A method for converting insoluble phosphorus using microorganisms of the genus Enterobacter and a high-efficiency phosphorus-dissolving agent and its application

Abstract

This invention relates to the fields of environmental protection, resource utilization, and agricultural biotechnology, specifically to a method for converting insoluble phosphorus using *Enterobacter horneri* microorganisms, as well as a highly efficient phosphorus-solubilizing agent and its applications. The highly efficient phosphorus-solubilizing agent comprises phosphorus-solubilizing bacteria, bone char, and a culture medium; the phosphorus-solubilizing bacteria is *Enterobacter horneri* HS-6. By combining *Enterobacter horneri* HS-6 with bone char, both the phosphorus-solubilizing ability of *Enterobacter horneri* HS-6 and the phosphorus resources of bone char can be fully utilized. The surface of bone char provides a carrier for the growth and reproduction of the microorganisms, and its insoluble phosphorus can be converted into soluble phosphorus. This phosphorus-solubilizing agent shows great promise in the preparation of bio-fertilizers, agricultural soil improvement, bone char phosphorus resource recovery, and the promotion of crop growth. This method for converting insoluble phosphorus using *Enterobacter horneri* microorganisms not only effectively improves the utilization efficiency of phosphorus resources and reduces dependence on limited phosphate rock resources, but also has significant implications for promoting sustainable agricultural development.

Description

Technical Field

[0001] This invention relates to the fields of environmental protection, resource utilization, and agricultural biotechnology, and specifically to a method for converting insoluble phosphorus using Enterobacter spp. microorganisms, as well as a highly efficient phosphorus-solubilizing agent and its application. Background Technology

[0002] In the global agricultural and ecological fields, the rational utilization and sustainable development of phosphorus resources are facing unprecedented challenges. Phosphorus, as one of the three essential nutrients for plant growth, plays a crucial role in many physiological processes, including photosynthesis, energy metabolism, signal transduction, and genetic material synthesis. However, on the one hand, global phosphate rock reserves are limited and unevenly distributed. With the continuous expansion of agricultural production and the widespread demand for phosphorus in the industrial sector, the mining rate of phosphate rock resources is accelerating, facing the risk of increasing depletion. On the other hand, the current over-reliance on and irrational use of phosphate fertilizers in agricultural production has led to a series of serious environmental problems. Large amounts of phosphate fertilizers that are not absorbed and utilized by plants enter water bodies through surface runoff, causing severe eutrophication, resulting in massive algal blooms, water quality deterioration, and disruption of the balance of aquatic ecosystems. Therefore, finding a sustainable way to utilize phosphorus resources is urgently needed.

[0003] Bone char, a unique phosphorus-containing material, is widely available and rich in phosphorus, attracting significant attention from researchers. Typically obtained from animal bones through high-temperature pyrolysis and carbonization, bone char is a biochar material with a rich porous structure and a large specific surface area. Its phosphorus content is much higher than that of biochar prepared from other raw materials. However, the phosphorus in bone char exists primarily in the form of insoluble phosphates, making it difficult for plant roots to directly absorb and utilize it. If the insoluble phosphorus in bone char can be fully converted into soluble phosphorus, making it available to plants, not only can the efficient utilization of phosphorus resources be achieved, but also dependence on traditional phosphate fertilizers can be reduced, thereby lowering agricultural production costs and environmental pressure.

[0004] Microorganisms play a vital role in the natural cycle of matter, and many microorganisms possess unique phosphorus-solubilizing abilities, commonly known as phosphate-solubilizing bacteria (PSBs). PSBs can secrete organic acids such as acetic acid, lactic acid, malic acid, oxalic acid, succinic acid, citric acid, and gluconic acid, thus enabling them to dissolve phosphorus. The use of phosphate-solubilizing bacteria to convert insoluble phosphorus into soluble phosphorus has received increasing attention in recent years. However, the development of phosphate-solubilizing bacteria still needs further expansion to enrich resources and promote soil improvement and agricultural development. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, the first objective of this invention is to provide a highly efficient phosphate-solubilizing agent. This highly efficient phosphate-solubilizing agent expands the resources of phosphate-solubilizing agents by combining bone char with Enterobacter holmieae, thereby improving the efficiency of converting insoluble phosphorus in bone char into soluble phosphorus.

