Composite microbial solid bacterial fertilizer as well as preparation method and application thereof

Abstract

The invention relates to the field of environment and agricultural biotechnology, in particular to a compound microbial solid bacterial fertilizer and a preparation method and application thereof, and the compound microbial solid bacterial fertilizer comprises enterobacter hormaechei HS6, pantoea agglomerans CT3, a culture medium and an organic matrix. Enterobacter hormaechei HS6 and pantoea agglomerans CT3 are compounded, a culture medium is used for promoting growth and metabolism, meanwhile, an organic matrix is used for further promoting growth of strains and serves as a carrier of the strains to form the solid bacterial fertilizer, insoluble phosphorus in soil can be synergistically and efficiently converted into soluble phosphorus, the utilization rate of phosphorus in the soil is effectively increased, and the soil quality is improved. The method reduces the use amount of chemical phosphate fertilizer, increases other nutritional ingredients for soil, improves the physicochemical properties and fertility of soil, promotes the green and sustainable development of agricultural production, and has a good application prospect.

Description

Technical Field

[0001] The invention relates to the fields of environment and agricultural biotechnology, and in particular to a composite microbial solid fertilizer and a preparation method and application thereof. Background Art

[0002] At present, in order to improve the situation of lack of effective phosphorus in the soil during agricultural production, my country often adopts the method of applying chemical phosphorus fertilizers. Among them, chemical phosphorus fertilizers are mostly phosphates. However, due to the special chemical properties of phosphates and the specific physical and chemical properties of soils, the utilization rate of chemical phosphorus fertilizers applied to fields in the current season is only 10% to 25%. A large amount of phosphorus sources in the soil mostly exist in the form of insoluble and invalid phosphates such as Ca-P, Al-P, and Fe-P, resulting in insufficient effective phosphorus content in the soil. In addition, deposited insoluble phosphates can also cause problems of soil compaction and decreased fertility, and the soil phosphorus sources in some areas are also unevenly distributed.

[0003] Phosphate-dissolving bacterial fertilizer has the advantages of low cost, low risk, good effect, no pollution to the environment and full utilization of potential phosphorus source resources. In the existing technology, single phosphate-dissolving bacterial fertilizer is widely used in vegetable, fruit tree, tobacco and grain production, while the preparation process and practical application of compound phosphate-dissolving bacterial fertilizer are still less studied, and there is a lack of systematic research on its impact on soil fertility and nutrients. Summary of the invention

[0004] In order to overcome the deficiencies of the prior art, the first object of the present invention is to provide a composite microbial solid fertilizer. The composite microbial solid fertilizer can effectively improve the utilization rate of phosphorus in the soil, reduce the use of chemical phosphorus fertilizers, improve the physical and chemical properties of the soil and the soil fertility, and promote the green and sustainable development of agricultural production.

[0005] 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 composite microbial solid fertilizer. The preparation method is simple and easy to operate. The prepared composite microbial solid fertilizer can effectively improve the utilization rate of phosphorus in the soil, reduce the use of chemical phosphorus fertilizers, 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 object of the present invention is to provide an application of a composite microbial solid fertilizer.

[0007] In order to achieve the first object of the above invention, the technical solution adopted by the present invention is as follows:

[0008] The present invention provides a composite microbial solid fertilizer, comprising Enterobacter holmesii HS6, Pantoea agglomerans CT3, a culture medium and an organic matrix;

[0009] The classification name of the Enterobacter hormaechei HS6 is Enterobacter hormaechei, which was 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;

[0010] The classification name of the Pantoea agglomerans CT3 is Pantoea agglomerans, which has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on January 8, 2025, with a deposit number of CGMCCNO.: 33335;

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

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

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

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

[0015] Among them, the organic matrix contains rich nutrients. On the one hand, it can promote the growth of Enterobacter HS6 and Pantoea agglomerans CT3. On the other hand, it can serve as a carrier of Enterobacter HS6 and Pantoea agglomerans CT3 to form solid bacterial fertilizer. In addition, it can also increase nutrients for the soil and improve the physical and chemical properties of the soil.

[0016] In addition, the culture medium was also used to promote the growth and metabolism of Enterobacter hallii HS6 and Pantoea agglomerans CT3.

