Method for converting indissolvable phosphorus by using enterobacter hormaechei microorganisms, high-efficiency phosphate-solubilizing bacterial agent and application of high-efficiency phosphate-solubilizing bacterial agent

Abstract

The invention relates to the field of environment, resource utilization and agricultural biotechnology, in particular to a method for converting insoluble phosphorus by using enterobacter hormaechei microorganisms, a high-efficiency phosphate-solubilizing bacterial agent and application, and the high-efficiency phosphate-solubilizing bacterial agent comprises phosphate-solubilizing bacteria, bone black and a culture medium; the phosphate solubilizing bacteria are enterobacter hormaechei HS-6. By combining the enterobacter hormaechei HS-6 with the bone black, the phosphorus dissolving capacity of the enterobacter hormaechei HS-6 can be utilized, the phosphorus resource of the bone black can be fully utilized, the surface of the bone black can provide a growth and reproduction carrier for strains, and insoluble phosphorus can be converted into soluble phosphorus. The phosphate-solubilizing bacterial agent has good prospects in the application of biological bacterial fertilizer preparation, agricultural soil improvement, bone charcoal phosphorus resource recovery and crop growth promotion. The method for converting the insoluble phosphorus by using the enterobacter hormaechei microorganisms not only can effectively improve the utilization efficiency of phosphorus resources and reduce the dependence on limited phosphorite resources, but also has important significance for promoting agricultural sustainable development.

Description

Technical Field

[0001] The invention relates to the fields of environment, resource utilization and agricultural biotechnology, and in particular to a method for transforming insoluble phosphorus using microorganisms of the genus Enterobacter, a high-efficiency phosphorus-dissolving bacterial agent and an application thereof. Background Art

[0002] In the global agricultural and ecological fields, the rational utilization and sustainable development of phosphorus resources are facing unprecedented challenges. As one of the three essential nutrients for plant growth, phosphorus plays a key role in many physiological processes such as photosynthesis, energy metabolism, signal transduction, and genetic material synthesis. However, on the one hand, the global phosphate resources are limited and unevenly distributed. With the continuous expansion of agricultural production and the extensive demand for phosphorus in the industrial field, the mining speed of phosphate resources is accelerating and facing the risk of depletion. On the other hand, the current excessive dependence on and unreasonable use of phosphate fertilizers in agricultural production have led to a series of severe environmental problems. A large amount of phosphate fertilizers that are not absorbed and utilized by plants enter the water body with surface runoff, causing serious eutrophication of water bodies, resulting in the massive reproduction of algae, deterioration of water quality, and destruction of the balance of aquatic ecosystems. Therefore, it is urgent to find a sustainable way to utilize phosphorus resources.

[0003] Bone char, as a unique phosphorus-containing material, has a wide range of sources and is rich in phosphorus, which has attracted widespread attention from scientific researchers. Bone char is usually obtained by high-temperature pyrolysis and carbonization of animal bones. It is a biochar material with rich pore structure and 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 mostly exists in the form of insoluble phosphates, which makes it difficult for the phosphorus in it to be directly absorbed and utilized by plant roots. If the insoluble phosphorus in bone char can be fully converted into soluble phosphorus so that it can be used by plants, it can not only achieve efficient utilization of phosphorus resources, but also reduce dependence on traditional phosphorus fertilizers, thereby reducing agricultural production costs and environmental pressures.

[0004] Microorganisms play an important role in the material cycle of nature. Many microorganisms have unique phosphorus-dissolving abilities and are commonly referred to as phosphate-dissolving bacteria (PSB). PSB can secrete organic acids such as acetic acid, lactic acid, malic acid, oxalic acid, succinic acid, citric acid, and gluconic acid, thereby having the ability to dissolve phosphorus. The use of phosphate-dissolving bacteria to convert insoluble phosphorus into soluble phosphorus has received increasing attention in recent years. However, the development of phosphate-dissolving bacteria needs to be further expanded to enrich the resources of phosphate-dissolving bacteria and promote soil improvement and agricultural development. 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 highly efficient phosphate-dissolving bacteria agent. The highly efficient phosphate-dissolving bacteria agent expands the resources of phosphate-dissolving bacteria agents by combining bone char with Enterobacter hallii, and improves the efficiency of converting insoluble phosphorus in bone char into soluble phosphorus.

