Bacillus megaterium LLH-B2 and application thereof

Abstract

The application relates to the technical field of biology, and particularly discloses a bacillus megaterium LLH-B2 and application thereof. The bacillus megaterium LLH-B2 has been preserved in the China Center Type Culture Collection of Wuhan University in Wuhan, China on November 14, 2022, and is named bacillus megaterium LLH-B2, with a preservation number of CCTCC NO: M 20221787. The bacillus megaterium LLH-B2 can survive stably for a long time in a sandy soil drought environment, can efficiently treat heavy metals in the sandy soil, can improve soil fertility, and can promote plant growth.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and more specifically, to a Bacillus megaterium LLH-B2 and its applications. Background Technology

[0002] With rapid industrialization and urbanization, as well as the irrational use of fertilizers and pesticides in agricultural production, heavy metal pollution has become a major challenge to the sustainable development of global agriculture. Heavy metals (such as cadmium, lead, and copper) enter the soil through mining, electroplating, chemical emissions, and the overuse of pesticides and fertilizers, accumulating continuously and insidiously. This not only disrupts the balance of the soil ecosystem but also threatens human health through the food chain, posing a serious threat to both ecosystem security and human health.

[0003] Microbial remediation boasts advantages such as low cost, environmental friendliness, and strong sustainability, and can synergistically improve soil fertility, gradually becoming a research hotspot in the field of soil heavy metal remediation. Among them, Bacillus spp. (…) Bacillus spp. Due to its strong resistance, ease of cultivation, and mature genetic manipulation, *Bacillus amyloliquefaciens* has shown significant potential in heavy metal adsorption and transformation, as well as plant growth promotion. For example, *Bacillus amyloliquefaciens* (… Bacillus amyloliquefaciens Bacillus subtilis ( ) has the potential to enhance lead adsorption by secreting metal-binding proteins. Bacillus subtilis It has the potential to induce the activity of plant antioxidant enzymes to alleviate cadmium stress. However, existing studies have mostly focused on single heavy metal pollution scenarios, and there is a lack of systematic verification on the long-term stability of the strain in complex soil environments, its interaction mechanism with plants, and its field application effects.

[0004] Bacillus megaterium ( Bacillus megaterium As a common type of soil probiotic, *Bacillus megaterium* possesses characteristics such as rapid reproduction, salt tolerance, high temperature tolerance, and strong stress resistance, playing an important role in soil ecological regulation. Existing research has shown that some *Bacillus megaterium* strains can influence the speciation of heavy metals and reduce their bioavailability by secreting extracellular polymers to adsorb heavy metal ions or by altering the soil microenvironment through their metabolic activities (such as regulating pH and redox potential). Furthermore, *Bacillus megaterium* also has the potential to enhance plant tolerance to heavy metal stress by synthesizing plant hormones and inducing systemic resistance.

[0005] However, regarding the aforementioned technologies, the inventors have discovered that current screening of *Bacillus megaterium* strains with highly efficient heavy metal adsorption and conversion capabilities and significant plant growth promotion is still insufficient, particularly in the field of heavy metal remediation in sandy soils. Due to the coarse particles, high porosity, low organic matter content, and poor water and fertilizer retention capacity of sandy soils, heavy metals are easily migrated, significantly increasing the difficulty of remediation. Conventional *Bacillus* strains often suffer from long remediation cycles and low efficiency when used to treat sandy soils due to factors such as low soil fertility, high heavy metal migration, and insufficient drought tolerance of the strains themselves. Therefore, screening strains that can adapt to sandy soil environments and efficiently remediate heavy metal pollution is of great significance for promoting the development of heavy metal pollution remediation technologies for sandy soils. Summary of the Invention

[0006] The purpose of this application is to provide a Bacillus megaterium LLH-B2 strain and its application in the remediation of heavy metal soil pollution. The Bacillus megaterium LLH-B2 strain can survive stably for a long time in arid sandy soil environments, effectively remediating heavy metal pollution in sandy soils while simultaneously improving soil fertility and promoting plant growth.