[0017] The composite microbial solid fertilizer is formed by combining Enterobacter hallii HS6 and Pantoea agglomerans CT3, and using culture medium to promote growth and metabolism. At the same time, the organic matrix is ​​used to further promote the growth of the bacteria and serve as a carrier of the bacteria to form solid fertilizer. It can synergistically convert the insoluble phosphorus in the soil into soluble phosphorus efficiently, add other nutrients to the soil and improve the physical and chemical properties of the soil.

[0018] In order to achieve the second purpose of the above invention, the technical solution adopted by the present invention is as follows:

[0019] The present invention provides a method for preparing a composite microbial solid fertilizer, comprising the following steps:

[0020] S1. Pretreatment of organic matrix: crushing and sieving the organic matrix and then sterilizing it at high temperature and high pressure to obtain an organic matrix carrier;

[0021] S2. Activation culture: Enterobacter holmesii HS6 and Pantoea agglomerans CT3 were inoculated into LB medium respectively, and then placed in a constant temperature shaker at 28°C and cultured at 170 r / min-190 r / min for 24 h-48 h, and then centrifuged. The sediments were diluted with sterile water to obtain two activated bacterial suspensions;

[0022] S3. Prepare a liquid composite phosphate-dissolving bacterial agent: use an inoculation loop to dip bacterial liquid from the two activated bacterial suspensions, and then inoculate them into LB liquid culture medium respectively, and then place them in a constant temperature shaker at 28°C and shake at 170r / min-190r / min to culture until the logarithmic growth phase to obtain HS6 bacterial liquid and CT3 bacterial liquid, and then mix the HS6 bacterial liquid and CT3 bacterial liquid to form a liquid composite phosphate-dissolving bacterial agent;

[0023] S4, preparing solid bacterial fertilizer: adding the liquid composite phosphate-dissolving bacterial agent obtained in step S3 to the organic matrix carrier obtained in step S1, and then culturing at 28° C. for 2 days to 4 days to obtain the composite microbial solid bacterial fertilizer.

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

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

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

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

[0028] In step S4, the organic matrix carrier obtained in step S1 is placed in a sterile polyethylene bag, the liquid composite phosphate-dissolving bacteria agent obtained in step S3 is added to the organic matrix carrier in the sterile polyethylene bag, the bag is sealed after mixing, 5 to 8 ventilation holes are pierced on the surface of the polyethylene bag with a sterilized needle, and then another layer of polyethylene bag is put in and 5 to 8 ventilation holes are pierced, and then the bag is cultured at 28°C for 2d-4d to obtain the composite microbial solid fertilizer.

[0029] Among them, two layers of sterile polyethylene bags can provide a better environment for the cultivation of Enterobacter HS6 and Pantoea agglomerans CT3, which is beneficial to the growth of Enterobacter HS6 and Pantoea agglomerans CT3.

[0030] In order to achieve the third purpose of the above invention, the technical solution adopted by the present invention is as follows:

[0031] The present invention provides application of the composite microbial solid fertilizer described above in agricultural soil improvement.

[0032] Specifically, the composite microbial solid fertilizer is applied to the soil, and the synergistic effect of Enterobacter hullii HS6, Pantoea agglomerans CT3 and organic matrix is ​​utilized to promote the transformation and release of insoluble phosphorus in the soil, thereby increasing the effective phosphorus content in the soil and promoting plant growth.

[0033] The present invention provides the use of the composite microbial solid fertilizer described above in improving soil fertility.

[0034] Specifically, applying the composite microbial solid fertilizer to the soil can improve the pH and electrical conductivity of the soil, increase the effective phosphorus content and the quick-acting potassium content in the soil, and Enterobacter hullii HS6 and Pantoea agglomerans CT3 promote the growth of the strains themselves by converting nitrogen elements in the organic matrix into alkaline nitrogen, thereby improving soil fertility.

[0035] The present invention provides the application of the composite microbial solid fertilizer described above in promoting plant growth.

