A strain of Brevibacillus russii and its application

Abstract

The invention belongs to the field of biotechnology, and provides a strain of Brevibacillus russelii and an application thereof. The Brevibacillus russelii is named as YJY23-07 and has a deposit number of CGMCC NO.26981. The strain can use 1,2-dichloropropane 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-dichloropropane, and can be harmlessly treated to prevent it from entering the soil or groundwater environment to cause long-term pollution. The application of the strain can avoid the disadvantages of high cost and easy secondary pollution caused by the existing treatment method, has no side effects on operators, has good application value in actual production, and is of great significance for the efficient purification of 1,2-dichloropropane in seawater, soil and industrial wastewater.

Description

Technical Field

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

[0002] With the development of modern chemical synthesis technology, a large number of artificially synthesized organic chlorides are widely used in many fields such as the chemical industry, pharmaceutical industry, printing and dyeing industry, and agriculture. Organic chlorides have high density, low water solubility, and strong mobility in the soil. Therefore, once these compounds enter the soil, they can eventually enter the surface water and be discharged into the sea through soil leaching, or penetrate into the groundwater environment. Among them, 1,2-dichloropropane is a typical chloroalkane compound with stable chemical properties and toxicity. It has toxic effects such as pathogenicity and carcinogenicity. It often enters the body through the respiratory tract, digestive tract, skin, etc. and causes poisoning. It is now generally used as a soil fumigant and soil insecticide. Therefore, its degradation has been widely and deeply studied.

[0003] Organic pollutants in water, especially organochlorinated hydrocarbons, are difficult to remove effectively due to their low solubility and high interfacial tension using conventional treatment methods, such as extraction. They cannot be fundamentally controlled and are difficult to degrade using general physical and chemical methods. Biodegradation can convert toxic substances into non-toxic substances through the action of microorganisms, with low investment and no secondary pollution. Therefore, it is increasingly important to make full use of microbial degradation technology.

[0004] In order to find efficient and environmentally friendly treatment technologies, domestic and foreign scholars have studied the microbial degradation of chlorinated hydrocarbons. The anaerobic degradation of chlorinated alkanes is mainly catalyzed by organic halogen-respiring bacteria. At present, a variety of organic halogen-respiring bacteria that can degrade chlorinated alkanes have been isolated from contaminated sites. The main bacterial genera are: Dehalococcoides, Dehalobacter, Desulfitobacterium, Geobacter, Sulfurospirillum, etc. Their dehalogenation reactions are mostly carried out under anaerobic conditions, and they are all heterotrophic bacteria. It is difficult to separate a single strain. In the process of application, due to the low-carbon characteristics of seawater and groundwater, it is often necessary to supplement carbon sources for strengthening. There are fewer types of dehalogenating microorganisms and their degradation effect on 1,2-dichloropropane is weak. In order to further cut off the pollution of pollutants such as 1,2-dichloropropane to seawater, groundwater and soil, timely in-situ remediation of water sources or soil surfaces contaminated by such substances is an efficient method for removing chlorinated alkanes from groundwater, while solving the above defects of organic halogen-respiring bacteria.

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

[0006] In view of the shortcomings of the above-mentioned technology, the present invention provides a strain of Brevibacillus ruderii and its application. Brevibacillus reuszeri ), the strain was named YJY23-07, and the deposit number was CGMCCNO. 26981. The strain can use 1,2-dichloropropane 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-dichloropropane to treat it harmlessly, thereby blocking it from entering the soil or groundwater environment to cause long-term pollution. The application of the strain can avoid the disadvantages of the existing treatment method such as high cost and easy secondary pollution, has no side effects on operators, has good application value in actual production, and is of great significance for the efficient purification of 1,2-dichloropropane in seawater, soil and industrial wastewater.

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

[0008] Firstly, five strains that can use 1,2-dichloropropane as the sole carbon source were isolated from samples taken near a sewage treatment plant in Binzhou City, Shandong Province, and were separated and purified respectively.

[0009] Five candidate strains were cultured in culture medium with different concentrations of 1,2-dichloropropane as the sole carbon source. Finally, a strain with high degradation efficiency, rapid growth and stable propagation characteristics was screened out and named YJY23-07.

[0010] The strain has the following morphological characteristics: Gram-negative short rod, with round, flat, neatly edged, light yellow, rough, opaque colonies on LB medium, and a colony size of about 1-3 mm. The growth temperature is 15-45°C, and it grows in the pH range of 6-9. It is catalase positive, oxidase, nitrate reduction, and starch hydrolysis are all negative, and it uses glucose, galactose, and mannose to produce acid.

