A compound microbial solid fertilizer, its preparation method and application

Abstract

This invention relates to the fields of environmental science and agricultural biotechnology, specifically to a compound microbial solid fertilizer, its preparation method, and its application. The compound microbial solid fertilizer comprises *Enterobacter holmium* HS6, *Pantotheca acuminata* CT3, a culture medium, and an organic matrix. By combining *Enterobacter holmium* HS6 and *Pantotheca acuminata* CT3, utilizing the culture medium to promote growth and metabolism, and further promoting the growth of the microorganisms and using the organic matrix as a carrier to form a solid fertilizer, it can synergistically convert insoluble phosphorus in the soil into soluble phosphorus, effectively improving the utilization rate of phosphorus in the soil, reducing the amount of chemical phosphate fertilizer used, increasing other nutrients in the soil, improving soil physicochemical properties and fertility, promoting the green and sustainable development of agricultural production, and has excellent application prospects.

Description

Technical Field

[0001] This invention relates to the fields of environmental and agricultural biotechnology, specifically to a compound microbial solid fertilizer, its preparation method, and its application. Background Technology

[0002] Currently, in order to improve the deficiency of available phosphorus in agricultural production, my country often uses the application of chemical phosphate fertilizers. These chemical phosphate fertilizers are mostly phosphates. However, due to the unique chemical properties of phosphates and the specific physicochemical properties of soil, the utilization rate of chemical phosphate fertilizers applied to the field is only 10%–25% in the current season. A large amount of phosphorus in the soil exists in the form of insoluble, unavailable phosphates such as Ca-P, Al-P, and Fe-P, resulting in insufficient available phosphorus content in the soil. Furthermore, deposited insoluble phosphates can cause soil compaction and decreased fertility, and in some areas, the distribution of phosphorus sources in the soil is uneven.

[0003] Phosphate-solubilizing bacterial fertilizers have advantages such as low cost, low risk, good effect, no environmental pollution, and full utilization of potential phosphorus resources. In the current technology, single phosphate-solubilizing bacterial fertilizers are widely used in vegetable, fruit tree, tobacco and grain production, while research on the preparation process and practical application of compound phosphate-solubilizing bacterial fertilizers is still limited, and there is a lack of systematic research on their impact on soil fertility and nutrients. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, the first objective of this invention is to provide a compound microbial solid fertilizer. This compound microbial solid fertilizer can effectively improve the utilization rate of phosphorus in the soil, reduce the amount of chemical phosphate fertilizer used, improve the physical and chemical properties of the soil and soil fertility, and promote the green and sustainable development of agricultural production.

[0005] To overcome the shortcomings of the prior art, the second objective of this invention is to provide a method for preparing a compound microbial solid fertilizer. This method is simple and easy to operate, and the resulting compound microbial solid fertilizer can effectively improve the utilization rate of phosphorus in the soil, reduce the amount of chemical phosphate fertilizer used, improve the physical and chemical properties of the soil and soil fertility, and promote the green and sustainable development of agricultural production.

[0006] The third objective of this invention is to provide an application of a compound microbial solid fertilizer.

[0007] To achieve the first objective of the invention, the technical solution adopted by the present invention is as follows:

[0008] This invention provides a compound microbial solid fertilizer, comprising Enterobacter holmium HS6, Pantotheca cumulus CT3, culture medium, and organic matrix;

[0009] The classification name of the Enterobacter hormaechei HS6 is Enterobacter hormaechei, which was deposited at the China General Microbiological Culture Collection Center on October 19, 2023, with the accession number CGMCC NO.: 28680;

[0010] The aforementioned Pantoea agglomerans CT3 is classified as Pantoea agglomerans and was deposited on January 8, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.: 33335.

[0011] The organic matrix is ​​at least one of wheat bran or rice straw.

[0012] Furthermore, the 16S rDNA gene sequence of the *Enterobacter holmieae* HS6 is shown in SEQ ID No. 1;

[0013] The 16S rDNA gene sequence of the CT3 clump is shown in SEQ ID No. 2.

[0014] Furthermore, the particle size of the organic matrix is ​​such that it passes through a 30-50 mesh sieve.

[0015] The organic matrix contains abundant nutrients, which can promote the growth of Enterobacter holmie HS6 and Pantotheca clumps CT3, and can also serve as a carrier for Enterobacter holmie HS6 and Pantotheca clumps CT3 to form solid microbial fertilizer. In addition, it can increase the nutrients in the soil and improve the soil's physical and chemical properties.

