Phosphorus-solubilizing bacterial agent with high available phosphorus conversion amount, preparation method of phosphorus-solubilizing bacterial agent, biological bacterial fertilizer and application

Abstract

The invention relates to the field of environment, resource utilization and agricultural biotechnology, in particular to a phosphate-solubilizing bacterial agent with high available phosphorus conversion amount and a preparation method thereof, a biological bacterial fertilizer and application, the phosphate-solubilizing bacterial agent with high available phosphorus conversion amount comprises phosphate-solubilizing bacteria, bone black and a culture medium, and the phosphate-solubilizing bacteria are pantoea agglomerans CT-3. The bone black is used for preparing the phosphate-solubilizing bacterial agent with high available phosphorus conversion amount, so that the full utilization of bone black phosphorus resources can be greatly promoted, and the efficient dissolution of insoluble phosphorus in the bone black is realized. According to the phosphate-solubilizing bacterial agent with the high available phosphorus conversion amount, the pantoea agglomerans CT-3 is combined with the bone black, so that the phosphate-solubilizing capacity of the pantoea agglomerans CT-3 can be utilized, and the phosphorus resource of the bone black can be fully utilized. The phosphate-solubilizing bacterial agent with the high available phosphorus conversion amount has good prospects in application to preparation of biological bacterial fertilizer, application to agricultural soil improvement, application to bone black phosphorus resource recovery and application to promotion of crop growth.

Description

Technical Field

[0001] The invention relates to the fields of environment, resource utilization and agricultural biotechnology, and in particular to a phosphate-dissolving bacteria agent with high effective phosphorus conversion rate, a preparation method thereof, a biological bacterial fertilizer and applications thereof. Background Art

[0002] Phosphorus accounts for about 0.2% to 10.8% of plant dry weight and is an important essential macronutrient for crops and global food security. However, less than 0.1% of inorganic and organic phosphorus in the soil is available for plant absorption, which cannot meet the needs of plants or microorganisms. Due to the lack of phosphorus, it is estimated that 5.7 billion hm2 2 Crop productivity in agricultural soils is limited. It is reported that at least 70% of phosphorus fertilizer enters the soil and is fixed in the soil, making it difficult for crops to absorb and use it.

[0003] Bone char is a material rich in phosphorus. It has a wide range of sources and is often made from waste bones of livestock and poultry through oxygen-limited thermal cracking and carbonization. Compared with biochar prepared from other raw materials, bone char has a higher phosphorus content, but it mainly exists in the form of insoluble phosphates such as calcium phosphates. The most common ones are hydroxyapatite (Ca 5 (PO 4 ) 3 OH), tricalcium phosphate (Ca 3 (PO 4 ) 2 ), etc., which are difficult to be effectively absorbed by plants. 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 pressure.

[0004] Microorganisms play an important role in maintaining and regulating phosphorus traits through inorganic phosphorus dissolution and organic phosphorus mineralization, thereby improving the phosphorus availability of plants. This has important practical significance and application value for solving the problem of phosphorus resource utilization and promoting sustainable agricultural development.

[0005] 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 still needs to be strengthened and expanded to enrich the resources of phosphate-dissolving microbial agents and promote soil improvement and agricultural development. Summary of the invention

[0006] In order to overcome the shortcomings of the prior art, the first object of the present invention is to provide a phosphate-dissolving bacteria agent with a high effective phosphorus conversion rate. The phosphate-dissolving bacteria agent with a high effective phosphorus conversion rate expands the resources of phosphate-dissolving bacteria agents by combining bone char with phosphate-dissolving bacteria, and improves the efficiency of converting insoluble phosphorus in bone char into soluble phosphorus.

[0007] 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 with a high effective phosphorus conversion rate. The preparation method can realize the efficient conversion of insoluble phosphorus in bone char into soluble phosphorus. The prepared phosphate-dissolving bacteria agent expands the resources of phosphate-dissolving bacteria agents and improves the efficiency of converting insoluble phosphorus in bone char into soluble phosphorus.

