Bacillus megaterium-containing microbial fertilizer for soil improvement and preparation method thereof

Abstract

The invention discloses a bacillus megaterium-containing microbial fertilizer for soil improvement and a preparation method thereof, and belongs to the technical field of fertilizers, the bacillus megaterium-containing microbial fertilizer for soil improvement is prepared from the following raw materials in parts by weight: 35-50 parts of an immobilized microbial agent, 3-5 parts of a water-retaining agent, 10-15 parts of humic acid and 2-5 parts of trehalose; the immobilized microbial agent is amino MOF (Metal Organic Framework) modified charcoal loaded bacillus megaterium; the microbial fertilizer is prepared from the immobilized microbial agent, the water-retaining agent, humic acid and trehalose, is high in pertinence and outstanding in performance in soil improvement, on one hand, available phosphorus in reclamation soil is activated, needed nutritional ingredients can be provided for plants, on the other hand, soil organic matter can be activated and updated, and the soil fertility is improved. The soil fertility is improved and the plant nutrient requirements are met.

Description

Technical Field

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

[0002] Coal mining has seriously caused ecological environment problems such as ecosystem degradation, land resource damage, and soil quality decline. If reclamation and environmental restoration are not paid attention to, the mining area will eventually become desolate and the environment will continue to deteriorate, resulting in serious economic recession. Land reclamation can reduce the impact of coal mining on the ecological environment and turn unutilizable land into utilizable land.

[0003] The land in coal mining subsidence areas usually undergoes engineering reclamation first. After engineering reclamation, the subsoil layer of the soil covers the topsoil layer, resulting in poor soil nutrients and prominent problems of "lack of nitrogen 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 phosphate fertilizers. However, most of the phosphorus in calcareous soils is fixed by cations such as Ca 2+ and Mg 2+ in the soil into non-available forms that are difficult for plants to utilize and accumulate in the soil, resulting in low crop yields.

[0004] Under this background, microbial inoculants, as a kind of 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, the existing microbial fertilizers lack specificity, resulting in poor application effects in the improvement of reclaimed soils. Summary of the Invention

[0005] The present invention provides a microbial fertilizer for soil improvement containing Bacillus megaterium and a preparation method thereof, which can solve the problem of poor application effects of existing microbial fertilizers in the improvement of reclaimed soils.

[0006] The purpose of the present invention can be achieved through the following technical solutions: A microbial fertilizer for soil improvement containing Bacillus megaterium, comprising the following raw materials in parts by weight: 35 - 50 parts of immobilized inoculant, 3 - 5 parts of water-retaining agent, 10 - 15 parts of humic acid, 2 - 5 parts of trehalose.

[0007] The immobilized inoculant is amino-MOF modified biochar loaded with Bacillus megaterium.

[0008] The Bacillus megaterium is classified and named as Bacillus megaterium YJ004, deposited at the China Center for Type Culture Collection on November 22, 2021, with the deposit number CCTCC NO: M 20211463, and its 16S rDNA sequence is as shown in SEQ ID NO.1.

[0009] The water retention agent is a water-absorbing resin carrying phosphate groups.

[0010] As a preferred technical solution of the present invention, the preparation method of the immobilized microbial agent is as follows: Add the amino-MOF modified biochar to the Bacillus megaterium fermentation broth, then place it on a shaker and oscillate and adsorb at a temperature of 25-35 °C for 12-36 h. Finally, centrifuge, collect the precipitate, and air-dry naturally to obtain the immobilized microbial agent.

[0011] As a preferred technical solution of the present invention, the dosage ratio of the amino-MOF modified biochar to the Bacillus megaterium fermentation broth is 1 g: 10-30 mL.

[0012] As a preferred technical solution of the present invention, the rotation speed of the shaker is 100-200 rpm.

[0013] As a preferred technical solution of the present invention, the centrifugation is carried out at 25 °C and 1500 r / min for 5 min.

[0014] As a preferred technical solution of the present invention, the preparation method of the Bacillus megaterium fermentation broth is as follows: Inoculate Bacillus megaterium into a liquid medium and culture it at 25-40 °C for 24-48 h to obtain a Bacillus megaterium fermentation broth with a Bacillus megaterium content of 2×10 7 cfu / mL - 2×10 8 cfu / mL.

