1×10 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 volume percentage of the culture medium.

[0025] In order to achieve the third purpose of the above invention, the technical solution adopted by the present invention is as follows:

[0026] The invention provides a biological bacterial fertilizer, which is prepared by utilizing the above-mentioned phosphate-dissolving bacterial agent for degrading hydroxyapatite.

[0027] Among them, the phosphate-dissolving bacteria agent for degrading hydroxyapatite combines Pantoea agglomerans CT3 with hydroxyapatite, which can not only utilize the phosphate-dissolving ability of Pantoea agglomerans CT3, but also make full use of the phosphorus resources of hydroxyapatite. Therefore, it has a good prospect for application in the preparation of biological fertilizer.

[0028] In order to achieve the fourth object of the above invention, the technical solution adopted by the present invention is as follows:

[0029] The present invention provides application of the above-mentioned phosphate-dissolving bacteria agent for degrading hydroxyapatite in agricultural soil improvement.

[0030] Specifically, the phosphate-dissolving bacteria agent that degrades hydroxyapatite is applied to the soil, and the effect of Pantoea agglomerans CT3 is utilized to promote the dissolution and release of phosphorus in the soil, thereby improving the effective phosphorus content in the soil and promoting plant growth.

[0031] The present invention provides application of the above-mentioned phosphate-dissolving bacteria agent for degrading hydroxyapatite in the recovery of phosphate rock resources.

[0032] Specifically, applying the phosphate-dissolving bacteria agent for degrading hydroxyapatite to the biological dissolution of waste phosphate ore or low-grade phosphate ore (hydroxyapatite) can improve the recovery rate of phosphate ore resources.

[0033] The present invention provides the use of the above-mentioned phosphate-dissolving bacteria agent for degrading hydroxyapatite in promoting wheat growth.

[0034] Among them, the phosphate-dissolving bacteria agent for degrading hydroxyapatite combines phosphate-dissolving bacteria with phosphate rock, which can not only utilize the phosphate-dissolving ability of the bacteria, but also 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 recycling rate of phosphorus resources, but also reduces the environmental pollution of traditional chemical treatment methods, and can improve agricultural soil, which can increase the effective phosphorus content in agricultural soil by 10.90 times. It can also promote wheat growth when used as a biological fertilizer, so it has a good application prospect in the above applications.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] (1) A phosphate-dissolving bacteria agent for degrading hydroxyapatite of the present invention comprises phosphate-dissolving bacteria, hydroxyapatite and a culture medium; the phosphate-dissolving bacteria is Pantoea agglomerans CT3. By using hydroxyapatite to prepare the phosphate-dissolving bacteria agent for degrading hydroxyapatite, it is possible to greatly promote the full utilization of phosphate rock resources and achieve the dissolution of insoluble phosphorus in phosphate rock. The phosphate-dissolving bacteria agent for degrading hydroxyapatite combines Pantoea agglomerans CT3 with hydroxyapatite, thereby not only utilizing the phosphate-dissolving ability of Pantoea agglomerans CT3 but also fully utilizing the phosphorus resources of hydroxyapatite, and the surface of hydroxyapatite can also provide a carrier for the growth and reproduction of the bacteria. Therefore, the phosphate-dissolving bacteria agent for degrading hydroxyapatite not only expands the resources of phosphate-dissolving bacteria, improves the utilization efficiency of phosphate rock resources, reduces the waste of phosphate rock resources during use, and can promote plant growth, but also has good application effect and environmental friendliness.

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

[0038] (3) A biological fertilizer of the present invention is prepared by using the above-mentioned phosphate-dissolving bacteria agent for degrading hydroxyapatite, so it can promote the dissolution of insoluble phosphorus and has a good application effect. Among them, the phosphate-dissolving bacteria agent for degrading hydroxyapatite combines Pantoea agglomerans CT3 with hydroxyapatite, which can not only utilize the phosphate-dissolving ability of Pantoea agglomerans CT3, but also make full use of the phosphorus resources of hydroxyapatite, and thus has a good prospect for application in the preparation of biological fertilizer.