[0016] In addition, the culture medium is also used to promote the growth and metabolism of Enterobacter holmieae HS6 and Pantotheca clumps CT3.

[0017] This compound microbial solid fertilizer combines Enterobacter holmium HS6 and Pantotheca cumulus CT3, and uses a culture medium to promote growth and metabolism. At the same time, the organic matrix further promotes the growth of the strains and serves as a carrier for the strains to form solid fertilizer. It can synergistically convert insoluble phosphorus in the soil into soluble phosphorus, increase other nutrients in the soil and improve the soil's physical and chemical properties.

[0018] To achieve the second objective of the invention, the technical solution adopted by the present invention is as follows:

[0019] This invention provides a method for preparing a compound microbial solid fertilizer, comprising the following steps:

[0020] S1. Organic matrix pretreatment: The organic matrix is ​​crushed and sieved, and then sterilized under high temperature and high pressure to obtain the organic matrix carrier.

[0021] S2. Activation culture: Enterobacter holmieae HS6 and Pantotheca clumps CT3 were inoculated into LB medium and then placed in a constant temperature shaker at 28℃ with shaking at 170r / min~190r / min for 24h~48h. After centrifugation, the sediment was diluted with sterile water to obtain two activated bacterial suspensions.

[0022] S3. Preparation of liquid compound phosphate-solubilizing agent: Dip the bacterial suspension from the two activated bacterial suspensions with an inoculation loop, then inoculate them into LB liquid medium, and then place them in a constant temperature shaker at 28℃ and shake at 170r / min~190r / min until the logarithmic growth phase to obtain HS6 bacterial suspension and CT3 bacterial suspension. Then mix HS6 bacterial suspension and CT3 bacterial suspension to form liquid compound phosphate-solubilizing agent.

[0023] S4. Preparation of solid microbial fertilizer: The liquid compound phosphate-solubilizing agent obtained in step S3 is added to the organic matrix carrier obtained in step S1, and then cultured at 28℃ for 2-4 days to obtain the compound microbial solid fertilizer.

[0024] Furthermore, in step S1, the organic matrix is ​​pulverized and passed through a 30-50 mesh sieve; the conditions for high temperature and high pressure sterilization are: 121℃, 104KPa, 20min.

[0025] Furthermore, in step S2, the centrifugation conditions are: centrifugation at 4°C for 10 min; diluting the sediment with sterile water to an effective viable bacterial count of 1×10⁻⁶. 9 CFU / mL ~9×10 9 CFU / mL; and / or

[0026] In step S3, HS6 bacterial solution and CT3 bacterial solution are mixed at a volume ratio of 1:1 to form a liquid composite phosphate-solubilizing agent.

[0027] Furthermore, in step S4, the liquid composite phosphate-solubilizing agent accounts for 25% to 35% of the mass percentage of the organic matrix carrier; and / or

[0028] In step S4, the organic matrix carrier obtained in step S1 is placed into a sterile polyethylene bag, and the liquid composite phosphate-solubilizing agent prepared in step S3 is added to the organic matrix carrier in the sterile polyethylene bag. After mixing, the bag is sealed, and 5 to 8 ventilation holes are punched on the surface of the polyethylene bag with a sterile needle. Then, another polyethylene bag is placed on top and 5 to 8 ventilation holes are punched. The bag is then cultured at 28°C for 2-4 days to obtain the composite microbial solid fertilizer.

[0029] The use of two layers of sterile polyethylene bags provides a better environment for the cultivation of Enterobacter holmie HS6 and Pantotheca clumps CT3, thereby promoting their growth.

[0030] To achieve the third objective of the invention, the technical solution adopted by the present invention is as follows:

[0031] This invention provides the application of the aforementioned compound microbial solid fertilizer in agricultural soil improvement.

[0032] Specifically, the compound microbial solid fertilizer is applied to the soil, and the synergistic effect of Enterobacter holmium HS6, Pantotheca cumulus CT3 and organic matrix promotes the conversion and release of insoluble phosphorus in the soil, thereby increasing the available phosphorus content in the soil and promoting plant growth.

[0033] This invention provides the application of the aforementioned compound microbial solid fertilizer in improving soil fertility.

[0034] Specifically, applying the compound microbial solid fertilizer to the soil can improve the soil's pH and electrical conductivity, increase the content of available phosphorus and available potassium in the soil, and improve soil fertility by converting nitrogen in the organic matrix into alkaline nitrogen.

[0035] This invention provides the application of the aforementioned compound microbial solid fertilizer in promoting plant growth.