[0036] Among them, the composite microbial solid fertilizer combines Enterobacter hussini HS6 and Pantoea agglomerans CT3, and cooperates with organic matrix at the same time. It can utilize the synergistic effect of Enterobacter hussini HS6, Pantoea agglomerans CT3 and organic matrix to increase the effective phosphorus content and fast-acting potassium content in the soil, and can also reduce the use of chemical phosphate fertilizers. It can improve agricultural soil, improve soil fertility, and promote plant growth. Therefore, it has good application prospects in the above applications.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] (1) The composite microbial solid fertilizer of the present invention is prepared by combining Enterobacter hussini HS6 and Pantoea agglomerans CT3, and using a culture medium to promote growth and metabolism. Meanwhile, an organic matrix is ​​used to further promote the growth of the bacteria and serve as a carrier of the bacteria to form a solid fertilizer. The solid fertilizer can synergistically convert the insoluble phosphorus in the soil into soluble phosphorus, effectively improve the utilization rate of phosphorus in the soil, reduce the use of chemical phosphorus fertilizers, add other nutrients to the soil, improve the physical and chemical properties of the soil and the soil fertility, promote the green and sustainable development of agricultural production, and has a good application prospect.

[0039] (2) The preparation method of the composite microbial solid fertilizer of the present invention has the characteristics of being simple and easy to operate, and the prepared composite microbial solid fertilizer can effectively improve the utilization rate of phosphorus in the soil, reduce the use of chemical phosphorus fertilizers, improve the physical and chemical properties of the soil and soil fertility, and promote the green and sustainable development of agricultural production.

[0040] (4) Application of a composite microbial solid fertilizer of the present invention. Since the composite microbial solid fertilizer is prepared by combining Enterobacter hussini HS6, Pantoea agglomerans CT3 and an organic matrix, the synergistic effect of Enterobacter hussini HS6, Pantoea agglomerans CT3 and the organic matrix is ​​utilized to promote the conversion and release of insoluble phosphorus in the soil, thereby increasing the effective phosphorus content and the quick-acting potassium content in the soil, and reducing the use of chemical phosphorus fertilizers. It can improve agricultural soil, improve soil fertility and promote plant growth. Therefore, the composite microbial solid fertilizer has a good application prospect in the above-mentioned applications and promotes plant growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] 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.

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

[0043] Figure 2 It is a scanning electron microscope image of Pantoea agglomerans CT3 of the present invention.

[0044] Figure 3 This is a graph showing the pH values ​​of three organic matrices.

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

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

[0047] Figure 6 This is a test result diagram of the effect of the composite microbial solid fertilizer of the present invention on the pH value of soil.

[0048] Figure 7 This is a test result diagram of the effect of the composite microbial solid fertilizer of the present invention on soil conductivity.

[0049] Figure 8 This is a test result diagram of the effect of the composite microbial solid fertilizer of the present invention on the available phosphorus content in the soil.

[0050] Fig. 9 This is a test result diagram of the effect of the composite microbial solid fertilizer of the present invention on the available potassium content in the soil.

[0051] Fig.10 This is a test result diagram of the effect of the composite microbial solid fertilizer of the present invention on the alkaline nitrogen content of the soil. DETAILED DESCRIPTION

[0052] 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.

[0053] 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.

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

[0055] The culture medium formula described in the following examples is as follows:

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

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

[0058] NBRIP medium (g / L): glucose 10 g, (NH 4 ) 2 SO 4 0.5g, NaCl 0.3g, MgSO 4 7H 2 O0.3g, FeSO 4 7H 2 O 0.03g, MnSO 4 ·2H 2 O 0.03g, Ca 3 (PO 4 ) 2 5g, KCl 0.3g, lecithin 0.2g, pH 7.2~7.4.

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

[0060] Example 1

[0061] A composite microbial solid fertilizer, comprising Enterobacter hormaechei HS6, Pantoea agglomerans CT3, a culture medium and an organic matrix; the classification name of Enterobacter hormaechei HS6 is Enterobacter hormaechei, which has been deposited in the General Microbiological Center of China National Microbiological Culture Collection 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 classification name of Pantoea agglomerans CT3 is Pantoea agglomerans, which has been deposited in the General Microbiological Center of China National Microbiological Culture Collection 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.