[0039] (4) The application of a phosphate-dissolving bacteria agent for degrading hydroxyapatite of the present invention is that the phosphate-dissolving bacteria agent for degrading hydroxyapatite combines phosphate-dissolving bacteria with phosphate rock, which can not only utilize the phosphate-dissolving ability of the strain, but also provide a carrier for the strain to grow and reproduce through the surface of the phosphate rock, thereby improving the dissolution efficiency of the phosphate rock. This not only helps to improve the recycling rate of phosphorus resources, but also reduces the environmental pollution of traditional chemical treatment methods, and can improve agricultural soil, which can increase the effective phosphorus content in agricultural soil by 10.90 times. It can also promote wheat growth when used as a biological fertilizer. Therefore, it has a good application prospect in the application of agricultural soil improvement, the application in the recovery of phosphate rock resources and the application in promoting wheat growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0041] Figure 1 This is an observation diagram of the formation of a phosphate-dissolving circle by Pantoea agglomerans CT3 of the present invention on a culture medium containing hydroxyapatite.

[0042] Figure 2 It is a scanning electron microscope image of Pantoea agglomerans CT3 of the present invention.

[0043] Figure 3 This is a graph showing the results of testing the phosphorus solubilizing ability of Pantoea agglomerans CT3 of the present invention on hydroxyapatite of different sizes at different time periods.

[0044] Figure 4 It is a scanning electron microscope image of hydroxyapatite of different sizes before and after the 168h phosphorus dissolution treatment of Pantoea agglomerata CT3 of the present invention.

[0045] Figure 5 It is a graph showing the test results of the effective phosphorus content in the soil before and after applying the phosphate-dissolving bacteria agent and bacterial suspension for degrading hydroxyapatite prepared in Examples 1, 2 and 3 of the present invention.

[0046] Figure 6 It is a graph showing the test results of the root length and stem length of wheat grown in the original soil using the phosphate-dissolving bacteria agent and bacterial suspension for degrading hydroxyapatite prepared in Example 1, Example 2 and Example 3 of the present invention.

[0047] Figure 7It is a graph showing the test results of the above-ground fresh weight and underground fresh weight of wheat grown in original soil using the phosphate-dissolving bacteria agent and bacterial suspension for degrading hydroxyapatite prepared in Example 1, Example 2 and Example 3 of the present invention.

[0048] Figure 8 It is a graph showing the test results of the chlorophyll a and chlorophyll b contents of wheat grown in original soil using the phosphate-dissolving bacteria agent and bacterial suspension for degrading hydroxyapatite prepared in Example 1, Example 2 and Example 3 of the present invention. DETAILED DESCRIPTION

[0049] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0050] The terms used in the embodiments of the present invention are only for the purpose of describing specific embodiments, and are not intended to limit the present invention. In the present invention, the singular forms "a", "said" and "the" used in the embodiments and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0051] Unless otherwise specified, the 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, Ca 3 (PO 4 ) 2 5g, MgSO 4 0.1g, (NH 4 ) 2 SO 4 0.5g, glucose 15g, chloramphenicol 50mg, streptomycin 50mg, pH 6.8~7.0.

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

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

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

[0057] NBRIP medium (g / L): glucose 10 g, (NH 4 ) 2 SO4 0.5g, NaCl 0.3g, MgSO 4 7H 2 O0.3g, FeSO 4 7H 2 O 0.03g, MnSO 4 ·2H 2 O 0.03g, Ca 3 (PO 4 ) 2 5g, KCl 0.3g, lecithin 0.2g, pH 7.2~7.4.

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

[0059] Among them, hydroxyapatite, also known as hydroxyapatite, basic calcium phosphate, is the natural mineralization of calcium apatite, and its molecular formula is usually written as (Ca 10 (PO 4 ) 6 (OH) 2 ) in the form of.

[0060] Example 1

[0061] A phosphate-dissolving bacteria agent for degrading hydroxyapatite, comprising phosphate-dissolving bacteria, hydroxyapatite and culture medium; the phosphate-dissolving bacteria is Pantoea agglomerans CT3; wherein the classification name of Pantoea agglomerans CT3 is Pantoea agglomerans, which has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on January 8, 2025, with a deposit number of CGMCC NO.: 33335, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The scanning electron microscope image of Pantoea agglomerans CT3 is as follows: Figure 2 shown.