[0008] The third object of the present invention is to provide a biological fertilizer.

[0009] The fourth object of the present invention is to provide an application of a phosphate-dissolving bacteria agent with a high effective phosphorus conversion rate.

[0010] In order to achieve the first object of the above invention, the technical solution adopted by the present invention is as follows:

[0011] The present invention provides a phosphate-dissolving bacteria agent with high effective phosphorus conversion rate, comprising phosphate-dissolving bacteria, bone charcoal and culture medium; the phosphate-dissolving bacteria is Pantoea agglomerans CT-3, which belongs to bacteria;

[0012] The classification name of the Pantoea agglomerans CT-3 is Pantoea agglomerans, which was deposited in the General Microbiological Center of the China Culture Collection Administration on January 8, 2025, with the deposit number CGMCCNO.: 33335.

[0013] Furthermore, the 16S rDNA gene sequence of Pantoea agglomerans CT-3 is shown in SEQ ID No.1.

[0014] Furthermore, the particle size of the bone char is 30nm-2μm. Among them, Pantoea agglomerans CT-3 has a good effect of converting insoluble phosphorus into soluble phosphorus for bone char with a particle size of 30nm-2μm.

[0015] By using bone char to prepare the phosphate-dissolving bacteria agent with high effective phosphorus conversion rate, the full utilization of bone char phosphorus resources can be greatly promoted.

[0016] Among them, the phosphate-dissolving bacteria agent with high effective phosphorus conversion rate can not only utilize the phosphorus-dissolving ability of Pantoea agglomerans CT-3, but also make full use of the phosphorus resources of bone charcoal by combining Pantoea agglomerans CT-3 with bone charcoal. The surface of bone charcoal can also provide a carrier for the growth and reproduction of bacteria. In addition, the culture medium is used to promote the growth and metabolism of Pantoea agglomerans CT-3.

[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 with a high effective phosphorus conversion rate, comprising the following steps:

[0019] S1. Preparation of bone char: freeze-dry the animal bones, perform thermal cracking and cooling, and then perform ball milling to obtain micron-nano multi-grade bone char; the animal bones can be selected from pig bones, cow bones or sheep bones.

[0020] S2. Activation of Pantoea agglomerans: Pantoea agglomerans CT-3 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;

[0021] S3, preparing a phosphate-dissolving bacteria agent: sterilize the micron-nano multi-grade bone char prepared in step S1 by irradiation and add it to NBRIP-P culture medium to obtain a reaction culture medium, then inoculate the active bacterial liquid into the reaction culture medium, and then place it in a constant temperature shaker at 28°C and shake and culture at 180 r / min for 3 days to obtain the phosphate-dissolving bacteria agent with a high effective phosphorus conversion rate.

[0022] Further, 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 after cooling, a large piece of bone char is obtained, and then the large piece of bone char is ball-milled at 440rpm to 460rpm for 4h to 6h to obtain micron-nano multi-grade bone char; and / or

[0023] 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 the zirconium oxide particles to the bulk bone char is (15-20):1; the mass ratio of the ethanol to the bulk bone char is (1.5-2):1.

[0024] 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

[0025] 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

[0026] The radiation sterilization dose is 3.0 kGy to 6.0 kGy;

[0027] The inoculation amount of the active bacterial liquid in the reaction culture medium is 1% of the volume percentage of the reaction culture medium.

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

[0029] The invention provides a biological bacterial fertilizer, which is prepared by utilizing the above-mentioned phosphate-dissolving bacterial agent with high effective phosphorus conversion rate.

[0030] Among them, the phosphate-dissolving bacteria agent with high effective phosphorus conversion rate combines Pantoea agglomerans CT-3 with bone char, which can not only utilize the phosphate-dissolving ability of Pantoea agglomerans CT-3, but also make full use of the phosphorus resources of bone char. Therefore, it has a good prospect for application in the preparation of biological fertilizer.

