A bacillus tequilensis and use thereof

Abstract

This invention relates to the field of biodegradation technology, and proposes a type of Bacillus tergentii and its applications. Bacillus tergentii is a species of Bacillus tergentii (…). Bacillus tequilensis MB-HN-3, deposited at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, Beichen West Road, Chaoyang District, Beijing, on November 4, 2024, with accession number CGMCC No. 32479. This technical solution addresses the problem of poor lignite degradation ability of other microbial strains in related technologies.

Description

Technical Field

[0001] This invention relates to the field of biodegradation technology, specifically to a type of Bacillus tekiria and its applications. Background Technology

[0002] Humic acid is a high-molecular-weight organic compound formed by the decomposition of plant and animal remains by microorganisms in complex environments. It is widely distributed in soil, water bodies, peat, lignite, and weathered coal. my country has relatively abundant coal humic acid resources. According to statistics, lignite reserves are 130.3 billion tons, and weathered coal reserves are approximately 100 billion tons. The primary humic acid content in these low-rank coals is about 10% to 80%, exhibiting high biological activity. Due to their low degree of coalification, fewer fused-ring structures, more fatty chain structures, and higher lignin-like structures, their structure is closer to that of original plant remains, making them easier for microorganisms to degrade. However, due to the special structure and properties of lignite, microorganisms face harsh environmental conditions such as nutrient deficiency, high osmotic pressure, and low oxygen during the degradation process. Publicly reported microbial strains have low degradation rates for lignite. Therefore, screening and cultivating microbial strains with good lignite degradation effects is of great significance. Summary of the Invention

[0003] This invention proposes a strain of Bacillus tekirae and its application, which solves the problem that other microbial strains have poor ability to degrade lignite in related technologies.

[0004] The technical solution of the present invention is as follows: This invention proposes a type of Bacillus tergentii, wherein the Bacillus tergentii is Bacillus tergentii ( Bacillus tequilensis MB-HN-3, deposited at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, Beichen West Road, Chaoyang District, Beijing, on November 4, 2024, with accession number CGMCC No. 32479.

[0005] As a further technical solution, the *Bacillus tekirii* ( Bacillus tequilensis The 16S rDNA gene sequence of MB-HN-3 is shown in SEQ ID NO.1.

[0006] The present invention also proposes a microbial agent, wherein the microbial agent comprises the aforementioned Bacillus tekirae ( Bacillus tequilensis MB-HN-3.

[0007] As a further technical solution, the microbial agent may also include, but is not limited to, one or more of other strains, carriers, nutrients, and adjuvants.

[0008] As a further technical solution, when the microbial agent includes other strains, the other strains include, but are not limited to, one or more of nitrogen-fixing bacteria, Bacillus, actinomycetes, phosphate-solubilizing bacteria, and phosphate-solubilizing fungi.

[0009] As a further technical solution, when the microbial agent includes a carrier, the carrier includes, but is not limited to, one or more of diatomaceous earth, montmorillonite, kaolin, white carbon, light calcium carbonate, rice husk, sawdust, starch, polyvinyl alcohol, and polyethylene glycol.

[0010] As a further technical solution, when the microbial agent includes nutrients, the nutrients include, but are not limited to, one or more of the following: salts, minerals, amino acids, sugars, yeast extracts, and trace elements.

[0011] As a further technical solution, when the microbial agent includes adjuvants, the adjuvants include, but are not limited to, one or more of solvents, pH adjusters, dispersants, emulsifiers, preservatives, humectants, and wetting agents.

[0012] As a further technical solution, the microbial agent contains Bacillus tergentii ( Bacillus tequilensis The viable count of MB-HN-3 is 10. 7 ~10 11 CFU / g or 10 7 ~10 11 CFU / mL.

[0013] This invention also proposes the application of Bacillus tekirulatus or a microbial agent in the degradation of lignite.

[0014] As a further technical solution, the lignite does not require any pretreatment during the degradation process.

[0015] The present invention also proposes the application of Bacillus tekirulatus or a microbial agent in the fight against pathogenic fungi.