[0006] To overcome the shortcomings of the prior art, the second objective of this invention is to provide a method for converting insoluble phosphorus using Enterobacter spp. This method can not only effectively improve the utilization efficiency of phosphorus resources and reduce dependence on limited phosphate rock resources, but also has important significance for promoting sustainable agricultural development.

[0007] The third objective of this invention is to provide an application of a highly efficient phosphate-solubilizing agent.

[0008] To achieve the first objective of the invention, the technical solution adopted by the present invention is as follows:

[0009] This invention provides a highly efficient phosphate-solubilizing agent, comprising phosphate-solubilizing bacteria, bone char, and culture medium; wherein the phosphate-solubilizing bacteria is Enterobacter HS-6;

[0010] The Enterobacter hormaechei strain HS-6 was deposited on October 19, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.: 28680.

[0011] Furthermore, the 16S rDNA gene sequence of the *Enterobacter holmieae* HS-6 is shown in SEQ ID No. 1.

[0012] Furthermore, the particle size of the bone char is 30 nm to 2 μm. Enterobacter horneri HS-6 exhibits a good effect on converting insoluble phosphorus into soluble phosphorus in bone char with a particle size of 30 nm to 2 μm.

[0013] Furthermore, the preparation method of the highly efficient phosphate-solubilizing agent includes the following steps:

[0014] S1. Preparation of bone char: Animal bones are freeze-dried, then thermally pyrolyzed and cooled, then ball-milled, and then irradiated and sterilized to obtain micron- and nano-scale bone char.

[0015] S2. Activation of Enterobacter holmieae: Enterobacter holmieae HS-6 was inoculated into LB medium and then cultured in a constant temperature shaker at 28℃ and 180r / min for 16h to allow OD to be activated. 600 The concentration was 0.6–0.8, resulting in an active bacterial solution;

[0016] S3. Preparation of phosphate-solubilizing bacterial agent: The micron-nano multi-level bone char obtained in step S1 is added to NBRIP-P medium to obtain a reaction medium. Then, the active bacterial solution is inoculated into the reaction medium and placed in a constant temperature shaker at 28°C with shaking at 180 r / min to obtain the high-efficiency phosphate-solubilizing bacterial agent.

[0017] Further, in step S1, animal bones are freeze-dried at -65℃ to -55℃ for 40 to 55 hours, then thermally pyrolyzed at 440℃ to 460℃ for 100 to 140 minutes. After cooling, large pieces of bone char are obtained, and then the large pieces of bone char are ball-milled at 440 rpm to 460 rpm for 4 to 6 hours to obtain micron- to nano-scale bone char; and / or

[0018] In the ball mill, zirconium oxide particles are used to ball mill large pieces of bone char, and ethanol is added as a grinding aid; the weight ratio of zirconium oxide particles to large pieces of bone char is (15-20):1; the mass ratio of ethanol to large pieces of bone char is (1.5-2):1.

[0019] The irradiation sterilization dose is 3.0 kGy to 6.0 kGy.

[0020] Furthermore, in step S2, the effective viable count of the obtained active bacterial solution is 1×10⁻⁶. 10 CFU / mL ~9×10 10 CFU / mL; and / or

[0021] In step S3, the mass-to-volume ratio of the bone char to the NBRIP-P culture medium is 0.5% to 2.5%; and / or

[0022] The inoculation amount of the active bacterial solution in the reaction medium is 1% of the volume percentage of the reaction medium; and / or

[0023] In step S3, the active bacterial solution is inoculated into the reaction culture medium and then placed in a constant temperature shaker at 28°C with shaking at 180 r / min for 3 to 7 days to obtain the high-efficiency phosphate-solubilizing agent.

[0024] This invention, by using bone char to prepare this highly efficient phosphate-solubilizing agent, can greatly promote the full utilization of bone char phosphorus resources.

[0025] This highly efficient phosphate-solubilizing agent combines *Enterobacter holmieae* HS-6 with bone char, utilizing both the phosphate-solubilizing ability of *Enterobacter holmieae* HS-6 and the phosphorus resources of bone char. The surface of the bone char provides a carrier for the growth and reproduction of the bacteria, while *Enterobacter holmieae* HS-6 converts its insoluble phosphorus into soluble phosphorus. Additionally, the culture medium promotes the growth and metabolism of *Enterobacter holmieae* HS-6.