[0015] As a preferred technical solution of the present invention, the liquid medium is prepared from the following raw materials by 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.

[0016] 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. The mass ratio of biochar, FeCl3·6H20, and 2-aminoterephthalic acid is 0.12 g: 0.54-1.62 g: 0.18-0.54 g.

[0017] As a preferred technical solution of the present invention, the preparation method of the amino-MOF modified biochar includes the following steps: Biochar was ultrasonically dispersed in N,N-dimethylformamide, and then FeCl3·6H20 and 2-aminoterephthalic acid were added. After stirring for 1-2 h, a mixed solution was obtained. The mixed solution was transferred to a reaction kettle and reacted at 120 °C for 24 h. After the reaction, it was cooled to room temperature, filtered, and the filter cake was washed successively with absolute ethanol and deionized water, and finally dried.

[0018] As a preferred technical solution of the present invention, the biochar is the combustion ash carbon 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.

[0019] 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-(phosphonooxy)ethyl 2-methylacrylate, sodium hydroxide, and deionized water.

[0020] As a preferred technical solution of the present invention, the preparation method of the water-retaining agent includes the following steps: Under the condition of ice-water bath, acrylic acid was added to deionized water, and sodium hydroxide was added for neutralization with a neutralization degree of 70%. Then acrylamide and 2-(phosphonooxy)ethyl 2-methylacrylate were added. After stirring evenly, the temperature was raised to 75-80 °C under nitrogen protection, and N,N'-methylenebisacrylamide and potassium persulfate were added. After holding and stirring for 2 h, the reaction product was washed with deionized water, dried, crushed and sieved through a 40-60 mesh sieve to obtain the water-retaining agent.

[0021] As a preferred technical solution of the present invention, the molar ratio of acrylic acid, acrylamide, and 2-(phosphonooxy)ethyl 2-methylacrylate is 8:1-1.5:0.5-1, the dosage of N,N'-methylenebisacrylamide is 0.05% of the total mass of acrylic acid, acrylamide, and 2-(phosphonooxy)ethyl 2-methylacrylate, and potassium persulfate is 2 times the mass of N,N'-methylenebisacrylamide.

[0022] The preparation method of the above microbial fertilizer for soil improvement containing Bacillus megaterium includes the following steps: According to the formula ratio, the immobilized inoculant, water-retaining agent, humic acid, and trehalose were stirred evenly.

[0023] The beneficial effects of the present invention: 1. The Bacillus megaterium provided by the present invention was sampled from the reclamation test field of Quercus nuttallii forest land in Guangde, Anhui Province, and then subjected to high-throughput sequencing, and then the dominant strain was screened by streak isolation. It exhibits excellent phosphorus-solubilizing effects 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.

[0024] 2. The microbial fertilizer of the present invention is composed of an immobilized inoculant, a water-retaining agent, humic acid, and trehalose. It has strong pertinence and performs outstandingly in soil improvement. On the one hand, it activates the available phosphorus in the reclaimed soil and can provide the necessary nutrients for plants. On the other hand, it can activate and renew the soil organic matter, improve soil fertility, and meet the nutrient requirements of plants.

[0025] 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. Moreover, compared with activated carbon as a carrier, the average pore diameter of amino-MOF modified biochar has increased, and the specific surface area is larger, which is more conducive to adsorbing Bacillus megaterium. In addition, the cell wall surface of Bacillus megaterium carries negatively charged functional groups such as carboxyl 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 combination of Bacillus megaterium and the carrier. Therefore, the adsorption capacity of the amino-MOF modified biochar provided by the present invention for Bacillus megaterium is significantly improved, which is beneficial to increasing the loading amount of active bacteria in the microbial fertilizer, thereby enhancing its soil improvement efficiency.

