Phosphorus-containing ore phosphate-solubilizing bacterial agent, preparation method thereof, 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 ore-containing phosphate-solubilizing bacterial agent, a preparation method thereof, a biological bacterial fertilizer and application, the phosphate ore-containing phosphate-solubilizing bacterial agent comprises phosphate-solubilizing bacteria, hydroxyapatite and a culture medium, and the phosphate-solubilizing bacteria are enterobacter hormaechei HS-6. The hydroxyapatite is used for preparing the phosphate ore-containing phosphate-solubilizing bacterial agent, so that the phosphate ore resource can be greatly promoted to be fully utilized, and the dissolution of insoluble phosphorus in the phosphate ore is realized. According to the phosphate-containing ore phosphate-solubilizing bacterial agent, the enterobacter hormaechei HS-6 is combined with the hydroxyapatite, so that the phosphate-solubilizing capacity of the enterobacter hormaechei HS-6 can be utilized, and the phosphorus resource of the hydroxyapatite can be fully utilized. The phosphate-containing ore phosphate-solubilizing bacterial agent has good prospects in application to preparation of biological bacterial fertilizer, application to agricultural soil improvement, application to phosphate ore resource recovery and application to promotion of wheat growth.

Description

Technical Field

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

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

[0003] As an important phosphorus-containing mineral resource, phosphate rock is widely used in agriculture and chemical industry. The natural form of phosphate minerals is apatite, including fluoroapatite, hydroxyapatite and carbon fluoroapatite. However, the traditional phosphate rock treatment process has some obvious disadvantages, especially when treating low-grade phosphate rock, the traditional beneficiation method has high cost, low efficiency, large consumption of chemical agents and serious environmental pollution. Therefore, there is an urgent need for a more environmentally friendly and low-cost phosphate rock treatment technology. In recent years, biomineralization technology has been applied to the treatment of phosphate rock as an emerging green environmental protection technology, in which phosphate-dissolving bacteria can effectively dissolve phosphorus in phosphate rock through their metabolites (such as organic acids, acid phosphatase, etc.), thereby improving the utilization efficiency of phosphorus resources. As a microbial resource, phosphate-dissolving bacteria can dissolve phosphate rock through metabolism under mild conditions, and is a phosphate rock treatment method with great potential.

[0004] Microbial agents are an important carrier widely used in agriculture, mining, environmental governance and other fields, and have received increasing attention in recent years. However, the development of phosphate-dissolving microbial agents and the further full utilization of phosphate resources need to be strengthened and expanded, 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 object of the present invention is to provide a phosphate-containing ore phosphate-dissolving bacteria agent. The phosphate-containing ore phosphate-dissolving bacteria agent expands the phosphate-dissolving bacteria agent resources by combining phosphate ore with phosphate-dissolving bacteria, improves the dissolution efficiency of phosphate ore, 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 phosphate ore. 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 dissolution efficiency of phosphate ore, 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 phosphate-containing ore.

[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-containing ore phosphate-dissolving bacteria agent, comprising phosphate-dissolving bacteria, hydroxyapatite and a culture medium; the phosphate-dissolving bacteria is Enterobacter holmesii HS-6, which belongs to bacteria;

[0011] The classification name of the Enterobacter hormaechei HS-6 is Enterobacter hormaechei, and it was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on October 19, 2023, with the deposit number CGMCC NO.: 28680.

[0012] Furthermore, the 16S rDNA gene sequence of the Enterobacter huxleyi HS-6 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, Enterobacter holmii HS-6 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 phosphorus-containing ore phosphate-dissolving bacteria agent, the full utilization of phosphate ore resources can be greatly promoted.

[0016] The phosphate-containing ore phosphate-dissolving bacteria agent combines Enterobacter hollii HS-6 with hydroxyapatite, which can not only utilize the phosphate-dissolving ability of Enterobacter hollii HS-6, 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 Enterobacter hollii HS-6.