[0007] In a first aspect, this application provides a Bacillus megaterium LLH-B2 strain, which adopts the following technical solution: A strain of Bacillus megaterium LLH-B2 was deposited on November 14, 2022, at the China Center for Type Culture Collection, Wuhan University, China, and named Bacillus megaterium (LLH-B2). Bacillus megaterium ) LLH-B2, accession number CCTCC NO: M 20221787.

[0008] By adopting the above technical solution, the strain Bacillus megaterium LLH-B2 screened in this application has excellent heavy metal detoxification effect, and has a highly efficient heavy metal adsorption and transformation effect on soils contaminated with heavy metals such as cadmium, lead, and copper. It can effectively reduce their bioavailability and improve the resistance of the planted crops to abiotic stress.

[0009] Furthermore, the strain Bacillus megaterium LLH-B2 screened in this application has excellent drought resistance and can adapt to the environment of sandy soil that is dry, loosely structured, and has a high risk of heavy metal migration. It can survive stably for a long time in the dry environment of sandy soil, and its stability under high concentration of pollution demonstrates its stress resistance advantage. It can effectively treat heavy metal pollution in sandy soil, while improving soil fertility and promoting plant growth.

[0010] Secondly, this application provides an application of Bacillus megaterium LLH-B2 in the remediation of heavy metal contaminated soil.

[0011] Optionally, the total cadmium content in the heavy metal contaminated soil is ≤250mg / kg, the total lead content is ≤1200mg / kg, and the total copper content is ≤500mg / kg.

[0012] By adopting the above technical solution, the Bacillus megaterium LLH-B2 strain screened in this application can tolerate Cd. 2+ Pb 2+ and Cu 2+ The concentrations of 250 mg / L, 1200 mg / L and 500 mg / L, respectively, achieved removal rates of 98.2% for heavy metals cadmium, 92.8% for lead and 90.0% for copper. Compared with other publicly preserved Bacillus megaterium strains, these strains showed stronger targeting for the remediation of heavy metals cadmium, lead and copper.

[0013] Thirdly, this application provides a biological liquid fertilizer, which adopts the following technical solution: A biological liquid fertilizer comprising a bacterial solution of Bacillus megaterium LLH-B2, wherein the bacterial solution is made from Bacillus megaterium LLH-B2.

[0014] Optionally, the bacterial concentration of the Bacillus megaterium LLH-B2 bacterial suspension is ≥10. 9 cfu / mL.

[0015] By adopting the above technical solution, the bacterial concentration of Bacillus megaterium LLH-B2 in the bacterial culture is ≥10. 9 When used to prepare bio-liquid fertilizer, cfu / mL not only performs heavy metal remediation but also enhances soil fertility through microbial metabolic activities (such as nitrogen fixation and phosphorus solubilization). It has a significant effect on the remediation of sandy soils contaminated with heavy metals, can stably survive in relatively dry sandy soils, effectively adsorb and transform heavy metal ions, reduce the degree of soil pollution, regulate the pH of sandy soil, reduce the degree of soil salinization, increase soil organic matter content, and effectively increase crop yield, enhance plant tolerance, and promote crop growth.

[0016] Optionally, by weight, the raw materials include 15-25 parts of Bacillus megaterium LLH-B2 bacterial culture, 1-3 parts of ammonium dihydrogen phosphate, 0.5-1.5 parts of dipotassium hydrogen phosphate, 0.5-1.5 parts of sodium bicarbonate, 0.5-0.8 parts of calcium chloride, 0.05-0.1 parts of zinc sulfate, 0.2-0.5 parts of magnesium sulfate, 2-5 parts of sucrose, 3-4 parts of humic acid, 4-5 parts of chitosan composite microspheres, 1-1.5 parts of carboxymethyl nanocellulose, and 300-400 parts of water.

[0017] Optionally, the raw materials for the chitosan composite microspheres include chitosan, polyvinyl alcohol, FeCl3·6H2O and FeCl2·4H2O.