[0016] The present invention also proposes the application of Bacillus tekirulatus or a microbial agent in salt-alkali tolerant environments.

[0017] This invention also proposes the application of Bacillus tekirulatus or a microbial agent in the production of extracellular polysaccharides and the improvement of soil aggregate structure.

[0018] The present invention also proposes the application of Bacillus tekirulatus or a microbial agent in the degradation of organic matter.

[0019] As a further technical solution, the organic matter includes one or more of cellulose, protein, lipids, and starch.

[0020] Applying Bacillus tekirae MB-HN-3 or microbial agents containing Bacillus tekirae MB-HN-3 to degrade cellulose can promote the efficient and pollution-free degradation of cellulose, facilitating the treatment and recycling of cellulose-containing wastes, such as agricultural waste (e.g., plant straw). Furthermore, based on the excellent ability of Bacillus tekirae MB-HN-3 to degrade proteins, lipids, inorganic phosphorus, and starch, Bacillus tekirae MB-HN-3 or microbial agents containing Bacillus tekirae MB-HN-3 can effectively degrade wastes or organic fertilizers containing at least one of cellulose, protein, fat, inorganic phosphorus, and starch, improving the treatment efficiency and comprehensive utilization of organic matter containing complex components.

[0021] The working principle and beneficial effects of this invention are as follows: In this invention, *Bacillus tekirae* was isolated from soil in Jianfeng Town, Ledong County, Hainan Province. Bacillus tequilensis The strain MB-HN-3 does not require any pretreatment of lignite when degrading it. After being cultured at 30°C with shaking for 7 days, and then treated at 120°C for 20 minutes, the humic acid release rate reached 9.46%, which improved the degradation rate of lignite.

[0022] Bacillus tekirulatus ( Bacillus tequilensis The MB-HN-3 strain not only has the ability to degrade lignite to produce humic acid, but also has the ability to produce extracellular polysaccharides, promote the formation of soil aggregates, and degrade lipids, proteins, cellulose, starch and inorganic phosphorus. It is a salt-tolerant and alkali-tolerant multifunctional strain with broad application prospects. Attached Figure Description

[0023] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0024] Figure 1 This is a diagram of the color reduction reaction experiment of strain MB-HN-3 in Example 1; In the image: a is aniline blue; b is azure B; c is Coomassie Brilliant Blue; Figure 2 This is a comparison image of the supernatant after high-temperature treatment in the shake flask experiment of MB-HN-3 strain degrading lignite in Example 2; In the image: the left image is the control group, and the right image is the MB-HN-3 strain; Figure 3 This is the phylogenetic tree of the MB-HN-3 strain in Example 3; Figure 4 The morphology of the MB-HN-3 strain in Example 4 on R2A medium; Figure 5 The resistance of the MB-HN-3 strain in Example 5 to different pathogenic fungi; In the diagram: a represents the causal agent of cotton wilt; b represents the causal agent of wheat sheath blight; c represents the causal agent of wheat root rot; d represents the causal agent of gray mold; e represents the causal agent of pear black spot; f represents the causal agent of apple leaf spot; g represents the causal agent of wheat scab; h represents the causal agent of tomato early blight; i represents the causal agent of wheat stem base rot. Figure 6 The growth of the MB-HN-3 strain in Example 6 under different conditions; In the diagram: a represents 45℃; b represents pH 5; c represents pH 10; d represents the addition of 5% NaCl; e represents the addition of 10% NaCl; f represents the addition of 5% KNO3; g represents the addition of 10% KNO3. Figure 7 The degradation ability of the MB-HN-3 strain in Example 7 on lipids, proteins, cellulose, starch, and inorganic phosphorus substances; In the diagram: a is a lipid culture medium; b is a protein culture medium; c is CMC culture medium; d is a soluble protein culture medium; e is an inorganic phosphorus culture medium. Figure 8 The effect of different treatments on the proportion of water-stable aggregates (>0.25 mm) in soil in Example 9.

