Microbial fertilizer for soil improvement containing Bacillus megaterium and preparation method thereof

Abstract

The present invention discloses a soil-improving microbial fertilizer containing Bacillus megaterium and a preparation method thereof, belonging to the technical field of fertilizers. The soil-improving microbial fertilizer containing Bacillus megaterium comprises the following raw materials in parts by weight: 35-50 parts of an immobilized bacterial agent, 3-5 parts of a water-retaining agent, 10-15 parts of humic acid, and 2-5 parts of trehalose; the immobilized bacterial agent is amino-MOF-modified biochar loaded with Bacillus megaterium; the water-retaining agent is a water-absorbing resin carrying a phosphate group. The microbial fertilizer of the present invention comprises the immobilized bacterial agent, the water-retaining agent, the humic acid, and the trehalose, has strong targeting, and performs outstandingly in soil improvement. On the one hand, it activates available phosphorus in reclaimed soil to provide required nutrients for plants; on the other hand, it activates and regenerates soil organic matter, improves soil fertility, and meets plant nutrient requirements.

Description

Technical Field

[0001] The invention belongs to the technical field of fertilizers, and in particular relates to a microbial fertilizer for soil improvement containing Bacillus megaterium and a preparation method thereof. Background Art

[0002] Land in coal mining subsidence areas is usually first reclaimed by engineering. After the reclamation, the subsoil layer is covered on the topsoil layer, resulting in poor soil nutrients and prominent problems of "lack of ammonia and phosphorus". In order to quickly restore the fertility of the reclaimed soil and increase crop yields, farmers usually apply a large amount of inorganic phosphorus fertilizer. However, most of the phosphorus in calcareous soil is consumed by Ca in the soil. 2+ Mg 2+ Isocations are fixed in an ineffective state that is difficult for plants to utilize and accumulate in the soil, resulting in low crop yields.

[0003] In this context, microbial agents, as a highly efficient, green and environmentally friendly biological fertilizer, play an important role in improving soil microbial diversity, increasing soil nutrients, and promoting plant growth. However, existing microbial fertilizers are not very targeted, resulting in poor application effects in soil reclamation and improvement. Summary of the Invention

[0004] The present invention provides a soil improvement microbial fertilizer containing Bacillus megaterium and a preparation method thereof, which can solve the problem of poor application effect of microbial fertilizers in the prior art in improving reclaimed soil.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] A soil improving microbial fertilizer containing Bacillus megaterium comprises the following raw materials in parts by weight:

[0007] 35-50 parts of immobilized bacteria agent, 3-5 parts of water retaining agent, 10-15 parts of humic acid, 2-5 parts of trehalose.

[0008] The immobilized bacterial agent is amino MOF-modified biochar loaded with Bacillus megaterium.

[0009] The Bacillus megaterium is classified and named Bacillus megaterium YJ004, and the preservation location is China Center for Type Culture Collection. The preservation time is November 22, 2021, and the preservation number is CCTCC NO: M 20211463. Its 16SrRDNA sequence is shown in SEQ ID NO.1.

[0010] The water-retaining agent is a water-absorbing resin carrying a phosphate group.

[0011] As a preferred technical solution of the present invention, the preparation method of the immobilized bacterial agent is as follows:

[0012] The amino-MOF-modified biochar was added to the fermentation broth of Bacillus megaterium, and then placed on a shaker for oscillation adsorption at a temperature of 25-35°C for 12-36 hours. Finally, the biochar was centrifuged, the precipitate was collected, and naturally dried to obtain an immobilized bacterial agent.

[0013] As a preferred technical solution of the present invention, the usage ratio of the amino MOF modified biochar and the Bacillus megaterium fermentation broth is 1 g: 10-30 mL.

[0014] As a preferred technical solution of the present invention, the shaking table has a rotation speed of 100-200 rpm.

[0015] As a preferred technical solution of the present invention, the centrifugation is carried out at 25° C. and 1500 r / min for 5 minutes.

[0016] As a preferred technical solution of the present invention, the preparation method of the Bacillus megaterium fermentation broth is as follows:

[0017] Bacillus megaterium was inoculated into liquid culture medium and cultured at 25-40°C for 24-48 hours to obtain a Bacillus megaterium content of 2×10 7 cfu / mL-2×10 8 cfu / mL of Bacillus megaterium fermentation broth.

[0018] As a preferred technical solution of the present invention, the liquid culture medium is prepared according to the following raw materials in the following mass ratio: 5 parts of sodium alginate, 5 parts of ammonium sulfate, 1 part of magnesium sulfate, 2 parts of dipotassium hydrogen phosphate, 0.01 part of ferrous sulfate, 20 parts of agar and 1000 parts of distilled water.