[0062] In this embodiment, the organic matrix is ​​wheat bran. Wheat bran contains rich nutrients, which can promote the growth of Enterobacter hussii HS6 and Pantoea agglomerans CT3 on the one hand, and can serve as a carrier of Enterobacter hussii HS6 and Pantoea agglomerans CT3 to form solid bacterial fertilizer on the other hand, and can also increase nutrients for the soil and improve the physical and chemical properties of the soil.

[0063] Among them, the scanning electron micrograph of Enterobacter huoyi HS6 is shown in Figure 1 As shown in the scanning electron micrograph of Pantoea agglomerata CT3, Figure 2 shown.

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

[0065] The 16S rDNA gene sequence of Pantoea agglomerans CT3 is shown in SEQ ID No. 2. The length of the gene sequence is 1411.

[0066] The preparation method of the composite microbial solid fertilizer comprises the following steps:

[0067] S1. Pretreatment of organic matrix: crushing the organic matrix, passing it through a 40-mesh sieve, and then sterilizing it at high temperature and high pressure to obtain an organic matrix carrier; wherein the conditions for high temperature and high pressure sterilization are: 121° C., 104 KPa, 20 min;

[0068] S2. Activation culture: Enterobacter HS6 and Pantoea agglomerans CT3 were inoculated into LB medium respectively, and then placed in a constant temperature shaker at 28°C and shaken at 170r / min~190r / min for 24h~48h, and then centrifuged at 4°C for 10min. The sediment was diluted with sterile water to an effective viable count of 1×109 CFU / mL~9×10 9 CFU / mL, and two activated bacterial suspensions were obtained;

[0069] S3. Prepare a liquid composite phosphate-dissolving agent: use an inoculation loop to dip bacterial liquid from the two activated bacterial suspensions, and then inoculate them into LB liquid culture medium respectively, and then place them in a constant temperature shaker at 28°C and shake at 170r / min-190r / min to culture until the logarithmic growth phase to obtain HS6 bacterial liquid and CT3 bacterial liquid, and then mix the HS6 bacterial liquid and the CT3 bacterial liquid in a volume ratio of 1:1 to form a liquid composite phosphate-dissolving agent;

[0070] S4, prepare solid bacterial fertilizer: put the organic matrix carrier obtained in step S1 into a sterile polyethylene bag, add the liquid composite phosphate-dissolving bacteria agent obtained in step S3 into the organic matrix carrier of the sterile polyethylene bag, seal it after mixing, pierce 5 ventilation holes on the surface of the polyethylene bag with a sterilized needle, put another layer of polyethylene bag and pierce 5 ventilation holes, and then culture it at 28°C for 3 days to obtain the composite microbial solid bacterial fertilizer. In this embodiment, the mass percentage of the liquid composite phosphate-dissolving bacteria agent in the organic matrix carrier is 30%;

[0071] Example 2

[0072] A composite microbial solid fertilizer, the difference between this embodiment and embodiment 1 is that in this embodiment, the organic matrix is ​​rice straw. The remaining conditions and methods of this embodiment are the same as those of embodiment 1.

[0073] Example 3

[0074] A composite microbial solid fertilizer, the difference between this embodiment and embodiment 1 is that, in this embodiment, the organic matrix is ​​a composition of wheat bran and rice straw. The remaining conditions and methods of this embodiment are the same as those of embodiment 1.

[0075] Example 4

[0076] A composite microbial solid fertilizer, the difference between this embodiment and embodiment 1 is that in this embodiment, in step S1, the organic matrix is ​​crushed and passed through a 30-mesh sieve; in step S4, 6 ventilation holes are pierced on the surface of the polyethylene bag, and then cultured at 28° C. for 2 days; in step S4, the mass percentage of the liquid composite phosphate-dissolving bacteria agent to the organic matrix carrier is 25%. The remaining conditions and methods of this embodiment are the same as those of embodiment 1.

[0077] Example 5

[0078] A composite microbial solid fertilizer, the difference between this embodiment and embodiment 1 is that in this embodiment, in step S1, the organic matrix is ​​crushed and passed through a 50-mesh sieve; in step S4, 8 ventilation holes are pierced on the surface of the polyethylene bag, and then cultured at 28° C. for 4 days; in step S4, the mass percentage of the liquid composite phosphate-dissolving bacteria agent in the organic matrix carrier is 35%. The remaining conditions and methods of this embodiment are the same as those of embodiment 1.