[0026] 4. The present invention introduces a water-retaining agent into the microbial fertilizer. The water-retaining agent is a water-absorbing resin carrying phosphate groups. 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 soil surface water, and can also provide a suitable humidity environment for the growth, reproduction, and metabolic activities of Bacillus megaterium. In addition, the phosphate groups in the water-retaining agent carry negative charges and can form electrostatic interactions with amino-MOF modified biochar. On the one hand, it can reduce the loss of Bacillus megaterium, and on the other hand, it enables the two to cooperate with each other when adsorbing water to form a more stable hydration layer. When the soil water evaporates, it can reduce the loss of water, maintain the moist state of the soil, and improve the water-retaining performance of the soil. Detailed implementation mode

[0027] In order to make the technical problems to be solved, technical solutions, and beneficial effects of the present application clearer, the following further details the present application in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0028] In the specification of the embodiments of the present application, the weights of the relevant components mentioned not only can refer to the specific contents of each component, but also can represent the proportional relationships of the weights between each component. Therefore, as long as the contents of the relevant components in the specification of the embodiments of the present application are enlarged or reduced proportionally, they are within the scope disclosed in the specification of the embodiments of the present application. Specifically, the mass described in the specification of the embodiments of the present application can be mass units well-known in the chemical industry such as μg, mg, g, kg, etc.

[0029] All kinds of raw materials, reagents, instruments, equipment, etc. used in the present application can be obtained through market purchase or can be prepared by existing methods.

[0030] Preparation Example 1

[0031] This preparation example provides an amino-MOF modified biochar, and the preparation method is as follows: Disperse 0.12 g of biochar in 60 mL of N,N-dimethylformamide by ultrasonic treatment, then add 0.54 g of FeCl3·6H20 and 0.18 g of 2-aminoterephthalic acid, stir for 1 h to obtain a mixed solution, transfer the mixed solution to a reaction kettle, react at 120 °C for 24 h, after the reaction is completed, cool to room temperature, filter, wash the filter cake successively with absolute ethanol and deionized water, and finally dry it.

[0032] The process for preparing the biochar is as follows: Wash the rice husks 3 times with deionized water, dry them at 80 °C for 24 h, then add them to a tubular furnace, under a nitrogen atmosphere, heat up at a heating rate of 3 °C / min to 750 °C, hold for 2 h, and then cool to room temperature to obtain biochar.

[0033] Preparation Example 2

[0034] This preparation example provides an amino-MOF modified biochar, and the preparation method is as follows: Disperse 0.12 g of biochar in 70 mL of N,N-dimethylformamide by ultrasonic treatment, then add 1.08 g of FeCl3·6H20 and 0.36 g of 2-aminoterephthalic acid, stir for 1.5 h to obtain a mixed solution, transfer the mixed solution to a reaction kettle, react at 120 °C for 24 h, after the reaction is completed, cool to room temperature, filter, wash the filter cake successively with absolute ethanol and deionized water, and finally dry it.

[0035] The process for preparing the biochar is the same as that in Preparation Example 1.

[0036] Preparation Example 3

[0037] This preparation example provides an amino-MOF modified biochar, and the preparation method is as follows: Disperse 0.12 g of biochar ultrasonically in 80 mL of N,N-dimethylformamide, then add 1.62 g of FeCl3·6H20 and 0.54 g of 2-aminoterephthalic acid, stir for 2 h to obtain a mixed solution. Transfer the mixed solution to a reaction kettle, react at 120 °C for 24 h. After the reaction, cool to room temperature, filter, wash the filter cake successively with absolute ethanol and deionized water, and finally dry it.

[0038] The preparation process of the biochar is the same as that in Preparation Example 1.

[0039] Control Example 1

[0040] This control example provides an amino-MOF modified biochar. Compared with Preparation Example 1, the difference is that 2-aminoterephthalic acid in Preparation Example 1 is replaced with an equimolar amount of terephthalic acid.

[0041] Control Example 2

[0042] This control example is biochar, and the preparation process of the biochar is the same as that in Preparation Example 1.

[0043] For the products in Preparation Examples 1 - 3 and Control Examples 1 - 2, measure the specific surface area and average pore diameter using a specific surface area and pore size analyzer, and then calculate the microbial adsorption rate. The specific process is as follows: (1) Inoculate Bacillus megaterium (classified and named Bacillus megaterium YJ004, deposited at the China Center for Type Culture Collection, deposit number CCTCC NO: M 20211463) into a liquid medium, and culture at 37 °C for 24 h to obtain a Bacillus megaterium fermentation broth with a Bacillus megaterium content of 4.5×10 7 cfu / mL of Bacillus megaterium fermentation broth.

[0044] (2) The liquid medium is prepared according to the following mass ratio of raw materials: 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.