[0036] This compound microbial solid fertilizer combines Enterobacter holmium HS6 and Pantotheca cumulus CT3 with an organic matrix. By utilizing the synergistic effect of Enterobacter holmium HS6, Pantotheca cumulus CT3 and the organic matrix, it can increase the available phosphorus and available potassium content in the soil, reduce the use of chemical phosphate fertilizers, improve agricultural soil, enhance soil fertility, and promote plant growth. Therefore, it has good application prospects in the above applications.

[0037] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0038] (1) The present invention provides a compound microbial solid fertilizer by combining Enterobacter holmium HS6 and Pantotheca cumulus CT3, and using a culture medium to promote growth and metabolism. At the same time, an organic matrix is ​​used to further promote the growth of the strain and serve as a carrier for the strain to form a solid fertilizer. This can synergistically convert insoluble phosphorus in the soil into soluble phosphorus, effectively improve the utilization rate of phosphorus in the soil, reduce the amount of chemical phosphate fertilizer used, increase other nutrients in the soil, improve the physical and chemical properties of the soil and soil fertility, promote the green and sustainable development of agricultural production, and has a good application prospect.

[0039] (2) The preparation method of the compound microbial solid fertilizer of the present invention has the characteristics of simple preparation method and easy operation, and the prepared compound microbial solid fertilizer can effectively improve the utilization rate of phosphorus in the soil, reduce the amount of chemical phosphate fertilizer used, improve the physical and chemical properties of the soil and soil fertility, and promote the green and sustainable development of agricultural production.

[0040] (4) The application of the compound microbial solid fertilizer of the present invention is as follows: the compound microbial solid fertilizer combines Enterobacter HS6, Pantotheca CT3 and organic matrix, and utilizes the synergistic effect of Enterobacter HS6, Pantotheca CT3 and organic matrix to promote the conversion and release of insoluble phosphorus in the soil, thereby increasing the effective phosphorus content and available potassium content in the soil. It can also reduce the use of chemical phosphate fertilizer, improve agricultural soil, improve soil fertility and promote plant growth. Therefore, it has good application prospects in the above applications and promotes plant growth. Attached Figure Description

[0041] 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.

[0042] Figure 1 This is a scanning electron microscope image of Enterobacter HS6 of the present invention.

[0043] Figure 2 This is a scanning electron microscope image of the CT3 cluster of pantothecin bacteria according to the present invention.

[0044] Figure 3 This is a graph showing the pH test results for three organic matrices.

[0045] Figure 4 This is a graph showing the results of moisture content testing for three organic matrices.

[0046] Figure 5 This is a graph showing the test results of the water absorption rate of three organic matrices.

[0047] Figure 6 This is a graph showing the test results of the effect of the compound microbial solid fertilizer of the present invention on soil pH.

[0048] Figure 7 This is a graph showing the test results of the effect of the compound microbial solid fertilizer of the present invention on soil electrical conductivity.

[0049] Figure 8 This is a graph showing the test results of the effect of the compound microbial solid fertilizer of the present invention on the available phosphorus content of the soil.

[0050] Figure 9 This is a graph showing the test results of the effect of the compound microbial solid fertilizer of the present invention on the available potassium content in the soil.

[0051] Figure 10 This is a graph showing the test results of the effect of the compound microbial solid fertilizer of the present invention on the content of soil alkaline nitrogen. Detailed Implementation

[0052] 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.

[0053] 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.

[0054] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0055] The culture medium formulations described in the following examples are as follows:

[0056] LB medium (g / L): 10g peptone, 5g yeast extract, 10g NaCl, pH 7.0-7.2.

[0057] LB solid medium: Add 18g to 20g of agar powder to every 1L of LB medium.

[0058] NBRIP medium (g / L): glucose 10g, (NH4)2SO4 0.5g, NaCl 0.3g, MgSO4·7H2O 0.3g, FeSO4·7H2O 0.03g, MnSO4·2H2O 0.03g, Ca3(PO4)2 5g, KCl 0.3g, lecithin 0.2g, pH 7.2~7.4.

[0059] Solid medium for NBRIP: Add 18g to 20g of agar powder to every 1L of NBRIP medium.

[0060] Example 1

[0061] A compound microbial solid fertilizer comprises *Enterobacter hormaechei* HS6, *Pantoea agglomerans* CT3, culture medium, and organic substrate. *Enterobacter hormaechei* HS6 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. *Pantoea agglomerans* CT3 was deposited on January 8, 2025, at the same center with accession number CGMCC NO. 33335, also located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0062] In this embodiment, the organic substrate is wheat bran. Wheat bran is rich in nutrients, which can promote the growth of Enterobacter holmieae HS6 and Pantotheca clumps CT3, and can also serve as a carrier for Enterobacter holmieae HS6 and Pantotheca clumps CT3 to form solid microbial fertilizer. In addition, it can also increase the nutrients in the soil and improve the soil's physical and chemical properties.