[0079] Comparative Example 1

[0080] A composite microbial solid fertilizer, the difference between this comparative example and Example 1 is that in this comparative example, the organic matrix is ​​corn cobs. The remaining conditions and methods of this comparative example are the same as those of Example 1.

[0081] Experimental testing:

[0082] (I) Detection of the effective viable bacteria count of composite microbial solid fertilizers using different organic matrices

[0083] The effective live bacteria 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 number of viable bacteria in composite microbial solid fertilizers using different organic matrices

[0085] Example Organic matrix Effective viable 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 uses wheat bran as the organic matrix, and Example 2 uses rice straw as the organic matrix, and the effective viable bacteria counts are 1.32×10 10 CFU / g and 1.08×10 10 CFU / g, both have high effective viable bacteria counts and meet the standards for agricultural microbial fertilizers. However, in Comparative Example 1, corn cobs were used as the organic matrix, and its effective viable bacteria count was only 2.90×10 7 CFU / g, which is difficult to meet the agricultural microbial fertilizer standards. It shows that the composite microbial solid fertilizer prepared by the organic matrix wheat bran and / or rice straw in the present invention in coordination with Enterobacter holsteinii HS6 and Pantoea agglomerans CT3 can better promote the conversion of insoluble phosphorus in the soil into soluble phosphorus, thereby increasing the content of effective phosphorus in the soil, reducing the use of chemical phosphorus fertilizers, improving agricultural soil, improving 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 the wheat bran, rice straw and corn cob respectively used in Example 1, Example 2 and Comparative Example 1 were tested. The tests of the above three basic physical and chemical properties are as follows.

[0089] (1) pH value detection

[0090] Weigh 15g of organic matrix into a 50mL beaker, add distilled water in an appropriate proportion according to the type of organic matrix, stir evenly with a glass rod, let stand for 30 minutes to obtain a sample suspension, and use a pH meter to measure the pH value of the sample suspension. The experiment was repeated three times, and the average of the three experimental results was taken as the final pH value. The pH value test results of wheat bran, rice straw and corn cob are as follows: Figure 3 shown.

[0091] The pH value of the organic matrix as a carrier is an important factor affecting bacterial survival, and different bacteria have different optimal growth pH. Figure 3 It can be seen that the pH value of rice straw is the lowest, at 6.25; the pH value of corn cobs is the highest, at 7.93; and the pH value of wheat bran is 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 the growth and reproduction of bacteria.

[0092] According to the effective live bacterial count test results of the composite microbial solid fertilizers prepared from wheat bran, rice straw and corn cobs respectively, the most suitable pH range for the growth of Enterobacter hussini HS6 and Pantoea agglomerans CT3 of the present invention is between 6 and 7, which indicates that the organic matrix wheat bran and rice straw used in the present invention can promote the growth of Enterobacter hussini HS6 and Pantoea agglomerans CT3.

[0093] (2) Moisture content detection

[0094] Place the empty aluminum box in a drying oven at 105°C for 30 minutes to ensure that there is no moisture in the aluminum box. Take out the aluminum box and cool it to room temperature. Weigh its mass and record it. Weigh 20.00g of organic matrix that has passed a 1-mesh test sieve and place it in the aluminum box. After weighing the total mass, place it in a drying oven and dry it at 105°C for 5 hours to ensure that the moisture in the organic matrix is ​​completely evaporated. Take out the aluminum box and cool it at room temperature for 20 minutes. Weigh the total mass of the aluminum box and the organic matrix again. The moisture content is calculated according to the following formula. Perform three repeated experiments. The average of the three experimental results is taken as the final moisture content, and the value is expressed in %. Among them, the calculation formula of the moisture content is shown in Formula I.

[0095]

[0096] In formula I: w is the moisture content of the organic matrix (%); m 1 is the mass of the organic matrix and the aluminum box, in grams (g); m 2 is the mass of the organic matrix and aluminum box after drying, in grams (g); m 0 is the mass of the empty aluminum box in grams (g).

[0097] Among them, the test results of moisture content of wheat bran, rice straw and corn cob are as follows: Figure 4 shown.

