Brevibacillus brevis, microbial inoculum, application of brevibacillus brevis and microbial inoculum and preparation method of microbial inoculum

Abstract

The invention discloses brevibacillus brevis, a microbial inoculum, applications of the brevibacillus brevis and the microbial inoculum, and a preparation method of the microbial inoculum. The strain is preserved in the China Center for Type Culture Collection (CCTCC) and is named as Brevibacillus sp. BSB-3, and the preservation number of the strain is CCTCC NO: M 2025263. The maximum tolerance concentration of the strain to inorganic phosphorus reaches 2000 mg / L, the removal rate of soluble phosphorus can reach 65.91% or above after the strain is cultured for 7 days in a liquid culture medium, and the accumulation amount of phosphorus-accumulating particles in bacterial cells is remarkably higher than that of conventional phosphorus-accumulating bacteria. In addition, the strain can keep stable phosphorus accumulating activity within the pH range of 5.0-8.0 and the temperature range of 20-35 DEG C and under different carbon source and nitrogen source systems, and is especially suitable for high-phosphorus wastewater treatment and black and odorous water body remediation.

Description

Technical Field

[0001] The invention belongs to the field of environmental biotechnology, and specifically relates to a strain of brevis bacillus, a bacterial agent, their application and a method for preparing the bacterial agent. Background Art

[0002] Phosphorus is one of the main limiting factors for eutrophication of water bodies. 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 secondary pollution. Biological phosphorus removal technology has gradually become the mainstream direction of sewage treatment due to its advantages such as economy, environmental protection and sustainability.

[0003] The core of biological phosphorus removal technology relies on a special type of microorganisms called phosphorus accumulating organisms (PAOs). This type of microorganism can absorb and store phosphorus in excess, and plays a key role in the biological phosphorus removal process. At present, the reported phosphorus accumulating bacteria are mainly concentrated in the genera of Acinetobacter, Pseudomonas, Tetrasphaera, etc., among which Acinetobacter has been widely studied due to its efficient phosphorus accumulation ability. However, these strains still face many challenges in practical applications, such as poor environmental adaptability, insufficient stability, and phosphorus accumulation efficiency is easily affected by external conditions. Therefore, screening and developing new and efficient phosphorus accumulation strains has become the focus of current research.

[0004] The present invention isolates a strain of Brevibacillus with high-efficiency phosphorus accumulation characteristics from the bottom mud of the simulated black and smelly water body of Huameitan, Xiangtan University, Yuhu District, Xiangtan City, Hunan Province. The discovery of this strain not only enriches the resource library of phosphorus accumulation bacteria, but also provides a new candidate strain for the development of efficient and stable biological phosphorus removal technology. Compared with traditional phosphorus accumulation 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 formation ability helps to improve the survival rate and persistence of the strain in the sewage treatment system. In addition, the study of the phosphorus accumulation mechanism of this strain may also provide a new perspective for revealing the metabolic diversity of Brevibacillus, and further expand its application range in the fields of environmental remediation and biotechnology. The discovery of this strain not only enriches the resource library of phosphorus accumulation bacteria, but also provides a new candidate strain for the development of efficient biological phosphorus removal technology, which has important theoretical significance and application prospects. Summary of the invention

[0005] In order to solve the defects and shortcomings of the prior art, the primary purpose of the present invention is to provide a strain of Brevibacillus sp. BSB-3 with efficient phosphorus accumulation characteristics; the strain is named Brevibacillus sp. BSB-3, and the preservation number is: CCTCC NO: M 2025264. The strain exhibits significant phosphorus accumulation ability under laboratory conditions, and its phosphorus absorption efficiency is significantly higher than that of conventional phosphorus accumulation strains. Preliminary studies have shown that the strain can not only efficiently absorb phosphorus under aerobic conditions, but also exhibit strong environmental adaptability and stability, and can maintain high phosphorus accumulation activity under a wide temperature, pH range and nutritional conditions. In addition, it makes it more advantageous in the sewage treatment process and can maintain activity in harsh environments, thereby improving the stability and reliability of the biological phosphorus removal process.

[0006] The second aspect of the present invention aims to provide the use of the Bacillus brevis for accumulating phosphorus.

[0007] Furthermore, the phosphorus includes soluble phosphates, such as potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, and the like.

[0008] Furthermore, the Bacillus brevis strain can accumulate phosphorus in the pH range of 5.0-8.0, the temperature range of 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 polyphosphate medium containing different phosphorus sources (monopotassium phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate), carbon sources (glucose, sodium acetate, sodium lactate) and nitrogen sources (ammonium chloride, ammonium sulfate, urea), and can form significant colonies (colony diameter ≥ 2 mm) within 24 hours.