[0045] (3) Using the products in Preparation Examples 1 - 3 and Control Examples 1 - 2 as carriers, according to the dosage ratio of the carrier to the Bacillus megaterium fermentation broth of 1 g:10 mL, add 2 g of the carrier to 20 mL of the Bacillus megaterium fermentation broth respectively, oscillate and adsorb at 25 °C for 24 h, then centrifuge at 25 °C and 1500 r / min for 5 min, take the supernatant, measure the viable bacteria count in the supernatant by the plate counting method, and calculate the adsorption rate according to the formula adsorption rate (%) = (X0 - X1) / X0 × 100%, where X0 is the viable bacteria count in the original fermentation broth and X1 is the viable bacteria count in the supernatant; The test results are shown in Table 1: Table 1

[0046] Analysis of the recorded data in Table 1 shows that, compared with Comparative Example 2, the amino-MOF modified biochars in Preparation Examples 1 - 3 have larger specific surface areas, average pore diameters, and adsorption rates. Among them, Preparation Example 2 has the best comprehensive performance. From the test results of Preparation Example 1 and Comparative Example 1, it can be seen that when 2-aminoterephthalic acid in the preparation process of the amino-MOF modified biochar is replaced with an equimolar amount of terephthalic acid, the adsorption effect of the resulting product on the bacterial solution deteriorates significantly.

[0047] Example 1 Isolation and Identification of Bacillus megaterium

[0048] I. Isolation of Bacteria

[0049] Soil samples were collected from the reclamation test field of Quercus nuttallii in Guangde, Anhui Province. The soil microbial community was analyzed by high-throughput sequencing technology to screen out the target strain. The specific operations are as follows: Weigh 10 g of the soil sample and add it to a conical flask containing 90 mL of sterile normal saline and glass beads. Shake it on a shaker at 180 rpm for 30 min, and then treat it in a 75 °C water bath for 15 minutes to inhibit the growth of miscellaneous bacteria to obtain a mixed solution. Perform ten-fold serial dilutions on the mixed solution. Take 100 μL of the diluted solution and spread it evenly on an LB plate medium (nutrient broth solid plate). Then place the plate in a constant temperature incubator at 30 °C for 48 h, and pick out the formed single suspected Bacillus megaterium colonies. Purify the colonies on a fresh LB plate by the streaking method multiple times until no miscellaneous bacteria are observed under the microscope, and a strain with morphological and characteristic features consistent with Bacillus megaterium is successfully isolated and named YJ004.

[0050] II. Identification of Bacteria

[0051] (I) Identification Based on Morphological and Physiological and Biochemical Characteristics

[0052] According to the standard methods in "Bergey's Manual of Determinative Bacteriology" (Eighth Edition) and "Manual of Systematic Identification of Common Bacteria", the morphological characteristics, cultural characteristics, and physiological and biochemical characteristics of Bacillus megaterium YJ004 were systematically analyzed. The results are as follows: Morphological and Physiological and Biochemical Characteristics of the Strain: After culturing on LB medium at 30 °C for 48 h, the colonies are milky white, round, with a moist and smooth surface, regular edges, and a relatively large diameter (3 - 5 mm), which conform to the typical morphological characteristics of Bacillus megaterium. Under the microscope, the bacterial cells are straight rods, Gram-positive, with thick and large rod-shaped cells (1.2 - 1.5 μm × 2.0 - 4.0 μm), and can form oval terminal spores.

[0053] Physiological and biochemical characteristics of the strain: Gelatin liquefaction: positive (+), catalase activity: positive (+), V-P reaction: negative (﹣), indole formation: negative (﹣), citrate utilization: positive (+), glucose fermentation: positive (+), gas production, starch hydrolysis: positive (+), nitrate reduction: positive (+); Salt tolerance: Grows well under 3% NaCl condition, and growth is limited under 7% NaCl condition.

[0054] These results are highly consistent with the typical characteristics of Bacillus megaterium, indicating that this strain is Bacillus megaterium.

[0055] (2) 16S rDNA test

[0056] Extract the DNA of strain YJ004, amplify the 16S rRNA gene and sequence it. The obtained sequence is shown as SEQ ID NO.1. Then, through comparison with the GenBank database, the homology between strain YJ004 and Bacillus megaterium strain ATCC 14581 (accession number NR 074540.1) reaches 99.5%, and it is confirmed as Bacillus megaterium.