[0098] The moisture content of the organic matrix as a carrier is one of the key factors affecting the survival of bacteria. Generally, the moisture content must be less than 30% to prepare bacterial fertilizer. Too high a moisture content will fill the void space in the carrier with water, hindering the circulation of air and causing the carrier to mold. Too low a moisture content will not meet the basic conditions for bacterial survival, resulting in reduced bacterial activity or death.

[0099] Depend on Figure 4 It can be seen that among the three organic matrices, corn cob has the highest moisture content, which is 9.55%, rice straw has the lowest moisture content, which is 7.37%, and wheat bran has a moisture content of 8.79%. This shows that the high moisture content of corn cob is not suitable for the growth of Enterobacter holmesii HS6 and Pantoea agglomerans CT3. The moisture content of wheat bran and rice straw can meet the moisture content standard as a carrier of solid bacterial fertilizer.

[0100] (3) Water absorption test

[0101] Weigh 100g of organic matrix and put it into a polyethylene bag. Use a pipette to add sterile water to the polyethylene bag in a 5mL volume gradient. Stir and mix with a glass rod until the organic matrix remains moist, loose and non-agglomerated. Record the total mass of sterile water used to reach this state. This mass is the maximum amount of liquid carried by 100g of organic matrix. The water absorption rate is calculated according to the following formula II. The experiment is repeated three times. The average of the three experimental results is taken as the final water absorption rate, and the value is expressed in %.

[0102]

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

[0104] Among them, the test results of water absorption rate of wheat bran, rice straw and corn cob are as follows: Figure 5 shown.

[0105] The water absorption rate of organic matrix as a carrier is an important parameter for evaluating the quality of bacterial fertilizer, which directly affects the growth of inoculated microorganisms. Within a certain range, a carrier with strong water absorption capacity can provide a more moist living environment for attached bacteria for a relatively long time, which is essential for maintaining bacterial activity and growth.

[0106] Depend on Figure 5It can be seen that the water absorption rate of wheat bran and rice straw is 100%, which has a suitable water absorption capacity, thereby promoting the growth of Enterobacter HS6 and Pantoea agglomerans CT3. The water absorption rate of corn cobs is 300%. Too high a water absorption rate will fill the void space in the corn cobs with water, hindering the circulation of air, causing the corn cobs to mold, which is not conducive to the growth of Enterobacter HS6 and Pantoea agglomerans CT3.

[0107] (III) Detection of the effects of composite microbial solid fertilizer on the physical and chemical properties of soil

[0108] The composite microbial solid fertilizer prepared in Example 1 was used to perform four fertilization treatments on the soil to detect the effect of the composite microbial solid fertilizer of the present invention on the physical and chemical properties of the soil. The specific design scheme of the fertilization treatment experiment is shown in Table 2 below.

[0109] Table 2 Fertilization treatment experimental design table

[0110] Experimental Group Fertilization treatment CK No fertilizer applied T1 Base fertilizer 20g T2 Base fertilizer 20g + 10d topdressing 2g T3 Base fertilizer 20g + 10d topdressing 4g

[0111] According to the experimental design in Table 2, the soil was fertilized respectively, and then the effects of the composite 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 respectively.

[0112] Among them, the effect of the composite microbial solid fertilizer of the present invention on the pH value of the soil is as follows: Figure 6 As shown. Figure 6 It can be seen that the composite microbial solid fertilizer of the present invention was subjected to three fertilization treatments of T1, T2 and T3, and observed from 0d to 21d. Compared with the case where no fertilizer (CK) was applied, the pH value of the soil was reduced, indicating that the composite microbial solid fertilizer of the present invention can alleviate the soil alkalinization to a certain extent, thereby facilitating plant growth.