[0019] As a preferred technical solution of the present invention, the raw materials for preparing the amino MOF-modified biochar include biochar, FeCl3·6H20, and 2-aminoterephthalic acid, and the mass ratio of biochar, FeCl3·6H20, and 2-aminoterephthalic acid is 0.12g:0.54-1.62g:0.18-0.54g.

[0020] As a preferred technical solution of the present invention, the preparation method of amino MOF modified biochar comprises the following steps:

[0021] The biochar was ultrasonically dispersed in N,N-dimethylformamide, and then FeCl3·6H20 and 2-aminoterephthalic acid were added and stirred for 1-2 hours to obtain a mixed solution. The mixed solution was transferred to a reactor and reacted at 120°C for 24 hours. After the reaction, it was cooled to room temperature and filtered. The filter cake was washed with anhydrous ethanol and deionized water in sequence and finally dried.

[0022] As a preferred technical solution of the present invention, the biochar is ash charcoal from the combustion of biomass, and the biomass is selected from at least one of corn straw, sorghum straw, sunflower straw, rice straw, soybean straw, and rice husk.

[0023] As a preferred technical solution of the present invention, the raw materials for preparing the water-retaining agent include acrylic acid, acrylamide, N,N'-methylenebisacrylamide, potassium persulfate, 2-methyl-2-acrylic acid-2-(phosphonooxy)ethyl ester, sodium hydroxide, and deionized water.

[0024] As a preferred technical solution of the present invention, the preparation method of the water-retaining agent comprises the following steps:

[0025] Under ice-water bath conditions, acrylic acid was added to deionized water, and sodium hydroxide was added for neutralization to a neutralization degree of 70%. Then, acrylamide and 2-methyl-2-acrylic acid-2-(phosphonooxy)ethyl ester were added, and the mixture was stirred evenly. The temperature was raised to 75-80°C under nitrogen protection, N,N'-methylenebisacrylamide and potassium persulfate were added, and the mixture was stirred and reacted for 2 hours. The reaction product was washed with deionized water, dried, and crushed to pass through a 40-60 mesh sieve to obtain a water retaining agent.

[0026] As a preferred technical solution of the present invention, the molar ratio of acrylic acid, acrylamide and 2-methyl-2-acrylate-2-(phosphonooxy)ethyl ester is 8:1-1.5:0.5-1, the amount of N,N'-methylenebisacrylamide is 0.05% of the total mass of acrylic acid, acrylamide and 2-methyl-2-acrylate-2-(phosphonooxy)ethyl ester, and the amount of potassium persulfate is twice the mass of N,N'-methylenebisacrylamide.

[0027] The method for preparing the soil-improving microbial fertilizer containing Bacillus megaterium comprises the following steps:

[0028] According to the formula ratio, mix the immobilized bacteria agent, water-retaining agent, humic acid and trehalose evenly.

[0029] Beneficial effects of the present invention:

[0030] 1. The Bacillus megaterium provided by the present invention was sampled from a reclamation test field of the Guangde Nata oak forest in Anhui Province, and then the dominant strain was screened through high-throughput sequencing and then streaking isolation. It exhibits excellent phosphate solubilization in the improvement of reclaimed soil, can quickly repair reclaimed soil, and can also release plant growth hormones (such as IAA), enhance plant root development, improve crop resistance and increase crop yield.

[0031] 2. The microbial fertilizer of the present invention is composed of immobilized bacterial agents, water-retaining agents, humic acid, and trehalose. It is highly targeted and performs outstandingly in soil improvement. On the one hand, it activates the available phosphorus in the reclaimed soil and can provide the required nutrients for plants. On the other hand, it can activate and renew soil organic matter, improve soil fertility, and meet the nutrient needs of plants.

[0032] 3. In order to improve the stability and activity of Bacillus megaterium in reclaimed soil, the present invention uses amino MOF-modified biochar to load Bacillus megaterium. Amino MOF-modified biochar can provide an effective shelter for Bacillus megaterium and improve the adaptability of Bacillus megaterium to the living environment. Compared with activated carbon as a carrier, the average pore size of amino MOF-modified biochar is improved, the specific surface area is larger, and it is more conducive to the adsorption of Bacillus megaterium. In addition, the cell wall surface of Bacillus megaterium carries negatively charged functional groups such as carboxylic acid groups and phosphate groups, while the amino groups on the surface of amino MOF-modified biochar are positively charged in aqueous solution, which can promote the binding of Bacillus megaterium to the carrier. Therefore, the amino MOF-modified biochar provided by the present invention has a significantly improved adsorption capacity for Bacillus megaterium, which is beneficial to increase the loading amount of active bacteria in microbial fertilizers, thereby enhancing its soil improvement efficiency.