[0025] Instructions for the Preservation of Biological Materials The Bacillus tergentii provided by this invention ( Bacillus tequilensis MB-HN-3, deposited at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 32479, deposited on November 4, 2024. Detailed Implementation

[0026] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0027] Example 1 Screening of microorganisms capable of degrading lignite 1. Soil sampling Soil samples for this experiment were collected from Jianfeng Town, Ledong County, Hainan Province.

[0028] 2. Screening of target microorganisms After diluting the soil sample, spread it on the screening medium (10g yeast extract, 20g glucose, 0.1g aniline blue, 18g agar, 1L distilled water) and incubate at 30℃ for 2 days. Select strains that can change the color of the medium and incubate them on R2A medium (0.5g yeast extract, 0.5g tryptone, 0.5g peptone, 0.5g glucose, 0.5g soluble starch, 0.3g dipotassium hydrogen phosphate, 0.3g sodium pyruvate, 15g agar, 1L water) at 30℃ for 2 days. 3. Microbial Function Replication Validation After the strain was extracted and purified, it was inoculated onto three different screening media containing aniline blue, azure B, and Coomassie brilliant blue (10g yeast extract, 20g glucose, 0.1g aniline blue / azure B / Coomassie brilliant blue, 18g agar, and 1L distilled water). The color reduction reaction of the strain was observed, and functional rescreening was performed.

[0029] Taking the MB-HN-3 strain as an example, the color reduction reaction in different culture media is as follows: Figure 1 As shown.

[0030] Example 2 Lignite Shake Flask Degradation Test The strains selected above were inoculated into 250 mL shake flasks containing 0.25 g lignite in 50 mL of nutrient-deficient R2A medium (0.5 g yeast extract, 0.75 g peptone, 0.5 g glucose, 0.3 g dipotassium hydrogen phosphate, 0.3 g sodium pyruvate, 1 L water, pH 7.0). A negative control without inoculation was also included. The flasks were cultured at 30 °C with shaking for 7 days, followed by treatment at 120 °C for 20 min. The humic acid content in the supernatant was measured again, and the conversion rate was calculated. The experimental results for the MB-NM-3 strain and the control are as follows: Figure 2 As shown. The determination and calculation formula of humic acid are carried out in accordance with the industry standard "NYT1971-2010 Determination of Humic Acid Content in Water-Soluble Fertilizers".

[0031] Besides the inherent differences in oxidation levels among different types of lignite, pretreatment significantly impacts microbial degradation. The lignite used in this experiment underwent no pretreatment, increasing the difficulty of microbial degradation, and the reaction system was 5g / 1000mL. Furthermore, the determination method involved measuring the humic acid content in the supernatant and then calculating its weight. This method excludes fulvic acid and other substances that cannot be precipitated by acid. Additionally, to facilitate calculation and comparison, factors such as the moisture content of the lignite were not excluded, resulting in a lower release rate compared to other literature. However, using a consistent determination method within the same system, the data in this experiment are comparable, but cannot be directly compared with data from other experiments.

[0032] The test results are as follows: This experiment screened six strains with lignite degradation function from soil samples collected from Jianfeng Town, Ledong County, Hainan Province. Through lignite shake-flask degradation experiments, one strain, MB-HN-3, was obtained with high efficiency in degrading lignite. The specific degradation results are shown in Table 1.

[0033] Table 1. Effects of the strains on lignite degradation

[0034] As shown in Table 1, the humic acid release rate of strain MB-HN-3 was 9.46%, which was higher than that of strains MB-HN-1, MB-HN-2, MB-HN-4, MB-HN-5, and MB-HN-6. This indicates that strain MB-HN-3 had the best degradation effect on lignite. Therefore, strain MB-HN-3 was identified and preserved.