[0011] The inventors sequenced the 16S rDNA of the bacterium and compared the 16S rDNA sequence with the NCBI database. The results of molecular identification showed that the bacterium belongs to Brevibacillus ruckerii ( Brevibacillus reuszeri ).

[0012] The inventor biologically preserved it and deposited it in the General Microbiology Center of China Microbiological Culture Collection Administration in March 2023. The strain was named YJY23-07 and the preservation number was CGMCC No. 26981.

[0013] After obtaining the above strain, the inventors also provided a corresponding method for preparing a bacterial agent, comprising the following steps:

[0014] 1) Activation of bacterial strains: Move the test tube strain of Brevibacillus ruderi to room temperature for activation for 4h-8h.

[0015] 2) Seed solution preparation: The activated test tube strains were inoculated into the corresponding liquid culture medium, 1 test tube strain was inoculated into 50-100 mL beef extract peptone medium, and the seed solution was obtained by shaking culture for 12-18 hours. The culture conditions were: temperature 32-37°C, shaking speed 100-200 r / min;

[0016] The beef extract peptone culture medium has a formula of: by weight (w / w): 5% beef extract, 10% peptone, 5% NaCl, pH 7.0, and the balance is water.

[0017] 3) Fermentation: Inoculate the seed liquid of Brevibacillus russii in a volume percentage of 1-5% into the corresponding fermentation medium for liquid deep fermentation to obtain a fermentation liquid; the culture conditions are: temperature 32-37° C., rotation speed 100-200 r / min, and culture time 12-18 h;

[0018] The amount of bacteria in the fermentation liquid was 0.5×10 8 -8×10 9 cfu / mL.

[0019] 4) Drying and crushing: The fermentation liquid obtained by the above fermentation is adsorbed, dried and crushed to obtain a single bacterial agent with an effective live bacterial count of more than 10 billion / g.

[0020] The fermentation medium formula in step (3) is as follows: by weight (w / w): soybean cake powder 1-3%, glucose 1-3%, corn steep liquor dry powder 1-3%, peptone 0.1-0.5%, magnesium sulfate 0.01-0.05%, sodium chloride 0-0.5%, ammonium sulfate 0.1-0.6%, potassium dihydrogen phosphate 0.01-0.1%, calcium carbonate 0.1-1%, pH 7.0-8.0, and the balance is water.

[0021] The inventors also claim to protect the application of the above-mentioned Brevibacillus russii, which is to degrade 1,2-dichloropropane. The specific application scope includes the biochemical treatment of seawater, soil or other wastewater containing 1,2-dichloropropane.

[0022] The specific application method is: the fermentation liquid of Brevibacillus russii or its bacterial agent is put into the treatment system, which can effectively withstand the impact of 1,2-dichloropropane pollutants, can use the organic matter in the biochemical system as nutrients for its own growth, and quickly degrade the organic matter.

[0023] Preferably, the effective viable count of Brevibacillus russii in the treatment system is ensured to be 10 5 -108 cfu / mL, more preferably 0.1-6×10 7 cfu / mL, which enables the strain to quickly adapt to the environment, metabolize and grow with 1,2-dichloropropane as the carbon source, and effectively exert the strain's 1,2-dichloropropane degradation effect.

[0024] Through application experiments, it was shown that the above-mentioned Brevibacillus russii can be continuously cultured and applied in seawater containing 1,2-dichloropropane, and the degradation effect is stable.

[0025] In specific applications, the fermentation liquid of Brevibacillus russii or its bacterial agent can be added to seawater, soil or sewage containing 1,2-dichloropropane. 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.

[0026] Preferably, the initial concentration of 1,2-dichloropropane is below 600 mg / L, and more preferably, the initial concentration of 1,2-dichloropropane in the biochemical system is below 300 mg / L.

[0027] The application conditions of the strain of the present invention are: temperature 28-37°C, preferably 33°C; pH 6-9, preferably pH 7.

[0028] 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 wastewater, waste gas and soil containing 1,2-dichloropropane, has very good degradation characteristics for 1,2-dichloropropane, is simple and efficient in application, and the strain and method of the present invention significantly improve the treatment efficiency of 1,2-dichloropropane wastewater.