[0113] Among them, the effect of the composite microbial solid fertilizer of the present invention on soil conductivity is as follows: Figure 7 As shown. Figure 7It can be seen that the composite microbial solid fertilizer of the present invention was subjected to three fertilization treatments of T1, T2 and T3, and observed from 0d to 21d. Compared with the case where no fertilizer (CK) was applied, the electrical conductivity of the soil was improved. This is because the decomposition activities of Enterobacter hussi HS6 and Pantoea agglomerans CT3 in the composite microbial solid fertilizer of the present invention will produce phosphates, ammonium salts, etc., which will increase the soil conductivity and reach a maximum value of 239.5 μs / cm on the 7th day, which is 1.7 times higher than that of the CK group. This may be because the produced inorganic salts are dissolved and distributed in the soil and are relatively stable on the 7th day. In addition, on the 14th and 21st days after fertilization, the conductivity of groups T1, T2 and T3 all decreased. This may be because Enterobacter HS6 and Pantoea agglomerans CT3 consumed the nutrients of the organic matrix wheat bran as a carrier, and needed to absorb inorganic salts from the soil environment to maintain normal life activities, which led to a decrease in inorganic salts in the soil, thereby reducing the conductivity. However, the conductivity of groups T1, T2 and T3 was still higher than that of group CK, indicating that the composite microbial solid fertilizer of the present invention can increase the inorganic salt content of the soil, thereby providing nutrients for plants and promoting plant growth.

[0114] Among them, the effect of the composite microbial solid fertilizer of the present invention on the available phosphorus content in the soil is as follows: Figure 8 As shown. Figure 8 It can be seen that the composite microbial solid fertilizer of the present invention is respectively subjected to three kinds of fertilization treatments of T1, T2 and T3, and observed from 0d to 21d, and the available phosphorus content in the soil is significantly increased relative to the situation where the fertilizer (CK) is not applied. Among them, the available phosphorus content of the 7th day applied by the base fertilizer reaches the highest value, which is 68.5mg / kg, which is 17.4 times higher than that of the CK group, but the content of the T1 group drops rapidly at the 14th day, which may be because the T1 group does not carry out topdressing at the 10th day, and the growth of Enterobacter HS6 and Pantoea agglomerans CT3 needs to absorb phosphorus nutrients from the soil to maintain its own metabolism, but at this time relative to the CK group, the available phosphorus content of the T1 group is still relatively high. It shows that the composite microbial solid fertilizer of the present invention can efficiently convert the insoluble phosphorus in the soil into soluble phosphorus, effectively improve the utilization rate of phosphorus in the soil, reduce the use of chemical phosphorus fertilizers, improve soil physical and chemical properties and soil fertility, promote the green and sustainable development of agricultural production, and have a good application prospect.

[0115] Among them, the effect of the composite microbial solid fertilizer of the present invention on the available potassium content of the soil is as follows: Fig. 9 As shown. Fig. 9It can be seen that the composite microbial solid fertilizer of the present invention is subjected to three kinds of fertilization treatments of T1, T2 and T3 respectively, and observed from 0d to 21d, relative to the situation that the fertilizer (CK) is not applied, the content of available potassium in the soil is significantly increased. The T1 group reaches a maximum value of 55.1mg / kg at the 7th day, the T2 group reaches a maximum value of 56.2mg / kg at the 14th day, and the T3 group reaches a maximum value of 58.1mg / kg at the 14th day. It is shown that the composite microbial solid fertilizer of the present invention can greatly increase the available potassium content in the soil, and increases with the increase of the quality of topdressing, so the composite microbial solid fertilizer of the present invention can increase nutrients for the soil, improve soil physical and chemical properties and soil fertility, promote the green sustainable development of agricultural production, and have a good application prospect. In addition, the available potassium content of the CK group continues to decline, which may be because the microorganisms existing in the soil absorb and utilize available potassium, thereby further showing that the composite microbial solid fertilizer of the present invention can greatly increase the available potassium content in the soil, and then increase potassium source nutrients for the soil, improve soil physical and chemical properties and soil fertility, promote plant growth, and promote the green sustainable development of agricultural production.

[0116] Among them, the effect of the composite microbial solid fertilizer of the present invention on the soil alkaline nitrogen content is as follows: Fig.10 As shown. Fig.10 It can be seen that the composite microbial solid fertilizer of the present invention is subjected to three kinds of fertilization treatments of T1, T2 and T3 respectively, and the alkaline nitrogen content of 0d, 7d, 14d and 21d is measured respectively, and the alkaline nitrogen content in the soil increases relative to the case where the fertilizer (CK) is not applied. However, as time goes by, the alkaline nitrogen content of the soil of the T1, T2 and T3 groups decreases significantly at the 7th, 14th and 21st days compared with the 0th day, because the organic nitrogen in the soil is required for growth and reproduction in the early stage by Enterobacter hussi HS6 and Pantoea agglomerans CT3. Among them, the alkaline nitrogen content of the T1, T2 and T3 groups at the 7th, 14th and 21st days is compared, and the alkaline nitrogen content first decreases and then increases, which may be caused by the direct and indirect nitrogen fixation of phosphate-soluble bacteria, and can convert nitrogen in the atmosphere into organic nitrogen that can be directly absorbed and utilized by crops.