[0033] 4. The present invention introduces a water-retaining agent into the microbial fertilizer. The water-retaining agent is a water-absorbing resin carrying a phosphate group. When the microbial fertilizer is added to the reclaimed soil, the water-retaining agent can absorb water and retain fertilizer, improve the soil aggregate structure, inhibit the evaporation of water on the soil surface, and provide a suitable humidity environment for the growth, reproduction, and metabolic activities of Bacillus megaterium. In addition, the phosphate group in the water-retaining agent carries a negative charge and can form an electrostatic interaction with the amino-MOF-modified biochar. On the one hand, it can reduce the loss of Bacillus megaterium, and on the other hand, it can enable the two to cooperate with each other in water adsorption to form a more stable hydration layer. When the soil water evaporates, it can reduce water loss, keep the soil moist, and improve the soil's water retention performance. DETAILED DESCRIPTION

[0034] In order to make the technical problems, technical solutions and beneficial effects to be solved by this application more clearly understood, the present application 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 this application and are not intended to limit this application.

[0035] The weights of the relevant components mentioned in the examples of this application may not only refer to the specific content of each component, but also represent the weight ratio between the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the examples of this application, it is within the scope disclosed in the examples of this application. Specifically, the mass described in the examples of this application may be a mass unit known in the chemical industry, such as μg, mg, g, kg, etc.

[0036] Various raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

[0037] Preparation Example 1

[0038] This preparation example provides an amino-MOF-modified biochar, and the preparation method is as follows:

[0039] 0.12 g of biochar was ultrasonically dispersed in 60 mL of N,N-dimethylformamide, and then 0.54 g of FeCl3·6H20 and 0.18 g of 2-aminoterephthalic acid were added and stirred for 1 h to obtain a mixed solution. The mixed solution was transferred to a reactor and reacted at 120°C for 24 h. After the reaction, it was cooled to room temperature and filtered. The filter cake was washed with anhydrous ethanol and deionized water in sequence and finally dried.

[0040] The biochar preparation process is as follows:

[0041] The rice husk was washed three times with deionized water, dried at 80°C for 24 h, and then added to a tubular furnace. Under a nitrogen atmosphere, the temperature was increased at a rate of 3°C / min to 750°C, kept warm for 2 h, and then cooled to room temperature to obtain biochar.

[0042] Preparation Example 2

[0043] This preparation example provides an amino-MOF-modified biochar, and the preparation method is as follows:

[0044] 0.12 g of biochar was ultrasonically dispersed in 70 mL of N,N-dimethylformamide, and then 1.08 g of FeCl3·6H20 and 0.36 g of 2-aminoterephthalic acid were added and stirred for 1.5 h to obtain a mixed solution. The mixed solution was transferred to a reactor and reacted at 120°C for 24 h. After the reaction, it was cooled to room temperature and filtered. The filter cake was washed with anhydrous ethanol and deionized water in sequence and finally dried.

[0045] The biochar preparation process is the same as that of Preparation Example 1.

[0046] Preparation Example 3

[0047] This preparation example provides an amino-MOF-modified biochar, and the preparation method is as follows:

[0048] 0.12 g of biochar was ultrasonically dispersed in 80 mL of N,N-dimethylformamide, and then 1.62 g of FeCl3·6H20 and 0.54 g of 2-aminoterephthalic acid were added and stirred for 2 h to obtain a mixed solution. The mixed solution was transferred to a reactor and reacted at 120°C for 24 h. After the reaction, it was cooled to room temperature and filtered. The filter cake was washed with anhydrous ethanol and deionized water in sequence and finally dried.

[0049] The biochar preparation process is the same as that of Preparation Example 1.

[0050] Comparative Example 1

[0051] This comparative example provides an amino-MOF-modified biochar. Compared with Preparation Example 1, the difference is that the 2-aminoterephthalic acid in Preparation Example 1 is replaced by an equal molar amount of terephthalic acid.

[0052] Comparative Example 2

[0053] This control example is biochar, and the biochar preparation process is the same as that of Preparation Example 1.

[0054] The specific surface area and average pore size of the products in Preparation Examples 1 to 3 and Control Examples 1 and 2 were measured using a specific surface area and pore size analyzer, and then the microbial adsorption rate was calculated. The specific process is as follows:

[0055] (1) Bacillus megaterium (classified as Bacillus megaterium YJ004, deposited at China Center for Type Culture Collection, with a deposit number of CCTCC NO: M 20211463) was inoculated into liquid culture medium and cultured at 37°C for 24 h. The Bacillus megaterium content was 4.5×10 7 cfu / mL of Bacillus megaterium fermentation broth.

[0056] (2) The liquid culture medium was prepared according to the following mass ratios: 5 parts sodium alginate, 5 parts ammonium sulfate, 1 part magnesium sulfate, 2 parts dipotassium hydrogen phosphate, 0.01 parts ferrous sulfate, 20 parts agar, and 1000 parts distilled water.