[0018] Optionally, the method for preparing the chitosan composite microspheres includes the following steps: S1: Chitosan is added to an acetic acid solution and stirred to prepare a chitosan acetic acid solution. Polyvinyl alcohol is dissolved in water and stirred to prepare a polyvinyl alcohol aqueous solution. Then, the chitosan acetic acid solution and the polyvinyl alcohol aqueous solution are mixed to obtain a mixture. S2: FeCl3·6H2O and FeCl2·4H2O are used as solutes, and the molar ratio is 2:1 to prepare Fe 3+ / Fe 2+ Iron salt solution; S3: Add the iron salt solution dropwise to the mixture, stir, sonicate, and then use a syringe with a needle inner diameter of 100μm to drip into the NaOH and sodium citrate mixture under the action of a dual-channel injection peristaltic pump. After continuous stirring, let it stand, collect the microspheres by centrifugation, wash, and freeze-dry to obtain the product.

[0019] By adopting the above technical solution, the chitosan composite microspheres prepared by this application using chitosan, polyvinyl alcohol, FeCl3·6H2O and FeCl2·4H2O as raw materials have a porous structure. Chitosan and polyvinyl alcohol provide a stable mechanical structure for the porous microspheres, which can enhance the water retention capacity of the soil.

[0020] Fe 3+ / Fe 2+ The co-precipitation of magnetic Fe3O4 particles can attract Cd 2+ Pb 2+ Cu 2+ The microspheres coordinate and complex with heavy metal ions, forming a porous structure that provides a high specific surface area, significantly increasing the exposure density of active sites and further enhancing the protection against Cd. 2+ Pb 2+ Cu 2+ The attraction of heavy metal ions not only increases the contact area between Bacillus megaterium LLH-B2 and heavy metal ions, promoting the adsorption and transformation of heavy metal ions by Bacillus megaterium LLH-B2 and effectively mitigating the migration of heavy metal ions in sandy soil, but also accelerates the adsorption and transformation of heavy metal ions by Bacillus megaterium LLH-B2, thereby improving remediation efficiency.

[0021] In addition, Fe3O4 particles can slowly release Fe 3+ / Fe 2+ As an essential micronutrient for plants, it promotes chlorophyll synthesis and further promotes crop growth.

[0022] In summary, this application has the following beneficial effects: 1. The strain Bacillus megaterium LLH-B2 screened in this application has excellent drought resistance and tolerance to high concentrations of heavy metal ions. When remediating heavy metals in sandy soil, it breaks through the survival and function bottleneck of conventional microorganisms in overcoming the drought, high porosity, low organic matter content and water and fertilizer retention capacity of sandy soil environment. It shows a stable stress resistance advantage in sandy soil with high concentration of heavy metal pollution, and can more effectively treat heavy metal pollution in sandy soil.

[0023] 2. When the strain Bacillus megaterium LLH-B2 selected in this application is used to prepare biological liquid fertilizer, it can effectively adsorb and transform heavy metal ions, reduce the degree of soil pollution, regulate the pH of sandy soil, reduce the degree of soil salinization, increase the soil organic matter content, effectively increase crop yield, enhance plant tolerance, and promote crop growth.

[0024] 3. This application uses Bacillus megaterium LLH-B2 in the preparation of bio-liquid fertilizer, supplementing nutrients such as nitrogen, carbon, potassium, and calcium sources. Chitosan composite microspheres with a porous structure and high specific surface area are prepared using chitosan, polyvinyl alcohol, FeCl3·6H2O, and FeCl2·4H2O as raw materials. 3+ / Fe 2+ Magnetic Fe3O4 particles were co-precipitated inside the microspheres, which can attract Cd. 2+ Pb 2+ Cu 2+ The presence of heavy metal ions and their coordination not only increases the contact area between Bacillus megaterium LLH-B2 and heavy metal ions, promoting the adsorption and transformation of heavy metal ions by Bacillus megaterium LLH-B2 and effectively mitigating the migration of heavy metal ions in sandy soil, but also accelerates the adsorption and transformation of heavy metal ions by Bacillus megaterium LLH-B2, thereby improving remediation efficiency. Detailed Implementation

[0025] The present application will be further described in detail below with reference to specific embodiments. For the specific methods or materials used in the embodiments, those skilled in the art can make conventional substitutions based on the technical concept of the present invention and existing technologies, and are not limited to the specific descriptions in the embodiments of the present invention. The equipment and reagents selected in the present invention can be selected from any commercially available type.