[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 bacteria agent for phosphate-containing ore, comprising the following steps:

[0019] S1. Activation culture: streak the refrigerated Enterobacter HS-6 on an LB medium plate, and activate and culture it at 28°C for 48 hours to obtain activated culture colonies;

[0020] S2. Prepare bacterial suspension: pick a single colony from the activated cultured colonies with an inoculation loop and inoculate it into LB liquid culture 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;

[0021] S3. Preparation of bacterial agent: using NBRIP medium as the culture medium, and using hydroxyapatite as the phosphorus source of the NBRIP 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 present invention provides a biological bacterial fertilizer which is prepared by utilizing the above-mentioned phosphorus-dissolving bacterial agent of phosphorus-containing ore.

[0027] Among them, the phosphate-containing ore phosphate-dissolving bacteria agent combines Enterobacter holmii HS-6 with hydroxyapatite, which can not only utilize the phosphate-dissolving ability of Enterobacter holmii HS-6, 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 the use of the above-mentioned phosphate-containing ore phosphate-dissolving bacteria agent in agricultural soil improvement.

[0030] Specifically, the phosphate-containing ore phosphate-dissolving bacteria agent is applied to the soil, and the effect of Enterobacter holmesii HS-6 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 the use of the above-mentioned phosphate-containing ore phosphate-dissolving bacteria agent in phosphate ore resource recovery.

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

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

[0034] Among them, the phosphate-containing ore-dissolving bacteria agent combines phosphate-dissolving bacteria with phosphate ore, 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 ore, thereby improving the dissolution efficiency of the phosphate ore. 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 13.38 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-containing ore phosphate-dissolving bacteria agent of the present invention comprises phosphate-dissolving bacteria, hydroxyapatite and a culture medium, wherein the phosphate-dissolving bacteria is Enterobacter hussii HS-6. By using hydroxyapatite to prepare the phosphate-containing ore phosphate-dissolving bacteria agent, it is possible to greatly promote the full utilization of phosphate ore resources and achieve the dissolution of insoluble phosphorus in phosphate ore. The phosphate-containing ore phosphate-dissolving bacteria agent combines Enterobacter hussii HS-6 with hydroxyapatite, thereby not only utilizing the phosphate-dissolving ability of Enterobacter hussii HS-6, 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-containing ore phosphate-dissolving bacteria agent not only expands the phosphate-dissolving bacteria agent resources and improves the dissolution efficiency of phosphate ore, but also has good application effect and environmental friendliness.

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

[0038] (3) The biological fertilizer of the present invention is prepared by using the above-mentioned phosphorus-dissolving bacteria agent containing phosphorus ore, so it can promote the dissolution of insoluble phosphorus and has a good application effect. Among them, the phosphorus-dissolving bacteria agent containing phosphorus ore combines Enterobacter holmii HS-6 with hydroxyapatite, which can not only utilize the phosphorus-dissolving ability of Enterobacter holmii HS-6, but also make full use of the phosphorus resources of hydroxyapatite, so it has a good prospect for application in the preparation of biological fertilizer.

[0039] (4) Application of a phosphate-containing ore-dissolving bacteria agent of the present invention. Since the phosphate-containing ore-dissolving bacteria agent combines phosphate-dissolving bacteria with phosphate ore, it 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 ore, thereby improving the dissolution efficiency of the phosphate ore. This not only helps to improve the recovery 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 13.38 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 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 the Enterobacter HS-6 of the present invention on a culture medium containing hydroxyapatite.

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

[0043] Figure 3 It is a graph showing the results of the detection of the phosphorus dissolving ability of the Enterobacter HS-6 of the present invention on hydroxyapatite of different sizes at different time periods.

[0044] Figure 4 The present invention shows scanning electron microscope images of hydroxyapatite of different sizes before and after the 168-hour phosphorus dissolution treatment of Enterobacter holmii HS-6.

[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-containing ore phosphate-dissolving bacteria agent and bacterial suspension prepared in Examples 1, 2 and 3 of the present invention.

[0046] Figure 6It is a graph showing the test results of root length and stem length of wheat using the phosphate-dissolving bacteria agent and bacterial suspension prepared by Examples 1, 2 and 3 of the present invention and wheat without any treatment.

[0047] Figure 7 It is a graph showing the test results of the above-ground fresh weight and underground fresh weight of wheat using the phosphate-dissolving bacteria agent and bacterial suspension prepared using phosphate-containing ore according to Example 1, Example 2 and Example 3 of the present invention and without any treatment.