[0063] Among them, the scanning electron microscope image of Enterobacter holmieae HS6 is shown below. Figure 1 As shown. Scanning electron micrograph of CT3 clusters of pantothenic bacteria, as shown. Figure 2 As shown.

[0064] The 16S rDNA gene sequence of Enterobacter holmieae HS6 is shown in SEQ ID No. 1. The gene sequence length is 1476.

[0065] The 16S rDNA gene sequence of *Pantotheca cumulus* CT3 is shown in SEQ ID No. 2. The gene sequence length is 1411.

[0066] The preparation method of this compound microbial solid fertilizer includes the following steps:

[0067] S1. Organic matrix pretreatment: The organic matrix is ​​crushed and passed through a 40-mesh sieve, and then subjected to high temperature and high pressure sterilization to obtain the organic matrix carrier; the conditions for high temperature and high pressure sterilization are: 121℃, 104KPa, 20min.

[0068] S2. Activation Culture: Enterobacter holmieae HS6 and Pantotheca cumulus CT3 were inoculated into LB medium and cultured at 170-190 rpm for 24-48 hours in a constant temperature shaker at 28°C. The culture was then centrifuged at 4°C for 10 minutes, and the sediment was diluted with sterile water to a viable count of 1 × 10⁻⁶. 9 CFU / mL ~9×10 9CFU / mL, two activated bacterial suspensions were obtained;

[0069] S3. Preparation of liquid composite phosphate-solubilizing agent: Dip the bacterial suspension from the two activated bacterial suspensions with an inoculation loop, then inoculate them into LB liquid medium, and then place them in a constant temperature shaker at 28℃ and shake at 170r / min~190r / min until the logarithmic growth phase to obtain HS6 bacterial suspension and CT3 bacterial suspension. Then mix HS6 bacterial suspension and CT3 bacterial suspension at a volume ratio of 1:1 to form liquid composite phosphate-solubilizing agent.

[0070] S4. Preparation of solid microbial fertilizer: The organic matrix carrier obtained in step S1 is placed in a sterile polyethylene bag. The liquid composite phosphate-solubilizing agent prepared in step S3 is added to the organic matrix carrier in the sterile polyethylene bag. After mixing, the bag is sealed. Five ventilation holes are punched on the surface of the polyethylene bag with a sterile needle. Another polyethylene bag is then placed inside and five ventilation holes are punched. The bag is then incubated at 28°C for 3 days to obtain the composite microbial solid fertilizer. In this embodiment, the liquid composite phosphate-solubilizing agent accounts for 30% of the mass percentage of the organic matrix carrier.

[0071] Example 2

[0072] A compound microbial solid fertilizer is disclosed in this embodiment. The difference between this embodiment and Example 1 is that the organic substrate in this embodiment is rice straw. All other conditions and methods in this embodiment are the same as in Example 1.

[0073] Example 3

[0074] A compound microbial solid fertilizer is disclosed in this embodiment. The difference between this embodiment and Example 1 is that the organic substrate in this embodiment is a combination of wheat bran and rice straw. The remaining conditions and methods in this embodiment are the same as in Example 1.

[0075] Example 4

[0076] A compound microbial solid fertilizer is disclosed. The difference between this embodiment and Embodiment 1 is that in this embodiment, in step S1, the organic matrix is ​​pulverized and passed through a 30-mesh sieve; in step S4, six ventilation holes are punched on the surface of the polyethylene bag, and then it is cultured at 28°C for 2 days; in step S4, the liquid compound phosphate-solubilizing agent accounts for 25% of the mass percentage of the organic matrix carrier. All other conditions and methods in this embodiment are the same as in Embodiment 1.

[0077] Example 5

[0078] A compound microbial solid fertilizer is disclosed. The difference between this embodiment and Embodiment 1 is that in this embodiment, in step S1, the organic matrix is ​​pulverized and passed through a 50-mesh sieve; in step S4, eight ventilation holes are punched on the surface of the polyethylene bag, and then it is cultured at 28°C for 4 days; in step S4, the liquid compound phosphate-solubilizing agent accounts for 35% of the mass percentage of the organic matrix carrier. All other conditions and methods in this embodiment are the same as in Embodiment 1.