[0006] In order to overcome the shortcomings of the prior art, the second purpose of the present invention is to provide a method for converting insoluble phosphorus using microorganisms of the genus Enterobacter. This method for converting insoluble phosphorus using microorganisms of the genus Enterobacter 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 the sustainable development of agriculture.

[0007] The third object of the present invention is to provide an application of a highly efficient phosphate-dissolving bacteria agent.

[0008] In order to achieve the first object of the above invention, the technical solution adopted by the present invention is as follows:

[0009] The present invention provides a high-efficiency phosphate-dissolving bacteria agent, comprising phosphate-dissolving bacteria, bone charcoal and culture medium; the phosphate-dissolving bacteria is Enterobacter hallii HS-6;

[0010] 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.

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

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

[0013] Furthermore, the preparation method of the highly efficient phosphate-dissolving bacteria agent comprises the following steps:

[0014] S1. Preparation of bone char: freeze-drying animal bones, thermal cracking and cooling, ball milling, and irradiation sterilization to obtain micron-nano multi-grade bone char;

[0015] S2. Activation of Enterobacter holmesii: Enterobacter holmesii HS-6 was inoculated into LB medium and then placed in a constant temperature oscillator at 28°C and 180 rpm for 16 h to increase the OD 600 0.6-0.8, to obtain active bacterial solution;

[0016] S3, preparing a phosphate-dissolving agent: adding the micron-nano multi-grade bone char prepared in step S1 to the NBRIP-P culture medium to obtain a reaction culture medium, then inoculating the active bacterial liquid into the reaction culture medium, and then placing it in a constant temperature shaker at 28° C. and shaking and culturing at 180 r / min to obtain the highly efficient phosphate-dissolving agent.

[0017] Furthermore, in step S1, the animal bone is freeze-dried at -65°C to -55°C for 40h to 55h, and then thermally cracked at 440°C to 460°C for 100min to 140min, and then cooled to obtain a large piece of bone char, which is then ball-milled in a ball mill at 440rpm to 460rpm for 4h to 6h to obtain micron-nano multi-grade bone char; and / or

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

[0019] The radiation sterilization dosage is 3.0 kGy to 6.0 kGy.

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

[0021] In step S3, the mass 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 liquid in the reaction medium is 1% of the volume percentage of the reaction medium; and / or

[0023] In step S3, the active bacterial liquid is inoculated into a reaction culture medium, and then placed in a constant temperature shaker at 28° C. and shaken at 180 r / min for 3 to 7 days to obtain the highly efficient phosphate-dissolving bacterial agent.

[0024] The present invention can greatly promote the full utilization of bone char phosphorus resources by using bone char to prepare the high-efficiency phosphate-dissolving bacterial agent.

[0025] Among them, the highly efficient phosphate-dissolving bacteria agent can not only utilize the phosphate-dissolving ability of Enterobacter HS-6 but also make full use of the phosphorus resources of bone char by combining Enterobacter HS-6 with bone char. The surface of bone char can provide a carrier for the growth and reproduction of bacteria on the one hand, and can be converted into soluble phosphorus by Enterobacter HS-6 on the other hand. In addition, the culture medium is used to promote the growth and metabolism of Enterobacter HS-6.