[0048] Figure 8 It is a graph showing the test results of chlorophyll a and chlorophyll b content of wheat using the phosphate-dissolving bacteria agent and bacterial suspension prepared by Examples 1, 2 and 3 of the present invention and wheat without any treatment. 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 SO 4 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-containing ore phosphate-dissolving bacteria agent, comprising phosphate-dissolving bacteria, hydroxyapatite and culture medium; the phosphate-dissolving bacteria is Enterobacter hormaechei HS-6; wherein the classification name of Enterobacter hormaechei HS-6 is Enterobacter hormaechei, which has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on October 19, 2023, with a deposit number of CGMCC NO.: 28680, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The scanning electron microscope image of Enterobacter hormaechei HS-6 is as follows: Figure 2 shown.

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

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

[0064] The preparation method of the phosphate-containing ore phosphate-dissolving bacteria agent comprises the following steps:

[0065] S1. Activation culture: streak the refrigerated Enterobacter HS-6 on an LB medium plate, and activate and culture it at 28°C for 48 hours to obtain activated culture colonies;

[0066] S2. Prepare bacterial suspension: pick a single colony from the activated cultured colonies with an inoculation loop and inoculate it into LB liquid culture 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 10 CFU / mL~9×10 10 CFU / mL;

[0067] S3. Preparation of bacterial agent: using NBRIP medium as the culture medium, and using hydroxyapatite as the phosphorus source of the NBRIP medium to obtain a culture medium containing hydroxyapatite, controlling the pH of the culture medium to be 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 and culturing at 180 r / min for 2 days to obtain the phosphate-containing phosphate-dissolving bacterial agent.

[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 bacteria agent for phosphate-containing ore, 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 bacteria agent for phosphate-containing ore, 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 phosphate-containing ore, the difference between this embodiment and embodiment 1 is that in step S3 of preparing the bacterial agent, 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 phosphate-containing ore, the difference between this embodiment and embodiment 1 is that in step S3 of preparing the bacterial agent in this embodiment, 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 bacteria agent containing phosphate ore obtained by any one of Examples 1 to 5.

[0080] Experimental testing:

[0081] Experimental Example 1: Detection of whether Enterobacter HS-6 has the ability 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 medium containing hydroxyapatite, and then apply the strain Enterobacter holliformis HS-6 on the surface of the medium containing hydroxyapatite, and then culture it at 28℃ for 3d to 5d. Then it can be found that Enterobacter holliformis HS-6 can produce obvious phosphate dissolution rings on the medium containing hydroxyapatite, such as Figure 1 As shown, it is demonstrated that Enterobacter HS-6 has the ability to dissolve hydroxyapatite.

[0083] Experimental Example 2 Detection of the Phosphate Dissolving Capacity of Enterobacter HS-6 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 3It can be seen that when 0.1μm to 1μm hydroxyapatite is used, the amount of dissolved phosphorus reaches 89.00mg / L in 48h, and the maximum amount of dissolved phosphorus can reach 105.16mg / L in 168h. When 75μm to 90μm hydroxyapatite is used, the amount of dissolved phosphorus reaches 48.96mg / L in 48h, and the maximum amount of dissolved phosphorus can reach 71.75mg / L in 168h. When 200μm to 212μm hydroxyapatite is used, the amount of dissolved phosphorus reaches 51.47mg / L in 48h, and the maximum amount of dissolved phosphorus can reach 62.97mg / L in 168h.

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

[0087] In addition, the hydroxyapatite of 0.1μm-1μm, 75μm-90μm, and 200μm-212μm after 168h phosphorus dissolution treatment by Enterobacter HS-6 were tested by scanning electron microscopy. The test results are as follows: Figure 4 shown.

[0088] Specifically, after Enterobacter HS-6 was allowed to react with hydroxyapatite of different sizes for 168 hours, the supernatant was removed, and then 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 Enterobacter HS-6 for 168h, Figure c is the SEM image of hydroxyapatite after 75μm~90μm hydroxyapatite was treated by Enterobacter HS-6 for 168h, and Figure d is the SEM image of hydroxyapatite after 200μm~212μm hydroxyapatite was treated by Enterobacter HS-6 for 168h.