[0079] Comparative Example 1

[0080] A compound microbial solid fertilizer is disclosed. The difference between this comparative example and Example 1 is that the organic substrate in this comparative example is corn cob. All other conditions and methods in this comparative example are the same as in Example 1.

[0081] Experimental testing:

[0082] (I) Detection of the number of viable bacteria in compound microbial solid fertilizers using different organic substrates

[0083] The effective viable counts of the composite microbial solid fertilizers prepared in Example 1, Example 2 and Comparative Document 1 were tested respectively, and the test results are shown in Table 1.

[0084] Table 1. Effective viable bacteria count of composite microbial solid fertilizers using different organic substrates.

[0085] Example organic matrix Effective viable bacteria count Example 1 wheat bran <![CDATA[1.32×10 10 CFU / g]]> Example 2 rice straw <![CDATA[1.08×10 10 CFU / g]]> Comparative Example 1 corn cob <![CDATA[2.90×10 7 CFU / g]]>

[0086] As shown in Table 1, Example 1 used wheat bran as the organic substrate, and Example 2 used rice straw as the organic substrate, with effective viable bacteria counts of 1.32 × 10⁻⁶ and 1.32 × 10⁻⁶, respectively. 10 CFU / g and 1.08×10 10 All samples showed high CFU / g viable counts, meeting the standards for agricultural microbial fertilizers. However, Comparative Example 1, using corn cob as the organic substrate, had only 2.90 × 10⁻⁶ viable counts. 7 The CFU / g level is insufficient to meet the standards for agricultural microbial fertilizers. This indicates that the compound microbial solid fertilizer prepared by the organic matrix of wheat bran and / or rice straw in this invention, in synergy with Enterobacter holmium HS6 and Pantothecin CT3, can better promote the conversion of insoluble phosphorus in the soil into soluble phosphorus, thereby increasing the content of available phosphorus in the soil, reducing the use of chemical phosphate fertilizers, improving agricultural soil, enhancing soil fertility, and promoting plant growth.

[0087] (II) Testing the basic physicochemical properties of three organic matrices

[0088] The pH, moisture content, and water absorption rate of wheat bran, rice straw, and corn cob used in Examples 1, 2, and 1 were tested, respectively. The three basic physicochemical properties were tested as follows.

[0089] (1) pH value detection

[0090] Weigh 15g of organic matrix into a 50mL beaker. Add distilled water in an appropriate ratio according to the type of organic matrix. Stir evenly with a glass rod and let stand for 30 minutes to obtain a sample suspension. Measure the pH value of the sample suspension using a pH meter. The experiment was repeated three times, and the average of the three results was taken as the final pH value. The pH values ​​of wheat bran, rice straw, and corn cob are shown below. Figure 3 As shown.

[0091] The pH value of the organic substrate, used as a carrier, is a crucial factor affecting bacterial survival; different bacteria have different optimal pH values ​​for growth. Figure 3 As can be seen, rice straw has the lowest pH value at 6.25; corn cob has the highest pH value at 7.93; while wheat bran has a pH value of 6.72, which is closer to neutral conditions. Neutral conditions are an ideal growth environment for many microorganisms, providing stable conditions that are conducive to bacterial growth and reproduction.

[0092] Based on the results of the effective viable count test of the compound microbial solid fertilizers prepared from wheat bran, rice straw and corn cob respectively, the optimal pH range for the growth of Enterobacter holmieae HS6 and Pantotheca cumulus CT3 of the present invention is between 6 and 7. This indicates that the organic substrates wheat bran and rice straw used in the present invention can promote the growth of Enterobacter holmieae HS6 and Pantotheca cumulus CT3.

[0093] (2) Moisture content detection

[0094] Place the empty aluminum box in a drying oven at 105℃ for 30 minutes to ensure there is no moisture inside. Remove the aluminum box and cool it to room temperature, then weigh and record its mass. Weigh 20.00g of organic matrix that has passed through a 1-mesh sieve and place it in the aluminum box. Weigh the total mass and then place it in the drying oven at 105℃ for 5 hours to ensure complete evaporation of moisture from the organic matrix. Remove the aluminum box and cool it to room temperature for 20 minutes, then weigh the total mass of the aluminum box and organic matrix again. The moisture content is calculated using the following formula. Three repeated experiments are performed, and the average of the three experimental results is taken as the final moisture content, expressed as a percentage (%). The formula for calculating the moisture content is shown in Formula I.

[0095]

[0096] In Formula I: w is the moisture content of the organic matrix (%); m1 is the mass of the organic matrix and aluminum box in grams (g); m2 is the mass of the dried organic matrix and aluminum box in grams (g); m0 is the mass of the empty aluminum box in grams (g).

