A strain of Bacillus brevis and its application

Abstract

The invention belongs to the field of biotechnology, and provides a strain of Bacillus brevis and an application thereof. The strain of Bacillus brevis is named YJY23-08, and the deposit number is CGMCC No. 26982. The strain can use 1,2,3-trichloropropane as the only carbon source to achieve its degradation. The strain or its bacterial agent is put into seawater, soil or sewage containing 1,2,3-trichloropropane, and can be harmlessly treated to further block the way for it to further enter the environment and cause long-term pollution. The application of the strain can avoid the shortcomings of low efficiency and easy secondary pollution caused by the existing treatment method, has good application value in actual production, and is of great significance for the efficient purification of 1,2,3-trichloropropane in seawater, soil and sewage.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering and provides a strain of Bacillus brevis and application thereof. Background Art

[0002] Due to the extensive and large-scale use of 1,2,3-trichloropropane, it has become one of the common organic pollutants in the environment. The natural decay rate constant of 1,2,3-trichloropropane in shallow and deep groundwater is low (the degradation half-life is as long as 680±180 years under the environmental conditions of 25°C and pH=7). It has a strong migration ability in the soil, which allows it to gradually penetrate into the groundwater environment and persist in the groundwater environment for a long time, becoming one of the persistent toxic and harmful organic pollutants that are difficult to degrade in groundwater, causing serious organochlorine pollution. At the same time, after being washed away by rainfall, 1,2,3-trichloropropane will also enter the river and then flow into the sea, resulting in an increase in the content of 1,2,3-trichloropropane in seawater.

[0003] In recent years, researchers have conducted relevant research on the degradation and transformation of 1,2,3-trichloropropane. It includes biodegradation and non-biodegradation. Non-biodegradation methods are physical and chemical methods, while biodegradation mainly utilizes 1,2,3-trichloropropane through the metabolic activities of anaerobic or aerobic microorganisms to degrade it into harmless substances. Although the physical remediation method has certain cost advantages, its remediation efficiency is low; the technology of catalytic dechlorination in the chemical method is relatively mature, but some metal ions are required as catalysts in its catalytic process. These catalysts are often costly and extremely unfriendly to the environment; and the above-mentioned physical and chemical methods have poor treatment effects on seawater. In contrast, biological remediation has the advantages of high efficiency, greenness, environmental protection and sustainability. It is a practical and increasingly popular and valued remediation technology.

[0004] In order to cut off the pollution of water and soil by pollutants such as 1,2,3-trichloropropane, it is a better method to promptly carry out in situ remediation of water sources and soil surfaces contaminated by such substances. The biodegradation of 1,2,3-trichloropropane can occur under both anaerobic and aerobic conditions, but the microbial degradation in the natural environment is extremely slow. At present, there are few wild-type microbial strains that can directly utilize 1,2,3-trichloropropane in an aerobic environment, and they are all in the basic research stage. With the development of synthetic biology technology, it has provided a powerful tool for the construction of aerobic strains that can efficiently degrade 1,2,3-trichloropropane. The aerobic biodegradation bacteria of 1,2,3-trichloropropane reported so far are mostly genetically engineered bacteria. Although genetic engineering provides new methods and approaches for the construction of microorganisms that are difficult to degrade organic halogen pollutants such as 1,2,3-trichloropropane, the stability and safety of genetically engineered strains cannot be ignored.

[0005] Therefore, how to obtain efficient 1,2,3-trichloropropane-degrading strains that are adaptable to sewage, seawater or soil environments has become an urgent problem to be solved in this field. Summary of the invention

[0006] In view of the deficiencies in the above-mentioned technologies, the present invention provides a strain of Bacillus brevis and its application. The strain of Bacillus brevis is named as YJY23-08 and its deposit number is CGMCC No. 26982. The strain can degrade 1,2,3-trichloropropane with 1,2,3-trichloropropane as the only carbon source. The strain or its bacterial agent is put into seawater, soil or sewage containing 1,2,3-trichloropropane to render it harmless, thereby blocking the way for it to further enter the environment and cause long-term pollution. The application of the strain can avoid the shortcomings of low efficiency and easy secondary pollution caused by the existing treatment method, has good application value in actual production, and is of great significance for the efficient purification of 1,2,3-trichloropropane in seawater, soil and sewage.

