A Brevibacillus strain, microbial agent and their applications and preparation method of the microbial agent

Abstract

The present invention discloses a Brevibacillus strain, a bacterial agent and their applications and a preparation method of the bacterial agent. The strain is preserved in the China Center for Type Culture Collection (CCTCC), named Brevibacillus sp. BSB-3, and the preservation number is CCTCC NO: M 2025263. The maximum tolerance concentration of the strain to inorganic phosphorus reaches 2000 mg / L. In a liquid medium, after 7 days of culture, the removal rate of soluble phosphorus can reach more than 65.91%, and the accumulation amount of polyphosphate granules in the bacterial cells is significantly higher than that of conventional polyphosphate-accumulating bacteria. In addition, the strain can maintain stable polyphosphorus activity within the ranges of pH 5.0-8.0, temperature 20-35 °C and different carbon source and nitrogen source systems, and is particularly suitable for the treatment of high-phosphorus wastewater and the restoration of black and odorous water bodies.

Description

Technical Field

[0001] The present invention belongs to the field of environmental biotechnology, and specifically relates to a Brevibacillus strain, a bacterial agent, their applications, and a preparation method of the bacterial agent. Background Art

[0002] Phosphorus is one of the main limiting factors for water eutrophication. How to efficiently remove phosphorus from sewage has become a research hotspot in the field of environmental science and engineering. Although the traditional chemical phosphorus removal method has significant effects, it has problems such as high cost and easy generation of secondary pollution. The biological phosphorus removal technology has gradually become the mainstream direction of sewage treatment due to its advantages of economy, environmental protection, and sustainability.

[0003] The core of the biological phosphorus removal technology depends on a special type of microorganism called Phosphate Accumulating Organisms (PAOs). These microorganisms can absorb and store phosphorus in excess and play a key role in the biological phosphorus removal process. Currently, the reported PAOs mainly focus on genera such as Acinetobacter, Pseudomonas, and Tetrasphaera. Among them, Acinetobacter has been widely studied due to its high phosphorus accumulation ability. However, these strains still face many challenges in practical applications, such as poor environmental adaptability, insufficient stability, and the phosphorus accumulation efficiency being easily affected by external conditions. Therefore, screening and developing new and efficient phosphorus-accumulating strains has become the focus of current research.

[0004] A Brevibacillus strain with high phosphorus accumulation characteristics was isolated from the sediment of a simulated black and odorous water body in Huameitan of Xiangtan University, YuHu District, Xiangtan City, Hunan Province. The discovery of this strain not only enriches the resource library of phosphorus-accumulating bacteria but also provides a new candidate strain for developing efficient and stable biological phosphorus removal technology. Compared with traditional phosphorus-accumulating bacteria, the unique physiological characteristics of Brevibacillus make it have greater potential in practical applications. For example, its high-temperature resistance makes it suitable for high-temperature wastewater treatment, and its spore-forming ability helps to improve the survival rate and persistence of the strain in the sewage treatment system. In addition, the research on the phosphorus accumulation mechanism of this strain may also provide a new perspective for revealing the metabolic diversity of the genus Brevibacillus and further expanding its application scope in the fields of environmental remediation and biotechnology. The discovery of this strain not only enriches the resource library of phosphorus-accumulating bacteria but also provides a new candidate strain for developing efficient biological phosphorus removal technology, having important theoretical significance and application prospects. Summary of the Invention

[0005] To address the deficiencies and drawbacks of the existing technologies, the primary objective of the present invention is to provide a Brevibacillus sp. BSB-3 strain with efficient phosphorus-accumulating characteristics. This strain is named Brevibacillus sp. BSB-3 and has a preservation number of CCTCC NO: M 2025264. Under laboratory conditions, this strain exhibits remarkable phosphorus-accumulating ability, and its phosphorus absorption efficiency is significantly higher than that of conventional phosphorus-accumulating strains. Preliminary studies have shown that this strain can not only efficiently absorb phosphorus under aerobic conditions but also demonstrates strong environmental adaptability and stability, capable of maintaining high phosphorus-accumulating activity within a wide range of temperatures, pH values, and nutrient conditions. Additionally, this makes it more advantageous in the sewage treatment process, being able to maintain its activity in harsh environments, thereby enhancing the stability and reliability of the biological phosphorus removal process.