[0026] In order to achieve the second purpose of the above invention, the technical solution adopted by the present invention is as follows:

[0027] The present invention provides a method for converting insoluble phosphorus using microorganisms of the genus Enterobacter. The method for converting insoluble phosphorus using the above-mentioned efficient phosphorus-dissolving bacterial agent comprises the following steps:

[0028] S1. Conversion of insoluble phosphorus in bone char: In the highly efficient phosphate-dissolving bacteria, Enterobacter holsteinii HS-6 converts insoluble phosphorus in bone char into soluble phosphorus during the culture process in the culture medium; the culture process is carried out in a constant temperature shaker at 28° C. and 180 r / min for 3 to 7 days;

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

[0030] The present invention discloses a method for converting insoluble phosphorus by using microorganisms of the genus Enterobacter. The method comprises the following steps: combining Enterobacter HS-6 with bone char in a culture medium so that Enterobacter HS-6 can efficiently convert insoluble phosphorus in the bone char into soluble phosphorus, greatly improving the utilization efficiency of phosphorus resources in the bone char, and obtaining a highly efficient phosphorus-dissolving bacterial agent. The highly efficient phosphorus-dissolving bacterial agent is then applied to soil, and Enterobacter HS-6 further converts the insoluble phosphorus in the soil into soluble phosphorus, thereby increasing the soluble phosphorus content in the soil, promoting the growth of crops, greatly reducing the use of chemical phosphorus fertilizers, and improving agricultural soil. Therefore, the method has a good application prospect.

[0031] In order to achieve the third purpose of the above invention, the technical solution adopted by the present invention is as follows:

[0032] The invention provides an application of a high-efficiency phosphate-dissolving bacteria agent as a biological fertilizer or in the preparation of a biological fertilizer.

[0033] Among them, this high-efficiency phosphate-dissolving bacteria agent combines Enterobacter hornii HS-6 with bone char, which can not only utilize the phosphate-dissolving ability of Enterobacter hornii HS-6, but also make full use of the phosphorus resources of bone char. Therefore, it has a good prospect for use as a biological fertilizer or for the preparation of biological fertilizer.

[0034] The invention provides an application of a high-efficiency phosphate-dissolving bacteria agent in improving agricultural soil or promoting the growth of crops.

[0035] Specifically, the highly efficient phosphate-dissolving bacteria agent is applied to the soil, and the action of Enterobacter hullii HS-6 is utilized to promote the transformation and release of insoluble phosphorus in the soil. The soluble phosphorus transformed from bone char by the highly efficient phosphate-dissolving bacteria agent can also greatly increase the soluble phosphorus content in the soil, thereby synergistically improving the effective phosphorus content in the soil and promoting plant growth.

[0036] The invention provides application of a high-efficiency phosphate-dissolving bacteria agent in the recovery of bone char phosphorus resources.

[0037] Specifically, the high-efficiency phosphate-dissolving bacteria agent is applied to convert the insoluble phosphorus in bone char into soluble phosphorus, which can improve the recovery rate of bone char phosphorus resources.

[0038] In addition, the high-efficiency phosphate-dissolving bacteria agent combines phosphate-dissolving bacteria with bone char, 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 bone char, thereby improving the efficiency of converting the insoluble phosphorus in the bone char into soluble phosphorus. This not only helps to improve the recycling rate of phosphorus resources, but also reduces the environmental pollution of traditional chemical treatment methods, improves agricultural soil, and can promote the growth of crops when used as a biological fertilizer. Therefore, it has a good application prospect in the above applications.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] (1) A highly efficient phosphate-dissolving bacteria agent of the present invention comprises phosphate-dissolving bacteria, bone charcoal and a culture medium; the phosphate-dissolving bacteria is Enterobacter hollandii HS-6. Not only does it expand the resources of phosphate-dissolving bacteria agents, but it can also greatly promote the full utilization of bone char phosphorus resources, achieve the dissolution of insoluble phosphorus in bone char, and greatly improve the efficiency of converting insoluble phosphorus in bone char into soluble phosphorus. By combining Enterobacter hollandii HS-6 with bone char, the highly efficient phosphate-dissolving bacteria agent can not only utilize the phosphate-dissolving ability of Enterobacter hollandii HS-6, but also fully utilize the phosphorus resources of bone char. On the one hand, the surface of bone char can provide a carrier for the growth and reproduction of the bacteria, and on the other hand, Enterobacter hollandii HS-6 can convert its insoluble phosphorus into soluble phosphorus. In addition, the culture medium is used to promote the growth and metabolism of Enterobacter hollandii HS-6.