[0026] To achieve the second objective of the invention, the technical solution adopted by the present invention is as follows:

[0027] This invention provides a method for converting insoluble phosphorus using *Enterobacter hooligans* microorganisms, utilizing the aforementioned highly efficient phosphate-solubilizing agent, comprising the following steps:

[0028] S1. Conversion of insoluble phosphorus in bone char: In the highly efficient phosphate-solubilizing bacterial agent, Enterobacter horneri HS-6 converts insoluble phosphorus in bone char into soluble phosphorus during the culture process; the culture process is carried out in a constant temperature shaker at 28℃ with shaking at 180r / min for 3 to 7 days.

[0029] S2. Conversion of insoluble phosphorus in soil: The highly efficient phosphorus-solubilizing agent is applied to the soil, and Enterobacter holmieae HS-6 further converts the insoluble phosphorus in the soil into soluble phosphorus.

[0030] This invention discloses a method for converting insoluble phosphorus using *Enterobacter horneri* microorganisms. By combining *Enterobacter horneri* HS-6 with bone char in a culture medium, *Enterobacter horneri* HS-6 can efficiently convert insoluble phosphorus in bone char into soluble phosphorus, significantly improving the utilization efficiency of phosphorus resources in bone char and yielding a highly efficient phosphorus-solubilizing agent. This highly efficient phosphorus-solubilizing agent is then applied to the soil, where *Enterobacter horneri* HS-6 further converts insoluble phosphorus in the soil into soluble phosphorus, resulting in a high soluble phosphorus content in the soil. This effectively promotes crop growth, significantly reduces the use of chemical phosphate fertilizers, and improves agricultural soil. Therefore, this method has excellent application prospects.

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

[0032] This invention provides a highly efficient phosphate-solubilizing agent for use as a bio-fertilizer or for its application in the preparation of bio-fertilizers.

[0033] This highly efficient phosphorus-solubilizing agent combines Enterobacter holmieae HS-6 with bone char, which can utilize both the phosphorus-solubilizing ability of Enterobacter holmieae HS-6 and the phosphorus resources of bone char. Therefore, it has a promising future as a bio-fertilizer or for the preparation of bio-fertilizers.

[0034] This invention provides an application of a highly efficient phosphate-solubilizing agent in agricultural soil improvement or crop growth promotion.

[0035] Specifically, when the highly efficient phosphorus-solubilizing agent is applied to the soil, the action of Enterobacter holmieae HS-6 is utilized to promote the conversion and release of insoluble phosphorus in the soil. In addition, the soluble phosphorus converted from bone char by the highly efficient phosphorus-solubilizing agent can also greatly increase the content of soluble phosphorus in the soil, thereby synergistically improving the available phosphorus content in the soil and promoting plant growth.

[0036] This invention provides an application of a highly efficient phosphate-solubilizing agent in the recovery of bone char phosphorus resources.

[0037] Specifically, applying the highly efficient phosphorus-solubilizing agent to bone char converts insoluble phosphorus into soluble phosphorus, thereby improving the recovery rate of phosphorus resources in bone char.

[0038] Furthermore, this highly efficient phosphate-solubilizing bacterial agent combines phosphate-solubilizing bacteria with bone char, utilizing both the phosphate-solubilizing ability of the bacteria and providing a growth and reproduction carrier for the bacteria on the surface of the bone char, thereby improving the efficiency of converting insoluble phosphorus in bone char into soluble phosphorus. This not only helps improve the recovery and utilization rate of phosphorus resources but also reduces environmental pollution from traditional chemical treatment methods, improves agricultural soil, and promotes crop growth when used as a bio-fertilizer. Therefore, it has excellent application prospects in all these applications.

[0039] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0040] (1) A highly efficient phosphate-solubilizing agent of the present invention comprises phosphate-solubilizing bacteria, bone char, and culture medium; wherein the phosphate-solubilizing bacteria is Enterobacter holmieae HS-6. This not only expands the resources of phosphate-solubilizing agents but also greatly promotes the full utilization of phosphorus resources in bone char, realizing the dissolution of insoluble phosphorus in bone char and significantly improving the efficiency of converting insoluble phosphorus in bone char into soluble phosphorus. This highly efficient phosphate-solubilizing agent combines Enterobacter holmieae HS-6 with bone char, utilizing both the phosphate-solubilizing ability of Enterobacter holmieae HS-6 and the phosphorus resources of bone char. The surface of bone char provides a carrier for the growth and reproduction of bacteria, and allows Enterobacter holmieae HS-6 to convert its insoluble phosphorus into soluble phosphorus. Furthermore, the culture medium promotes the growth and metabolism of Enterobacter holmieae HS-6.