[0026] Example The culture medium formulation used in the examples is as follows: Nutrient agar medium: 10 g / L peptone, 5 g / L sodium chloride, 3 g / L beef powder, 15 g / L agar, solvent is deionized water, pH is 7.0, selected from Beijing Luqiao nutrient agar commercial medium; LB liquid medium: sodium chloride 10 g / L, yeast extract 4 g / L, tryptone 15 g / L, solvent: deionized water, pH 7.2; PDA solid medium: peeled potato 200 g / L, glucose 20 g / L, agar 15 g / L, solvent: deionized water, pH 5.6.

[0027] Example 1: Isolation and Screening of Bacillus megaterium from Soil 1. Soil sample: Rhizosphere soil from soybean planting area in Guhe Street, Laixi City, Qingdao, Shandong Province.

[0028] In the soybean planting area of ​​Guhe Street, Laixi City, Qingdao, Shandong Province, several representative sampling points were selected, with each sampling point spaced 50-100m apart. Centered on the soybean plant at each sampling point, the soil around the plant was carefully dug with a sterile shovel to a depth of about 10-20cm to expose the root system. The loose soil attached to the root surface was then gently shaken off, and the rhizosphere soil close to the root surface was collected with a sterile brush. The soil was mixed evenly and placed on a sterile plastic sheet or paper. After air drying, the soil sample was reduced to about 50g using the quartering method. After collection, it was stored at 4℃ for later use.

[0029] 2. Preparation of soil dilution solution: Take 10g of collected soil sample and add it to 90mL of sterile water. Shake at 30℃ and 130rpm for 15min to form a suspension. Dilute the suspension serially to prepare 10... -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 Prepare a diluted solution (multiplied by 1:1) for later use.

[0030] 3. Initial screening: Take 10 μL of the above-mentioned different dilutions and spread them evenly on sterile nutrient agar medium. Perform three parallel replicates for each dilution. Incubate at 30°C for 3 days. Select strains with fast growth rate or clear zone around the strain and isolate them using the streak method. Then, aseptically transfer them to new nutrient agar medium for re-culturing to obtain the initial screening strains.

[0031] 4. Secondary screening and purification: The primary screening strains were inoculated into LB liquid medium and cultured in a shaker at 30℃ and 180r / min for 1 day to obtain the screening strains. The screening strains were then purified and stored.

[0032] 5. Identification of the purified strain LLH-B2: After Gram staining, capsule staining and spore staining, the morphology of the purified strain was observed under a microscope. It was found that Bacillus megaterium LLH-B2 is a short rod-shaped, Gram-positive bacterium with spores. The spores are elliptical with blunt ends and are motile. The colonies formed on nutrient agar are round with milky white edges, about 2-5 mm in diameter, and have relatively neat edges.

[0033] Morphological and physiological identification confirmed that this strain belongs to the genus *Bacillus*. It was deposited on November 14, 2022, at the China Center for Type Culture Collection, Wuhan University, China, and named *Bacillus megaterium*. Bacillus megaterium )LLH-B2, accession number CCTCC NO: M 20221787.

[0034] Example 2: Heavy metal resistance of Bacillus megaterium LLH-B2 Solutions of cadmium chloride, lead chloride, and copper sulfate were prepared separately and added to LB liquid medium to obtain Cd. 2+ Final concentrations of 0, 50, 100, 150, 200, 250, 300, 350, and 400 mg / L, Pb 2+ Final concentrations of 0, 200, 400, 600, 800, 1000, 1200, 1400, and 1600 mg / L, Cu 2+ Final concentrations are 0, 100, 200, 300, 400, 500, 600, 700, and 800 mg / L; After culturing the Bacillus megaterium LLH-B2 obtained in Example 1 in LB liquid medium at 30°C and 130 rpm for 12 h, the seed culture was inoculated at a 2% inoculum into the above-prepared medium containing different concentrations of Cd. 2+ Pb 2+ and Cu 2+ After culturing under the same conditions for 48 hours in each culture medium, the growth and survival of the strains in each medium were tested. It was found that the growth of *Bacillus megaterium* LLH-B2 strain was significantly inhibited above a certain concentration. *Bacillus megaterium* LLH-B2 strain can tolerate Cd... 2+ Pb 2+ and Cu 2+ The concentrations were 250 mg / L, 1200 mg / L and 500 mg / L, respectively.