[0041] (2) The present invention discloses a method for converting insoluble phosphorus using microorganisms of the genus Enterobacter. By combining Enterobacter HS-6 with bone char in a culture medium, Enterobacter HS-6 can efficiently convert insoluble phosphorus in bone char into soluble phosphorus, which can greatly improve the utilization efficiency of phosphorus resources in bone char, and obtain a highly efficient phosphate-dissolving bacterial agent, and then the highly efficient phosphate-dissolving bacterial agent is applied to the soil, and Enterobacter HS-6 further converts the insoluble phosphorus in the soil into soluble phosphorus, thereby increasing the soluble phosphorus content in the soil, which can well promote the growth of crops, greatly reduce the use of chemical phosphate fertilizers, and improve agricultural soil, so the method has a good application prospect. Therefore, the method for converting insoluble phosphorus using microorganisms of the genus Enterobacter can not only effectively improve the utilization efficiency of phosphorus resources and reduce the dependence on limited phosphate rock resources, but also has important significance for promoting the sustainable development of agriculture.

[0042] (3) Application of a highly efficient phosphate-dissolving bacteria agent of the present invention. Since the highly efficient phosphate-dissolving bacteria agent combines phosphate-dissolving bacteria with bone char, it 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 bone char, thereby improving the efficiency of converting the insoluble phosphorus in the bone char into soluble phosphorus. This not only helps to improve the recycling rate of phosphorus resources, but also reduces the environmental pollution of traditional chemical treatment methods, improves agricultural soil, and promotes the growth of crops when used as a biological fertilizer. Therefore, it has a good application prospect in the above applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] 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.

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

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

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

[0047] Figure 4 This is a graph showing the results of detecting the dehydrogenase activity of Enterobacter holmii HS-6 of the present invention to different amounts of bone charcoal applied. DETAILED DESCRIPTION

[0048] 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.

[0049] 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.

[0050] Unless otherwise specified, the 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): peptone 10 g, yeast extract 5 g, NaCl 10 g, pH 7.0-7.2.

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

[0054] NBRIP-P medium (g / L): glucose 10g, MgCl 2 6H 2 O 5g, MgSO 4 7H 2 O 0.25g, FeSO 4 7H 2 O 0.01g, KCl 0.2g, (NH 4 ) 2 SO 4 0.1g, pH 7.0~7.4.

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

[0056] Example 1

[0057] A highly efficient phosphate-dissolving agent, comprising phosphate-dissolving bacteria, bone charcoal 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 1 shown.

[0058] 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.

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

[0060] The preparation method of the high-efficiency phosphate-dissolving bacteria agent comprises the following steps:

[0061] S1. Preparation of bone char: freeze-dry the animal bones at -60°C for 48 hours, and then pyrolyze at 450°C for 120 minutes to obtain large pieces of bone char after cooling. Then, the large pieces of bone char were ball-milled at 450 rpm for 5 hours to obtain micron-nano multi-grade bone char.

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

[0063] S2. Activation of Enterobacter holmesii: Enterobacter holmesii HS-6 was inoculated into LB medium and then placed in a constant temperature oscillator at 28°C and 180 rpm for 16 h to increase the OD 600 is 0.6 to 0.8, and an active bacterial solution is obtained; wherein the effective number of live bacteria in the active bacterial solution is 1×10 10 CFU / mL~9×10 10 CFU / mL;

[0064] S3, preparing a phosphate-dissolving agent: adding the micron-nano multi-grade bone char prepared in step S1 to the NBRIP-P culture medium to obtain a reaction culture medium, then inoculating the active bacterial liquid into the reaction culture medium, and then placing it in a constant temperature shaker at 28° C. and shaking and culturing at 180 r / min for 3 days to obtain the highly efficient phosphate-dissolving agent.