[0041] (2) This invention discloses a method for converting insoluble phosphorus using *Enterobacter horneri* microorganisms. By combining *Enterobacter horneri* HS-6 with bone char in a culture medium, *Enterobacter horneri* HS-6 can efficiently convert insoluble phosphorus in bone char into soluble phosphorus, significantly improving the utilization efficiency of phosphorus resources in bone char and yielding a highly efficient phosphorus-solubilizing agent. This highly efficient phosphorus-solubilizing agent is then applied to the soil, where *Enterobacter horneri* HS-6 further converts insoluble phosphorus in the soil into soluble phosphorus, resulting in a high soluble phosphorus content in the soil. This effectively promotes crop growth, significantly reduces the use of chemical phosphate fertilizers, and improves agricultural soil. Therefore, this method has excellent application prospects. Thus, this method for converting insoluble phosphorus using *Enterobacter horneri* microorganisms not only effectively improves the utilization efficiency of phosphorus resources and reduces dependence on limited phosphate rock resources, but also has significant implications for promoting sustainable agricultural development.

[0042] (3) The application of the high-efficiency phosphate-solubilizing agent of the present invention, because the high-efficiency phosphate-solubilizing agent combines phosphate-solubilizing bacteria with bone char, can utilize the phosphate-solubilizing ability of the bacteria and provide a carrier for the growth and reproduction of the bacteria through the surface of bone char, thereby improving the efficiency of converting insoluble phosphorus in bone char into soluble phosphorus. This not only helps to improve the recovery and utilization rate of phosphorus resources, but also reduces environmental pollution from traditional chemical treatment methods, improves agricultural soil, and promotes crop growth when used as a bio-fertilizer. Therefore, it has excellent application prospects in all the above applications. Attached Figure Description

[0043] 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.

[0044] Figure 1 This is a scanning electron microscope image of Enterobacter HS-6 of the present invention.

[0045] Figure 2 This is a scanning electron microscope image of Enterobacter HS-6 of the present invention after reaction with bone char.

[0046] Figure 3 This is a graph showing the results of the phosphorus-solubilizing ability of Enterobacter HS-6 of the present invention under different amounts of bone char application.

[0047] Figure 4 This is a graph showing the results of dehydrogenase activity detection of Enterobacter holmieae HS-6 with different amounts of bone char applied according to the present invention. Detailed Implementation

[0048] 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.

[0049] 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.

[0050] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0051] The culture medium formulations described in the following examples are as follows:

[0052] LB medium (g / L): 10g peptone, 5g yeast extract, 10g NaCl, pH 7.0-7.2.

[0053] LB solid medium: Add 18g to 20g of agar powder to every 1L of LB medium.

[0054] NBRIP-P medium (g / L): glucose 10g, MgCl2·6H2O 5g, MgSO4·7H2O 0.25g, FeSO4·7H2O 0.01g, KCl 0.2g, (NH4)2SO4 0.1g, pH 7.0~7.4.

[0055] NBRIP-P solid medium: Add 18g to 20g of agar powder to every 1L of NBRIP-P medium.

[0056] Example 1

[0057] A highly efficient phosphate-solubilizing agent includes phosphate-solubilizing bacteria, bone char, and culture medium; the phosphate-solubilizing bacteria is *Enterobacter hormaechei* HS-6; wherein *Enterobacter hormaechei* HS-6 is classified as *Enterobacter hormaechei*, and 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 1 As shown.

[0058] The 16S rDNA gene sequence of Enterobacter holmieae HS-6 is shown in SEQ ID No. 1. The gene sequence length is 1476.

[0059] In this embodiment, the particle size of bone char is 30 nm to 2 μm.

[0060] The preparation method of this highly efficient phosphate-solubilizing agent includes the following steps:

[0061] S1. Preparation of bone char: Animal bones were freeze-dried at -60℃ for 48h, then thermally pyrolyzed at 450℃ for 120min. After cooling, large pieces of bone char were obtained. The large pieces of bone char were then ball-milled at 450rpm for 5h to obtain micron- and nano-level bone char.

[0062] In the ball mill, zirconium oxide particles are used to ball mill large bone char pieces, and ethanol is added as a grinding aid; the weight ratio of zirconium oxide particles to large bone char pieces is 18:1; the mass ratio of ethanol to large bone char pieces is 1.5 to 2:1; and the irradiation sterilization dose is 5.0 kGy.