[0035] Example 3 Strain identification 1. Inoculate a single colony into 5 mL of R2A liquid (0.5 g yeast extract, 0.5 g tryptone, 0.75 g peptone, 0.5 g glucose, 0.5 g soluble starch, 0.3 g dipotassium hydrogen phosphate, 0.3 g sodium pyruvate, 1 L water) and incubate at 30 °C for 24 h to obtain seed culture solution; 2. Take 1 mL of seed culture medium and inoculate it into 100 mL of R2A liquid, and incubate at 37℃ and 220 r / min for 16 hours; 3. Centrifuge at 5000 r / min for 10 minutes and discard the supernatant; 4. After centrifugation and washing with 10 mL TE, dissolve the bacterial cells with 10 mL TE, mix well, and store at -20℃ for later use. 5. Take 3.5 mL of bacterial suspension, add 184 μL of 10% SDS (w / v), mix well, add 37 μL of 10 mg / mL proteinase K, mix well, and incubate at 37°C for 1 hour; 6. Add 740 μL of 5 mol / L NaCl, then add 512 μL of CTAB / NaCl, mix well, and incubate at 65°C for 10 minutes; 7. Add an equal volume of chloroform / isoamyl alcohol (volume ratio 24:1), mix well, centrifuge at 10000 r / min for 5 minutes, and retain the supernatant; 8. Add an equal volume of a mixture of phenol:chloroform:isoamyl alcohol (volume ratio 25:24:1) to the supernatant, mix well, centrifuge at 10000 r / min for 5 minutes, and retain the supernatant; 9. Add 0.6 times the volume of isopropanol, mix well, centrifuge at 10000 r / min for 5 minutes, collect the DNA precipitate, and wash the DNA precipitate with 70% ethanol by centrifugation. 10. Dissolve the DNA in 1 mL TE buffer, add RNase A to a final concentration of 20 μg / mL, and store at 4°C.

[0036] 2.2 Amplification and Sequencing PCR amplification of 16S rDNA was performed using universal primers 27f (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492r (5'-GGTTACCTTGTTACGACTT-3'). The PCR reaction mixture consisted of 20 μL Taq PCR Master Mix, 0.8 μL of each primer, 1 μL of DNA template, and 17.4 μL ddH2O. The PCR conditions were: 94℃ pre-denaturation for 30 s; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 60 s, 35 cycles, followed by a final extension at 72℃ for 10 min. The PCR products were subjected to 1.0% (w / v) agarose gel electrophoresis. After agarose gel electrophoresis, the PCR products were recovered, purified, and sequenced (Shanghai Sangon Biotech Co., Ltd.). Homologous sequences were searched for in GenBank using Blast search based on the obtained 16S rDNA sequence. A phylogenetic tree was constructed using MEGA 7.0 software; the phylogenetic tree is shown below. Figure 3 As shown.

[0037] Analysis of the 16S rDNA sequence of strain MB-HN-3 showed that strain MB-HN-3 is related to Bacillus tekirae (Bacillus tekirae). Bacillus tequilensis The homology is as high as 100%; the 16S rDNA sequence of strain MB-HN-3 is shown in SEQ ID NO.1.

[0038] Example 4 Observation of strain morphology The selected MB-HN-3 strain was inoculated onto R2A plates and incubated at 30°C for 48 hours. The size, shape, color, gloss, raised shape, transparency, and edge characteristics of the colonies were observed.

[0039] Result: As Figure 4 As shown, the bacterial strain grew on R2A medium for 2 days. The colonies were round with irregular edges, white and opaque, slightly raised, moist and glossy, and 3-4 mm in diameter.

[0040] Example 5 Antifungal properties of strains The selected and sequenced safe functional strains were subjected to a plate confrontation test with existing fungi in the laboratory. 8 mm fungal discs were punched out and inoculated into the center of PDA medium (200 g potato, 20 g glucose, 15-20 g agar, 1000 mL water). In a cross pattern, MB-HN-3 (abbreviated as HN-3) was picked up with a 10 μL inoculation loop and inoculated 2.5 cm away from the pathogenic fungus. The mixture was incubated at 28 °C, and the resistance of the functional strains to the pathogenic fungus was determined.

[0041] Existing fungi include: *Fusarium wiltii* (the causal agent of cotton wilt). Fusarium oxysporum Wheat sheath blight pathogen ( Rhizoctonia cereals ), wheat root rot fungus ( Bipolaris sorokiniana ), gray mold ( Botrytis cinerea ), pear black spot fungus ( Alternaria alternate Apple spot leaf spot pathogen ( Alternaria brassicae ), wheat scab ( Fusahum graminearum Early blight of tomato ( Altemaria solani ), wheat stem rot fungus ( Fusarium pseudograminearum ).