[0035] Example 3: Test of the removal effect of Bacillus megaterium LLH-B2 on cadmium, lead, and copper. Experimental group: Bacillus megaterium LLH-B2 obtained in Example 1 was inoculated into PDA solid medium at a 5% inoculum size and cultured at 30℃ and pH 7.0 for 10-12 hours to obtain activated bacteria. Then, it was inoculated into 100 mL of Cd-containing medium at a 1% inoculum size. 2+ Pb 2+ and Cu 2+ The samples were incubated at a constant temperature of 30°C in LB liquid medium with concentrations of 50 mg / L, 100 mg / L and 50 mg / L, respectively. Control group: LB liquid culture medium containing the same concentration of heavy metal ions without bacterial cells was set up as a blank control group; The experimental and control groups were each set up in triplicate. Samples were taken sequentially at adsorption times of 1, 3, 7, and 10 days, centrifuged, and the supernatant was collected, filtered, sterilized, and the Cd concentration in the solution before and after removal was determined. 2+ Pb 2+ and Cu 2+ Calculate Cd based on the change in concentration. 2+ Pb 2+ and Cu 2+ The removal rate was recorded in Table 1. The average of the three parallel replicates for each group was taken as the final result, and the final result was recorded in Table 1. Removal rate R (%) = (C0 - Ct) / C0 × 100%, where C0 is the initial concentration of heavy metal ions in the solution, and Ct is the concentration of metal ions in the solution after the bacterial cell treatment time t (d).

[0036] Table 1

[0037] The data in Table 1 show that LLH-B2 achieved removal rates of 98.2%, 92.8%, and 90.0% for heavy metals cadmium, lead, and copper, respectively. This indicates that Bacillus megaterium LLH-B2 has excellent removal effects on heavy metals cadmium, lead, and copper.

[0038] Example 4: Preparation of Bacillus megaterium LLH-B2 bacterial culture The method for preparing Bacillus megaterium LLH-B2 bacterial suspension includes the following steps: S1: The Bacillus megaterium LLH-B2 obtained in Example 1 was inoculated into PDA solid medium at an inoculation rate of 5%, and cultured at 30°C and pH 7.0 for 10-12 hours to obtain activated bacteria; S2: The activated bacteria were transferred to LB liquid medium at an inoculum of 8% and cultured at 30°C and 130 rpm until the logarithmic cycle. The cell counts were then performed directly under a microscope (BX53, Olympus China Co., Ltd.) using a hemocytometer, and the cell density was recorded. The final cell count was ≥10⁻⁶. 9 At a concentration of cfu / mL, the supernatant was separated by centrifugation to obtain a Bacillus megaterium LLH-B2 bacterial suspension, with an effective bacterial count of 5.5 × 10⁻⁶. 9 cfu / mL.