[0057] (3) Using the products of Preparation Example 1-Preparation Example 3 and Control Example 1-Control Example 2 as carriers, 2 g of the carrier was added to 20 mL of Bacillus megaterium fermentation broth at a ratio of 1 g:10 mL of the carrier to the Bacillus megaterium fermentation broth, and the mixture was shaken and adsorbed at 25°C for 24 h. The mixture was then centrifuged at 25°C and 1500 r / min for 5 min. The supernatant was taken and the number of viable bacteria in the supernatant was measured by the plate counting method. The adsorption rate was calculated according to the formula adsorption rate (%) = (X0-X1) / X0×100%, where X0 is the number of viable bacteria in the original fermentation broth and X1 is the number of viable bacteria in the supernatant.

[0058] The test results are shown in Table 1:

[0059] Table 1

[0060]

[0061] Analysis of the data recorded in Table 1 shows that compared with Control Example 2, the amino-MOF-modified biochars in Preparation Examples 1 to 3 have larger specific surface areas, average pore sizes, and adsorption rates, among which Preparation Example 2 has the best comprehensive performance. From the test results of Preparation Example 1 and Control Example 1, it can be seen that when 2-aminoterephthalic acid in the preparation process of amino-MOF-modified biochar is replaced with an equal molar amount of terephthalic acid, the adsorption effect of the obtained product on the bacterial liquid is significantly worse.

[0062] Example 1 Isolation and identification of Bacillus megaterium

[0063] 1. Isolation of bacterial strains

[0064] Soil samples were collected from a reclamation pilot field of Quercus nata woodland in Guangde, Anhui Province. High-throughput sequencing was used to analyze the soil microbial community and screen for target strains. The specific procedures were as follows:

[0065] Weigh 10 g of soil sample and add it to a conical flask containing 90 mL of sterile physiological saline and glass beads. Oscillate on a shaker at 180 rpm for 30 min, then place in a 75°C water bath for 15 min to inhibit the growth of bacteria to obtain a mixed solution.

[0066] The mixture was diluted tenfold, and 100 μL of the dilution was evenly spread on LB plate medium (nutrient broth solid plate). The plate was then placed in a 30°C constant temperature incubator for 48 hours, and a single suspected Bacillus megaterium colony was picked;

[0067] The colonies were purified multiple times on fresh LB plates by the streak method until no bacterial contamination was observed under a microscope. A strain with morphology and characteristics consistent with those of Bacillus megaterium was successfully isolated and named YJ004.

[0068] 2. Strain Identification

[0069] (1) Identification based on morphological and physiological and biochemical characteristics

[0070] According to the standard methods in the Bergey's Manual of Bacterial Identification (8th Edition) and the Manual of Systematic Identification of Common Bacteria, a systematic analysis of the morphological characteristics, cultural characteristics, and physiological and biochemical properties of Bacillus megaterium YJ004 was conducted. The results are as follows:

[0071] Morphological and physiological and biochemical characteristics of the strain:

[0072] After 48 hours of incubation at 30°C on LB medium, colonies appeared milky white, round, with a smooth, moist surface, neat edges, and a large diameter (3-5 mm), typical of Bacillus megaterium. Microscopically, the bacteria were straight rod-shaped, Gram-positive, and thickly rod-shaped (1.2-1.5 μm × 2.0-4.0 μm), forming oval terminal spores.

[0073] Physiological and biochemical characteristics of the strain:

[0074] Gelatin liquefaction: positive (+), catalase activity: positive (+), VP reaction: negative (-), indole formation: negative (-), citrate utilization: positive (+), glucose fermentation: positive (+), gas production, starch hydrolysis: positive (+), nitrate reduction: positive (+);

[0075] Salt tolerance: It grows well under 3% NaCl conditions, but its growth is restricted under 7% NaCl conditions.

[0076] These results were highly consistent with the typical characteristics of Bacillus subtilis, indicating that the strain was Bacillus subtilis.

[0077] (2) 16S rDNA test

[0078] DNA from strain YJ004 was extracted, and the 16S rRNA gene was amplified and sequenced. The resulting sequence is shown in SEQ ID NO. 1. Subsequently, comparison with the GenBank database revealed that strain YJ004 shared 99.5% homology with Bacillus megaterium strain ATCC 14581 (accession number NR074540.1), confirming its identity as Bacillus megaterium.

[0079] Based on the above characteristics, the strain YJ004 was named Bacillus megaterium YJ004 and deposited in the China Center for Type Culture Collection on November 22, 2021, with the preservation number CCTCC NO: M 20211463, and the preservation address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0080] Example 2

[0081] This embodiment provides a soil-improving microbial fertilizer containing Bacillus megaterium, comprising the following raw materials in parts by weight:

[0082] 35 parts of immobilized bacteria agent, 3 parts of water retaining agent, 10 parts of humic acid, and 2 parts of trehalose.