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

[0066] Example 2

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

[0068] Example 3

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

[0070] Example 4

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

[0072] Example 5

[0073] A highly efficient phosphate-dissolving bacteria agent. The difference between this embodiment and embodiment 1 is that, in this embodiment, step S1, preparation of bone char: freeze-drying pig bones at -65°C for 40 hours, then thermally cracking at 440°C for 140 minutes, cooling to obtain large pieces of bone char, and then ball-milling the large pieces of bone char at 440 rpm for 6 hours to obtain micron-nano multi-grade bone char.

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

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

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

[0077] Example 6

[0078] A highly efficient phosphate-dissolving bacteria agent. The difference between this embodiment and embodiment 1 is that, in this embodiment, step S1, preparation of bone char: freeze-drying pig bones at -55°C for 55 hours, then thermally cracking at 460°C for 100 minutes, cooling to obtain large pieces of bone char, and then ball-milling the large pieces of bone char at 460 rpm for 4 hours to obtain micron-nano multi-grade bone char.

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

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

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

[0082] Example 7

[0083] The method of using microorganisms of the genus Enterobacter to transform insoluble phosphorus of the present invention comprises the following steps:

[0084] S1. Conversion of insoluble phosphorus in bone char: In the highly efficient phosphate-dissolving bacteria, Enterobacter holsteinii HS-6 converts insoluble phosphorus in bone char into soluble phosphorus during the culture process; the culture process is to culture in a constant temperature shaker at 28°C and 180r / min for 3 to 7 days;

[0085] S2. Conversion of insoluble phosphorus in soil: Apply high-efficiency phosphate-dissolving bacteria to the soil, and Enterobacter holmesii HS-6 further converts the insoluble phosphorus in the soil into soluble phosphorus.

[0086] Example 8

[0087] The application of a highly efficient phosphate-dissolving bacteria agent of the present invention, specifically, a highly efficient phosphate-dissolving bacteria agent prepared by the present invention can be used as a biological fertilizer or used to prepare biological fertilizer. The highly efficient phosphate-dissolving bacteria agent can not only utilize the phosphate-dissolving ability of Enterobacter HS-6, but also make full use of the phosphorus resources of bone char by combining Enterobacter HS-6 with bone char, so it has a good prospect to be used as a biological fertilizer or used to prepare biological fertilizer. In addition, the highly efficient phosphate-dissolving bacteria agent prepared by the present invention can be applied to agricultural soil improvement or to promote the growth of crops. The highly efficient phosphate-dissolving bacteria agent is applied to the soil, and the effect of Enterobacter HS-6 is used to promote the conversion and release of insoluble phosphorus in the soil, and the soluble phosphorus converted from bone char by the highly efficient phosphate-dissolving bacteria agent can also greatly increase the content of soluble phosphorus in the soil, thereby synergistically improving the effective phosphorus content in the soil and promoting plant growth. In addition, a highly efficient phosphate-dissolving bacteria agent of the present invention can be applied to the recovery of bone char phosphorus resources. The highly efficient phosphate-dissolving bacteria agent is applied to the conversion of insoluble phosphorus in bone char into soluble phosphorus, which can improve the recovery rate of bone char phosphorus resources.

[0088] Experimental testing:

[0089] (I) Scanning electron microscope detection

[0090] Among them, the scanning electron micrograph of the strain Enterobacter holmesii HS-6 of the present invention is shown in Figure 1 .in, Figure 1 (b) Yes Figure 1 (a) The enlarged view of the part in the dotted box. Figure 1 As shown, Enterobacter hallii HS-6 is a rod-shaped bacterium 1 μm in length.