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

[0032] The present invention provides application of the above-mentioned phosphate-dissolving bacteria agent with high effective phosphorus conversion rate in agricultural soil improvement.

[0033] Specifically, the phosphate-dissolving bacteria agent with a high effective phosphorus conversion rate is applied to the soil, and the action of Pantoea agglomerans CT-3 is utilized to promote the conversion and release of insoluble phosphorus in the soil, thereby improving the effective phosphorus content in the soil and promoting plant growth.

[0034] The present invention provides application of the above-mentioned phosphate-dissolving bacteria agent with high effective phosphorus conversion rate in bone char phosphorus resource recovery.

[0035] Specifically, the phosphate-dissolving bacteria agent with a high effective phosphorus conversion rate is applied to convert the insoluble phosphorus in bone char into soluble phosphorus, which can improve the recovery rate of bone char phosphorus resources.

[0036] The present invention provides the use of the above-mentioned phosphate-dissolving bacteria agent with high effective phosphorus conversion rate in promoting the growth of crops.

[0037] Among them, the phosphate-dissolving bacteria agent with high effective phosphorus conversion rate combines phosphate-dissolving bacteria with bone char, which can not only utilize the phosphorus-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, and can also improve agricultural soil. It can also promote the growth of crops when used as a biological fertilizer. Therefore, it has a good application prospect in the above applications.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] (1) A phosphate-dissolving bacteria agent with a high effective phosphorus conversion rate of the present invention comprises phosphate-dissolving bacteria, bone charcoal and a culture medium; the phosphate-dissolving bacteria is Pantoea agglomerans CT-3. By combining bone charcoal with phosphate-dissolving bacteria to prepare the phosphate-dissolving bacteria agent with a high effective phosphorus conversion rate, not only the phosphate-dissolving bacteria agent resources are expanded, but also the full utilization of bone char phosphorus resources can be greatly promoted, the dissolution of insoluble phosphorus in bone charcoal can be achieved, and the efficiency of converting insoluble phosphorus in bone charcoal into soluble phosphorus is greatly improved. The phosphate-dissolving bacteria agent with a high effective phosphorus conversion rate combines Pantoea agglomerans CT-3 with bone charcoal, which can not only utilize the phosphate-dissolving ability of Pantoea agglomerans CT-3, but also fully utilize the phosphorus and calcium resources of bone charcoal, and the surface of bone charcoal can also provide a carrier for the growth and reproduction of the bacteria. Therefore, the phosphate-dissolving bacteria agent with a high effective phosphorus conversion rate not only expands the phosphate-dissolving bacteria agent resources, but also improves the utilization efficiency of bone char phosphorus resources, reduces the waste of bone char phosphorus resources during use, and can further promote plant growth.

[0040] (2) The method for preparing a phosphate-dissolving agent with a high effective phosphorus conversion rate of the present invention realizes resource recycling. Since bone char is usually a product of waste such as animal bones, the use of Pantoea agglomerans CT-3 to convert phosphorus in bone char is a secondary utilization of waste resources, which helps to realize the recycling of resources, reduce dependence on primary phosphate resources, and meet the requirements of sustainable development. The method has the characteristics of simple preparation method and easy operation, and the prepared phosphate-dissolving agent expands the resources of phosphate-dissolving agents, improves the utilization efficiency of bone char phosphorus resources, and has good application effect and environmental friendliness.

[0041] (3) The present invention provides a method for preparing a phosphate-dissolving bacteria agent with a high effective phosphorus conversion rate. Since Pantoea agglomerans CT-3 has a strong phosphorus-dissolving ability and bone charcoal is rich in insoluble phosphorus, after the reaction of Pantoea agglomerans CT-3 with bone charcoal, a large amount of insoluble phosphorus in the bone charcoal can be converted into soluble phosphorus in a short period of time, and the soluble phosphorus conversion efficiency is high. The soluble phosphorus amount in 72 hours reaches 4907.65 mg / L, and the effect of converting insoluble phosphorus into soluble phosphorus is stable. The soluble phosphorus amount in 168 hours can also reach 2702.02 mg / L, which improves the effectiveness and durability of phosphorus, and further has the advantage of a high effective phosphorus conversion rate. Therefore, the prepared phosphate-dissolving bacteria agent has a good application prospect.