[0062] The 16S rDNA gene sequence of Pantoea agglomerans 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 the phosphate-dissolving bacteria agent for degrading hydroxyapatite comprises the following steps:

[0065] S1. Activation culture: Pantoea agglomerans CT3 was inoculated on an LB medium plate and activated and cultured at 28°C for 48 hours to obtain activated culture colonies;

[0066] S2. Prepare bacterial suspension: Use an inoculation loop to pick a single colony from the activated cultured colonies and inoculate it into LB liquid culture medium, then place it in a 28°C constant temperature shaker at 180 r / min and culture until OD 600 =0.7, and obtain bacterial suspension;

[0067] S3. Preparation of bacterial agent: using hydroxyapatite to replace the phosphorus source of NBRIP culture medium to obtain a culture medium containing hydroxyapatite, controlling the pH of the culture medium to 7.1, inoculating the bacterial suspension into the culture medium containing hydroxyapatite, and then placing the culture medium in a constant temperature shaker at 28°C and shaking at 180 r / min for 2 days to obtain a phosphate-dissolving bacterial agent containing phosphate ore.

[0068] Among them, hydroxyapatite was used to replace the phosphorus source in the NBRIP medium (i.e., hydroxyapatite was used to replace Ca 3 (PO 4 ) 2 ), the concentration of hydroxyapatite in the culture medium is 5 g / L;

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

[0070] Example 2

[0071] A phosphate-dissolving bacterial agent for degrading hydroxyapatite, the difference between this embodiment and embodiment 1 is that in this embodiment, the particle size of hydroxyapatite is 75 μm to 90 μm. The remaining conditions and methods of this embodiment are the same as those of embodiment 1.

[0072] Example 3

[0073] A phosphate-dissolving bacterial agent for degrading hydroxyapatite, the difference between this embodiment and embodiment 1 is that in this embodiment, the particle size of hydroxyapatite is 200 μm to 212 μm. The remaining conditions and methods of this embodiment are the same as those of embodiment 1.

[0074] Example 4

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

[0076] Example 5

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

[0078] Example 6

[0079] A biological fertilizer is prepared by using a phosphate-dissolving bacterial agent for degrading hydroxyapatite obtained in any one of Examples 1 to 5.

[0080] Experimental testing:

[0081] Experimental Example 1: Detection of the ability of Pantoea agglomerans CT3 to dissolve hydroxyapatite

[0082] Hydroxyapatite is used to replace the phosphorus source in the solid culture medium of PVK (i.e., to replace Ca 3 (PO 4 ) 2 ) to form a culture medium containing hydroxyapatite, and then apply Pantoea agglomerans CT3 on the surface of the culture medium containing hydroxyapatite, and then culture it at 28°C for 3 to 5 days. Then it can be observed that Pantoea agglomerans CT3 can produce obvious phosphate dissolution circles on the culture medium containing hydroxyapatite, such as Figure 1 As shown, it was demonstrated that Pantoea agglomerans CT3 had the ability to dissolve hydroxyapatite.

[0083] Experimental Example 2 Detection of the Phosphate Dissolving Capacity of Pantoea agglomerans CT3 on Hydroxyapatite of Different Sizes

[0084] The phosphorus source of NBRIP medium (i.e., replacing Ca 3 (PO 4 ) 2 ), respectively forming a culture medium containing hydroxyapatite, inoculating the bacterial suspension prepared in Example 1 into the culture medium containing hydroxyapatite at an inoculum volume ratio of 1%, and then placing it in a constant temperature shaker at 28°C and shaking at 180r / min for 7 days, and detecting the effective phosphorus content every day. The detection results of the effective phosphorus content can be found in Figure 3 .

[0085] Depend on Figure 3 It can be seen that when 0.1μm to 1μm hydroxyapatite is used, the amount of dissolved phosphorus reaches 69.284mg / L in 24h, and the dissolved phosphorus reaches a peak of 94.205mg / L in 168h. When 75μm to 90μm hydroxyapatite is used, the amount of dissolved phosphorus is only 1.059mg / L in 24h, and the dissolved phosphorus reaches a peak of 87.304mg / L in 108h. When 200μm to 212μm hydroxyapatite is used, the amount of dissolved phosphorus is only 6.572mg / L in 24h, and the dissolved phosphorus reaches a peak of 69.011mg / L in 144h.