[0091] In addition, the scanning electron microscopy of Enterobacter HS-6 after reacting with bone charcoal can be seen in Figure 2 ,in Figure 2 (b) Figure 2 (a) Enlarged view of the dotted box. Figure 2 It can be seen that Enterobacter holmii HS-6 is adsorbed on the surface of bone charcoal, and the morphology of Enterobacter holmii HS-6 is complete and plump, indicating that bone charcoal provides favorable conditions for the growth and physiological state of Enterobacter holmii HS-6.

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

[0093] Prepare samples: Add the micron-nano multi-grade bone char prepared in Example 1 to the NBRIP-P culture medium to obtain a reaction culture medium, and then inoculate the active bacterial liquid prepared in Example 1 into the reaction culture medium to prepare samples, wherein the inoculation amount of the active bacterial liquid in the reaction culture medium is 1% of the volume percentage of the reaction culture medium, and the mass volume ratio of bone char to the NBRIP-P culture medium is set to 0.5%, 1%, 2% and 2.5%, respectively, to prepare four samples. In addition, without adding bone charcoal, only 1% of Enterobacter holmesii HS-6 is inoculated in the NBRIP-P culture medium (the volume percentage of Enterobacter holmesii HS-6 in the NBRIP-P culture medium is 1%) as a blank control. Three parallel samples are set.

[0094] The above samples were placed in a constant temperature shaker at 28°C and cultured at 180r / min for 168h, and samples were taken every 24h for effective phosphorus content testing. Figure 3 .

[0095] Depend on Figure 3 In the table, “1%PSB6+0.5%BC” represents the sample in which the mass volume ratio of bone char to NBRIP-P culture medium is 0.5%, “1%PSB6+1%BC” represents the sample in which the mass volume ratio of bone char to NBRIP-P culture medium is 1%, “1%PSB6+2%BC” represents the sample in which the mass volume ratio of bone char to NBRIP-P culture medium is 2%, and “1%PSB6+2.5%BC” represents the sample in which the mass volume ratio of bone char to NBRIP-P culture medium is 2.5%.

[0096] Depend on Figure 3It can be seen that after the reaction of Enterobacter HS-6 with bone charcoal, the 72h solubilized phosphorus amounts of the "1% PSB6 + 0.5% BC" sample, "1% PSB6 + 1% BC" sample, "1% PSB6 + 2% BC" sample and "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. Among the four samples, the 72h solubilized phosphorus amount of the "1% PSB6 + 1% BC" sample was the highest, reaching 5872.64 mg / L, and the "1% PSB6 + 0.5% BC" sample was second only to it; but with the increase of time, the "1% PSB6 + 0.5% BC" sample showed a higher solubilized phosphorus amount than other samples, for example, the "1% PSB6 + The solubilized phosphorus amounts of the "0.5% BC" sample, "1% PSB6+1% BC" sample, "1% PSB6+2% BC" sample and "1% PSB6+2.5% BC" sample after 168 hours were 4308.89 mg / L, 3283.78 mg / L, 1883.51 mg / L and 1863.06 mg / L respectively. It can be seen that the solubilized phosphorus amount of the "1% PSB6+0.5% BC" sample after 168 hours was still as high as 4308.89 mg / L, which indicates that in the "1% PSB6+0.5% BC" sample, the 1% inoculation amount of Enterobacter HS-6 and 0.5% bone charcoal have a more sufficient effect, can more efficiently convert the insoluble phosphorus in bone charcoal into soluble phosphorus, and have excellent conversion stability, and can continuously and efficiently convert the insoluble phosphorus into soluble phosphorus. It can be seen that the efficient phosphate-dissolving bacteria agent of the present invention can efficiently and stably convert phosphorus in bone char into soluble phosphorus through Enterobacter holmii HS-6, has excellent phosphate-dissolving ability, and can provide sufficient soluble phosphorus for the growth of crops. The efficient phosphate-dissolving bacteria agent or the biological fertilizer prepared by the phosphate-dissolving bacteria agent of the present invention will be a new agricultural material for the sustainable development of green agriculture.