[0006] The second objective of the present invention is to provide the application of the above-mentioned Brevibacillus sp. for phosphorus accumulation.

[0007] Furthermore, the phosphorus mentioned above includes soluble phosphates. For example: potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, etc.

[0008] Even further, the Brevibacillus sp. strain can accumulate phosphorus within the range of pH 5.0 - 8.0, temperature 20 - 35 °C, and under different carbon source and nitrogen source systems.

[0009] The carbon source includes at least one of glucose, sodium acetate, and sodium lactate, and the nitrogen source includes at least one of ammonium chloride, ammonium sulfate, and urea.

[0010] Under laboratory conditions, the BSB-3 strain of the present invention can grow in a phosphorus-accumulating culture medium containing different phosphorus sources (potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate), carbon sources (glucose, sodium acetate, sodium lactate), and nitrogen sources (ammonium chloride, ammonium sulfate, urea), and significant colonies (colony diameter ≥ 2 mm) can be formed within 24 hours.

[0011] Meanwhile, in liquid shake flask culture, BSB-3 has the highest utilization efficiency for sodium lactate and ammonium chloride (the K value is 20% - 40% higher than other carbon-nitrogen source combinations), indicating that its metabolic pathway may preferentially rely on the synergistic effect of specific carbon and nitrogen sources, and has strong adaptability to a wide range of nitrogen sources, carbon sources, and phosphorus sources. Therefore, it is suitable for sewage treatment systems with complex components, especially capable of maintaining stable phosphorus removal performance even in an environment with fluctuating carbon-nitrogen-phosphorus ratios.

[0012] The growth rate of this bacterium is moderate in liquid and solid polyphosphorus media. In the liquid fermentation medium, it reaches the logarithmic growth phase within 6 - 24 hours of shaking culture on a shaker, and the polyphosphorus medium changes from clear to turbid. On the solid polyphosphorus medium, the colony morphology is slightly irregularly round, with a wavy edge, showing typical diffusible characteristics. The surface of the colony is rough and moist, presenting an obvious dull feeling, and the color is semi - transparent grayish - white. The strain shows high diffusibility and forms a uniform film - like growth on the surface of the solid medium, gradually expanding and covering the entire medium plane. The 16S rDNA gene sequence of the strain has been sequenced and analyzed. Through sequence alignment analysis, this bacterium is identified as Brevibacillus.

[0013] In solid and liquid polyphosphorus media, Brevibacillus sp. BSB - 3 can grow within 24 hours and form obvious colonies. By staining PolyP in its body with Loeffler's methylene blue stain, it can be clearly seen that it is stained blue. In the solid polyphosphorus medium with a dissolved total phosphorus concentration of 20 - 2000 mg / L, BSB - 3 can grow. In the shake - flask experiment of the liquid polyphosphorus medium, by changing experimental conditions such as culture temperature, carbon source, nitrogen source, and pH, the degradation efficiency of BSB - 3 for dissolved phosphate changes little, and the highest removal rate can reach 65.92%.

[0014] The purpose of the third aspect of the present invention is to provide a bacterial agent, and the bacterial agent contains the Brevibacillus sp. BSB - 3 described above.

[0015] The purpose of the fourth aspect of the present invention is to provide a preparation method of the bacterial agent, including the following steps:

[0016] (a) Inoculate Brevibacillus sp. BSB - 3 into LB medium or polyphosphorus medium for amplification culture;

[0017] (b) Collect the bacterial cells and mix them with a carrier to prepare a preparation.