[0042] (4) The biological fertilizer of the present invention is prepared by using the above-mentioned phosphate-dissolving bacteria agent with high effective phosphorus conversion rate, so it can promote the conversion of insoluble phosphorus into soluble phosphorus and has a good application effect. Among them, the phosphate-dissolving bacteria agent with high effective phosphorus conversion rate combines Pantoea agglomerans CT-3 with bone char, which can not only utilize the phosphate-dissolving ability of Pantoea agglomerans CT-3, but also fully utilize the phosphorus resources and calcium resources of bone char, so it has a good prospect for application in the preparation of biological fertilizer.

[0043] (4) Application of a phosphate-dissolving bacteria agent with high effective phosphorus conversion rate of the present invention, because the phosphate-dissolving bacteria agent with high effective phosphorus conversion rate combines phosphate-dissolving bacteria with bone char, can not only utilize the phosphorus-dissolving ability of the strain, but also provide a carrier for growth and reproduction for the strain through the surface of 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, and can also improve agricultural soil. It can also promote the growth of crops when used as a biological fertilizer. Therefore, it has a good application prospect in the application of agricultural soil improvement, the application of bone char phosphorus resource recovery and the application of promoting the growth of crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] 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.

[0045] Figure 1 It is a scanning electron microscope image of Pantoea agglomerans CT-3 of the present invention.

[0046] Figure 2 This is a scanning electron microscope image of the Pantoea agglomerans CT-3 of the present invention after the reaction with bone charcoal.

[0047] Figure 3 This is a graph showing the results of testing the phosphorus solubilizing ability of Pantoea agglomerata CT-3 of the present invention with different amounts of bone charcoal applied.

[0048] Figure 4 This is a graph showing the results of detecting the dehydrogenase activity of Pantoea agglomerata CT-3 of the present invention to different amounts of bone charcoal applied. 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] LB medium (g / L): peptone 10 g, yeast extract 5 g, NaCl 10 g, pH 7.0-7.2.

[0054] LB solid culture medium: Add 18g to 20g of agar powder per 1L of LB culture medium.

[0055] 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.

[0056] Solid culture medium of NBRIP-P: Add 18g to 20g of agar powder to every 1L of NBRIP-P culture medium.

[0057] Example 1

[0058] A phosphate-dissolving bacteria agent with high effective phosphorus conversion, comprising phosphate-dissolving bacteria, bone charcoal and culture medium; the phosphate-dissolving bacteria is Pantoea agglomerans CT-3; wherein the classification name of Pantoea agglomerans CT-3 is Pantoea agglomerans, which has been deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on January 8, 2025, with a deposit number of CGMCC NO.: 33335, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The scanning electron microscope image of Pantoea agglomerans CT-3 is as follows: Figure 1 shown.

[0059] The 16S rDNA gene sequence of Pantoea agglomerans CT-3 is shown in SEQ ID No. 1. The length of the gene sequence is 1411.

[0060] In this embodiment, the particle size of bone char is 30 nm to 2 μm.

[0061] The preparation method of the phosphate-dissolving bacteria agent with high effective phosphorus conversion rate comprises the following steps:

[0062] S1. Preparation of bone char: freeze-dry the pig bones at -60°C for 48 hours, and then pyrolyze them 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.

[0063] 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 18:1; the mass ratio of the ethanol to the large piece of bone char is 1.5:1;

[0064] S2. Activation of Pantoea agglomerans: Pantoea agglomerans CT-3 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;

[0065] S3, preparing a phosphate-dissolving bacteria agent: sterilize the micron-nano multi-grade bone char prepared in step S1 by irradiation and add it to NBRIP-P culture medium to obtain a reaction culture medium, then inoculate the active bacterial liquid into the reaction culture medium, and then place it in a constant temperature shaker at 28°C and shake and culture at 180 r / min for 3 days to obtain the phosphate-dissolving bacteria agent with a high effective phosphorus conversion rate.