[0097] The moisture content test results for wheat bran, rice straw, and corn cobs are as follows: Figure 4 As shown.

[0098] The moisture content of the organic substrate is a key factor affecting bacterial survival; generally, a moisture content below 30% is required for the preparation of microbial fertilizer. Excessive moisture content will fill the voids in the substrate with water, hindering air circulation and leading to mold growth. Conversely, insufficient moisture content will fail to meet the basic conditions for bacterial survival, resulting in reduced bacterial activity or even bacterial death.

[0099] Depend on Figure 4 It is evident that among the three organic substrates, corn cob had the highest moisture content at 9.55%, rice straw had the lowest at 7.37%, and wheat bran had a moisture content of 8.79%. This indicates that the high moisture content of corn cob is unsuitable for the growth of *Enterobacter holmieae* HS6 and *Pantotheca acuminata* CT3. The moisture content of wheat bran and rice straw meets the standards for use as carriers of solid microbial fertilizer.

[0100] (3) Water absorption rate test

[0101] Weigh 100g of organic matrix and place it in a polyethylene bag. Using a pipette, add sterile water to the polyethylene bag in 5mL volume gradients, stirring with a glass rod until the organic matrix remains moist, loose, and non-clumping. Record the total mass of sterile water used to achieve this state; this mass represents the maximum liquid volume that 100g of organic matrix can hold. The water absorption rate is calculated using Formula II. The experiment is repeated three times, and the average of the three results is taken as the final water absorption rate, expressed as a percentage (%).

[0102]

[0103] In Formula II: w is the water absorption rate of the organic matrix (%); M 湿 The total weight of the carrier after adding sterile water is expressed in grams (g); M 干 The total weight of the carrier before water is added is expressed in grams (g).

[0104] The test results for the water absorption rates of wheat bran, rice straw, and corn cobs are as follows: Figure 5 As shown.

[0105] The water absorption rate of the organic substrate as a carrier is an important parameter for evaluating the quality of microbial fertilizers, directly affecting the growth of inoculated microorganisms. Within a certain range, a carrier with strong water absorption capacity can provide a more humid living environment for the attached bacteria for a relatively long period of time, which is crucial for maintaining bacterial activity and growth.

[0106] Depend on Figure 5It is evident that wheat bran and rice straw have a water absorption rate of 100%, possessing suitable water absorption capacity, which promotes the growth of *Enterobacter holmieae* HS6 and *Pantotheca cum Caulis* CT3. In contrast, corn cobs have a water absorption rate of 300%. This excessively high water absorption rate causes the pores within the corn cob to become filled with moisture, hindering air circulation and leading to mold growth. This, in turn, is detrimental to the growth of *Enterobacter holmieae* HS6 and *Pantotheca cum Caulis* CT3.

[0107] (III) Detection of the effects of compound microbial solid fertilizer on the physicochemical properties of soil

[0108] The compound microbial solid fertilizer prepared in Example 1 was applied to soil for four different fertilization treatments to test the effects of the compound microbial solid fertilizer of the present invention on the physicochemical properties of the soil. The specific design scheme of the fertilization treatment experiments is shown in Table 2 below.

[0109] Table 2 Experimental Design of Fertilization Treatments

[0110] experimental group Fertilization treatment CK No microbial fertilizer applied T1 20g of base fertilizer T2 20g base fertilizer + 2g top dressing over 10 days T3 20g base fertilizer + 4g top dressing over 10 days

[0111] According to the experimental design in Table 2, the soil was fertilized and then the effects of the compound microbial solid fertilizer of the present invention on soil pH, soil electrical conductivity, soil available phosphorus content, soil available potassium content and soil alkaline nitrogen content were detected.

[0112] The effect of the compound microbial solid fertilizer of the present invention on soil pH is as follows: Figure 6 As shown. Figure 6 It can be seen that the compound microbial solid fertilizer of the present invention, when subjected to three fertilization treatments (T1, T2, and T3) and observed from 0 to 21 days, can reduce the soil pH value compared to the case without fertilizer application (CK). This indicates that the compound microbial solid fertilizer of the present invention can alleviate soil alkalization to a certain extent, thereby benefiting plant growth.