[0090] Depend on Figure 4 visible, Figure 3 (b) Hydroxyapatite is higher than that before treatment 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 E. coli HS-6 can fully contact with 0.1μm to 1μm hydroxyapatite, 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 E. coli HS-6 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 E. coli HS-6 were less effective in dissolving it than the 75μm to 90μm hydroxyapatite. Overall, the dissolution effect of E. coli HS-6 on 75μm to 90μm hydroxyapatite and 200μm to 212μm hydroxyapatite was not much different, but the dissolution affinity of 0.1μm to 1μm hydroxyapatite with E. coli HS-6 was stronger. This shows that E. coli HS-6 has better phosphorus dissolving ability for hydroxyapatite with smaller particle size.

[0091] Experimental Example 3 Application of Phosphate-dissolving Bacteria in Phosphate-containing Rocks to Improve Soil Physical and Chemical Properties

[0092] A method for improving agricultural soil by using a phosphate-containing ore phosphate-dissolving bacterial agent is described. The phosphate-containing ore phosphate-dissolving bacterial agents prepared in Examples 1, 2 and 3, and the bacterial suspension prepared in Example 1 are applied to the soil for 14 days. The effective phosphorus content of the soil after application of the four samples is detected, and the effective phosphorus content of the soil without any treatment is detected for comparison. The test results are as follows: Figure 5 shown.

[0093] Figure 5 In the figure, CK represents soil without any treatment, Enterobacter holmii represents the bacterial suspension prepared in Example 1, "Enterobacter holmii+0.1-1μm" represents the phosphate-dissolving bacteria agent for phosphate ore prepared in Example 1, "Enterobacter holmii+75-90μm" represents the phosphate-dissolving bacteria agent for phosphate ore prepared in Example 2, and "Enterobacter holmii+200-212μm" represents the phosphate-dissolving bacteria agent for phosphate ore prepared in Example 3.

[0094] Depend on Figure 5 The test results show that the phosphate-containing ore-dissolving bacteria agents prepared in Examples 1, 2 and 3, and the bacterial suspension prepared in Example 1, after being applied to the soil for 14 days, the effective phosphorus content increased significantly relative to the soil without any treatment. Among them, after the application of the phosphate-containing ore-dissolving bacteria agent (the particle size of hydroxyapatite is 0.1μm to 1μm) prepared in Example 1, the effective phosphorus content increased by 13.38 times. After the application of the phosphate-containing ore-dissolving bacteria agent (the particle size of hydroxyapatite is 75μm to 90μm) prepared in Example 2, the effective phosphorus content increased by 3.03 times. After the application of the phosphate-containing ore-dissolving bacteria agent (the particle size of hydroxyapatite is 200μm to 212μm) prepared in Example 3, the effective phosphorus content increased by 3.68 times. After the application of the bacterial suspension prepared in Example 1, the effective phosphorus content increased by 2.74 times.

[0095] After the phosphate-containing ore phosphate-dissolving bacterial agent prepared by the present invention is applied to the soil, on the one hand, the Halligen Enterobacter HS-6 can further convert the insoluble phosphorus in the hydroxyapatite in the bacterial agent into effective phosphorus, and on the other hand, the Halligen Enterobacter HS-6 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 the Halligen Enterobacter HS-6, but because the content of the insoluble phosphorus in the soil is limited, the effective phosphorus content is not increased much.

[0096] Depend on Figure 5 It can be seen from the test results that the application of the phosphate-containing ore-dissolving bacteria agent (the particle size of hydroxyapatite is 0.1μm to 1μm) prepared in Example 1 can greatly increase the effective phosphorus content in the soil, indicating 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 of plants. The higher the effective phosphorus content, the stronger the plant growth and the higher the yield. The phosphate-containing ore-dissolving bacteria agent 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, Enterobacter holmii HS-6 further converts the insoluble phosphorus in the original soil into effective phosphorus. Therefore, the phosphate-containing ore-dissolving bacteria agent prepared by the present invention can be applied to biological fertilizers and has a good application prospect.