[0042] Result: As Figure 5 It is known that the strain MB-HN-3 of the present invention has significant antagonistic effects on *Fusarium wilt*, *Rhizoctonia solani*, *Radix et al.*, *Botrytis cinerea*, *Hypericum glomeratum*, *Hypericum spp.*, *Hypericum spp.*, *Hypericum spp.*, *Fusarium wilt*, *Hypericum spp.*, and *Hypericum spp.*.

[0043] Example 6 Stress resistance determination of strains The selected strains were picked up with a 10 μL inoculation loop and inoculated onto R2A medium with conditions of 45℃, pH 5, pH 10, 5% NaCl, 10% NaCl, 5% KNO3, and 10% KNO3 (content calculated by w / v). The growth of the strains was observed, as well as whether the growth was inhibited and the growth rate.

[0044] Result: As Figure 6 It is known that the strain MB-HN-3 of the present invention can grow normally on R2A medium with conditions of 45℃, pH 5, pH 10, 5% NaCl, 5% KNO3, and 10% KNO3. Its growth is somewhat inhibited on R2A medium with 10% NaCl, but it can still grow.

[0045] Example 7 Determination of the strain's ability to degrade lipids, proteins, cellulose, starch, and inorganic phosphorus. The strain MB-HN-3, which had been cultured on R2A medium for 1 day, was inoculated onto media containing different substances. After incubation at 30°C for 2 days, the diameter of the degradation zone was measured (in mm). The results are as follows: Figure 7 As shown in Table 2.

[0046] Degradation capacity formula: Degradation capacity = (Diameter of degradation zone in functional culture medium in millimeters) + X X is a weighting coefficient, which is -2, -1, 0, 1, or 2 depending on the transparency of the silica ring of the strain. Formulas for various functional culture media: Lipid culture medium: 10 g / L glyceryl tartrate + R2A solid culture medium; Protein culture medium: 5 g / L skim milk powder + R2A solid culture medium; Cellulose (CMC) medium: 1.0 g dipotassium hydrogen phosphate, 0.25 g magnesium sulfate heptahydrate, 2.0 g yeast extract, 10.0 g agar powder, 2.0 g CMC, 1000 mL distilled water; Soluble starch medium: 5.0g beef extract, 10.0g peptone, 5.0g sodium chloride, 2.0g soluble starch, 20.0g agar powder, 1000mL distilled water; Soluble starch medium and CMC medium require iodine fumigation before testing.

[0047] Inorganic phosphorus culture medium: yeast 0.5g, glucose 10.0g, ammonium sulfate 0.5g, potassium chloride 0.02g, magnesium sulfate heptahydrate 0.1g, ferrous sulfate 0.2mg, manganese sulfate 4mg, agar powder 15.0g, tricalcium phosphate 5.0g (prepared separately), distilled water 1000mL.

[0048] Table 2. Degradation capabilities of MB-HN-3 for lipids, proteins, cellulose, starch, and inorganic phosphorus substances.

[0049] Result: From Figure 7 As can be clearly seen from Table 2, the strain MB-HN-3 of the present invention has a significant degradation effect on lipids, proteins, cellulose, starch, and inorganic phosphorus.

[0050] Example 8 Screening of extracellular polysaccharide production media Method for determining extracellular polysaccharides: ① Centrifuge the above fermentation broth at 3000 r / min for 10 min to remove bacterial cells; ② Take 2 ml of supernatant into a 50 ml centrifuge tube, add 3 times the volume of anhydrous ethanol, centrifuge at 4℃ for 1 h at 4000 r / min for 5 min, and discard the supernatant; ③ Wash the precipitate with 10 ml of 80% ethanol solution, centrifuge and discard the supernatant; ④ Dissolve the precipitate in water and transfer it to a 100 ml volumetric flask, and make up to volume; ⑤ Determine the extracellular polysaccharide content using the sulfuric acid-phenol method.