[0007] The specific technical solution provided by the present invention is:

[0008] First, three strains that can tolerate 1,2,3-trichloropropane were isolated from samples taken near a sewage treatment plant in Binzhou City, Shandong Province, and then purified. The three selected strains were cultured in culture media with different concentrations of 1,2,3-trichloropropane as the sole carbon source, and finally a strain with high degradation efficiency, rapid growth and stable propagation characteristics was selected and named YJY23-08.

[0009] The morphological characteristics of this strain are: the cells are rod-shaped, Gram-positive, can form spores, are elliptical to columnar, located in the center or slightly off the center of the bacteria, are mainly composed of peritrichous flagella, can move, and the colonies are flat, opaque, and yellow-gray.

[0010] The inventors sequenced the 16S rDNA of the strain and compared the 16S rDNA sequence with the NCBI database. The results of molecular identification showed that the strain belonged to Bacillus brevis ( Brevibacillus sp. ).

[0011] The inventor made a biological deposit and deposited it in the General Microbiology Center of China Microbiological Culture Collection Administration on March 31, 2023. The strain was named YJY23-08 and the deposit number was CGMCC No. 26982.

[0012] After obtaining the above strain, the inventors also provided a corresponding bacterial agent production method, comprising the following steps:

[0013] 1) Activation of bacterial strains: Move the Bacillus brevis test tube bacterial strains to room temperature for activation for 4h-8h.

[0014] 2) Seed solution preparation: inoculate the activated test tube bacteria into 50-100 mL beef extract peptone medium, and culture on a shaking table for 12-18 hours to obtain a first-level seed solution. The culture conditions are: temperature 32-37°C, shaker speed 100-200 r / min; inoculate the obtained first-level seed solution into beef extract peptone medium at a ratio of 5% (V / V), and culture under the same conditions for 16 hours to obtain a second-level seed solution;

[0015] The beef extract peptone culture medium has a formula of: 5% beef extract, 10% peptone, 5% NaCl, pH 7.0, and the balance is water, by weight percentage.

[0016] 3) Fermentation: The secondary seed liquid obtained above is inoculated into the fermentation medium at an inoculation rate of 1-5% by volume for liquid deep fermentation; the fermentation is stopped to obtain the 1,2,3-trichloropropane degrading bacterial agent (fermentation liquid), and the bacterial amount in the obtained fermentation liquid is 1×10 8 -9×10 9 cfu / mL;

[0017] The culture conditions are: temperature 32-37℃, dissolved oxygen controlled at around 30%, and culture time 12-18h.

[0018] The fermentation medium formula in step (3) is as follows: by weight percentage, maltodextrin 2-4%, sucrose 1-2%, soybean meal 3-5%, yeast powder 0.8-1.3%, ammonium sulfate 0.1-0.6%, dipotassium hydrogen phosphate 0.1-0.3%, potassium dihydrogen phosphate 0.1-0.3%, magnesium sulfate 0.01-0.1%, calcium chloride 0.05-0.1%, pH 7.0-8.0, and the balance is water.

[0019] The inventors also claim to protect the application of the above-mentioned Bacillus brevis in the degradation of 1,2,3-trichloropropane, and the specific application scope is the biochemical treatment of seawater or soil containing 1,2,3-trichloropropane or other wastewater containing 1,2,3-trichloropropane.

[0020] In specific applications, the fermentation liquid of Bacillus brevis or its bacterial agent can be added to seawater, soil or sewage containing 1,2,3-trichloropropane. After adding the bacterial agent, the initial effective live bacteria in the system is 10 5 -10 8 cfu / mL, more preferably 0.1-6×10 7 cfu / mL.

[0021] Preferably, the initial concentration of 1,2,3-trichloropropane in seawater, soil or sewage is below 600 mg / L, and more preferably, the initial concentration of 1,2,3-trichloropropane is below 300 mg / L. The corresponding optimal application conditions are: temperature 28-37°C, preferably 33°C; pH 6-9, preferably pH 7.

[0022] In summary, the beneficial effects of the present invention are as follows: the bacterial agent prepared by the strain provided by the present invention is applied to the biological treatment of seawater, soil or sewage containing 1,2,3-trichloropropane, has very good degradation characteristics for 1,2,3-trichloropropane, is simple and efficient in application, and the strain and method of the present invention significantly improve the treatment efficiency of 1,2,3-trichloropropane in seawater, soil or sewage.