[0018] The purpose of the fifth aspect of the present invention is to provide an application of the bacterial agent for phosphorus removal from sewage.

[0019] The described bacterial agent can remove phosphorus within the range of pH 5.0 - 8.0, temperature 20 - 35 °C, and different carbon source and nitrogen source systems.

[0020] The carbon source includes at least one of glucose, sodium acetate, and sodium lactate, and the nitrogen source includes at least one of ammonium chloride, ammonium sulfate, and urea.

[0021] The strain of the present invention grows well in a solid medium with potassium hydrogen phosphate and potassium dihydrogen phosphate, the most common inorganic orthophosphates in sewage, as the phosphorus source. After 24 hours of cultivation, it can utilize inorganic orthophosphate as a growth substance, and a large number of biological colonies grow after 1-3 days of cultivation. In a liquid medium, the strain of the present invention can efficiently utilize phosphate with a maximum concentration of 227 mg / L. The present invention also discloses the suitable growth conditions of the strain. It can survive in a wide range of pH and temperature, as well as in media with various carbon and nitrogen sources. The strain has the best growth and phosphorus removal efficiency at about 30 °C and pH 6.72, with sodium lactate-ammonium chloride system as the carbon and nitrogen sources. Due to the lack of efficient polyphosphate-accumulating bacteria resources with strong environmental adaptability and stability at home and abroad, the strain of the present invention was first isolated by the inventor from the sediment of a black and odorous water body. It shows significant tolerance to complex sewage environments (such as fluctuations in pH, temperature, carbon source, nitrogen source, etc.), and can efficiently enrich dissolved phosphate in sewage. It can be widely applied to fields such as advanced phosphorus removal from municipal sewage, phosphorus recovery from industrial wastewater, and restoration of eutrophic water bodies, providing high-quality bacterial resources for biological enhanced sewage treatment processes and having good application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : Growth of strain BSB-3 on a solid screening plate.

[0023] Figure 2 : Molecular biological phylogenetic tree of strain BSB-3.

[0024] Figure 3 : Microscopic examination diagrams of Poly-P staining and DAPI staining of strain BSB-3; among them Figure 3 A: Microscopic examination diagram of Poly-P staining of strain BSB-3; Figure 3 B: Microscopic examination diagram of DAPI staining of strain BSB-3.

[0025] Figure 4 : OD 600 and pH change diagrams of strain BSB-3 in liquid polyphosphate media with different initial pH, temperature, carbon source, and nitrogen source; among them Figure 4 A: OD 600 change diagram of strain BSB-3 at different initial pH; Figure 4 B: pH change diagram of strain BSB-3 at different initial pH; Figure 4 C: OD 600 change diagram of strain BSB-3 at different initial temperatures; Figure 4 D: pH change diagram of strain BSB-3 at different initial temperatures; Figure 4 E: OD 600 and pH change diagrams of strain BSB-3 at different initial nitrogen sources;Figure 4 F: OD of Strain BSB-3 with Different Initial Carbon Sources 600 and pH Variation Diagram

[0026] Description of the Biological Characteristics of the Deposited Bacteria

[0027] This bacterium is deposited in the China Center for Type Culture Collection (CCTCC) with the deposit number CCTCC M 2025264. Address: Wuhan University, Wuhan, China. Latin scientific name: Brevibacillus sp. BSB-3, Chinese name: Brevibacillus sp. BSB-3. This bacterium was deposited on February 20, 2025. Detailed Implementation Modes

[0028] The features and advantages of the present invention can be further understood through the following detailed description in conjunction with the accompanying drawings. The provided embodiments are only illustrative of the method of the present invention and do not limit the remaining content disclosed by the present invention in any way.