[0057] Based on the above characteristics, the strain YJ004 was named Bacillus megaterium YJ004 and was deposited in the China Center for Type Culture Collection on November 22, 2021, with the deposit number CCTCC NO: M 20211463, and the deposit address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0058] Example 2

[0059] This example provides a microbial fertilizer for soil improvement containing Bacillus megaterium, which includes the following raw materials in parts by weight: 35 parts of immobilized bacterium agent, 3 parts of water retaining agent, 10 parts of humic acid, 2 parts of trehalose.

[0060] The preparation method of the immobilized bacterium agent is as follows: Add 100 g of the amino-MOF modified biochar prepared in Preparation Example 1 to 1000 mL of the Bacillus megaterium fermentation broth, then place it on a shaker, with the shaker speed of 100 rpm, oscillate and adsorb at 25℃ for 12 h, and finally centrifuge at 25℃ and 1500 r / min for 5 min, collect the precipitate, and air dry it naturally to obtain the immobilized bacterium agent.

[0061] The preparation method of the Bacillus megaterium fermentation broth is as follows: Inoculate Bacillus megaterium YJ004 in Example 1 into a liquid medium and culture it at 25 °C for 24 h to obtain a Bacillus megaterium fermentation broth with a Bacillus megaterium content of 2×10 7 cfu / mL.

[0062] The liquid medium is prepared from the following raw materials by 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.

[0063] The preparation method of the water-retaining agent includes the following steps: Under ice-water bath conditions, add 0.8 mol of acrylic acid to 500 mL of deionized water, add sodium hydroxide for neutralization, with a neutralization degree of 70%. Then add 0.1 mol of acrylamide and 0.1 mol of 2-(phosphonooxy)ethyl 2-methyl-2-propenoate. After stirring evenly, heat to 75 °C under nitrogen protection, add 0.043 g of N,N'-methylenebisacrylamide and 0.086 g of potassium persulfate, keep warm and stir for 2 h. Wash the reaction product with deionized water, dry it, and crush it through a 40-mesh sieve to obtain the water-retaining agent.

[0064] The preparation method of the microbial fertilizer for soil improvement containing Bacillus megaterium includes the following steps: According to the formula ratio, add the immobilized bacterium agent, water-retaining agent, humic acid, and trehalose into a blender and stir at 100 r / min for 30 min at room temperature.

[0065] Example 3

[0066] This example provides a microbial fertilizer for soil improvement containing Bacillus megaterium, which includes the following raw materials by weight: 40 parts of immobilized bacterium agent, 4 parts of water-retaining agent, 12 parts of humic acid, 4 parts of trehalose.

[0067] The preparation method of the immobilized bacterium agent is as follows: Add 100 g of the amino-MOF modified biochar in Preparation Example 1 into 2000 mL of the Bacillus megaterium fermentation broth, then place it on a shaker, shake at a speed of 150 rpm and a temperature of 30 °C for 24 h for oscillating adsorption. Finally, centrifuge at 25 °C and 1500 r / min for 5 min, collect the precipitate, and air-dry it to obtain the immobilized bacterium agent.

[0068] The preparation method of the Bacillus megaterium fermentation broth is as follows: Inoculate Bacillus megaterium YJ004 in Example 1 into a liquid medium and culture it at 30 °C for 36 h to obtain a Bacillus megaterium fermentation broth with a Bacillus megaterium content of 6.5×10 7 cfu / mL.

[0069] The liquid medium is prepared from the following raw materials by 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.

[0070] The preparation method of the water retaining agent is the same as that of Example 2.

[0071] The preparation method of the above microbial fertilizer for soil improvement containing Bacillus megaterium is also the same as that of Example 2.

[0072] Example 4

[0073] This example provides a microbial fertilizer for soil improvement containing Bacillus megaterium, which includes the following raw materials by weight: 50 parts of immobilized bacteria agent, 5 parts of water retaining agent, 15 parts of humic acid, 5 parts of trehalose.

[0074] The preparation method of the immobilized bacteria agent is as follows: Add 100 g of the amino-MOF modified biochar prepared in Preparation Example 1 to 3000 mL of the Bacillus megaterium fermentation broth, then place it on a shaker. The shaker speed is 200 rpm, and it is oscillated and adsorbed at 35 °C for 36 h. Finally, it is centrifuged at 25 °C and 1500 r / min for 5 min, and the precipitate is collected and air-dried to obtain the immobilized bacteria agent.