[0086] Therefore, the Pantoea agglomerans CT3 of the present invention has a better phosphorus-dissolving ability for hydroxyapatite of 0.1 μm to 1 μm.

[0087] In addition, Pantoea agglomerata CT3 was used to examine hydroxyapatite of 0.1 μm to 1 μm, hydroxyapatite of 75 μm to 90 μm, and hydroxyapatite of 200 μm to 212 μm after 168 h of phosphorus dissolution treatment, as well as hydroxyapatite before phosphorus dissolution treatment, respectively, using scanning electron microscopy images. The test results are as follows: Figure 3 shown.

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

[0089] Figure 4 In the figure, Figure a is the hydroxyapatite before treatment, Figure b is the SEM image of hydroxyapatite after 0.1μm~1μm hydroxyapatite was treated by Pantoea agglomerans CT3 for 168h, Figure c is the SEM image of hydroxyapatite after 75μm~90μm hydroxyapatite was treated by Pantoea agglomerans CT3 for 168h, and Figure d is the SEM image of hydroxyapatite after 200μm~212μm hydroxyapatite was treated by Pantoea agglomerans CT3 for 168h.

[0090] Depend on Figure 4 visible, Figure 4 (b) The hydroxyapatite in the Figure 4 (a) The hydroxyapatite is more dispersed, and the edges of the hydroxyapatite are smoother and the pores are increased after treatment, indicating that the agglomerated Pantoea CT3 can fully contact with the hydroxyapatite of 0.1μm to 1μm, promoting the dissolution of hydroxyapatite and releasing phosphorus. Figure 4 (c) It was found that the surface of 75μm to 90μm hydroxyapatite became slightly smoother after treatment, but the flaky structure was still visible, indicating that the metabolites of Pantoea agglomerata CT3 were less soluble in it than in 0.1μm to 1μm hydroxyapatite. Figure 4 (d) It was found that the 200μm to 212μm hydroxyapatite after treatment was still accumulated in large quantities, with an uneven surface and slightly smooth edges, indicating that the metabolites of Pantoea agglomerans CT3 were less effective in dissolving it than those of 75μm to 90μm hydroxyapatite. Overall, there was little difference in the dissolution effect of Pantoea agglomerans CT3 on 75μm to 90μm hydroxyapatite and 200μm to 212μm hydroxyapatite, but the dissolution affinity of 0.1μm to 1μm hydroxyapatite with Pantoea agglomerans CT3 was stronger. This shows that Pantoea agglomerans CT3 has a better ability to dissolve phosphorus for 0.1μm to 1μm hydroxyapatite.

[0091] Experimental Example 3 Application of the phosphate-dissolving bacteria agent for degrading hydroxyapatite in improving soil physical and chemical properties

[0092] A phosphate-dissolving bacterial agent for degrading hydroxyapatite is used in agricultural soil improvement. The phosphate-dissolving bacterial agents for degrading hydroxyapatite prepared in Examples 1, 2 and 3, and the bacterial suspension prepared in Example 1 are applied to the soil respectively for 14 days, and the effective phosphorus content of the soil after the application of the above four samples is detected respectively, and the effective phosphorus content of the original soil is detected for comparison. The test results are as follows: Figure 5 shown.

[0093] Figure 5 In the figure, CK represents the original soil, Pantoea agglomerans represents the bacterial suspension prepared in Example 1, "Pantoea agglomerans+0.1-1 μm" represents the phosphate-dissolving bacteria agent for degrading hydroxyapatite prepared in Example 1, "Pantoea agglomerans+75-90 μm" represents the phosphate-dissolving bacteria agent for degrading hydroxyapatite prepared in Example 2, and "Pantoea agglomerans+200-212 μm" represents the phosphate-dissolving bacteria agent for degrading hydroxyapatite prepared in Example 3.