[0083] The preparation method of the immobilized bacterial agent is as follows:

[0084] 100 g of the amino-MOF-modified biochar of Preparation Example 1 was added to 1000 mL of Bacillus megaterium fermentation broth, and then placed on a shaker with a shaker speed of 100 rpm and an oscillation adsorption temperature of 25°C for 12 h. Finally, the mixture was centrifuged at 25°C and 1500 r / min for 5 min, the precipitate was collected, and naturally dried to obtain an immobilized bacterial agent.

[0085] The preparation method of the Bacillus megaterium fermentation broth is as follows:

[0086] The Bacillus megaterium YJ004 in Example 1 was inoculated into a liquid culture medium and cultured at 25°C for 24 h to obtain a Bacillus megaterium content of 2×10 7 cfu / mL of Bacillus megaterium fermentation broth.

[0087] The liquid culture medium is prepared according to the following raw materials in the following mass ratio: 5 parts of sodium alginate, 5 parts of ammonium sulfate, 1 part of magnesium sulfate, 2 parts of dipotassium hydrogen phosphate, 0.01 part of ferrous sulfate, 20 parts of agar and 1000 parts of distilled water.

[0088] The preparation method of the water-retaining agent comprises the following steps:

[0089] Under ice-water bath conditions, 0.8 mol of acrylic acid was added to 500 mL of deionized water, and sodium hydroxide was added for neutralization to a degree of neutralization of 70%. Then, 0.1 mol of acrylamide and 0.1 mol of 2-methyl-2-acrylic acid-2-(phosphonooxy)ethyl ester were added, and stirred evenly. The temperature was raised to 75 ° C under nitrogen protection, 0.043 g of N, N'-methylenebisacrylamide and 0.086 g of potassium persulfate were added, and the mixture was stirred and reacted for 2 h. The reaction product was washed with deionized water, dried, and crushed through a 40-mesh sieve to obtain a water-retaining agent.

[0090] The method for preparing the soil-improving microbial fertilizer containing Bacillus megaterium comprises the following steps:

[0091] According to the formula ratio, add the immobilized bacteria agent, water-retaining agent, humic acid and trehalose into the blender and stir at 100 r / min for 30 minutes at room temperature.

[0092] Example 3

[0093] This embodiment provides a soil-improving microbial fertilizer containing Bacillus megaterium, comprising the following raw materials in parts by weight:

[0094] 40 parts of immobilized bacteria agent, 4 parts of water retaining agent, 12 parts of humic acid, and 4 parts of trehalose.

[0095] The preparation method of the immobilized bacterial agent is as follows:

[0096] 100 g of the amino-MOF-modified biochar of Preparation Example 1 was added to 2000 mL of Bacillus megaterium fermentation broth, and then placed on a shaker with a shaker speed of 150 rpm and an oscillation adsorption temperature of 30°C for 24 h. Finally, it was centrifuged at 25°C and 1500 r / min for 5 min, the precipitate was collected, and naturally dried to obtain an immobilized bacterial agent.

[0097] The preparation method of the Bacillus megaterium fermentation broth is as follows:

[0098] The Bacillus megaterium YJ004 in Example 1 was inoculated into a liquid culture medium and cultured at 30°C for 36 h to obtain a Bacillus megaterium content of 6.5×10 7 cfu / mL of Bacillus megaterium fermentation broth.

[0099] The liquid culture medium is prepared according to the following raw materials in the following mass ratio: 5 parts of sodium alginate, 5 parts of ammonium sulfate, 1 part of magnesium sulfate, 2 parts of dipotassium hydrogen phosphate, 0.01 part of ferrous sulfate, 20 parts of agar and 1000 parts of distilled water.

[0100] The preparation method of the water-retaining agent is the same as that of Example 2.

[0101] The preparation method of the soil improving microbial fertilizer containing Bacillus megaterium is the same as that in Example 2.

[0102] Example 4

[0103] This embodiment provides a soil-improving microbial fertilizer containing Bacillus megaterium, comprising the following raw materials in parts by weight:

[0104] 50 parts of immobilized bacteria agent, 5 parts of water retaining agent, 15 parts of humic acid, and 5 parts of trehalose.

[0105] The preparation method of the immobilized bacterial agent is as follows:

[0106] 100 g of the amino-MOF-modified biochar of Preparation Example 1 was added to 3000 mL of Bacillus megaterium fermentation broth, and then placed on a shaker with a shaker speed of 200 rpm and an oscillation adsorption temperature of 35°C for 36 hours. Finally, it was centrifuged at 25°C and 1500 r / min for 5 minutes, the precipitate was collected, and naturally dried to obtain an immobilized bacterial agent.