[0075] The preparation method of the Bacillus megaterium fermentation broth is as follows: Inoculate the Bacillus megaterium YJ004 in Example 1 into the liquid medium and culture it at 37 °C for 48 h to obtain a Bacillus megaterium fermentation broth with a Bacillus megaterium content of 2×10 8 cfu / mL.

[0076] The liquid medium is prepared from the following raw materials by 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.

[0077] The preparation method of the water retaining agent is the same as that of Example 2.

[0078] The preparation method of the above microbial fertilizer for soil improvement containing Bacillus megaterium is also the same as that of Example 2.

[0079] Example 5

[0080] This example provides a microbial fertilizer for soil improvement containing Bacillus megaterium. Compared with Example 2, the difference is that the amino-MOF modified biochar in Example 2 is replaced with the product obtained in Preparation Example 2 with the same mass.

[0081] Example 6

[0082] This example provides a microbial fertilizer for soil improvement containing Bacillus megaterium. Compared with Example 2, the difference is that the amino-MOF modified biochar in Example 2 is replaced with the product obtained in Preparation Example 3 of the same mass.

[0083] Example 7

[0084] This example provides a microbial fertilizer for soil improvement containing Bacillus megaterium. Compared with Example 2, the difference is that the water-retaining agent is different. The preparation method of the water-retaining agent in this example includes the following steps: Under ice-water bath conditions, 0.8 mol of acrylic acid is added to 550 mL of deionized water, and sodium hydroxide is added for neutralization with a neutralization degree of 70%. Then, 0.15 mol of acrylamide and 0.05 mol of 2-(phosphonooxy)ethyl 2-methyl-2-propenoate are added. After stirring evenly, the temperature is raised to 78 °C under nitrogen protection. 0.039 g of N,N'-methylenebisacrylamide and 0.079 g of potassium persulfate are added, and the mixture is kept warm and stirred for reaction for 2 h. The reaction product is washed with deionized water, dried, and pulverized through a 40-mesh sieve to obtain the water-retaining agent.

[0085] Example 8

[0086] This example provides a microbial fertilizer for soil improvement containing Bacillus megaterium. Compared with Example 2, the difference is that the water-retaining agent is different. The preparation method of the water-retaining agent in this example includes the following steps: Under ice-water bath conditions, 0.8 mol of acrylic acid is added to 550 mL of deionized water, and sodium hydroxide is added for neutralization with a neutralization degree of 70%. Then, 0.13 mol of acrylamide and 0.07 mol of 2-(phosphonooxy)ethyl 2-methyl-2-propenoate are added. After stirring evenly, the temperature is raised to 80 °C under nitrogen protection. 0.04 g of N,N'-methylenebisacrylamide and 0.08 g of potassium persulfate are added, and the mixture is kept warm and stirred for reaction for 2 h. The reaction product is washed with deionized water, dried, and pulverized through a 60-mesh sieve to obtain the water-retaining agent.

[0087] Comparative Example 1

[0088] This example provides a microbial fertilizer for soil improvement containing Bacillus megaterium. Compared with Example 2, the difference is that the amino-MOF modified biochar in Example 2 is replaced with the product obtained in Comparative Example 1 of the same mass.

[0089] Comparative Example 2

[0090] This example provides a microbial fertilizer for soil improvement containing Bacillus megaterium. Compared with Example 2, the difference is that the amino-MOF modified biochar in Example 2 is replaced with the product obtained in Comparative Example 2 of the same mass.

[0091] Comparative Example 3

[0092] This example provides a microbial fertilizer for soil improvement containing Bacillus megaterium. Compared with Example 2, the difference is that 2-(phosphonooxy)ethyl 2-methyl-2-propenoate in Example 2 is replaced with an equimolar amount of acrylamide.

[0093] Comparative Example 4

[0094] This example provides a microbial fertilizer for soil improvement containing Bacillus megaterium. Compared with Example 2, the difference is that the water-retaining agent in Example 1 is removed.