[0023] The inventors have deposited the above strains, and the specific deposit information is as follows:

[0024] Storage time: March 31, 2023

[0025] Name of depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0026] Deposit number: CGMCC No.26982

[0027] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0028] Classification name: Bacillus brevis ( Brevibacillus sp. ). BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a microscope morphological photograph of the strain of the present invention;

[0030] Figure 2 The degradation curve of 1,2,3-trichloropropane in the treatment of 1,2,3-trichloropropane wastewater by the bacterial agent in Example 4 is shown;

[0031] Figure 3 This is the degradation curve of 1,2,3-trichloropropane in the bacterial agent in Example 6 during the 1,2,3-trichloropropane soil treatment. DETAILED DESCRIPTION

[0032] The above contents of the present invention are further described in detail below through specific examples, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies implemented based on the above contents of the present invention belong to the scope of the present invention. Unless otherwise specified, the following examples are all completed using conventional existing technologies, and the strains used in the following examples are all strains with a preservation number of CGMCC No. 26982. If the specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0033] The "dissolved oxygen" in the bacterial fermentation process involved in the following embodiments refers to the air saturation (%), which is a common method of expressing dissolved oxygen concentration in the fermentation industry. The "dissolved oxygen" in the aeration process of sewage treatment refers to the absolute concentration of dissolved oxygen in water, expressed in mg / L, which is a commonly used method of expressing dissolved oxygen in sewage treatment in the environmental protection industry.

[0034] Example 1 Obtaining strains:

[0035] Three strains that can tolerate 1,2,3-trichloropropane were isolated from samples taken near a sewage treatment plant in Binzhou City, Shandong Province, and were separated and purified. The three selected strains were cultured in culture media with different concentrations of 1,2,3-trichloropropane as the sole carbon source, and finally a strain with high degradation efficiency, rapid growth and stable propagation characteristics was selected and named YJY23-08.

[0036] The morphological characteristics of this strain are Figure 1 As shown: The cells are rod-shaped, Gram-positive, can form spores, are oval to columnar, located in the center or slightly to the side of the bacteria, are mainly composed of peritrichous flagella, can move, and the colonies are flat, opaque, and yellow-gray.

[0037] The inventors sequenced the 16S rDNA of the bacteria and compared the 16S rDNA sequence with the NCBI database. The results of molecular identification showed that the bacteria belonged to Bacillus brevis ( Brevibacillus sp. ).

[0038] The inventor deposited it in the General Microbiology Center of China Microorganism Culture Collection Administration on March 31, 2023, with the strain code YJY23-08 and the deposit number CGMCC No.26982.

[0039] Example 2 Preparation of Bacillus brevis bacterial agent:

[0040] 1) Activation of bacterial strains: Move the Bacillus brevis test tube bacterial strains to room temperature for activation for 4h-8h.

[0041] 2) Seed solution preparation: Inoculate a single tube of the activated test tube strain into 100 mL of beef extract peptone medium, and culture it in a shaking incubator overnight at 35°C and 180 r / min to obtain a first-level seed solution. Inoculate the obtained first-level seed solution into beef extract peptone medium at a ratio of 5% (V / V), and culture it for 16 h under the same conditions to obtain a second-level seed solution.

[0042] 3) Fermentation: The secondary seed liquid obtained above is inoculated into a fermentation medium at an inoculum amount of 5% by volume for liquid deep fermentation, the fermentation temperature is controlled at 35±1°C, the dissolved oxygen is about 30%, and the culture is carried out for 18 hours. When the dissolved oxygen rises and the pH drops, the fermentation is stopped to obtain the 1,2,3-trichloropropane degrading bacterial agent (fermentation liquid).

[0043] The beef extract peptone culture medium has a formula of: 5% beef extract, 10% peptone, 5% NaCl, pH 7.0, and the balance is water, by weight percentage.

[0044] The fermentation medium is composed of 3% maltodextrin, 2% sucrose, 3% soybean meal, 0.9% yeast powder, 0.2% ammonium sulfate, 0.2% dipotassium hydrogen phosphate, 0.2% potassium dihydrogen phosphate, 0.0125% magnesium sulfate, 0.05% calcium chloride, pH 7.4, and the balance is water.

[0045] The number of viable bacteria in the fermentation broth obtained in step (3) above was 6.8×10 9 cfu / mL.