[0029] Example 1 Enrichment, Screening of Polyphosphorus Microorganisms and Phosphorus Tolerance Thresholds under Different Concentrations of Phosphate

[0030] The experimental sediment was collected from the sediment of a simulated black-odor water body in Huameitan, Xiangtan University, YuHu District, Xiangtan City, Hunan Province. 20 g of fresh black-odor sediment was taken and added to a triangular flask with 200 mL of distilled water, sealed and shaken evenly on a shaker at 25 °C and 150 r·min - 1 for 1 hour. After natural sedimentation, a muddy water mixture containing a large number of free bacteria was obtained. After standing for 0.5 h, 0.5 mL of the supernatant was taken and added to the enrichment medium. The sample solution (10 mL) was transferred to a conical flask containing 150 mL of sterilized enrichment medium (composition of the enrichment medium: anhydrous sodium acetate 5 g / L, MgSO4·7H2O 0.5 g / L, (NH4)2SO4 2 g / L, NaCl 2.5 g / L, KH2PO4 0.2 g / L, K2HPO4 0.3 g / L, CaCl2 0.25 g / L, FeSO4 0.001 g / L, CuSO4 0.005 g / L) with a sterilized pipette and shaken on a shaker at 25 °C and 150 r·min - 1 for enrichment culture. When the bacteria reached the logarithmic growth phase, the supernatant was taken again and transferred to a new enrichment medium at an inoculation amount of 1% (v / v) for the second cycle of enrichment, and this was repeated 3 times.

[0031] Take 1 mL of the culture solution in the enrichment medium and inoculate it onto the solid screening medium (the composition of the screening medium is: yeast extract: 2 g / L, KH2PO4 0.4 g / L, K2HPO4 0.6 g / L, disodium hydrogen phosphate (Na2HPO4): 0.5 g / L, magnesium sulfate (MgSO4·7H2O): 0.2 g / L, glucose: 2 g / L, agar 18 g / L) by the dilution plating method. Repeat each treatment on 3 plates, then place the plates in an incubator at 28ºC for cultivation. After 2 days, record the number of strains on each plate and observe the colony characteristics of the microorganisms on the solid plates. According to Figure 1 the growth situation, it can be seen that BSB-3 can grow normally on the solid screening medium.

[0032] For the phosphorus-accumulating medium (sodium acetate 1.5 g / L, NH4Cl 0.2 g / L, MgSO4·7H2O 0.1 g / L, CaCl2·2H2O 0.03 g / L, NaCl 0.2 g / L, agar 18 g / L), add KH2PO4 and K2HPO4 mixed in a 1:1 molar ratio, and prepare phosphorus-accumulating media with phosphate gradients of 20, 100, 500, 1000, 1500, and 2000 mg / L for coating. Repeat each treatment on 3 plates, then place the plates in an incubator at 28°C for cultivation. After 2 days, record the number of strains on each plate and observe the colony characteristics of the microorganisms on the solid plates. After cultivation and observation, the BSB-3 strain shows typical colony morphology in the solid phosphorus-accumulating medium with a total phosphorus concentration of 20 - 2000 mg / L.

[0033] Identification of the strain in Example 2.

[0034] Inoculate the purified strain onto the solid LB medium (composition: yeast extract 5 g / L, peptone 10 g / L, NaCl 5 g / L, agar 18 g / L, pH = 7.0 - 7.2). After 2 days of cultivation, observe the colony morphological characteristics of the strain on the solid plate, then pick a little colony and observe the microscopic structure of the strain under an optical microscope, and record the morphological characteristics of the strain: the colony morphology is slightly irregularly round, the edge is wavy, with typical diffusivity characteristics. The surface of the colony is rough and moist, showing an obvious dull feeling, and the color is semi-transparent grayish white.

[0035] Bacterial 16S rDNA sequencing includes strain DNA extraction, in vitro amplification of 16S rRNA (using 16S universal primers 27F and 1492R), and gene sequencing. The sequence results obtained by the gene company are subjected to double-end splicing, analysis, and then brought into the NCBI database for BLAST gene sequence alignment. According to Figure 2Based on the morphological observation and the constructed phylogenetic tree, this strain was identified as Brevibacillus.