[0113] Among them, the effect of the compound microbial solid fertilizer of the present invention on soil electrical conductivity is as follows: Figure 7 As shown. Figure 7It is evident that the compound microbial solid fertilizer of the present invention, when subjected to three fertilization treatments (T1, T2, and T3) and observed from day 0 to day 21, all showed an increase in soil electrical conductivity compared to the control group (CK). This is because the decomposition activities of Enterobacter holmieae HS6 and Pantotheca cumulus CT3 in the compound microbial solid fertilizer of the present invention produce phosphates, ammonium salts, etc., which in turn increase the soil electrical conductivity, reaching a maximum value of 239.5 μs / cm on day 7, which is 1.7 times higher than the CK group. This may be because the inorganic salts produced dissolve and are relatively stable in the soil by day 7. In addition, the electrical conductivity of groups T1, T2, and T3 decreased on days 14 and 21 after fertilization. This may be because Enterobacter holmieae HS6 and Pantotheca agglomerata CT3 consumed all the nutrients in the organic substrate wheat bran, which served as the carrier, and needed to absorb inorganic salts from the soil environment to maintain normal life activities, thus reducing the amount of inorganic salts in the soil and consequently decreasing the electrical conductivity. However, the electrical conductivity of groups T1, T2, and T3 was still higher than that of the CK group, indicating that the compound microbial solid fertilizer of the present invention can increase the inorganic salt content of the soil, thereby providing nutrients for plants and effectively promoting plant growth.

[0114] Among them, the effect of the compound microbial solid fertilizer of the present invention on the available phosphorus content of the soil is as follows: Figure 8 As shown. Figure 8 As can be seen, the compound microbial solid fertilizer of the present invention, after three fertilization treatments (T1, T2, and T3) and observation from day 0 to day 21, showed a significant increase in the available phosphorus content in the soil compared to the control group (CK). The available phosphorus content reached its highest value of 68.5 mg / kg on day 7 after basal fertilization, 17.4 times higher than the CK group. However, the content in the T1 group decreased rapidly on day 14, possibly because no topdressing was applied to the T1 group on day 10. While *Enterobacter holmieae* HS6 and *Pantothecin CT3* require phosphorus nutrients from the soil to maintain their metabolism, the available phosphorus content in the T1 group was still relatively high compared to the CK group at this point. This indicates that the compound microbial solid fertilizer of the present invention can efficiently convert insoluble phosphorus in the soil into soluble phosphorus, effectively improving the utilization rate of phosphorus in the soil, reducing the amount of chemical phosphate fertilizer used, improving soil physicochemical properties and soil fertility, promoting the green and sustainable development of agricultural production, and has excellent application prospects.

[0115] The effect of the compound microbial solid fertilizer of the present invention on the available potassium content in the soil is as follows: Figure 9 As shown. Figure 9As can be seen, the compound microbial solid fertilizer of the present invention, applied under three fertilization treatments (T1, T2, and T3) and observed from day 0 to day 21, significantly increased the content of available potassium in the soil compared to the control group (CK). The T1 group reached its maximum value of 55.1 mg / kg on day 7, the T2 group reached its maximum value of 56.2 mg / kg on day 14, and the T3 group reached its maximum value of 58.1 mg / kg on day 14. This indicates that the compound microbial solid fertilizer of the present invention can significantly increase the content of available potassium in the soil, and this increase is proportional to the amount of fertilizer applied. Therefore, the compound microbial solid fertilizer of the present invention can increase soil nutrients, improve soil physicochemical properties and fertility, promote the green and sustainable development of agricultural production, and has excellent application prospects. Furthermore, the continuous decrease in available potassium content in the CK group may be due to the absorption and utilization of available potassium by microorganisms already present in the soil, further demonstrating that the compound microbial solid fertilizer of the present invention can greatly increase the content of available potassium in the soil, thereby increasing potassium source nutrients, improving soil physicochemical properties and fertility, promoting plant growth, and promoting the green and sustainable development of agricultural production.

[0116] The effect of the compound microbial solid fertilizer of the present invention on the content of soil alkaline nitrogen is as follows: Figure 10 As shown. Figure 10 As can be seen, the compound microbial solid fertilizer of the present invention was applied under three fertilization treatments (T1, T2, and T3), and the alkaline nitrogen content was measured at 0d, 7d, 14d, and 21d. Compared with the case without fertilizer application (CK), the alkaline nitrogen content in the soil increased in all three treatments. However, as time progressed, the alkaline nitrogen content in the soil of groups T1, T2, and T3 decreased significantly at 7d, 14d, and 21d compared to 0d. This is because Enterobacter holmieae HS6 and Pantotheca cumulus CT3 require organic nitrogen in the soil for growth and reproduction in their early stages. Among them, the alkaline nitrogen content of groups T1, T2, and T3 showed a trend of first decreasing and then increasing at 7d, 14d, and 21d. This may be due to the direct and indirect nitrogen fixation by phosphate-solidifying bacteria, which can convert atmospheric nitrogen into organic nitrogen that can be directly absorbed and utilized by crops.