[0066] In this embodiment, the radiation sterilization dose is 5.0 kGy, the mass volume ratio of bone char to NBRIP-P culture medium is 0.5%, and the inoculation amount of active bacterial liquid in the reaction culture medium is 1% of the volume percentage of the reaction culture medium.

[0067] Example 2

[0068] A phosphate-dissolving bacteria agent with high effective phosphorus conversion rate, 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 other conditions and methods of this embodiment are the same as those of embodiment 1.

[0069] Example 3

[0070] A phosphate-dissolving bacteria agent with high effective phosphorus conversion rate, 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 other conditions and methods of this embodiment are the same as those of embodiment 1.

[0071] Example 4

[0072] A phosphate-dissolving bacteria agent with high effective phosphorus conversion rate, 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 other conditions and methods of this embodiment are the same as those of embodiment 1.

[0073] Example 5

[0074] A phosphate-dissolving bacteria agent with high effective phosphorus conversion rate. 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 with a ball mill at 440 rpm for 6 hours to obtain micron-nano multi-grade bone char.

[0075] 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 the zirconium oxide particles to the bulk bone char is 15:1; the mass ratio of the ethanol to the bulk bone char is 1.7:1; and the dose of irradiation sterilization is 3.0 kGy.

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

[0077] Example 6

[0078] A phosphate-dissolving bacteria agent with high effective phosphorus conversion rate. 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 with a ball mill at 460rpm 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 large pieces of bone char, and ethanol is added as a grinding aid; the weight ratio of the zirconium oxide particles to the large pieces of bone char is 20:1; the mass ratio of the ethanol to the large pieces of bone char is 2:1; and the dose of irradiation sterilization is 6.0 kGy.

[0080] The remaining conditions and methods of this embodiment are the same as those of embodiment 1.

[0081] Example 7

[0082] A biological fertilizer is prepared by using a phosphate-dissolving bacteria agent with high effective phosphorus conversion rate obtained by any one of Examples 1 to 6.

[0083] Example 8

[0084] The application of a phosphate-dissolving bacteria agent with a high effective phosphorus conversion rate of the present invention, specifically, the phosphate-dissolving bacteria agent with a high effective phosphorus conversion rate prepared by the present invention can be applied to agricultural soil improvement, the phosphate-dissolving bacteria agent with a high effective phosphorus conversion rate is applied to the soil, and the action of Pantoea agglomerans CT-3 is utilized to promote the conversion and release of insoluble phosphorus in the soil, thereby improving the effective phosphorus content in the soil and promoting plant growth. In addition, the phosphate-dissolving bacteria agent with a high effective phosphorus conversion rate prepared by the present invention can be applied to bone char phosphorus resource recovery, the phosphate-dissolving bacteria agent with a high effective phosphorus conversion rate is applied to convert insoluble phosphorus in bone char into soluble phosphorus, and the recovery rate of bone char phosphorus resources can be improved. In addition, the phosphate-dissolving bacteria agent with a high effective phosphorus conversion rate prepared by the present invention can be applied to promote the growth of crops, the phosphate-dissolving bacteria agent with a high effective phosphorus conversion rate is applied to the soil where crops grow, and sufficient soluble phosphorus and soluble calcium can be provided to the crops, thereby promoting the growth of crops well.

[0085] Experimental testing:

[0086] (I) Scanning electron microscope detection

[0087] Among them, the scanning electron micrograph of the strain Pantoea agglomerata CT-3 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, Pantoea agglomerans CT-3 is a rod-shaped bacterium 1 μm in length.