[0029] The inventors have deposited the above strains, and the specific deposit information is as follows:

[0030] Storage time: March 31, 2023

[0031] Name of depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0032] Deposit number: CGMCC No.26981

[0033] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0034] Classification name: Brevibacillus ruderi Brevibacillus reuszeri ). BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1This is a photo of the colony morphology of the strain of the present invention on LB medium;

[0036] Figure 2 This is a degradation curve diagram of 1,2-dichloropropane in 1,2-dichloropropane wastewater treatment in Example 4;

[0037] Figure 3 This is a degradation curve of 1,2-dichloropropane in 1,2-dichloropropane soil treatment in Example 6. DETAILED DESCRIPTION

[0038] The above contents of the present invention are further described in detail below by means of specific implementation methods in the form of 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 prior art, and the strains used in the following examples are strains with a preservation number of CGMCC No. 26981. If the specific conditions are not indicated in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer is not indicated for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0039] 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.

[0040] Example 1 Obtaining strains:

[0041] Five strains that can utilize 1,2-dichloropropane as the sole carbon source were isolated from samples taken from a sewage treatment plant in Binzhou City, Shandong Province, and were separated and purified respectively.

[0042] Five candidate strains were cultured in culture medium with different concentrations of 1,2-dichloropropane as the sole carbon source. Finally, a strain with high degradation efficiency, rapid growth and stable propagation characteristics was screened out and named YJY23-07.

[0043] The strain has the following morphological characteristics: Gram-negative short rods, such as Figure 1 As shown, the colonies on LB medium are round, flat, with neat edges, light yellow, rough, opaque, and about 1-3 mm in size. The growth temperature is 15-45°C, and the pH range is 6-9. It is catalase positive, oxidase, nitrate reduction, and starch hydrolysis are all negative, and it uses glucose, galactose, and mannose to produce acid.

[0044] The inventors sequenced the 16S rDNA of the bacterium and compared the 16S rDNA sequence with the NCBI database. The results of molecular identification showed that the bacterium belongs to Brevibacillus ruckerii ( Brevibacillus reuszeri ).

[0045] The inventor biologically preserved it and deposited it in the General Microbiology Center of China Microbiological Culture Collection Administration in March 2023. The strain code is YJY23-07 and the preservation number is CGMCC No.26981.

[0046] Example 2 Preparation of Brevibacillus russii bacterial agent:

[0047] In the first step, the purified Brevibacillus ruckerii is inoculated on a test tube slant culture medium, and the test tube is placed in a constant temperature incubator and cultured until colonies grow on the culture medium to obtain a Brevibacillus ruckerii slant;

[0048] In the second step, the single colony obtained in the first step was transferred to 50-100 mL beef extract peptone liquid medium, and cultured overnight at 35°C and 185 r / min to obtain a first-level seed solution. The first-level seed solution was inoculated into LB medium at 10% (V / V), and cultured for 16 hours under the same conditions to obtain a second-level seed solution, which was stored at 4°C.

[0049] The beef extract peptone medium formula is: by weight percentage: 5% beef extract, 10% peptone, 5% NaCl, pH 7.0, the balance is water, adjust the pH value to 7.0, sterilize at 121°C for 20 minutes before use;

[0050] In the third step, the secondary seed liquid obtained in the second step is inoculated into the fermentation medium at 5% (V / V) for fermentation culture, the fermentation temperature is controlled at 35±1°C, the dissolved oxygen is about 30%, and the culture is carried out for 16-24 hours. When the dissolved oxygen rises and the pH drops, the fermentation is stopped to obtain the 1,2-dichloropropane degrading bacterial agent.

[0051] The viable bacterial count of the bacterial agent obtained in this example was 7.8×10 9 cfu / mL.

[0052] The fermentation medium is composed of 2% soybean cake powder, 2% glucose, 1.5% corn steep liquor powder, 0.2% peptone, 0.01% magnesium sulfate, 0.1% sodium chloride, 0.4% ammonium sulfate, 0.03% potassium dihydrogen phosphate, 0.1% calcium carbonate, pH 7.0-7.5, and the balance is water.

[0053] Example 3 Simulated application of bacterial agent in 1,2-dichloropropane wastewater treatment:

[0054] The 1,2-dichloropropane degradation bacterial agent was prepared according to the method of Example 2. The culture temperature was 33±1°C. The viable bacterial count of the obtained bacterial agent was 5×10 8 cfu / mL.