[0036] Example 3: Poly-P staining of the bacterial cells and DNA staining of the strain.

[0037] Take 1 - 2 μL of the bacterial liquid cultured to the logarithmic growth phase in the liquid polyphosphate medium, evenly coat it on a clean glass slide, and quickly dry it using the flame of an alcohol lamp. Drop Loeffler's methylene blue stain on the smear to ensure complete coverage of the smear. After staining for 10 minutes, gently rinse it with distilled water to remove the excess stain. Blot the water on the glass slide with absorbent paper and then examine it under an optical microscope. According to Figure 3 A It can be seen that the polyphosphate granules are stained dark blue.

[0038] Take 1 - 2 μL of the bacterial liquid cultured to the logarithmic growth phase in the liquid polyphosphate medium, evenly coat it on a clean glass slide, air-dry it at room temperature, drop 70% ethanol as a fixative to cover the smear, and let it stand for 15 minutes. Gently rinse it 3 times with PBS, 1 minute each time, to remove the residual fixative, and then air-dry it at room temperature. Drop 50 - 100 μL of DAPI stain on the fixed sample to ensure complete coverage of the sample area. Place the glass slide in a light-proof box and incubate it at room temperature for 15 minutes. Gently rinse it 3 times with PBS, 1 minute each time, to remove the unbound DAPI dye, and then mount the slide. Use a fluorescence microscope, select the blue fluorescence excitation filter, and examine it. According to Figure 3 B It can be seen that the bacterial DNA shows bright blue fluorescence.

[0039] Example 4: Study on the phosphorus removal efficiency and optimal growth conditions of the strain.

[0040] Inoculate the strain into the liquid LB medium (composition: yeast extract 5 g / L, peptone 10 g / L, NaCl 5 g / L, pH = 7.0 - 7.2) and pre-culture it to the logarithmic phase (24 h). Then standardize the initial bacterial liquid concentration to OD 600 ≈0.01, and inoculate it into the polyphosphate medium containing different initial KH2PO4 concentrations at 1% (v / v). The KH2PO4 concentration gradients are (0.1, 0.5, 1.0 g / L). The composition of the basal medium is as follows (sodium acetate 1.5 g / L, NH4Cl 0.2 g / L, MgSO4·7H2O 0.1 g / L, CaCl2·2H2O 0.03 g / L, NaCl 0.2 g / L), pH is 6.72. Set 3 biological replicates for each group. All cultures are shaken on a shaker at 30 °C and 150 rpm for 7 days, and the initial and final phosphate concentration values in the medium are detected.

[0041] After inoculating the strain into liquid LB medium and pre-culturing it to the logarithmic phase (24 h), the initial bacterial liquid concentration was standardized to OD 600 ≈0.01, and it was inoculated into the polyphosphate medium (sodium acetate 1.5 g / L, KH2PO4 0.1 g / L, NH4Cl 0.2 g / L, MgSO4·7H2O 0.1 g / L, CaCl2·2H2O 0.03 g / L, NaCl 0.2 g / L), with a pH of 6.72. Three biological replicates were set for each group. All cultures were shaken on a shaker at 30 °C and 150 rpm for 7 days, and OD 600 , pH, and phosphate concentration values were measured regularly. In addition, pH gradients (5.0 / 6.0 / 7.0 / 8.0), temperature gradients (20 / 25 / 30 / 35 °C), carbon sources (equal weight replacement of glucose / sodium acetate / sodium lactate), and nitrogen sources (equal weight replacement of ammonium chloride / ammonium sulfate / urea) were set in sequence; three biological replicates were set for each condition, and OD 600 , pH, and phosphate concentration were dynamically monitored; the results showed that the strain grew optimally under the conditions of 30 °C, pH 6.72, and the sodium lactate-ammonium chloride system, and the phosphate removal rate reached 65.9%. The research results can provide a theoretical basis for the application of polyphosphate-accumulating organisms in sewage treatment.