[0046] Example 3 Application of bacterial agent in the treatment of wastewater containing 1,2,3-trichloropropane:

[0047] The 1,2,3-trichloropropane-degrading bacterial agent was prepared again according to the method of Example 2. The culture temperature was 33±1°C. The viable bacterial count of the obtained bacterial agent was 1.3×10 9 cfu / mL.

[0048] The treated water comes from the wastewater of a pesticide and pharmaceutical intermediates park. The wastewater COD is about 1100mg / L, total salt is 4.5%, ammonia nitrogen is about 5mg / L, Cl - The content is about 10000 mg / L, the biodegradability is poor, it is used after five times dilution, the processing volume is 5L, and 1,2,3-trichloropropane is added to a concentration of 120 mg / L; then 1,2,3-trichloropropane liquid bacterial agent is added, and the initial bacterial count of the control system is about 2×10 6 cfu / mL, aeration treatment at room temperature made the dissolved oxygen above 2mg / L, and the sewage pH 6.0-8.0; after 24h of aeration treatment, the 1,2,3-trichloropropane content in the sewage was <1mg / L, and the degradation rate reached more than 99.9%.

[0049] Example 4 Application of bacterial agent in the treatment of wastewater containing 1,2,3-trichloropropane 2:

[0050] The 1,2,3-trichloropropane-degrading bacterial agent was prepared again according to the method of Example 2. The culture temperature was 32±1°C. The viable bacterial count of the obtained bacterial agent was 9×10 8 cfu / mL.

[0051] The treated water was derived from oilfield reinjection water, and 1,2,3-trichloropropane was artificially added to a concentration of 200 mg / L. The above-mentioned 1,2,3-trichloropropane degradation agent was added during the treatment, so that the initial bacterial count of the system was about 1.8×10 7 cfu / mL, treatment volume is 2L, temperature is 33℃, sewage pH is 7.5, COD is about 750mg / L, ammonia nitrogen is about 24mg / L, total nitrogen is 51mg / L, sampling is performed every 4h to detect the content of 1,2,3-trichloropropane in the system, and its degradation is shown in Figure 2 , the pollutants can be completely degraded within 32 hours.

[0052] Example 5 Application of bacterial agent in 1,2,3-trichloropropane wastewater treatment III:

[0053] The 1,2,3-trichloropropane-degrading bacterial agent was prepared again according to the method of Example 2. The culture temperature was 35±1°C. The viable bacterial count of the obtained bacterial agent was 6×10 9 cfu / mL.

[0054] The treated water source is the influent of the aerobic pool of the petrochemical wastewater treatment plant, with a treatment capacity of 10L. The above-mentioned 1,2,3-trichloropropane degradation agent is added to make the initial bacterial count of the system about 6×10 7 cfu / mL, aeration treatment at room temperature to make the dissolved oxygen above 2mg / L, sewage pH 6.0-8.0, 1,2,3-trichloropropane initial content below 5mg / L, COD about 1800mg / L, volatile phenol about 240mg / L, ammonia nitrogen about 95mg / L, sulfide about 8mg / L, inlet and outlet water once every 24h; at the same time, a certain amount of 1,2,3-trichloropropane was added when influent, and the concentration of 1,2,3-trichloropropane in the influent and effluent was tested (the results are shown in Table 1 below). The system was used continuously. According to the content of 1,2,3-trichloropropane in the effluent, it can be seen that the bacterial agent has a continuous degradation ability for 1,2,3-trichloropropane sewage.

[0055] Table 1

[0056] 0d 1d 2d 3d 4d 5d 6d 7d TCP content after water inlet (mg / L) 100 100 120 150 200 200 200 200 TCP content in effluent (mg / L) / ＜1.0 ＜1.0 ＜1.0 ＜1.0 ＜1.0 ＜1.0 ＜1.0 Degradation rate of 1,2,3-trichloropropane / >99% >99% >99% >99% >99% >99% >99% .

[0057] Example 6 Application of bacterial agent in saline-alkali soil treatment:

[0058] The test soil was collected from a vegetable planting base on the tidal flats of the Yellow River estuary in Dongying. Impurities such as gravel particles were removed, and the soil was naturally air-dried and passed through a 2 mm sieve. 1,2,3-Trichloropropane was added to the soil at a ratio of 300 mg / kg, mixed thoroughly, and balanced at room temperature in the dark for 3 days.