[0095] The water retention performance of the microbial fertilizers prepared in Examples 2 - 8 and Comparative Examples 1 - 4 was detected. The water retention rate was measured by the following method: At a temperature of 28°C, 200 g of reclaimed soil was added to 4 g of the sample, and the mixture was stirred evenly. The water content of the reclaimed soil was 4%. Then, 200 g of water was added to the evenly mixed sample, and the weight was measured and recorded as W. i After standing for 15 days, the weight was measured again and recorded as W. n . And the soil water evaporation rate was calculated. The soil water evaporation rate (%) = (W i - W n ) / 200 × 100%. The test results are shown in Table 2; (2) The microbial fertilizers prepared in Examples 2 - 8 and Comparative Examples 1 - 4 were added to the reclaimed soil, and their improvement effects on the reclaimed soil were detected through a pot experiment. The specific method is as follows: The test soil samples were collected from the soil around the coal mining subsidence area in Bengbu City, Anhui Province. The test vegetable was Aijiaohuang. Eleven soil samples were taken and the microbial fertilizers prepared in Examples 2 - 8 and Comparative Examples 1 - 4 were applied respectively. The application rate was 4 g / kg of soil (4‰). The pot containers were plastic pots with a size of 150 mm × 125 mm (upper diameter × height), and each pot was filled with 1.0 kg of air-dried reclaimed soil. The microbial fertilizers in each group were evenly mixed with the soil according to the above application rates and then filled into the pots. Before sowing, the soil was thoroughly watered. The germinated and white-emerged Aijiaohuang was sown into the pots, 6 seeds per pot. The soil was kept moist before emergence, and watered once every 2 - 3 days after emergence to maintain 40% of the maximum field water holding capacity. When 2 - 3 true leaves grew, thinning was carried out, with a seedling spacing of 3 - 4 cm, and 2 plants were left in each pot. After thinning, the soil was watered immediately once. After 40 days, the harvest was carried out, and soil and plant samples were collected. The soil organic matter, available nitrogen, phosphorus, and potassium contents were measured, and the vegetable biomass was measured. The results are shown in Table 2: Table 2

[0096] Analysis of the recorded data in Table 2 shows that, compared with Example 2, the reclaimed soil improved by the microbial fertilizer in Example 3 and Example 4 has a lower soil water evaporation rate, higher organic matter and available phosphorus contents in the soil, and higher fresh and dry weights of vegetables. Therefore, the improvement effect is better; From the test results of Example 2, Example 5 and Example 6, it can be seen that, under the condition that the microbial fertilizer formula and preparation process remain unchanged, when using the amino-MOF modified biochar loaded with Bacillus megaterium YJ004 prepared by Preparation Example 1, Preparation Example 2, and Preparation Example 3 to prepare the microbial fertilizer, 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 diameter, which can load more Bacillus megaterium YJ004 and provide a comfortable living environment for it, enabling it to play a role in phosphorus solubilization and other functions efficiently; From the test results of Example 2, Example 7 and Example 8, it can be seen that during the preparation of the water-retaining agent, reducing the content of 2-(phosphonooxy)ethyl 2-methylacrylate will cause a decrease in the water-retaining performance of the prepared microbial fertilizer and a slightly worse improvement effect on the reclaimed soil; From the test results of Example 2 and Comparative Example 1, it can be seen that when using the MOF modified biochar loaded with Bacillus megaterium YJ004 prepared by Control Example 1 to prepare the microbial fertilizer, due to the lack of amino groups, its water-retaining performance decreases significantly, and the organic matter and available phosphorus contents in the reclaimed soil also decrease significantly; From the test results of Example 2 and Comparative Example 2, it can be seen that when using the biochar loaded with Bacillus megaterium YJ004 prepared by Control Example 2 to prepare the microbial fertilizer, due to limited loading performance and the inability to interact with the water-retaining agent, its water-retaining performance decreases significantly, and the organic matter and available phosphorus contents in the reclaimed soil also decrease significantly; From the test results of Example 2 and Comparative Example 3, it can be seen that replacing 2-(phosphonooxy)ethyl 2-methylacrylate with an equimolar amount of acrylamide during the preparation of the water-retaining agent will cause a decrease in the water-retaining performance of the microbial fertilizer, and the organic matter and available phosphorus contents in the reclaimed soil also decrease significantly From the test results of Example 2 and Comparative Example 4, it can be seen that omitting the use of the water-retaining agent of the present invention will significantly increase the soil water evaporation rate, reduce the organic matter and available phosphorus contents in the soil, and also result in lower fresh and dry weights of vegetables, with a poor soil improvement effect.