[0107] The preparation method of the Bacillus megaterium fermentation broth is as follows:

[0108] The Bacillus megaterium YJ004 in Example 1 was inoculated into a liquid culture medium and cultured at 37°C for 48 h to obtain a Bacillus megaterium content of 2×10 8 cfu / mL of Bacillus megaterium fermentation broth.

[0109] The liquid culture medium is prepared according to the following raw materials in the following mass ratio: 5 parts of sodium alginate, 5 parts of ammonium sulfate, 1 part of magnesium sulfate, 2 parts of dipotassium hydrogen phosphate, 0.01 part of ferrous sulfate, 20 parts of agar and 1000 parts of distilled water.

[0110] The preparation method of the water-retaining agent is the same as that of Example 2.

[0111] The preparation method of the soil improving microbial fertilizer containing Bacillus megaterium is the same as that in Example 2.

[0112] Example 5

[0113] This embodiment provides a soil improvement microbial fertilizer containing Bacillus megaterium. Compared with Example 2, the difference is that the amino MOF-modified biochar in Example 2 is replaced by the product obtained in Preparation Example 2 of equal mass.

[0114] Example 6

[0115] This embodiment provides a soil improvement microbial fertilizer containing Bacillus megaterium. Compared with Example 2, the difference is that the amino MOF-modified biochar in Example 2 is replaced by the product obtained in Preparation Example 3 of equal mass.

[0116] Example 7

[0117] This embodiment provides a soil-improving microbial fertilizer containing Bacillus megaterium. Compared with Example 2, the difference lies in the use of a different water-retaining agent. The preparation method of the water-retaining agent in this embodiment includes the following steps:

[0118] Under ice-water bath conditions, 0.8 mol of acrylic acid was added to 550 mL of deionized water, and sodium hydroxide was added for neutralization to a neutralization degree of 70%. Then, 0.15 mol of acrylamide and 0.05 mol of 2-methyl-2-acrylic acid-2-(phosphonooxy)ethyl ester were added. After stirring evenly, the temperature was raised to 78 ° C under nitrogen protection, 0.039 g of N, N'-methylenebisacrylamide and 0.079 g of potassium persulfate were added, and the mixture was stirred and reacted for 2 h. The reaction product was washed with deionized water, dried, and crushed through a 40-mesh sieve to obtain a water-retaining agent.

[0119] Example 8

[0120] This embodiment provides a soil-improving microbial fertilizer containing Bacillus megaterium. Compared with Example 2, the difference lies in the use of a different water-retaining agent. The preparation method of the water-retaining agent in this embodiment includes the following steps:

[0121] Under ice-water bath conditions, 0.8 mol of acrylic acid was added to 550 mL of deionized water, and sodium hydroxide was added for neutralization to a neutralization degree of 70%. Then, 0.13 mol of acrylamide and 0.07 mol of 2-methyl-2-acrylic acid-2-(phosphonooxy)ethyl ester were added. After stirring evenly, the temperature was raised to 80°C under nitrogen protection, 0.04 g of N,N'-methylenebisacrylamide and 0.08 g of potassium persulfate were added, and the mixture was stirred and reacted for 2 h. The reaction product was washed with deionized water, dried, and crushed through a 60-mesh sieve to obtain a water-retaining agent.

[0122] Comparative Example 1

[0123] This embodiment provides a soil improvement microbial fertilizer containing Bacillus megaterium. Compared with Example 2, the difference is that the amino MOF-modified biochar in Example 2 is replaced by the product obtained in Control Example 1 of equal mass.

[0124] Comparative Example 2

[0125] This embodiment provides a soil improvement microbial fertilizer containing Bacillus megaterium. Compared with Example 2, the difference is that the amino MOF-modified biochar in Example 2 is replaced by the product obtained in Control Example 2 of equal mass.

[0126] Comparative Example 3

[0127] This embodiment provides a soil improvement microbial fertilizer containing Bacillus megaterium. Compared with Example 2, the difference is that the 2-methyl-2-acrylic acid-2-(phosphonooxy)ethyl ester in Example 2 is replaced by an equimolar amount of acrylamide.

[0128] Comparative Example 4

[0129] This embodiment provides a soil improving microbial fertilizer containing Bacillus megaterium. Compared with Example 2, the difference is that the water retaining agent of Example 1 is removed.