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

[0098] Prepare samples: Add the micron-nano multi-grade bone char prepared in Example 1 to the NBRIP-P culture medium to obtain a reaction culture medium, and then inoculate the active bacterial liquid prepared in Example 1 into the reaction culture medium to prepare samples, wherein the inoculation amount of the active bacterial liquid in the reaction culture medium is 1% of the volume percentage of the reaction culture medium, and the mass volume ratio of bone char to the NBRIP-P culture medium is set to 0.5%, 1%, 2% and 2.5%, respectively, to prepare four samples. In addition, without adding bone charcoal, only 1% of Enterobacter holmesii HS-6 is inoculated in the NBRIP-P culture medium (the volume percentage of Enterobacter holmesii HS-6 in the NBRIP-P culture medium is 1%) as a blank control. Three parallel samples are set.

[0099] The above samples were placed in a 28°C constant temperature shaker at 180 r / min for 168 h, and samples were taken every 24 h to test the dehydrogenase activity. Figure 4 .

[0100] Depend on Figure 4 In the table, “1%PSB6+0.5%BC” represents the sample in which the mass volume ratio of bone char to NBRIP-P culture medium is 0.5%, “1%PSB6+1%BC” represents the sample in which the mass volume ratio of bone char to NBRIP-P culture medium is 1%, “1%PSB6+2%BC” represents the sample in which the mass volume ratio of bone char to NBRIP-P culture medium is 2%, and “1%PSB6+2.5%BC” represents the sample in which the mass volume ratio of bone char to NBRIP-P culture medium is 2.5%.

[0101] Depend on Figure 4 It can be seen that the metabolic activity of Enterobacter holmesii HS-6 changes dynamically. At the beginning, when there is a certain amount of insoluble phosphorus source in the environment, the lower effective phosphorus content stimulates Enterobacter holmesii HS-6 to increase the dehydrogenase activity and enhance its metabolic capacity to secrete more phosphorus-soluble substances to convert insoluble phosphorus into soluble phosphorus, thereby increasing the effective phosphorus content. The increase in effective phosphorus content will provide more energy and material basis for phosphorus-soluble bacteria, further promoting the growth of Enterobacter holmesii HS-6 and the increase of dehydrogenase activity, forming a positive feedback loop. At 72h, the amount of phosphorus solubilization reaches its peak. The excessively high effective phosphorus content in the bacterial solution may change the osmotic pressure in the cells and affect the normal physiological function of the cells. It will also perform negative feedback regulation, reducing the phosphorus solubilization ability and metabolic activity of Enterobacter holmesii HS-6, thereby reducing the dehydrogenase activity. At 168h, the "1% PSB6+0.5% BC" sample showed higher dehydrogenase activity than other samples. Therefore, the comprehensive Figure 3 and Figure 4 The "1% PSB6+0.5% BC" sample has a better phosphorus-dissolving ability, that is, the high-efficiency phosphorus-dissolving bacteria agent prepared in Example 1 can better realize the efficient conversion of the 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 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.

[0103]

Claims

1. A highly efficient phosphate-dissolving bacteria agent, characterized in that: It comprises phosphate-dissolving bacteria, bone charcoal and culture medium; the phosphate-dissolving bacteria is Enterobacter holmesii 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 highly efficient phosphate-dissolving bacteria agent as claimed in claim 1, characterized in that: The 16SrDNA gene sequence of the Enterobacter holmesii HS-6 is shown in SEQ ID No.

1.

3. A highly efficient phosphate-dissolving bacteria agent as claimed in claim 1, characterized in that: The particle size of the bone char is 30nm-2μm.