[0088] In addition, Pantoea agglomerans CT-3 was subjected to scanning electron microscopy after reacting with bone charcoal. Figure 2 ,in Figure 2 (b) Figure 2 (a) Enlarged view of the dotted box. Figure 2 It can be seen that Pantoea agglomerans CT-3 is adsorbed on the surface of bone charcoal, and the morphology of Pantoea agglomerans CT-3 is complete and plump, indicating that bone charcoal provides favorable conditions for the growth and physiological state of Pantoea agglomerans CT-3.

[0089] (II) Detection of the phosphate-dissolving capacity of Pantoea agglomerata CT-3 with different amounts of bone char

[0090] 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 Pantoea agglomerans CT-3 is inoculated in the NBRIP-P culture medium (the volume percentage of Pantoea agglomerans CT-3 in the NBRIP-P culture medium is 1%) as a blank control sample. Three parallel samples are set.

[0091] 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 to test the effective phosphorus content. Figure 3 .

[0092] Depend on Figure 3 In the table, “1% PSB3+0.5% BC” represents the sample in which the mass volume ratio of bone char to NBRIP-P culture medium is 0.5%, “1% PSB3+1% BC” represents the sample in which the mass volume ratio of bone char to NBRIP-P culture medium is 1%, “1% PSB3+2% BC” represents the sample in which the mass volume ratio of bone char to NBRIP-P culture medium is 2%, and “1% PSB3+2.5% BC” represents the sample in which the mass volume ratio of bone char to NBRIP-P culture medium is 2.5%.

[0093] Depend on Figure 3It can be seen that after the reaction of Pantoea agglomerata CT-3 with bone charcoal, the phosphorus solubility of the "1% PSB3 + 2% BC" sample reached a peak of 1071.35 mg / L at 48 hours, the phosphorus solubility of the "1% PSB3 + 2.5% BC" sample was 1898.41 mg / L at 72 hours, the phosphorus solubility of the "1% PSB3 + 1% BC" sample reached 4907.65 mg / L at 72 hours, and the phosphorus solubility of the "1% PSB3 + 0.5% BC" sample was 3456.80 mg / L at 72 hours. , as time goes by, the "1% PSB3+0.5% BC" sample shows a higher phosphorus solubility than other samples, and the phosphorus solubility at 168h is still as high as 2702.02mg / L, which shows that in the "1% PSB3+0.5% BC" sample, the 1% inoculation amount of Pantoea agglomerans CT-3 and 0.5% bone char have a more sufficient effect, can more efficiently convert the insoluble phosphorus in bone char into soluble phosphorus, and has excellent conversion stability, and can continuously and efficiently convert the insoluble phosphorus into soluble phosphorus. It can be seen that the phosphate-dissolving bacteria agent with a high effective phosphorus conversion amount of the present invention can efficiently and stably convert the phosphorus in bone char into soluble phosphorus through Pantoea agglomerans CT-3, has excellent phosphorus solubilizing ability, and can provide sufficient soluble phosphorus for the growth of crops. The phosphate-dissolving bacteria agent with a high effective phosphorus conversion amount or the biological fertilizer made from the phosphate-dissolving bacteria agent of the present invention will be a new agricultural material for the sustainable development of green agriculture.

[0094] (III) Detection of dehydrogenase activity of Pantoea agglomerata CT-3 in response to different amounts of bone char

[0095] 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 Pantoea agglomerans CT-3 is inoculated in the NBRIP-P culture medium (the volume percentage of Pantoea agglomerans CT-3 in the NBRIP-P culture medium is 1%) as a blank control sample. Three parallel samples are set.

[0096] 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 .

[0097] Depend on Figure 4In the table, “1% PSB3+0.5% BC” represents the sample in which the mass volume ratio of bone char to NBRIP-P culture medium is 0.5%, “1% PSB3+1% BC” represents the sample in which the mass volume ratio of bone char to NBRIP-P culture medium is 1%, “1% PSB3+2% BC” represents the sample in which the mass volume ratio of bone char to NBRIP-P culture medium is 2%, and “1% PSB3+2.5% BC” represents the sample in which the mass volume ratio of bone char to NBRIP-P culture medium is 2.5%.