[0130] (1) The water retention performance of the microbial fertilizers prepared in Examples 2 to 8 and Comparative Examples 1 to 4 was tested. The water retention rate was determined using the following method:

[0131] At a temperature of 28°C, add 200g of reclaimed soil to 4g of sample and stir to mix evenly. The moisture content of the reclaimed soil is 4%. Then add 200g of water to the mixed sample and weigh it. It is recorded as W. i After that, the sample was allowed to stand for 15 days and weighed again, which was recorded as W. n . And calculate the soil water evaporation rate, soil water evaporation rate (%) = (W i -W n ) / 200×100%, the test results are shown in Table 2;

[0132] (2) The microbial fertilizers prepared in Examples 2 to 8 and Comparative Examples 1 to 4 were added to the reclaimed soil, and their improvement effects on the reclaimed soil were tested through potting experiments. The specific method is as follows:

[0133] Soil samples were collected from soil surrounding a coal mining subsidence area in Bengbu City, Anhui Province. The vegetable tested was dwarf sedge. Eleven soil samples were treated with the microbial fertilizers prepared in Examples 2-8 and Comparative Examples 1-4 at a rate of 4 g / kg soil (4‰). The potting containers were 150 mm × 125 mm (upper diameter × height) plastic pots, each containing 1.0 kg of air-dried, reclaimed soil. Each microbial fertilizer, applied at the prescribed rate, was evenly mixed with the soil and potted thoroughly before sowing. Six dwarf sedge seeds, which had already germinated and turned white, were sown in the pots. The soil was kept moist from sowing until seedlings emerged. Water was applied every 2-3 days after emergence, maintaining a maximum field water capacity of 40%. Thinning was performed when two to three true leaves appeared, with seedlings spaced 3-4 cm apart, leaving two plants per pot. Water was then applied once after thinning. The plants were harvested 40 days later, and soil and plant samples were collected. The soil organic matter, available nitrogen, phosphorus and potassium contents and vegetable biomass were measured. The results are shown in Table 2:

[0134] Table 2

[0135]

[0136] Analysis of the data recorded in Table 2 shows that, compared with Example 2, the reclaimed soil improved by the microbial fertilizers obtained in Examples 3 and 4 has a lower soil moisture evaporation rate, higher organic matter and available phosphorus content in the soil, and higher fresh weight and dry weight of vegetables. Therefore, the improvement effect is better;

[0137] It can be seen from the test results of Example 2, Example 5 and Example 6 that when the microbial fertilizer formula and preparation process remain unchanged, the amino MOF-modified biochar prepared in Preparation Example 1, Preparation Example 2 and Preparation Example 3 is used to load Bacillus megaterium YJ004 to prepare the microbial feed. Preparation Example 2 has the best effect. The reason is that the amino MOF-modified biochar in Preparation Example 2 has a higher specific surface area and average pore size, can load more Bacillus megaterium YJ004 and provide it with a comfortable living environment, so that it can efficiently play the role of phosphate solubilization;

[0138] From the test results of Example 2, Example 7 and Example 8, it can be seen that when the content of 2-methyl-2-acrylic acid-2-(phosphonooxy)ethyl ester is reduced during the preparation of the water-retaining agent, the water-retaining performance of the prepared microbial fertilizer decreases, and the improvement effect on the reclaimed soil is slightly worse;

[0139] From the test results of Example 2 and Comparative Example 1, it can be seen that the MOF-modified biochar prepared in Comparative Example 1 loaded with Bacillus megaterium YJ004 was used to prepare the microbial feed. Due to the lack of amino groups, its water retention performance was significantly reduced, and the organic matter and available phosphorus content in the reclaimed soil were significantly reduced;

[0140] From the test results of Example 2 and Comparative Example 2, it can be seen that the use of the biochar prepared in Control Example 2 to load Bacillus megaterium YJ004 to prepare the microbial feed has a significant decrease in water retention performance due to limited loading performance and the inability to interact with the water-retaining agent, and the organic matter and available phosphorus content in the reclaimed soil are significantly reduced;

[0141] From the test results of Example 2 and Comparative Example 3, it can be seen that replacing 2-methyl-2-acrylic acid-2-(phosphonooxy)ethyl ester with an equimolar amount of acrylamide during the preparation of the water-retaining agent will lead to a decrease in the water-retaining performance of the microbial fertilizer, and the organic matter and available phosphorus content in the reclaimed soil will also be significantly reduced.

[0142] It can be seen from the test results of Example 2 and Comparative Example 4 that when the water-retaining agent of the present invention is omitted, the soil moisture evaporation rate is significantly increased, the organic matter and available phosphorus content in the soil are reduced, the fresh weight and dry weight of vegetables are also lower, and the soil improvement effect is poor.

[0143] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus.