[0117] 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.

[0118]

[0119]

Claims

1. A compound microbial solid fertilizer, characterized in that, Includes Enterobacter holmieae HS6, Pantotheca clumps CT3, culture medium, and organic matrix; The classification name of the Enterobacter hormaechei HS6 is Enterobacter hormaechei, which was deposited at the China General Microbiological Culture Collection Center on October 19, 2023, with the accession number CGMCC NO.28680. The classification name of the clump-forming pantoea CT3 is Pantoea agglomerans, which was deposited at the China General Microbiological Culture Collection Center on January 8, 2025, with the accession number CGMCC NO.33335. The organic matrix is ​​at least one of wheat bran or rice straw.

2. The compound microbial solid fertilizer as described in claim 1, characterized in that, The 16S rDNA gene sequence of *Enterobacter holmium* HS6 is shown in SEQ ID No. 1; The 16S rDNA gene sequence of the CT3 clump is shown in SEQ ID No.

2.

3. The compound microbial solid fertilizer as described in claim 1, characterized in that, The particle size of the organic matrix is ​​such that it passes through a 30-50 mesh sieve.

4. A method for preparing a compound microbial solid fertilizer according to any one of claims 1 to 3, characterized in that, Includes the following steps: S1. Organic matrix pretreatment: The organic matrix is ​​crushed and sieved, and then sterilized under high temperature and high pressure to obtain the organic matrix carrier. S2. Activation culture: Enterobacter holmieae HS6 and Pantotheca clumps CT3 were inoculated into LB medium and then placed in a constant temperature shaker at 28℃ with shaking at 170r / min~190r / min for 24h~48h. After centrifugation, the sediment was diluted with sterile water to obtain two activated bacterial suspensions. S3. Preparation of liquid compound phosphate-solubilizing agent: Dip the bacterial suspension from the two activated bacterial suspensions with an inoculation loop, then inoculate them into LB liquid medium, and then place them in a constant temperature shaker at 28℃ and shake at 170r / min~190r / min until the logarithmic growth phase to obtain HS6 bacterial suspension and CT3 bacterial suspension. Then mix HS6 bacterial suspension and CT3 bacterial suspension to form liquid compound phosphate-solubilizing agent. S4. Preparation of solid microbial fertilizer: The liquid compound phosphate-solubilizing agent obtained in step S3 is added to the organic matrix carrier obtained in step S1, and then cultured at 28℃ for 2-4 days to obtain the compound microbial solid fertilizer.

5. The method for preparing a compound microbial solid fertilizer as described in claim 4, characterized in that, In step S1, the organic matrix is ​​pulverized and passed through a 30-50 mesh sieve; the conditions for high temperature and high pressure sterilization are: 121℃, 104KPa, 20min.

6. The method for preparing a compound microbial solid fertilizer as described in claim 4, characterized in that, In step S2, the centrifugation conditions are: centrifugation at 4°C for 10 min; diluting the sediment with sterile water to an effective viable count of 1×10⁻⁶. 9 CFU / mL ~9×10 9 CFU / mL; and / or In step S3, HS6 bacterial solution and CT3 bacterial solution are mixed at a volume ratio of 1:1 to form a liquid composite phosphate-solubilizing agent.

7. The method for preparing a compound microbial solid fertilizer as described in claim 4, characterized in that, In step S4, the liquid composite phosphate-solubilizing agent accounts for 25% to 35% of the mass percentage of the organic matrix carrier; and / or In step S4, the organic matrix carrier obtained in step S1 is placed into a sterile polyethylene bag, and the liquid composite phosphate-solubilizing agent prepared in step S3 is added to the organic matrix carrier in the sterile polyethylene bag. After mixing, the bag is sealed, and 5 to 8 ventilation holes are punched on the surface of the polyethylene bag with a sterile needle. Then, another polyethylene bag is placed on top and 5 to 8 ventilation holes are punched. The bag is then cultured at 28°C for 2-4 days to obtain the composite microbial solid fertilizer.

8. The application of the compound microbial solid fertilizer according to any one of claims 1 to 3 in agricultural soil improvement.

9. The application of the compound microbial solid fertilizer according to any one of claims 1 to 3 in improving soil fertility.

10. The application of a compound microbial solid fertilizer according to any one of claims 1 to 3 in promoting plant growth.

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