[0094] Depend on Figure 5 The test results show that the phosphate-dissolving bacteria for degrading hydroxyapatite prepared in Example 1, Example 2 and Example 3, as well as the bacterial suspension prepared in Example 1, are applied to the soil for 14 days, and the effective phosphorus content is increased relative to the original soil. Among them, after the phosphate-dissolving bacteria for degrading hydroxyapatite prepared in Example 1 (the particle size of hydroxyapatite is 0.1μm to 1μm) is applied, the effective phosphorus content is increased by 10.90 times. After the phosphate-dissolving bacteria for degrading hydroxyapatite prepared in Example 2 (the particle size of hydroxyapatite is 75μm to 90μm) is applied, the effective phosphorus content is increased by 1.64 times. After the phosphate-dissolving bacteria for degrading hydroxyapatite prepared in Example 3 (the particle size of hydroxyapatite is 200μm to 212μm) is applied, the effective phosphorus content is increased by 7.16 times. After the bacterial suspension prepared in Example 1 is applied, the effective phosphorus content is increased by 3.53 times.

[0095] After the phosphate-dissolving bacterial agent for degrading hydroxyapatite prepared by the present invention is applied to the soil, on the one hand, Pantoea agglomerans CT3 can further convert the insoluble phosphorus in the hydroxyapatite in the bacterial agent into effective phosphorus, and on the other hand, Pantoea agglomerans CT3 can convert the insoluble phosphorus in the soil into effective phosphorus, thereby increasing the phosphorus content in the soil that can be absorbed by plants. In addition, the bacterial suspension prepared by the present invention can convert the insoluble phosphorus in the soil into effective phosphorus because it contains Pantoea agglomerans CT3, but because the content of insoluble phosphorus in the soil is limited, the effective phosphorus content is not increased much.

[0096] Depend on Figure 5It can be seen from the test results that the application of the phosphate-dissolving bacteria agent for degrading hydroxyapatite prepared in Example 1 (the particle size of hydroxyapatite is 0.1μm to 1μm) can greatly increase the effective phosphorus content in the soil, which shows that the hydroxyapatite of 0.1μm to 1μm contained in the bacteria agent can greatly increase the effective phosphorus content in the soil. The content of effective phosphorus in the soil is closely related to the growth condition of plants. The higher the effective phosphorus content, the stronger the plant growth and the higher the yield. The phosphate-dissolving bacteria agent for degrading hydroxyapatite prepared in Example 1 can fully and effectively convert the insoluble phosphorus in hydroxyapatite into soluble phosphorus that can be directly absorbed and utilized by plants. At the same time, Pantoea agglomerata CT3 further converts the insoluble phosphorus in the original soil into effective phosphorus. Therefore, the phosphate-dissolving bacteria agent for degrading hydroxyapatite prepared by the present invention can be applied to biological fertilizers and has a good application prospect.

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

[0098] A phosphate-dissolving bacteria agent for degrading hydroxyapatite is used in promoting the growth of wheat. The phosphate-dissolving bacteria agents for degrading hydroxyapatite prepared in Example 1, Example 2 and Example 3, and the bacterial suspension prepared in Example 1 are respectively used as fertilizers for wheat growth and applied to the soil for 14 days. The root length, stem length, aboveground fresh weight and underground fresh weight of the wheat are respectively detected.

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

[0100] Figures 6 to 8 In the figure, CK represents the original soil without adding any materials, Pantoea agglomerans represents the bacterial suspension prepared in Example 1, "Pantoea agglomerans+0.1-1 μm" represents the phosphate-dissolving bacteria agent for degrading hydroxyapatite prepared in Example 1, "Pantoea agglomerans+75-90 μm" represents the phosphate-dissolving bacteria agent for degrading hydroxyapatite prepared in Example 2, and "Pantoea agglomerans+200-212 μm" represents the phosphate-dissolving bacteria agent for degrading hydroxyapatite prepared in Example 3.

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

[0102] Figure 7 middle, Figure 7 (A) is the aboveground fresh weight test results, Figure 7 (B) is the fresh weight of underground parts. Figure 7 The test results show that the application of the phosphate-dissolving bacteria for degrading hydroxyapatite prepared in Example 1, Example 2 and Example 3, and the bacterial suspension prepared in Example 1, significantly increased the aboveground fresh weight and underground fresh weight of wheat relative to the original soil growth. Moreover, the increase in the aboveground fresh weight of wheat by the phosphate-dissolving bacteria for degrading hydroxyapatite prepared in Example 1, Example 2 and Example 3 was significantly higher than that by the bacterial suspension prepared in Example 1, especially the application of the phosphate-dissolving bacteria for degrading hydroxyapatite prepared in Example 1 (the particle size of hydroxyapatite was 0.1 μm to 1 μm), the increase in the aboveground fresh weight of wheat was the most significant. This shows that the application of the phosphate-dissolving bacteria for degrading hydroxyapatite prepared in the present invention has the characteristics of high efficiency in promoting the growth of wheat.