[0039] Example 5 A biological liquid fertilizer comprises 15 kg of Bacillus megaterium LLH-B2 bacterial solution obtained in Example 4, 2 kg of ammonium dihydrogen phosphate, 0.5 kg of dipotassium hydrogen phosphate, 1 kg of sodium bicarbonate, 0.5 kg of calcium chloride, 0.05 kg of zinc sulfate, 0.2 kg of magnesium sulfate, 2 kg of sucrose, 3.2 kg of humic acid, 4.5 kg of chitosan composite microspheres, 1 kg of carboxymethyl cellulose nanoparticles, and 300 kg of water; wherein the carboxymethyl cellulose is selected from Aladdin, DS = 0.7, 200-500 mPa·s; The raw materials for the chitosan composite microspheres include chitosan, polyvinyl alcohol, FeCl3·6H2O and FeCl2·4H2O. The chitosan is selected from Sinopharm Chemical Reagent Co., Ltd., 69047436, and the polyvinyl alcohol is selected from Sinopharm Chemical Reagent Co., Ltd., C183120025. The preparation method of chitosan composite microspheres includes the following steps: S1: Add 4g of chitosan to 96g of 0.2mol / L acetic acid solution, heat and stir in a 60℃ water bath for 6h to obtain chitosan acetic acid solution; dissolve 12g of polyvinyl alcohol in 88g of water, heat and stir in a 90℃ water bath for 6h to obtain polyvinyl alcohol aqueous solution; mix chitosan acetic acid solution and polyvinyl alcohol aqueous solution at a mass ratio of 3:1 to obtain mixed solution; S2: According to Fe 3+ and Fe 2+ A ferric salt solution was prepared by weighing FeCl3·6H2O and FeCl2·4H2O in a molar ratio of 2:1 and dissolving them in deionized water. The FeCl3·6H2O and FeCl2·4H2O were then added to deionized water. 3+ and Fe 2+ The concentrations were 0.1 mol / L and 0.05 mol / L, respectively; S3: Add 2 mL of the iron salt solution from step S2 dropwise to 10 mL of the mixture obtained in step S1 at a dropping rate of 1 mL / min. Stir at 500 rpm for 30 min to form a homogeneous sol mixture. Sonicate at 40 kHz for 10 min. Then, using a syringe with a 100 μm inner diameter needle and a dual-channel peristaltic pump, add a mixture of 1 mol / L NaOH and 0.1 mol / L sodium citrate solution dropwise. Stir continuously for 1.5 h, then allow to stand and age for 8 h to allow the amino groups of chitosan to react with the iron salts. 3+ / Fe 2+ Ionic cross-linking occurs, and Fe 3+ / Fe 2+ Fe3O4 particles were co-precipitated, and the microspheres were collected by centrifugation, washed, and then freeze-dried to obtain the final product.

[0040] Example 6 A biological liquid fertilizer differs from Example 5 in that the raw materials include 20 kg of Bacillus megaterium LLH-B2 bacterial solution obtained in Example 5, 1 kg of ammonium dihydrogen phosphate, 1 kg of dipotassium hydrogen phosphate, 0.5 kg of sodium bicarbonate, 0.7 kg of calcium chloride, 0.08 kg of zinc sulfate, 0.35 kg of magnesium sulfate, 4 kg of sucrose, 4 kg of humic acid, 4 kg of chitosan composite microspheres, 1.5 kg of carboxymethyl cellulose nanoparticles, and 350 kg of water. All other steps are the same as in Example 5.

[0041] Example 7 A biological liquid fertilizer differs from Example 5 in that the raw materials include 25 kg of Bacillus megaterium LLH-B2 bacterial solution obtained in Example 5, 3 kg of ammonium dihydrogen phosphate, 1.5 kg of dipotassium hydrogen phosphate, 1.5 kg of sodium bicarbonate, 0.8 kg of calcium chloride, 0.1 kg of zinc sulfate, 0.5 kg of magnesium sulfate, 5 kg of sucrose, 3 kg of humic acid, 5 kg of chitosan composite microspheres, 1.2 kg of carboxymethyl cellulose nanoparticles, and 400 kg of water. All other steps are the same as in Example 5.

[0042] Example 8: Test on the effect of biological liquid fertilizer on remediation of heavy metal sandy soil 1. Test location: Qingdao City, Shandong Province; 2. Crop variety: Melon; 3. The tested soil was sandy soil contaminated with heavy metals, and its physicochemical properties are shown in Table 2. Table 2 Physicochemical properties of the tested soils

[0043] 4. The specific method includes the following steps: Twenty experimental plots of equal area and with identical field management conditions were selected. Four plots were randomly chosen from each plot to conduct experiments on the experimental and control groups. Before sowing, normal land preparation, shallow tillage, and uniform machine sowing were performed. Base fertilizer was applied in a conventional manner, and the same field management methods were used. The following treatments were applied during the topdressing period: Control group: The only difference from Example 5 was the absence of Bacillus megaterium LLH-B2 bacterial culture; all other formulation components were the same. Experimental Group A: Bio-liquid fertilizer from Example 5; Experimental Group B: Bio-liquid fertilizer from Example 6; Experimental Group C: Bio-liquid fertilizer from Example 7; First, dilute the liquid fertilizer of the control group and the experimental group by 2 times with clean water. Then, transplant the melon seedlings by dipping their roots in the solution. After transplanting, apply the 1000-fold diluted solution of the compound fertilizer with irrigation water at a rate of 3 kg per mu. After the melons enter the fruit expansion stage, spray the control group and the experimental group AC with the 1000-fold diluted solution of the liquid fertilizer in sequence at a rate of 1 kg per mu. After entering the ripening stage, count the total yield of each group, calculate the average total yield and yield increase rate of each group, randomly pick 20 melons in each experimental area, calculate the average weight of a single fruit, and record the test results in Table 3. Test the effect of the use of liquid fertilizer on the physicochemical properties of the soil in the experimental area and record the results in Table 4.