[0051] This example describes the screening of culture media for MB-HN-3 to produce extracellular polysaccharides. The specific details are as follows: Based on the initial culture medium (1L, including 20g sucrose, 3g yeast extract, 2.6g KH2PO4, 0.5g CaCl2, 2g MgSO4, 1mg FeSO4, with the remainder being water), different liquid culture media containing sucrose and glucose were prepared at 20g / L to determine the optimal carbon source; different liquid cultures containing yeast extract, peptone, KNO3, and (NH4)2SO4 were prepared at 3g / L to determine the optimal nitrogen source.

[0052] Based on previous experimental results, sucrose and yeast extract are the most suitable carbon and nitrogen sources for MB-HN-3. Through experiments involving the addition of sucrose, yeast extract, and different amounts of organic salts, the optimal culture medium for MB-HN-3 to produce extracellular polysaccharides was preliminarily determined. The specific components of the different culture media are described below.

[0053] Culture medium 1: per 1L, it includes 45g sucrose, 3g yeast powder, 2.6g KH2PO4, 0.5g CaCl2, 2g MgSO4, 1mg FeSO4, and the remainder is water; Culture medium 2: per 1L, it includes 45g sucrose, 5g yeast powder, 2.6g KH2PO4, 0.5g CaCl2, 2g MgSO4, 1mg FeSO4, and the remainder is water; Culture medium 3: per 1L, it includes 45g sucrose, 5g yeast powder, 1.8g KH2PO4, 0.5g CaCl2, 2g MgSO4, 1mg FeSO4, and the remainder is water; Culture medium 4: per 1L, it includes 45g sucrose, 5g yeast powder, 1.8g KH2PO4, 0.5g CaCl2, 2g MgSO4, 1.5mg FeSO4, and the remainder is water; Culture medium 5: per 1L, it includes 45g sucrose, 5g yeast powder, 1.8g KH2PO4, 0.5g CaCl2, 3g MgSO4, 1.5mg FeSO4, and the remainder is water.

[0054] After culturing at 30℃ and 200r / min for 72h, the extracellular polysaccharide content of MB-HN-3 under different culture media is shown in Table 3.

[0055] Table 3. Extracellular polysaccharide content under different culture media

[0056] As shown in Table 3, the most suitable culture medium for MB-HN-3 to produce extracellular polysaccharides is formula 5: sucrose 45g, yeast powder 5g, KH2PO4 1.8g, CaCl2 0.5g, MgSO4 3g, FeSO4 1.5mg, H2O 1000mL, and the polysaccharide content can reach 12.00mg / mL.

[0057] Example 9 Effects of strains and extracellular polysaccharides on the aggregate structure of water-stable soil Soil aggregates are the basic units of soil structure and an important indicator for measuring the degree of soil erosion. Soil aggregates are generally divided into two main categories: large aggregates (>0.25 mm) and micro-aggregates (<0.25 mm). Water-stable aggregates (>0.25 mm) are generally referred to as soil aggregate structure and are used as an indicator for evaluating soil aggregates.

[0058] ① Experimental treatment The test strain was activated on R2A medium, and then the activated strain was inoculated into an extracellular polysaccharide-producing medium (Example 5). The culture was carried out at 30°C with shaking at 200 rpm for 48 h. The fermentation broth was then collected, and the cells were collected by centrifugation at 4000 rpm for 5 min. The cells were washed twice with sterile deionized water and resuspended in sterile deionized water until the OD600 was 0.005 (approximately 10⁶ / mL). This bacterial suspension was divided into two portions: one for live bacteria and the other for sterilization at 121°C for 30 min to obtain an inactivated bacterial suspension. These were then cultured and fermented using the medium described in Example 6 to extract extracellular polysaccharides, preparing liquids with different extracellular polysaccharide contents for later use.