[0097] It should be noted that in this text, relational terms such as first and second are only used 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 "include", "comprise" or any other variant thereof are intended to cover non-exclusive inclusion, such that a process, method, article or device comprising a series of elements not only includes those elements but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device.

[0098] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A microbial fertilizer for soil improvement containing Bacillus megaterium, characterized in that, It comprises 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 bacteria agent is Bacillus megaterium loaded on amino-MOF modified biochar; The water-retaining agent is a water-absorbing resin carrying phosphate groups; The Bacillus megaterium is classified and named as Bacillus megaterium YJ004, and the preservation location is the China Center for Type Culture Collection, with the preservation number CCTCC NO: M 20211463, and its 16SrRDNA sequence is as shown in SEQ ID NO.

1.

2. The preparation method of the microbial fertilizer for soil improvement containing Bacillus megaterium according to claim 1, characterized in that, It comprises the following steps: S1. Preparation of the immobilized bacteria agent: Add amino-MOF modified biochar into the Bacillus megaterium fermentation broth, then place it on a shaker and oscillate and adsorb at a temperature of 25 - 35°C for 12 - 36 h. Finally, centrifuge, collect the precipitate, and air-dry naturally to obtain the immobilized bacteria agent; S2. Preparation method of the water-retaining agent: Under the condition of an ice-water bath, add acrylic acid into deionized water, add sodium hydroxide for neutralization, and the degree of neutralization is 70%. Then add acrylamide and 2-(phosphonooxy)ethyl 2-methyl-2-propenoate, stir evenly, and heat up to 75 - 80°C under nitrogen protection. Add N,N'-methylenebisacrylamide and potassium persulfate, keep warm and stir for 2 h. Wash the reaction product with deionized water, dry, and pulverize and sieve through a 40 - 60 mesh sieve to obtain the water-retaining agent; S3. Stir the immobilized bacteria agent, water-retaining agent, humic acid, and trehalose evenly to obtain a microbial fertilizer for soil improvement containing Bacillus megaterium.

3. The preparation method of the microbial fertilizer for soil improvement containing Bacillus megaterium according to claim 2, characterized in that, In S1, the dosage ratio of the amino-MOF modified biochar to the Bacillus megaterium fermentation broth is 1 g:10 - 30 mL.

4. The preparation method of the microbial fertilizer for soil improvement containing Bacillus megaterium according to claim 2, characterized in that, The preparation method of the Bacillus megaterium fermentation broth is as follows: Inoculate Bacillus megaterium into a liquid medium and culture it at 25 - 40 °C for 24 - 48 h to obtain a Bacillus megaterium fermentation broth with a Bacillus megaterium content of 2×10 7 cfu / mL - 2×10 8 cfu / mL.

5. The preparation method of the microbial fertilizer for soil improvement containing Bacillus megaterium according to claim 2, characterized in that, 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 preparation method of the microbial fertilizer for soil improvement containing Bacillus megaterium according to claim 2, characterized in that, The preparation method of the amino-MOF modified biochar includes the following steps: Ultrasonically disperse biochar in N,N-dimethylformamide, then add FeCl3·6H20 and 2-aminoterephthalic acid, stir for 1 - 2 h to obtain a mixed solution. Transfer the mixed solution to a reaction kettle, react at 120°C for 24 h. After the reaction, cool to room temperature, filter, wash the filter cake successively with absolute ethanol and deionized water, and finally dry.

7. The preparation method of the microbial fertilizer for soil improvement containing Bacillus megaterium according to claim 2, characterized in that, In S2, the molar ratio of acrylic acid, acrylamide, and 2-(phosphonooxy)ethyl 2-methyl-2-propenoate is 8:1 - 1.5:0.5 - 1, the dosage of N,N'-methylenebisacrylamide is 0.05% of the sum of the masses of acrylic acid, acrylamide, and 2-(phosphonooxy)ethyl 2-methyl-2-propenoate, and potassium persulfate is 2 times the mass of N,N'-methylenebisacrylamide.

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