[0063] S2. Activation of Enterobacter holmieae: Enterobacter holmieae HS-6 was inoculated into LB medium and then cultured in a constant temperature shaker at 28℃ and 180r / min for 16h to allow OD to be activated. 600 The concentration was 0.6–0.8, resulting in an active bacterial solution; wherein the effective viable count of the active bacterial solution was 1 × 10⁻⁶. 10 CFU / mL ~9×10 10 CFU / mL;

[0064] S3. Preparation of phosphate-solubilizing bacteria: The micron- and nano-level bone char obtained in step S1 is added to NBRIP-P medium to obtain a reaction medium. Then, the active bacterial solution is inoculated into the reaction medium and placed in a constant temperature shaker at 28°C with shaking at 180 r / min for 3 days to obtain the high-efficiency phosphate-solubilizing bacteria.

[0065] In this embodiment, the mass-to-volume ratio of bone char to NBRIP-P medium is 0.5%; wherein, the inoculation amount of active bacterial solution in the reaction medium is 1% of the volume percentage of the reaction medium.

[0066] Example 2

[0067] A highly efficient phosphate-solubilizing agent. The difference between this embodiment and Example 1 is that in this embodiment, the mass-to-volume ratio of bone char to NBRIP-P culture medium is 1%. All other conditions and methods in this embodiment are the same as in Example 1.

[0068] Example 3

[0069] A highly efficient phosphate-solubilizing agent. The difference between this embodiment and Example 1 is that in this embodiment, the mass-to-volume ratio of bone char to NBRIP-P culture medium is 2%. All other conditions and methods in this embodiment are the same as in Example 1.

[0070] Example 4

[0071] A highly efficient phosphate-solubilizing agent. The difference between this embodiment and Example 1 is that in this embodiment, the mass-to-volume ratio of bone char to NBRIP-P culture medium is 2.5%. All other conditions and methods in this embodiment are the same as in Example 1.

[0072] Example 5

[0073] A highly efficient phosphate-solubilizing agent. The difference between this embodiment and Example 1 is that in this embodiment, step S1, preparation of bone char: pig bones are freeze-dried at -65℃ for 40h, then thermally pyrolyzed at 440℃ for 140min, and after cooling, large pieces of bone char are obtained. Then, the large pieces of bone char are ball-milled at 440rpm for 6h to obtain micron-nano multi-level bone char.

[0074] In the ball mill, zirconium oxide particles are used to ball mill large bone char pieces, and ethanol is added as a grinding aid; the weight ratio of zirconium oxide particles to large bone char pieces is 15:1; the mass ratio of ethanol to large bone char pieces is 1.7:1; and the irradiation sterilization dose is 3.0 kGy.

[0075] In step S3, the active bacterial solution is inoculated into the reaction medium and then placed in a constant temperature shaker at 28°C with shaking at 180 r / min for 5 days to obtain a highly efficient phosphate-solubilizing bacterial agent.

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

[0077] Example 6

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

[0079] In the ball mill, zirconium oxide particles are used to ball mill large bone char pieces, and ethanol is added as a grinding aid; the weight ratio of zirconium oxide particles to large bone char pieces is 20:1; the mass ratio of ethanol to large bone char pieces is 2:1; and the irradiation sterilization dose is 6.0 kGy.

[0080] In step S3, the active bacterial solution is inoculated into the reaction medium and then placed in a constant temperature shaker at 28°C with shaking at 180 r / min for 7 days to obtain a highly efficient phosphate-solubilizing bacterial agent.

[0081] The remaining conditions and methods in this embodiment are the same as in Embodiment 1.

[0082] Example 7

[0083] The present invention discloses a method for converting insoluble phosphorus using *Enterobacter hookieri* microorganisms, which utilizes a highly efficient phosphate-solubilizing agent of the present invention to convert insoluble phosphorus, comprising the following steps:

[0084] S1. Conversion of insoluble phosphorus in bone char: In the high-efficiency phosphate-solubilizing bacterial agent, Enterobacter horneri HS-6 converts insoluble phosphorus in bone char into soluble phosphorus during the culture process; the culture process is carried out in a constant temperature shaker at 28℃ with shaking at 180r / min for 3d to 7d.

[0085] S2. Transformation of insoluble phosphorus in soil: When a highly efficient phosphorus-solubilizing agent is applied to the soil, Enterobacter holmieae HS-6 further converts the insoluble phosphorus in the soil into soluble phosphorus.