[0044] Table 3

[0045] Table 4

[0046]

[0047] As shown in Table 3-4, heavy metal contaminated soil severely impacts crop growth, with most plants struggling to survive. The *Bacillus megaterium* LLH-B2 strain described in this application exhibits excellent drought resistance and root adhesion. Its application as a bio-liquid fertilizer in the remediation of heavy metal-contaminated sandy soil demonstrates significant effectiveness. It can stably survive in relatively dry sandy soil conditions, effectively adsorbing and transforming heavy metal ions, thereby reducing soil pollution levels.

[0048] Meanwhile, Bacillus megaterium LLH-B2 can regulate the pH of sandy soil, gradually making the soil more neutral, increasing the soil organic matter content, and effectively increasing crop yield and promoting crop growth.

[0049] In the control group, no Bacillus megaterium LLH-B2 was added, and the soil conditions remained severely polluted. The liquid fertilizer only enhanced the water retention of the sandy soil and ensured crop growth conditions, but its fertilization effect was not significant.

[0050] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A Bacillus megaterium LLH-B2 strain, characterized in that, The aforementioned Bacillus megaterium LLH-B2 was deposited on November 14, 2022, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, and named Bacillus megaterium (LLH-B2). Bacillus megaterium ) LLH-B2, accession number CCTCC NO: M 20221787.

2. A biological liquid fertilizer, characterized in that, The bacterial suspension containing *Bacillus megaterium* LLH-B2 as described in claim 1, by weight, comprises the following raw materials: 15-25 parts *Bacillus megaterium* LLH-B2 bacterial suspension, 1-3 parts ammonium dihydrogen phosphate, 0.5-1.5 parts dipotassium hydrogen phosphate, 0.5-1.5 parts sodium bicarbonate, 0.5-0.8 parts calcium chloride, 0.05-0.1 parts zinc sulfate, 0.2-0.5 parts magnesium sulfate, 2-5 parts sucrose, 3-4 parts humic acid, 4-5 parts chitosan composite microspheres, 1-1.5 parts carboxymethyl nanocellulose, and 300-400 parts water; the bacterial concentration of the *Bacillus megaterium* LLH-B2 bacterial suspension is ≥10. 9 cfu / mL; the raw materials for the chitosan composite microspheres include chitosan, polyvinyl alcohol, FeCl3·6H2O and FeCl2·4H2O; The preparation method of the chitosan composite microspheres includes the following steps: S1: Chitosan is added to an acetic acid solution and stirred to prepare a chitosan acetic acid solution. Polyvinyl alcohol is dissolved in water and stirred to prepare a polyvinyl alcohol aqueous solution. Then, the chitosan acetic acid solution and the polyvinyl alcohol aqueous solution are mixed to obtain a mixture. S2: FeCl3·6H2O and FeCl2·4H2O are used as solutes, and the molar ratio is 2:1 to prepare Fe 3 + / Fe 2 +Iron salt solution; S3: Add the iron salt solution dropwise to the mixture, stir, sonicate, and then use a syringe with a needle inner diameter of 100μm to drip into the NaOH and sodium citrate mixture under the action of a dual-channel injection peristaltic pump. After continuous stirring, let it stand, collect the microspheres by centrifugation, wash, and freeze-dry to obtain the product.

3. The application of Bacillus megaterium LLH-B2 as described in claim 1 in the remediation of heavy metal contaminated soil, characterized in that, The heavy metals include cadmium, lead, and / or copper.

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