[0011] At the same time, in liquid shake flask culture, BSB-3 has the highest utilization efficiency of sodium lactate and ammonium chloride (K value is 20%-40% higher than other carbon and 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 nitrogen, carbon and phosphorus sources. Therefore, it is suitable for wastewater treatment systems with complex components, especially in an environment with fluctuating carbon, nitrogen and phosphorus ratios, it can still maintain stable phosphorus removal performance.

[0012] The growth rate of this bacterium in liquid and solid polyphosphate medium is moderate. The liquid fermentation medium reaches the logarithmic growth phase within 6-24 hours of shaking culture on a shaker, and the polyphosphate medium changes from clear to turbid. On the polyphosphate solid medium, the colony morphology is slightly irregular and circular, with wavy edges, and has typical diffusion characteristics. The surface of the colony is rough and moist, with a distinct dull feeling, and the color is translucent grayish white. The strain shows high diffusion, forming 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, and the bacterium was identified as Brevibacillus through sequence comparison analysis.

[0013] Bacillus brevis BSB-3 can grow within 24 hours in both solid and liquid polyphosphate culture media and form obvious colonies. PolyP staining in its body with Methylene Blue stain can clearly show that it is stained blue. BSB-3 can grow in solid polyphosphate culture media with a dissolved total phosphorus concentration of 20-2000 mg / L. In the shake flask experiment of liquid polyphosphate culture media, by changing the experimental conditions such as culture temperature, carbon source, nitrogen source, pH, etc., the degradation efficiency of BSB-3 for dissolved phosphate changes little, and the maximum removal rate can reach 65.92%.

[0014] The third aspect of the present invention aims to provide a bacterial agent, wherein the bacterial agent contains the Brevibacillus sp. BSB-3.

[0015] The fourth aspect of the present invention aims to provide a method for preparing the bacterial agent, comprising the following steps:

[0016] (a) Brevibacillus sp. BSB-3 was inoculated into LB medium or polyphosphate medium for expansion;

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

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

[0019] The bacterial agent can remove phosphorus in the range of pH 5.0-8.0, temperature 20-35°C and under 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 culture medium with dipotassium hydrogen phosphate and potassium dihydrogen phosphate, the most common inorganic orthophosphates in sewage, as a phosphorus source. After 24 hours of culture, the strain can use the inorganic orthophosphate as a growth substance, and grow a large number of biological colonies after 1-3 days of culture. In a liquid culture medium, the strain of the present invention can efficiently use phosphate with a maximum concentration of 227 mg / L. The present invention also discloses suitable growth conditions for the strain, which can survive in a wide pH and temperature range, as well as in culture media with a variety of carbon sources and nitrogen sources. The strain has the best growth and phosphorus removal efficiency at 30°C, pH 6.72, and with a sodium lactate-ammonium chloride system as a carbon source and a nitrogen source. Due to the lack of efficient polyphosphate bacteria with strong environmental adaptability and stability at home and abroad, the strain of the present invention was isolated by the inventors from the bottom mud of black and odorous water bodies for the first time. It shows remarkable tolerance to complex sewage environment (such as fluctuations in pH, temperature, carbon source, nitrogen source, etc.), and can efficiently enrich dissolved phosphate in sewage. It can be widely used in the fields of deep phosphorus removal of municipal sewage, phosphorus recovery of industrial wastewater and restoration of eutrophic water bodies to reduce the phosphorus content of sewage, and provides high-quality strain resources for biologically enhanced sewage treatment processes with good application value. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0024] Figure 3 : Poly-P staining and DAPI staining microscopic examination of strain BSB-3; Figure 3 A: Poly-P staining microscopic examination of strain BSB-3; Figure 3 B: DAPI staining microscopic examination of strain BSB-3.

[0025] Figure 4 : OD of strain BSB-3 in liquid polyphosphate medium with different initial pH, temperature, carbon source and nitrogen source 600 and pH change diagram; Figure 4 A: OD of strain BSB-3 at different initial pH 600 ; Figure 4 B: pH change of strain BSB-3 at different initial pH; Figure 4 C: OD of strain BSB-3 at different initial temperatures 600 ; Figure 4 D: pH variation of strain BSB-3 at different initial temperatures; Figure 4 E: OD of strain BSB-3 under different initial nitrogen sources 600 and pH change graph; Figure 4 F: OD of strain BSB-3 under different initial carbon sources 600 and pH change graph.

[0026] Description of the biological characteristics of the deposited bacteria.

[0027] The bacteria is deposited in China Center for Type Culture Collection with the accession number CCTCC M 2025264. Address: Wuhan University, Wuhan, China. The Latin name is Brevibacillus sp. BSB-3, and the Chinese name is Brevibacillus sp. BSB-3. The bacteria was deposited on February 20, 2025. DETAILED DESCRIPTION

[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 embodiments provided are only illustrative of the method of the present invention and do not limit the rest of the content disclosed by the present invention in any way.