[0086] Example 8

[0087] The present invention discloses an application of a highly efficient phosphate-solubilizing agent. Specifically, the highly efficient phosphate-solubilizing agent prepared by the present invention can be used as a bio-fertilizer or for the preparation of bio-fertilizers. This highly efficient phosphate-solubilizing agent combines *Enterobacter holmierei* HS-6 with bone char, utilizing both the phosphate-solubilizing ability of *Enterobacter holmierei* HS-6 and the phosphorus resources of bone char. Therefore, it has excellent prospects for use as a bio-fertilizer or for the preparation of bio-fertilizers. Furthermore, the highly efficient phosphate-solubilizing agent prepared by the present invention can be applied to agricultural soil improvement or to promote crop growth. When applied to the soil, the *Enterobacter holmierei* HS-6 promotes the conversion and release of insoluble phosphorus in the soil, and the soluble phosphorus converted from bone char by the highly efficient phosphate-solubilizing agent can also greatly increase the soluble phosphorus content in the soil, thereby synergistically improving the available phosphorus content in the soil and promoting plant growth. Additionally, the highly efficient phosphate-solubilizing agent of the present invention can be applied to the recovery of phosphorus resources from bone char. By applying the highly efficient phosphate-solubilizing agent to bone char to convert insoluble phosphorus into soluble phosphorus, the recovery rate of phosphorus resources from bone char can be improved.

[0088] Experimental testing:

[0089] (I) Scanning electron microscopy detection

[0090] For a scanning electron microscope image of the *Enterobacter holmieae* strain HS-6 of this invention, please refer to [link to image]. Figure 1 .in, Figure 1 (b) is Figure 1 (a) A magnified view of the area within the dashed box. For example... Figure 1 As shown, Enterobacter holmieae HS-6 is a rod-shaped bacterium 1 μm long.

[0091] In addition, scanning electron microscopy was performed on *Enterobacter holmieae* HS-6 after reaction with bone char. Please refer to [link to relevant documentation]. Figure 2 ,in Figure 2 (b) is Figure 2 (a) Enlarged view of the area within the dashed box. (From...) Figure 2 As can be seen, Enterobacter holmieae HS-6 is adsorbed on the surface of bone char, and the morphology of Enterobacter holmieae HS-6 is intact and full, indicating that bone char provides favorable conditions for the growth and physiological state of Enterobacter holmieae HS-6.

[0092] (II) Detection of the phosphorus-solubilizing ability of Enterobacter HS-6 on different applied amounts of bone char

[0093] Sample Preparation: The micron- and nano-scale multi-level bone char prepared in Example 1 was added to NBRIP-P medium to obtain a reaction medium. Then, the active bacterial solution prepared in Example 1 was inoculated into the reaction medium to prepare samples. The inoculation amount of the active bacterial solution in the reaction medium was 1% of the reaction medium volume. The mass-to-volume ratio of bone char in the NBRIP-P medium was set to 0.5%, 1%, 2%, and 2.5%, respectively, resulting in four samples. Additionally, a blank control was prepared by inoculating 1% *Enterobacter holmieae* HS-6 (1% of the NBRIP-P medium volume) into the NBRIP-P medium without adding bone char. Three replicates were prepared for each sample.

[0094] The above samples were placed in a constant temperature shaker at 28℃ and shaken at 180 r / min for 168 h. Samples were taken every 24 h to detect the available phosphorus content. The results of the available phosphorus content detection can be found in [link to relevant documentation]. Figure 3 .

[0095] Depend on Figure 3 In the sample, "1%PSB6+0.5%BC" represents a sample with bone char comprising 0.5% of the NBRIP-P medium by mass volume, "1%PSB6+1%BC" represents a sample with bone char comprising 1% of the NBRIP-P medium by mass volume, "1%PSB6+2%BC" represents a sample with bone char comprising 2% of the NBRIP-P medium by mass volume, and "1%PSB6+2.5%BC" represents a sample with bone char comprising 2.5% of the NBRIP-P medium by mass volume.