4. A highly efficient phosphate-dissolving bacteria agent as claimed in claim 1, characterized in that: The preparation method of the high-efficiency phosphate-dissolving bacteria agent comprises the following steps: S1. Preparation of bone char: freeze-drying animal bones, thermal cracking and cooling, ball milling, and irradiation sterilization to obtain micron-nano multi-grade bone char; S2. Activation of Enterobacter holmesii: Enterobacter holmesii HS-6 was inoculated into LB medium and then placed in a constant temperature oscillator at 28°C and 180 rpm for 16 h to increase the OD 600 0.6-0.8, to obtain active bacterial solution; S3, preparing a phosphate-dissolving agent: adding the micron-nano multi-grade bone char prepared in step S1 to the NBRIP-P culture medium to obtain a reaction culture medium, then inoculating the active bacterial liquid into the reaction culture medium, and then placing it in a constant temperature shaker at 28° C. and shaking and culturing at 180 r / min to obtain the highly efficient phosphate-dissolving agent.

5. A highly efficient phosphate-dissolving bacteria agent as claimed in claim 4, characterized in that: In step S1, the animal bone is freeze-dried at -65°C to -55°C for 40h to 55h, and then thermally cracked at 440°C to 460°C for 100min to 140min, and then cooled to obtain a large piece of bone char, which is then ball-milled at 440rpm to 460rpm for 4h to 6h to obtain micron-nano multi-grade bone char; and / or In the ball mill, the large piece of bone char is ball-milled with zirconium oxide particles, and ethanol is added as a grinding aid; the weight ratio of the zirconium oxide particles to the large piece of bone char is (15-20):1; the mass ratio of the ethanol to the large piece of bone char is (1.5-2):1; The radiation sterilization dosage is 3.0 kGy to 6.0 kGy.

6. A highly efficient phosphate-dissolving bacteria agent as claimed in claim 4, characterized in that: In step S2, the effective viable bacteria count of the prepared active bacterial solution is 1×10 10 CFU / mL~9×10 10 CFU / mL; and / or In step S3, the mass volume ratio of the bone char to the NBRIP-P culture medium is 0.5% to 2.5%; and / or The inoculation amount of the active bacterial liquid in the reaction medium is 1% of the volume percentage of the reaction medium; and / or In step S3, the active bacterial liquid is inoculated into a reaction culture medium, and then placed in a constant temperature shaker at 28° C. and shaken at 180 r / min for 3 to 7 days to obtain the highly efficient phosphate-dissolving bacterial agent.

7. A method for converting insoluble phosphorus using microorganisms of the genus Enterobacter, characterized in that: The method of converting insoluble phosphorus using a highly efficient phosphorus-dissolving bacterial agent according to any one of claims 1 to 3 comprises the following steps: S1. Conversion of insoluble phosphorus in bone char: In the highly efficient phosphate-dissolving bacteria, Enterobacter holsteinii HS-6 converts insoluble phosphorus in bone char into soluble phosphorus during the culture process in the culture medium; the culture process is carried out in a constant temperature shaker at 28° C. and 180 r / min for 3 to 7 days; S2. Conversion of insoluble phosphorus in soil: The highly efficient phosphate-dissolving bacteria agent is applied to the soil, and Enterobacter holmii HS-6 further converts the insoluble phosphorus in the soil into soluble phosphorus.

8. Use of a high-efficiency phosphate-dissolving bacterial agent as claimed in any one of claims 1 to 6 as a biological fertilizer or in the preparation of a biological fertilizer.

9. Use of a high-efficiency phosphate-dissolving bacteria agent as described in any one of claims 1 to 6 in improving agricultural soil or promoting crop growth.

10. Use of a highly efficient phosphate-dissolving bacteria agent as claimed in any one of claims 1 to 6 in the recovery of bone char phosphorus resources.

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