[0144] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A soil improving microbial fertilizer containing Bacillus megaterium, characterized in that: It includes the following raw materials in parts by weight: 35-50 parts of immobilized bacteria agent, 3-5 parts of water-retaining agent, 10-15 parts of humic acid, 2-5 parts of trehalose; The immobilized bacterial agent is amino MOF-modified biochar loaded with Bacillus megaterium; The water-retaining agent is a water-absorbing resin carrying a phosphate group; The Bacillus megaterium is classified and named Bacillus megaterium YJ004, and is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20211463. The 16SrRDNA sequence thereof is shown in SEQ ID NO.1; The phosphate groups in the water retaining agent carry negative charges and can form electrostatic interactions with amino-MOF-modified biochar; The preparation method of the amino MOF-modified biochar comprises the following steps: The biochar was ultrasonically dispersed in N,N-dimethylformamide, and then FeCl3·6H20 and 2-aminoterephthalic acid were added and stirred for 1-2 hours to obtain a mixed solution. The mixed solution was transferred to a reactor and reacted at 120°C for 24 hours. After the reaction, it was cooled to room temperature and filtered. The filter cake was washed with anhydrous ethanol and deionized water in sequence and finally dried. Preparation method of water retaining agent: under ice water bath conditions, acrylic acid is added to deionized water, and sodium hydroxide is added for neutralization to a neutralization degree of 70%, and then acrylamide and 2-methyl-2-acrylic acid-2-(phosphonooxy)ethyl ester are added. After stirring evenly, the temperature is raised to 75-80°C under nitrogen protection, N,N'-methylenebisacrylamide and potassium persulfate are added, and the mixture is stirred and reacted for 2 hours. The reaction product is washed with deionized water, dried, and crushed to pass through a 40-60 mesh sieve to obtain a water retaining agent.

2. The method for preparing a soil improving microbial fertilizer containing Bacillus megaterium according to claim 1, wherein: The following steps are involved: S1. Preparation of immobilized bacterial agent: Add amino-MOF-modified biochar to Bacillus megaterium fermentation broth, then place on a shaker, shake and adsorb at 25-35°C for 12-36 hours, and finally centrifuge, collect the precipitate, and air-dry to obtain the immobilized bacterial agent; S2. Preparation method of a water-retaining agent: acrylic acid is added to deionized water in an ice-water bath, and sodium hydroxide is added for neutralization to a degree of neutralization of 70%. Then, acrylamide and 2-(phosphonooxy)ethyl 2-methyl-2-acrylate are added, and the mixture is stirred uniformly. The mixture is heated to 75-80° C. under nitrogen protection, and N,N'-methylenebisacrylamide and potassium persulfate are added. The mixture is stirred and reacted at this temperature for 2 hours. The reaction product is washed with deionized water, dried, and pulverized through a 40-60 mesh sieve to obtain a water-retaining agent. S3, mixing the immobilized bacterial agent, water-retaining agent, humic acid and trehalose to obtain a soil-improving microbial fertilizer containing Bacillus megaterium; The preparation method of the amino MOF-modified biochar comprises the following steps: The biochar was ultrasonically dispersed in N,N-dimethylformamide, and then FeCl3·6H20 and 2-aminoterephthalic acid were added and stirred for 1-2 hours to obtain a mixed solution. The mixed solution was transferred to a reactor and reacted at 120°C for 24 hours. After the reaction, it was cooled to room temperature and filtered. The filter cake was washed with anhydrous ethanol and deionized water in sequence and finally dried.

3. The method for preparing a soil improving microbial fertilizer containing Bacillus megaterium according to claim 2, wherein: The usage ratio of the amino MOF-modified biochar and the Bacillus megaterium fermentation broth in S1 is 1 g: 10-30 mL.

4. The method for preparing a soil improving microbial fertilizer containing Bacillus megaterium according to claim 2, wherein: The preparation method of the Bacillus megaterium fermentation broth is as follows: Bacillus megaterium was inoculated into liquid culture medium and cultured at 25-40°C for 24-48 hours to obtain a Bacillus megaterium content of 2×10 7 cfu / mL-2×10 8 cfu / mL of Bacillus megaterium fermentation broth.

5. The method for preparing a soil improving microbial fertilizer containing Bacillus megaterium according to claim 2, wherein: The raw materials for preparing the amino MOF-modified biochar include biochar, FeCl3·6H20, and 2-aminoterephthalic acid, and the mass ratio of biochar, FeCl3·6H20, and 2-aminoterephthalic acid is 0.12:0.54-1.62:0.18-0.

54.

6. The method for preparing a soil improving microbial fertilizer containing Bacillus megaterium according to claim 2, wherein: The molar ratio of acrylic acid, acrylamide and 2-methyl-2-acrylic acid-2-(phosphonooxy)ethyl ester in S2 is 8:1-1.5:0.5-1, the amount of N,N'-methylenebisacrylamide is 0.05% of the total mass of acrylic acid, acrylamide and 2-methyl-2-acrylic acid-2-(phosphonooxy)ethyl ester, and the amount of potassium persulfate is 2 times the mass of N,N'-methylenebisacrylamide.

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