[0103] Figure 8 In the formula, chla represents chlorophyll a and chlb represents chlorophyll b. Figure 8 The test results show that the chlorophyll content of wheat increases when the phosphate-dissolving bacteria agents for degrading hydroxyapatite prepared in Example 1, Example 2 and Example 3 are applied. The content of chlorophyll a in the bacterial suspension prepared in Example 1 is not significantly different from that in the original soil, but the content of chlorophyll b is significantly increased. Among the phosphate-dissolving bacteria agents for degrading hydroxyapatite prepared by the present invention, the phosphate-dissolving bacteria agent for degrading hydroxyapatite of Example 1 (the particle size of hydroxyapatite is 0.1 μm to 1 μm) has the most obvious increase in chlorophyll content.

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

[0105] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

[0106]

Claims

1. A phosphate-dissolving bacterial agent for degrading hydroxyapatite, characterized in that: It comprises phosphate-dissolving bacteria, hydroxyapatite and culture medium; the phosphate-dissolving bacteria is Pantoea agglomerans CT3; The classification name of the Pantoea agglomerans CT3 is Pantoea agglomerans, which was deposited in the General Microbiological Center of the China Culture Collection Administration on January 8, 2025, with the deposit number CGMCCNO.: 33335.

2. The phosphate-dissolving bacterial agent for degrading hydroxyapatite according to claim 1, characterized in that: The 16S rDNA gene sequence of the Pantoea agglomerans CT3 is shown in SEQ ID No.

1.

3. The phosphate-dissolving bacterial agent for degrading hydroxyapatite according to claim 1, characterized in that: The particle size of the hydroxyapatite is 0.1 μm to 212 μm.

4. The phosphate-dissolving bacterial agent for degrading hydroxyapatite according to claim 3, characterized in that: The particle size of the hydroxyapatite is 0.1 μm to 1 μm.

5. A method for preparing a phosphate-dissolving bacteria agent for degrading hydroxyapatite according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1. Activation culture: Pantoea agglomerata CT3 was inoculated on an LB medium plate and activated and cultured at 28°C for 24 h to 48 h to obtain activated culture colonies; S2. Prepare bacterial suspension: Use an inoculation loop to pick a single colony from the activated cultured colonies and inoculate it into LB liquid culture medium, then place it in a 28°C constant temperature shaker at 180 r / min and culture until OD 600 =0.7, and obtain bacterial suspension; S3. Preparation of bacterial agent: using hydroxyapatite to replace the phosphorus source of NBRIP culture medium to obtain a culture medium containing hydroxyapatite, controlling the pH of the culture medium to 7.0-7.2, inoculating the bacterial suspension into the culture medium containing hydroxyapatite, and then placing the culture medium in a constant temperature shaker at 28°C and shaking and culturing at 180r / min for 2 days to obtain the phosphate-containing phosphate-dissolving bacterial agent.

6. The method for preparing a phosphate-dissolving bacteria agent for degrading hydroxyapatite according to claim 5, characterized in that: The effective viable bacteria count of the bacterial suspension prepared in step S2 is 1×10 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 volume percentage of the culture medium.

7. A biological fertilizer, characterized in that: The invention is prepared by using the phosphate-dissolving bacteria agent for degrading hydroxyapatite as described in any one of claims 1 to 4.

8. Use of the phosphate-dissolving bacteria agent for degrading hydroxyapatite according to any one of claims 1 to 4 in agricultural soil improvement.

9. Use of the phosphate-dissolving bacteria agent for degrading hydroxyapatite as claimed in any one of claims 1 to 4 in the recovery of phosphate rock resources.

10. Use of the phosphate-dissolving bacteria agent for degrading hydroxyapatite as claimed in any one of claims 1 to 4 in promoting wheat growth.

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