[0029] Example 1 Enrichment and screening of polyphosphate microorganisms and phosphorus tolerance thresholds under different phosphate concentrations.

[0030] The experimental sediment was collected from the simulated black and smelly water body of Huameitan Lake, Xiangtan University, Yuhu District, Xiangtan City, Hunan Province. 20 g of fresh black and smelly sediment was collected, added with 200 mL of distilled water, sealed in a conical flask, and heated at 25 °C and 150 r min. - 1 The mixture was shaken for 1 hour on a shaker. 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 plate was spread with a sterilized pipette and 10 mL of the sample solution was transferred to a conical flask containing 150 mL of sterilized enrichment medium (enrichment medium ingredients: 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) and stirred at 25 °C and 150 r·min - 1 When the bacteria reached the logarithmic growth phase, the supernatant was transferred to a new enrichment medium at a 1% (v / v) inoculation volume for the second cycle of enrichment, and this was repeated three times.

[0031] Take 1 mL of the culture solution from the enrichment medium and inoculate it onto the solid screening medium (the ingredients of the screening medium are: 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) according to the dilution plate method. Spread it on each plate. Repeat 3 plates for each treatment, then place the plates in a 28ºC incubator for culture. After 2 days, record the number of strains on each plate and observe the colony characteristics of the microorganisms on the solid plate. According to Figure 1 It can be seen from the growth conditions that BSB-3 can grow normally on solid screening medium.

[0032] Polyphosphate medium (sodium acetate 1.5g / L, NH4Cl 0.2g / L, MgSO4·7H2O 0.1g / L, CaCl2·2H2O0.03g / L, NaCl 0.2g / L, agar 18g / L) was added with KH2PO4 and K2HPO4 mixed in a 1:1 molar ratio, and the phosphate gradient of 20, 100, 500, 1000, 1500 and 2000 mg / L was coated on the polyphosphate medium. Each treatment was repeated 3 times, and then the plates were placed in a 28℃ incubator for culture. After 2 days, the number of strains on each plate was recorded, and the colony characteristics of microorganisms on the solid plate were observed. After cultivation and observation, the BSB-3 strain showed typical colony morphology in solid polyphosphate medium with a total phosphorus concentration of 20-2000 mg / L.

[0033] Example 2 Identification of strains.

[0034] The purified strain was inoculated into solid LB medium (ingredients: yeast extract 5g / L, peptone 10 g / L, NaCl 5g / L, agar 18g / L, pH=7.0-7.2). After culturing for 2 days, the colony morphology of the strain on the solid plate was observed, and then some colonies were picked to observe the microstructure of the strain under an optical microscope, and the morphological characteristics of the strain were recorded: the colony morphology was slightly irregular and circular, with wavy edges, and had typical diffusion characteristics. The surface of the colony was rough and moist, showing a distinct dull feeling, and the color was translucent grayish white.

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

[0036] Example 3 Poly-P staining of bacteria and DNA staining of strains.

[0037] Take 1-2 µL of bacterial culture cultured in liquid polyphosphate medium until the logarithmic growth phase, evenly spread it on a clean slide, and quickly dry it with an alcohol burner flame. Add a drop of Lu's methylene blue stain on the smear to ensure that the entire smear is covered. After staining for 10 minutes, gently rinse with distilled water to remove excess stain. Use absorbent paper to dry the water on the slide and examine it under an optical microscope. Figure 3 A Polyphosphate particles can be seen stained dark blue.

[0038] Take 1-2 µL of bacterial liquid cultured in liquid polyphosphate medium to the logarithmic growth phase, evenly spread it on a clean glass slide, dry it naturally at room temperature, add 70% ethanol as a fixative to cover the smear, and let it stand for 15 minutes. Rinse gently with PBS three times for 1 minute each time to remove residual fixative, and dry it naturally at room temperature. Add 50-100 µL of DAPI staining solution to the fixed sample to ensure that the sample area is completely covered. Place the glass slide in a light-proof box and incubate at room temperature for 15 minutes. Rinse gently with PBS three times for 1 minute each time to remove unbound DAPI dye and then seal the slide. Use a fluorescence microscope and select a blue fluorescence excitation filter for microscopic examination. According to Figure 3 B: The bacterial DNA can be seen to exhibit bright blue fluorescence.

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

[0040] The strain was inoculated into liquid LB medium (ingredients: yeast extract 5 g / L, peptone 10 g / L, NaCl 5 g / L, pH = 7.0-7.2) and pre-cultured to the logarithmic phase (24 h). The initial bacterial concentration was standardized to OD 600 ≈0.01, inoculated into polyphosphate medium containing different initial KH2PO4 concentrations at 1% (v / v), KH2PO4 concentration gradient (0.1, 0.5, 1.0 g / L), basal medium composition as follows (sodium acetate 1.5 g / L, NH4Cl 0.2 g / L, MgSO47H2O 0.1 g / L, CaCl2·2H2O 0.03 g / L, NaCl 0.2 g / L), pH 6.72, three biological replicates for each group, all cultures were shaken on a shaker at 30°C and 150 rpm for 7 days, and the initial and final phosphate concentrations of the medium were detected.