[0096] Depend on Figure 3It is evident that after the reaction of Enterobacter horneri HS-6 with bone char, the phosphorus solubility of the "1% PSB6 + 0.5% BC" sample, the "1% PSB6 + 1% BC" sample, the "1% PSB6 + 2% BC" sample, and the "1% PSB6 + 2.5% BC" sample reached 5033.27 mg / L, 5872.64 mg / L, 4136.16 mg / L, and 2465.60 mg / L, respectively, after 72 hours. Although the "1% PSB6 + 1% BC" sample had the highest phosphorus solubility at 5872.64 mg / L, followed by the "1% PSB6 + 0.5% BC" sample, the "1% PSB6 + 0.5% BC" sample showed a higher phosphorus solubility over time compared to the other samples. The phosphorus dissolution rates of the "0.5% BC" sample, the "1% PSB6 + 1% BC" sample, the "1% PSB6 + 2% BC" sample, and the "1% PSB6 + 2.5% BC" sample after 168 h reached 4308.89 mg / L, 3283.78 mg / L, 1883.51 mg / L, and 1863.06 mg / L, respectively. It can be seen that the phosphorus dissolution rate of the "1% PSB6 + 0.5% BC" sample is still as high as 4308.89 mg / L after 168 h. This indicates that in the "1% PSB6 + 0.5% BC" sample, the 1% inoculum of Enterobacter horneri HS-6 and 0.5% bone char have a more complete interaction, which can more efficiently convert the insoluble phosphorus in bone char into soluble phosphorus and has excellent conversion stability, and can continuously and efficiently convert insoluble phosphorus into soluble phosphorus. Therefore, it is evident that the highly efficient phosphate-solubilizing bacterial agent of the present invention can efficiently and stably convert phosphorus in bone char into soluble phosphorus through Enterobacter horneri HS-6, exhibiting excellent phosphate-solubilizing ability and providing sufficient soluble phosphorus for crop growth. This highly efficient phosphate-solubilizing bacterial agent, or the bio-fertilizer made from the phosphate-solubilizing bacterial agent of the present invention, will be a new agricultural input for green and sustainable agricultural development.

[0097] (III) Detection of dehydrogenase activity of Enterobacter holmieae HS-6 in response to different amounts of bone char

[0098] Sample Preparation: The micron- and nano-scale multi-level bone char prepared in Example 1 was added to NBRIP-P medium to obtain a reaction medium. Then, the active bacterial solution prepared in Example 1 was inoculated into the reaction medium to prepare samples. The inoculation amount of the active bacterial solution in the reaction medium was 1% of the reaction medium volume. The mass-to-volume ratio of bone char in the NBRIP-P medium was set to 0.5%, 1%, 2%, and 2.5%, respectively, resulting in four samples. Additionally, a blank control was prepared by inoculating 1% *Enterobacter holmieae* HS-6 (1% of the NBRIP-P medium volume) into the NBRIP-P medium without adding bone char. Three replicates were prepared for each sample.

[0099] The above samples were incubated in a constant temperature shaker at 28℃ with shaking at 180 rpm for 168 h, and samples were taken every 24 h to detect dehydrogenase activity. For the results of the dehydrogenase activity detection, please refer to [link to relevant documentation]. Figure 4 .

[0100] Depend on Figure 4 In the sample, "1%PSB6+0.5%BC" represents a sample with bone char comprising 0.5% of the NBRIP-P medium by mass volume, "1%PSB6+1%BC" represents a sample with bone char comprising 1% of the NBRIP-P medium by mass volume, "1%PSB6+2%BC" represents a sample with bone char comprising 2% of the NBRIP-P medium by mass volume, and "1%PSB6+2.5%BC" represents a sample with bone char comprising 2.5% of the NBRIP-P medium by mass volume.

[0101] Depend on Figure 4 It is evident that the metabolic activity of *Enterobacter holmieae* HS-6 is dynamic. Initially, when a certain amount of insoluble phosphorus is present in the environment, the low available phosphorus content stimulates *Enterobacter holmieae* HS-6 to increase dehydrogenase activity, enhancing its metabolic capacity to secrete more phosphate-solubilizing substances to convert insoluble phosphorus into soluble phosphorus, thereby increasing the available phosphorus content. This increase in available phosphorus content provides more energy and material basis for phosphate-solubilizing bacteria, further promoting the growth of *Enterobacter holmieae* HS-6 and increasing dehydrogenase activity, forming a positive feedback loop. At 72 hours, the phosphate solubility reaches its peak. Excessively high available phosphorus content in the bacterial culture may alter intracellular osmotic pressure, affecting normal cellular physiological functions, and thus triggering negative feedback regulation, reducing the phosphate-solubilizing capacity and metabolic activity of *Enterobacter holmieae* HS-6, thereby decreasing dehydrogenase activity. At 168 hours, the "1% PSB6 + 0.5% BC" sample showed higher dehydrogenase activity than other samples. Therefore, considering... Figure 3 and Figure 4 The "1% PSB6 + 0.5% BC" sample has a superior phosphorus-solubilizing ability. That is, the highly efficient phosphorus-solubilizing agent prepared in Example 1 can better realize the efficient conversion of insoluble phosphorus in bone char into soluble phosphorus, better improve the utilization rate of phosphorus resources in bone char, and provide an efficient and stable phosphorus source for crop growth, thereby promoting crop growth.