[0059] The experiment included five treatment groups: inoculated with live bacteria, inoculated with inactivated bacteria, inoculated with 1.4 mg / g extracellular polysaccharide, inoculated with 2.8 mg / g extracellular polysaccharide, and a control group inoculated with an equal volume of sterile water. Each treatment was replicated in triplicate. Soil samples passing through a 0.25 mm sieve were weighed and placed into Petri dishes, labeled with the group number. 5 mL of sterile water, live bacterial suspension, inactivated bacterial suspension, and different concentrations of extracellular polysaccharide solution were added to their respective Petri dishes. The Petri dishes were placed on a light-controlled culture rack and incubated at 20–30 °C. During incubation, deionized water was added periodically to keep the soil surface moist. After 30 days of incubation, the treatments were administered.

[0060] ② Measurement method Weigh 20g of undisturbed soil from a petri dish, taking care to preserve the soil's original shape as much as possible. Place it in a graduated cylinder, and slowly add deionized water along the cylinder wall until saturated. Transfer the saturated soil sample to the top of a sieve placed in a bucket of water, and shake until soil particles on the sieve are clearly visible on the water surface and no longer pass through the sieve openings. Collect soil samples from the top and bottom of the sieve separately, dry them, and weigh them. Samples located on the top of the sieve are identified as >0.25mm water-stable aggregates, while samples located on the bottom of the sieve are identified as <0.25mm water-stable aggregates. The measurement results are shown in Table 4 and... Figure 8 As shown.

[0061] Table 4. Effects of different treatments on the proportion of water-stable aggregates (>0.25 mm) in soil

[0062] Note: * indicates a significant difference between the control and the treatment, and ** indicates a highly significant difference between the control and the treatment.

[0063] Results: Table 4 shows that, except for the treatment with inactivated bacteria, which showed a significant difference from the control, all other treatments showed highly significant differences from the control. The 2.8 mg / g extracellular polysaccharide treatment resulted in the greatest increase in the proportion of soil aggregates >0.25 mm, increasing it by 13.47 times compared to the control. The 1.4 mg / g extracellular polysaccharide treatment increased the proportion of soil aggregates >0.25 mm by 8.38 times compared to the control, and the inoculation with live bacteria treatment increased the proportion of >0.25 mm water-stable aggregates by 1.9 times compared to the control. This indicates that the MB-HN-3 strain and the extracellular polysaccharides produced by the strain can promote the formation of soil aggregates >0.25 mm.

[0064] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A type of Bacillus tekirae, characterized in that, The *Bacillus tekirae* is *Bacillus tekirae* ( Bacillus tequilensis MB-HN-3, deposited at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, Beichen West Road, Chaoyang District, Beijing, on November 4, 2024, with accession number CGMCC No. 32479.

2. The *Bacillus tekirii* strain according to claim 1, characterized in that, The Bacillus tergenta ( Bacillus tequilensis The 16S rDNA gene sequence of MB-HN-3 is shown in SEQ ID NO.

1.

3. A microbial inoculant, characterized in that, The microbial agent includes Bacillus tekiria as described in claim 1 or 2. Bacillus tequilensis MB-HN-3.

4. A microbial inoculant according to claim 3, characterized in that, The microbial agent contains Bacillus tergentis ( Bacillus tequilensis The viable count of MB-HN-3 is 10. 7 ~10 11 CFU / g or 10 7 ~10 11 CFU / mL.

5. The application of a microbial agent according to claim 3 or 4 in the degradation of lignite.

6. The application according to claim 5, characterized in that, During the degradation of lignite, the lignite does not require any pretreatment.

7. The application of a Bacillus tekirae according to claim 1 or 2 or a microbial agent according to claim 3 or 4 in the resistance to wheat sheath blight, wheat root rot, pear black spot, apple leaf spot, tomato early blight, or wheat stem base rot.

8. The application of a Bacillus tekirae according to claim 1 or 2 or a microbial agent according to claim 3 or 4 in the production of extracellular polysaccharides and improvement of soil aggregate structure.

9. The use of a Bacillus tekirae according to claim 1 or 2 or a microbial agent according to claim 3 or 4 in the degradation of lipids, proteins, cellulose, starch or inorganic phosphorus.

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