[0041] The strain was inoculated into liquid LB medium and pre-cultured to the logarithmic phase (24 h). The initial bacterial concentration was standardized to OD 600 ≈0.01, inoculated into polyphosphate medium (sodium acetate 1.5 g / L, KH2PO4 0.1 g / L, NH4Cl 0.2 g / L, MgSO4·7H2O0.1 g / L, CaCl2·2H2O 0.03 g / L, NaCl 0.2 g / L), pH 6.72, three biological replicates per group, all cultures were cultured in a shaker at 30°C, 150 rpm for 7 days, and OD was measured regularly 600 In addition, the pH gradient (5.0 / 6.0 / 7.0 / 8.0), temperature gradient (20 / 25 / 30 / 35°C), carbon source (glucose / sodium acetate / sodium lactate replaced by equal weight) and nitrogen source (ammonium chloride / ammonium sulfate / urea replaced by equal weight) were set in sequence; 3 biological replicates were performed for each condition, and OD was dynamically monitored. 600 , pH and phosphate concentration; the results showed that the strain grew best at 30℃, pH 6.72, and 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 bacteria in wastewater treatment.

[0042] Determine OD using spectrophotometry 600 The pH value of the culture medium was monitored by a calibrated pH meter (three-point calibration method, standard buffer pH 4.01, 6.86, 9.18), and the data were recorded. The soluble phosphorus content in the supernatant was detected by phospho-antimony molybdenum blue spectrophotometry. First, KH2PO4 was used to draw a phosphorus standard curve, and the fitting degree of the curve should reach 0.999. During the test, the bacterial solution was taken at 12000 r min -1 Centrifuge for 5 minutes, add the supernatant in the centrifuge tube to a graduated test tube, dilute to 50 mL, add 1 mL of ascorbic acid solution and mix well, add 2 mL after 30 seconds, let stand at room temperature for 15 minutes, measure the sample at 700 nm, and finally calculate the soluble phosphorus content based on the standard curve value and calculate the degradation characteristics. The μ value (specific growth rate) is calculated using the formula To quantify the growth rate of the strain in 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 data in Table 1 and the experimental phenomena show that the phosphorus removal efficiency of the strain is negatively correlated with the initial concentration. The phosphate removal efficiency of the strain decreases significantly with the increase of the initial concentration. The phosphate removal efficiency is the highest (65.6%) at low concentrations (22.7 mg / L), and drops sharply to 12.8% at high concentrations (227 mg / L). The strain has high efficiency biological removal potential at low phosphate concentrations (<50 mg / L) and is highly applicable to domestic sewage treatment.

[0045]

[0046] According to the data in Table 2 and the experimental phenomena, BSB-3 has the best growth under 30℃, pH 6.72, and sodium lactate-ammonium chloride system, and the phosphate removal rate is 65.92%. Moreover, under different experimental conditions, more than 40.83% of phosphate can be removed, but the change of culture conditions has a greater impact on the phosphate degradation efficiency.

Claims

1. A strain of Bacillus brevis, characterized in that: It is named Brevibacillus sp. BSB-3, and the preservation unit is China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 2025264.

2. The use of Bacillus brevis according to claim 1, characterized in that: Used to accumulate phosphorus.

3. The use according to claim 2, characterized in that: The phosphorus includes soluble phosphates.

4. The use according to claim 2, characterized in that: The brevis bacillus strain accumulates phosphorus in the range of pH 5.0-8.0, temperature 20-35°C and different carbon source and nitrogen source systems.

5. The use according to claim 4, characterized in that: Carbon sources include: At least one of glucose, sodium acetate and sodium lactate; the nitrogen source includes at least one of ammonium chloride, ammonium sulfate and urea.

6. A bacterial agent, characterized in that: The bacterial agent contains the Brevibacillus sp. BSB-3 according to claim 1.

7. The method for preparing the bacterial agent according to claim 6, characterized in that: The following steps are involved: (a) Brevibacillus sp. BSB-3 was inoculated into LB medium or polyphosphate medium for expansion; (b) Collecting the bacterial cells and mixing them with a carrier to prepare a preparation.

8. The use of the bacterial agent according to claim 6, characterized in that: Used for phosphorus removal in sewage.

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

10. The use according to claim 9, characterized in that: Carbon sources include: At least one of glucose, sodium acetate and sodium lactate; the nitrogen source includes at least one of ammonium chloride, ammonium sulfate and urea.

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