[0098] Depend on Figure 4 It can be seen that the metabolic activity of Pantoea agglomerans CT-3 changes dynamically. At the beginning, when there is a certain amount of insoluble phosphorus source in the environment, the lower effective phosphorus content stimulates Pantoea agglomerans CT-3 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 Pantoea agglomerans CT-3 and the increase of dehydrogenase activity, forming a positive feedback loop. At 72 hours, 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 functions of the cells. It will also perform negative feedback regulation to reduce the phosphorus-solubilizing ability and metabolic activity of Pantoea agglomerans CT-3, thereby reducing the dehydrogenase activity. Therefore, comprehensive Figure 3 and Figure 4 The "1% PSB3+0.5% BC" sample has a better phosphorus-dissolving ability, that is, the phosphorus-dissolving bacteria agent with a high effective phosphorus conversion rate 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.

[0099] 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.

[0100]

Claims

1. A phosphate-dissolving bacterial agent with high effective phosphorus conversion rate, characterized in that: It comprises phosphate-dissolving bacteria, bone charcoal and culture medium; the phosphate-dissolving bacteria is Pantoea agglomerans CT-3; The classification name of the Pantoea agglomerans CT-3 is Pantoea agglomerans, which was deposited in the General Microbiological Center of the China Culture Collection Administration on January 8, 2025, with the deposit number CGMCCNO.: 33335.

2. A phosphate-dissolving bacteria agent with high effective phosphorus conversion rate as claimed in claim 1, characterized in that: The 16S rDNA gene sequence of the Pantoea agglomerans CT-3 is shown in SEQ ID No.

1.

3. A phosphate-dissolving bacteria agent with high effective phosphorus conversion rate as claimed in claim 1, characterized in that: The particle size of the bone char is 30nm-2μm.

4. A method for preparing a phosphate-dissolving bacteria agent with high effective phosphorus conversion rate according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1. Preparation of bone char: freeze-drying animal bones, thermal cracking and cooling, and then ball milling to obtain micron-nano multi-grade bone char; S2. Activation of Pantoea agglomerans: Pantoea agglomerans CT-3 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 0.6-0.8, and an active bacterial solution is obtained; S3, preparing a phosphate-dissolving bacteria agent: sterilize the micron-nano multi-grade bone char prepared in step S1 by irradiation and add it to NBRIP-P culture medium to obtain a reaction culture medium, then inoculate the active bacterial liquid into the reaction culture medium, and then place it in a constant temperature shaker at 28°C and shake and culture at 180 r / min for 3 days to obtain the phosphate-dissolving bacteria agent with a high effective phosphorus conversion rate.

5. The method for preparing a phosphate-dissolving bacteria agent with high effective phosphorus conversion rate 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, zirconium oxide particles are used to ball-mill the bulk bone char, and ethanol is added as a grinding aid; the weight ratio of the zirconium oxide particles to the bulk bone char is (15-20):1; the mass ratio of the ethanol to the bulk bone char is (1.5-2):

1.

6. The method for preparing a phosphate-dissolving bacteria agent with high effective phosphorus conversion rate 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 radiation sterilization dose is 3.0 kGy to 6.0 kGy; The inoculation amount of the active bacterial liquid in the reaction culture medium is 1% of the volume percentage of the reaction culture medium.

7. A biological fertilizer, characterized in that: The invention is prepared by using a phosphate-dissolving bacteria agent with high effective phosphorus conversion rate as described in any one of claims 1 to 3.

8. Use of a phosphate-dissolving bacteria agent with a high effective phosphorus conversion rate as described in any one of claims 1 to 3 in agricultural soil improvement.

9. Use of a phosphate-dissolving bacteria agent with a high effective phosphorus conversion rate as described in any one of claims 1 to 3 in the recovery of bone char phosphorus resources.

10. Use of the phosphate-dissolving bacteria agent with high effective phosphorus conversion rate as described in any one of claims 1 to 3 in promoting the growth of crops.

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