[0102] 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.

[0103]

Claims

1. A highly efficient phosphate-solubilizing agent, characterized in that, It includes phosphate-solubilizing bacteria, bone char, and culture medium; the phosphate-solubilizing bacteria is Enterobacter holmieae HS-6; The classification name of the Enterobacter HS-6 is... Enterobacter hormaechei It was deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 19, 2023, with accession number CGMCC NO.28680.

2. The highly efficient phosphate-solubilizing agent as described in claim 1, characterized in that, The particle size of the bone char is 30 nm to 2 μm.

3. The highly efficient phosphate-solubilizing agent as described in claim 1, characterized in that, The preparation method of the highly efficient phosphate-solubilizing agent includes the following steps: S1. Preparation of bone char: Animal bones are freeze-dried, then thermally pyrolyzed and cooled, then ball-milled, and then irradiated and sterilized to obtain micron- and nano-level bone char. S2. Activation of Enterobacter holmieae: Enterobacter holmieae HS-6 was inoculated into LB medium and then cultured in a constant temperature shaker at 28℃ and 180r / min for 16h to allow OD to be activated. 600 The concentration was 0.6~0.8, resulting in an active bacterial solution; S3. Preparation of phosphate-solubilizing bacteria: The micron- and nano-level bone char obtained in step S1 is added to NBRIP-P medium to obtain a reaction medium. Then, the active bacterial solution is inoculated into the reaction medium and placed in a constant temperature shaker at 28°C with shaking at 180 r / min to obtain the high-efficiency phosphate-solubilizing bacteria.

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

1. The irradiation sterilization dose is 3.0 kGy to 6.0 kGy.

5. The highly efficient phosphate-solubilizing agent as described in claim 3, characterized in that, In step S2, the effective viable count of the prepared active bacterial solution is 1×10⁻⁶. 10 CFU / mL ~9×10 10 CFU / mL; In step S3, the mass-to-volume ratio of the bone char to the NBRIP-P culture medium is 0.5% to 2.5%. The inoculation amount of the active bacterial solution in the reaction medium is 1% of the volume of the reaction medium; In step S3, the active bacterial solution is inoculated into the reaction culture medium and then placed in a constant temperature shaker at 28°C with shaking at 180 r / min for 3 to 7 days to obtain the high-efficiency phosphate-solubilizing agent.

6. A method for converting insoluble phosphorus using *Enterobacter hooligans* microorganisms, characterized in that, The conversion of sparingly soluble phosphorus using a highly efficient phosphorus-solubilizing bacterial agent according to any one of claims 1 to 2 includes the following steps: S1. Conversion of insoluble phosphorus in bone char: In the highly efficient phosphate-solubilizing bacterial agent, Enterobacter HS-6 converts insoluble phosphorus in bone char into soluble phosphorus during the culture process; the culture process is carried out in a constant temperature shaker at 28℃ with shaking at 180r / min for 3 to 7 days. S2. Conversion of insoluble phosphorus in soil: The highly efficient phosphorus-solubilizing agent is applied to the soil, and Enterobacter holmieae HS-6 further converts the insoluble phosphorus in the soil into soluble phosphorus; The particle size of the bone char is 30 nm to 2 μm.

7. The application of the high-efficiency phosphate-solubilizing agent according to any one of claims 1 to 5 as a bio-fertilizer or in the preparation of bio-fertilizer.

8. The application of the highly efficient phosphate-solubilizing agent according to any one of claims 1 to 5 in agricultural soil improvement or crop growth promotion.

9. The application of the highly efficient phosphate-solubilizing agent according to any one of claims 1 to 5 in the recovery of bone char phosphorus resources; The particle size of the bone char is 30 nm to 2 μm.

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