[0117] 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.

[0118]

[0119]

Claims

1. A composite microbial solid fertilizer, characterized in that: Includes Enterobacter hallii HS6, Pantoea agglomerans CT3, culture medium and organic matrix; The classification name of the Enterobacter hormaechei HS6 is Enterobacter hormaechei, which was 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; The classification name of the Pantoea agglomerans CT3 is Pantoea agglomerans, which has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on January 8, 2025, with a deposit number of CGMCCNO.: 33335; The organic matrix is ​​at least one of wheat bran and rice straw.

2. The composite microbial solid fertilizer according to claim 1, characterized in that: The 16SrDNA gene sequence of the Enterobacter holmesii HS6 is shown in SEQ ID No. 1; The 16S rDNA gene sequence of the Pantoea agglomerans CT3 is shown in SEQ ID No.

2.

3. A composite microbial solid fertilizer according to claim 1, characterized in that: The particle size of the organic matrix is ​​a size that can pass through a 30-50 mesh sieve.

4. The method for preparing a composite microbial solid fertilizer according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1. Pretreatment of organic matrix: crushing and sieving the organic matrix and then sterilizing it at high temperature and high pressure to obtain an organic matrix carrier; S2. Activation culture: Enterobacter holmesii HS6 and Pantoea agglomerans CT3 were inoculated into LB medium respectively, and then placed in a constant temperature shaker at 28°C and cultured at 170 r / min-190 r / min for 24 h-48 h, and then centrifuged. The sediments were diluted with sterile water to obtain two activated bacterial suspensions; S3. Prepare a liquid composite phosphate-dissolving bacterial agent: use an inoculation loop to dip bacterial liquid from the two activated bacterial suspensions, and then inoculate them into LB liquid culture medium respectively, and then place them in a constant temperature shaker at 28°C and shake at 170r / min-190r / min to culture until the logarithmic growth phase to obtain HS6 bacterial liquid and CT3 bacterial liquid, and then mix the HS6 bacterial liquid and CT3 bacterial liquid to form a liquid composite phosphate-dissolving bacterial agent; S4, preparing solid bacterial fertilizer: adding the liquid composite phosphate-dissolving bacterial agent obtained in step S3 to the organic matrix carrier obtained in step S1, and then culturing at 28° C. for 2 days to 4 days to obtain the composite microbial solid bacterial fertilizer.

5. The method for preparing a composite microbial solid fertilizer according to claim 4, characterized in that: In step S1, the organic matrix is ​​crushed and then passed through a 30-50 mesh sieve; the conditions for high temperature and high pressure sterilization are: 121° C., 104 KPa, 20 min.

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

7. The method for preparing a composite microbial solid fertilizer according to claim 4, characterized in that: In step S4, the mass percentage of the liquid composite phosphate-dissolving bacteria agent to the organic matrix carrier is 25% to 35%; and / or In step S4, the organic matrix carrier obtained in step S1 is placed in a sterile polyethylene bag, the liquid composite phosphate-dissolving bacteria agent obtained in step S3 is added to the organic matrix carrier in the sterile polyethylene bag, the bag is sealed after mixing, 5 to 8 ventilation holes are pierced on the surface of the polyethylene bag with a sterilized needle, and then another layer of polyethylene bag is put in and 5 to 8 ventilation holes are pierced, and then the bag is cultured at 28°C for 2d-4d to obtain the composite microbial solid fertilizer.

8. Use of the composite microbial solid fertilizer according to any one of claims 1 to 3 in agricultural soil improvement.

9. Use of the composite microbial solid fertilizer according to any one of claims 1 to 3 in improving soil fertility.

10. Use of the composite microbial solid fertilizer according to any one of claims 1 to 3 in promoting plant growth.

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