[0059] A microbial group and a control group were set up, wherein the control group was not treated in any way, and the 1,2,3-trichloropropane-degrading bacterial agent prepared in Example 2 was added to the microbial group to make the live bacterial count of 1,2,3-trichloropropane-degrading bacteria in the soil reach 10 6 -10 7 cfu / g, the soil pH was controlled at 7.8, the water content was 10%-20%, and the salt content was 0.5%; 2 kg of soil was treated in each group and placed at room temperature away from light. Samples were collected every 2 days to test the content of 1,2,3-trichloropropane, and deionized water was sprayed on the soil regularly to maintain soil moisture. Figure 3 This is the soil treatment effect of 1,2,3-trichloropropane-degrading bacterial agent, which shows that it has a strong degradation ability in 1,2,3-trichloropropane-contaminated soil.

[0060] Example 7 Application of bacterial agent in degrading 1,2,3-trichloropropane in seawater:

[0061] The 1,2,3-trichloropropane-degrading bacterial agent was prepared again according to the method of Example 2. The culture temperature was 30±1°C. The viable bacterial count of the obtained bacterial agent was 4.5×10 8 cfu / mL.

[0062] The water quality source is the shallow water seawater near Dongying, Bohai Sea in September 2023. The water quality is as follows: pH 8.0, COD about 1.3 mg / L, ammonia nitrogen about 0.3 mg / L, and salt content 34.897 g / kg; 1,2,3-trichloropropane is artificially added to a concentration of 200 mg / L; the above-mentioned 1,2,3-trichloropropane degradation agent is added to make the initial bacterial count of the seawater system about 6×10 6 cfu / mL, the treated water volume was 1L, and the aeration treatment was performed at room temperature for 1h before being allowed to stand. After 24h, the system was stirred and the 1,2,3-trichloropropane content in the water was tested by sampling and was less than 1mg / L, indicating that the bacteria had a certain tolerance to salt and could achieve efficient and rapid treatment when 1,2,3-trichloropropane was contaminated in seawater.

[0063] The above embodiments are convenient for those skilled in the art to better understand the present invention. The implementation methods of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A strain of Bacillus brevis ( Brevibacillus sp. )YJY23-08, characterized by: The deposit number is CGMCC No. 26982, and it was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on March 31, 2023.

2. A method for preparing a microbial agent using the Bacillus brevis according to claim 1, characterized in that: The specific steps are as follows: 1) Activation of bacteria: Move the Bacillus brevis test tube bacteria to room temperature for activation for 4h-8h; 2) Seed solution preparation: inoculate the activated test tube bacteria into 50-100 mL beef extract peptone medium, and culture on a shaking table for 12-18 hours to obtain a first-level seed solution. The culture conditions are: temperature 32-37°C, and a shaking speed of 100-200 r / min; inoculate the obtained first-level seed solution into a beef extract peptone medium at a V / V ratio of 5%, and culture under the same conditions for 16 hours to obtain a second-level seed solution; 3) Fermentation: The secondary seed liquid obtained above is inoculated into the fermentation medium at an inoculation rate of 1-5% by volume for liquid deep fermentation; the fermentation is stopped to obtain the 1,2,3-trichloropropane degradation bacterial agent fermentation liquid, and the bacterial amount in the obtained fermentation liquid is 1×10 8 -9×10 9 cfu / mL; culture conditions are: temperature 32-37℃, dissolved oxygen controlled at 30%, and culture time 12-18h.

3. The method for preparing a microbial agent using Bacillus brevis according to claim 2, characterized in that: The beef extract peptone culture medium in step 2) has a formula of: 5% beef extract, 10% peptone, 5% sodium chloride, pH 7.0, and the balance is water.

4. The method for preparing a microbial agent using Bacillus brevis according to claim 2, characterized in that: In step 3), the fermentation medium formula is as follows: by weight percentage, maltodextrin 2-4%, sucrose 1-2%, soybean meal 3-5%, yeast powder 0.8-1.3%, ammonium sulfate 0.1-0.6%, dipotassium hydrogen phosphate 0.1-0.3%, potassium dihydrogen phosphate 0.1-0.3%, magnesium sulfate 0.01-0.1%, calcium chloride 0.05-0.1%, pH 7.0-8.0, and the balance is water.

5. The use of the Bacillus brevis according to claim 1 in the degradation of 1,2,3-trichloropropane, characterized in that: Biochemical treatment of seawater or soil containing 1,2,3-trichloropropane or other wastewater containing 1,2,3-trichloropropane.

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