[0097] Experimental Example 4 Application of Phosphate-dissolving Bacteria from Phosphate-containing Rocks in Promoting Wheat Growth

[0098] A phosphate-dissolving bacteria agent containing phosphate ore is used to promote the growth of wheat. The phosphate-dissolving bacteria agent containing phosphate ore 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 wheat without any treatment, Enterobacter holmii represents the bacterial suspension prepared in Example 1, "Enterobacter holmii+0.1-1μm" represents the phosphate-dissolving bacteria agent for phosphate ore prepared in Example 1, "Enterobacter holmii+75-90μm" represents the phosphate-dissolving bacteria agent for phosphate ore prepared in Example 2, and "Enterobacter holmii+200-212μm" represents the phosphate-dissolving bacteria agent for phosphate ore prepared in Example 3.

[0101] Depend on Figure 6 The test results show that, compared with the wheat without any treatment, there are no significant differences in the stem length and root length of the wheat treated with the phosphate-dissolving bacteria agent containing phosphate rock in Example 1, Example 2, and Example 3 and the bacterial suspension prepared in Example 1, but the stem length of the wheat treated with Example 1 and Example 3 increased by 3.97% and 3.31%, respectively, and the root length of the wheat treated with the phosphate-dissolving bacteria agent containing phosphate rock in Example 1, Example 2, and Example 3 increased by 25.14%, 13.33%, and 12.38%, respectively.

[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 compared with the wheat without any treatment, the aboveground fresh weight and underground fresh weight of the phosphate-containing phosphate-dissolving bacteria agent prepared in Example 1, Example 2 and Example 3, and the bacterial suspension prepared in Example 1 significantly increased. Moreover, the increase in the aboveground fresh weight of wheat by the phosphate-containing phosphate-dissolving bacteria agent 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-containing phosphate-dissolving bacteria agent prepared in Example 1 (the particle size of hydroxyapatite is 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-containing phosphate-dissolving bacteria agent 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 compared with the wheat without any treatment, the chla content of the wheat treated with the phosphate-containing phosphate-dissolving bacteria agent prepared in Example 1 and Example 3 increased significantly by 27.87% and 8.73%, and the chlb content increased significantly by 64.94% and 13.64%, respectively. There was no significant difference in the chlorophyll content of the wheat treated with the phosphate-containing phosphate-dissolving bacteria agent prepared in Example 2 and the bacterial suspension prepared in Example 1. Among the phosphate-containing phosphate-dissolving bacteria agents prepared by the present invention, the phosphate-containing phosphate-dissolving bacteria agent 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-containing ore phosphate-dissolving bacteria agent 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] Sequence Listing

[0107]

Claims

1. A phosphate-dissolving bacteria agent for phosphate-containing ore, characterized in that: It comprises phosphate-dissolving bacteria, hydroxyapatite and culture medium; the phosphate-dissolving bacteria is Enterobacter hallii HS-6; The classification name of the Enterobacter hormaechei HS-6 is Enterobacter hormaechei, and it was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on October 19, 2023, with the deposit number CGMCC NO.: 28680.

2. A phosphate-dissolving bacteria agent for phosphate-containing ore according to claim 1, characterized in that: The 16S rDNA gene sequence of the Enterobacter holmesii HS-6 is shown in SEQ ID No.

1.

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

4. A phosphate-dissolving bacteria agent for phosphate-containing ore as claimed in claim 3, characterized in that: The particle size of the hydroxyapatite is 0.1 μm to 1 μm.

5. The method for preparing a phosphate-dissolving bacteria agent for phosphate-containing ore according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1. Activation culture: streak the refrigerated Enterobacter HS-6 on an LB medium plate, and activate and culture it at 28°C for 48 hours to obtain activated culture colonies; S2. Prepare bacterial suspension: pick a single colony from the activated cultured colonies with an inoculation loop and inoculate it into LB liquid culture 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; S3. Preparation of bacterial agent: using NBRIP medium as the culture medium, and using hydroxyapatite as the phosphorus source of the NBRIP 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 phosphate-containing ore 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 phosphate-containing ore as described in any one of claims 1 to 4.

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

9. Use of the phosphate-containing ore-dissolving bacteria agent according to any one of claims 1 to 4 in phosphate ore resource recovery.

10. Use of the phosphate-containing ore phosphate-dissolving bacteria agent according to any one of claims 1 to 4 in promoting wheat growth.

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