[0042] The OD 600 value was measured using spectrophotometry (wavelength 600 nm, 1 cm light path cuvette, with sterile medium as the blank control). At the same time, the pH value change of the culture solution was monitored using a calibrated pH meter (three-point calibration method, standard buffer solutions pH 4.01, 6.86, 9.18). Samples were taken at regular intervals to detect and record data. The content of soluble phosphorus in the supernatant was detected using the phosphomolybdenum blue spectrophotometry method. First, a phosphorus standard curve was drawn using KH2PO4, and the fitting degree of the drawn curve should reach 0.999. During detection, the bacterial liquid was centrifuged at 12000 r min -1 for 5 min. The supernatant in the centrifuge tube was added to a graduated test tube, fixed to 50 mL, then 1 mL of ascorbic acid solution was added and mixed evenly. After 30 s, 2 mL was added, and after standing at room temperature for 15 min, the sample was measured at 700 nm. Finally, the soluble phosphorus content was calculated according to the standard curve value, and the degradation characteristics were calculated. The μ value (specific growth rate) was calculated using the formula to quantify the growth rate of the strain during the logarithmic growth phase and monitor the real-time impact of environmental changes on the growth rate of the strain to reflect the proliferation ability of the strain.

[0043]

[0044] The negative correlation between the phosphorus removal efficiency of the strain and the initial concentration was obtained through the data in Table 1 and the observation of experimental phenomena. The phosphorus removal efficiency of the strain for phosphate decreased significantly with the increase of the initial concentration. The phosphorus removal efficiency was the highest (65.6%) at a low concentration (22.7 mg / L), and dropped sharply to 12.8% at a high concentration (227 mg / L). This strain has high biological removal potential at low phosphate concentrations (<50 mg / L) and strong applicability to domestic sewage treatment.

[0045]

[0046] It was obtained through the data in Table 2 and the observation of experimental phenomena that BSB-3 had the best growth under the conditions of 30 °C, pH 6.72, and sodium lactate-ammonium chloride system, and the phosphate removal rate was 65.92%. And under different experimental conditions, the removal of phosphate could reach more than 40.83%, but the change of culture conditions had a great influence on the degradation efficiency of phosphate.

Claims

1. A Brevibacillus strain, characterized in that, Named Bacillus brevis Brevibacillus sp. BSB-3, with the deposit number: CCTCC NO: M 2025264.

2. Use of the Brevibacillus described in claim 1, characterized in that, For phosphorus accumulation; the phosphorus includes soluble phosphate.

3. The application according to claim 2, characterized in that, The Brevibacillus strain accumulates phosphorus within the ranges of pH 5.0 - 8.0, temperature 20 - 35 °C and under different carbon source and nitrogen source systems.

4. The application according to claim 3, characterized in that The carbon sources include: At least one of glucose, sodium acetate, and sodium lactate, and the nitrogen sources include: at least one of ammonium chloride, ammonium sulfate, and urea.

5. A bacterial agent, characterized in that, The bacterial agent contains the Brevibacillus brevis described in claim 1 Brevibacillus sp. BSB-3.

6. The preparation method of the microbial agent according to claim 5, characterized in that, Comprising the following steps: (a) Inoculate Bacillus brevis Brevibacillus sp. BSB-3 into LB medium or polyphosphate medium for amplification culture; (b) Collect the bacterial cells and mix them with a carrier to prepare a preparation.

7. Use of the microbial agent according to claim 5, characterized in that, For phosphorus removal from sewage, the phosphorus includes soluble phosphate.

8. The application according to claim 7, characterized in that The bacterial agent removes phosphorus within the ranges of pH 5.0 - 8.0, temperature 20 - 35 °C and under different carbon source and nitrogen source systems.

9. The application according to claim 8, characterized in that, The carbon sources include: At least one of glucose, sodium acetate, and sodium lactate, and the nitrogen sources include: at least one of ammonium chloride, ammonium sulfate, and urea.

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