[0055] Simulation application: The water source for treatment is the aerobic section influent of an agricultural wastewater treatment plant, with a treatment volume of 5L. 1,2-dichloropropane degradation bacteria agent is added to make the initial bacterial count of the system about 1×10 6 cfu / mL, aeration treatment at room temperature made the dissolved oxygen above 2mg / L, sewage pH 6.0-8.0, raw sewage COD about 6325mg / L, ammonia nitrogen about 439mg / L, 1,2-dichloropropane was added to a concentration of 180mg / L, after 24h, the 1,2-dichloropropane content in the sewage was <1mg / L, and the degradation rate reached more than 99.9%.

[0056] Example 4 Practical application of bacterial agent in 1,2-dichloropropane wastewater treatment:

[0057] The 1,2-dichloropropane degradation bacterial agent was prepared according to the method of Example 2. The culture temperature was 33±1°C, the dissolved oxygen was controlled at 30%, and the viable bacterial count of the obtained bacterial agent was 9×10 8 cfu / mL.

[0058] The source of treated water is oilfield reinjection water. 1,2-dichloropropane degradation bacteria agent is added to make the initial bacterial count of the system about 1.8×10 7 cfu / mL, treatment volume is 2L, temperature is 33℃, sewage pH is 7.5, COD is about 807mg / L, ammonia nitrogen is about 11mg / L, total nitrogen is 41mg / L, 1,2-dichloropropane concentration is artificially added to 200mg / L, sampling is performed every 4h to detect the 1,2-dichloropropane content in the system, and its degradation is shown in Figure 2 , the pollutants can be completely degraded within 28 hours.

[0059] Example 5 Practical application of bacterial agent in 1,2-dichloropropane wastewater treatment 2:

[0060] The 1,2-dichloropropane degradation bacterial agent was prepared according to the method of Example 2. The culture temperature was 35±1°C, the dissolved oxygen was controlled at 30%, and the viable bacterial count of the obtained bacterial agent was 5×10 9 cfu / mL.

[0061] The treated water source is the influent of the aerobic pool of the petrochemical wastewater treatment plant, with a treatment volume of 10L. 1,2-dichloropropane degradation bacteria agent is added to make the initial bacterial count of the system about 2.5×10 7cfu / mL, aeration treatment at room temperature to make the dissolved oxygen above 2mg / L, sewage pH6.0-8.0, 1,2-dichloropropane concentration below 5mg / L, COD about 2200mg / L, volatile phenol about 240mg / L, ammonia nitrogen about 100mg / L, sulfide about 10mg / L, water inlet and outlet once every 24h, at the same time, a certain amount of 1,2-dichloropropane was added when the water was influent, and the concentration of 1,2-dichloropropane 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-dichloropropane in the effluent, it can be considered that the bacterial agent has a continuous degradation ability for 1,2-dichloropropane sewage.

[0062] Table 1

[0063] .

[0064] Example 6 Practical application of bacterial agent in saline-alkali soil treatment:

[0065] 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-Dichloropropane was added to the soil at a ratio of 100 mg / kg, mixed thoroughly, and balanced at room temperature in the dark for 3 days.

[0066] A microbial group and a control group were set up. The control group was not treated with any treatment. A 1,2-dichloropropane-degrading bacterial agent was added to the microbial group to make the number of live bacteria of 1,2-dichloropropane-degrading bacteria in the soil reach 10 6 -10 7 CFU / g, soil pH value was 7.8, soil moisture content was 10%-20%, and salt content was 0.5%; 2kg of soil was treated in each group, and samples were collected every 3 days to detect the content of 1,2-dichloropropane. When the concentration of the microbial substrate dropped below 2mg / kg, 1,2-dichloropropane was added again and the addition concentration was increased. The soil was thoroughly mixed. The experiment lasted for 21 days. Deionized water was added to the soil regularly to maintain soil moisture. Figure 3 This is the soil treatment effect of 1,2-dichloropropane, which shows that it has a relatively stable and continuous degradation ability in the soil contaminated by 1,2-dichloropropane.

[0067] Example 7 Simulated application of 1,2-dichloropropane degradation bacterial agent in degrading 1,2-dichloropropane in seawater:

[0068] The 1,2-dichloropropane degradation bacterial agent was prepared again according to the method of Example 2. The culture temperature was 30±1°C, the dissolved oxygen was controlled at 30%, and the viable bacterial count of the obtained bacterial agent was 8×10 8 cfu / mL.

[0069] Simulation application: The water quality source is the shallow water near Dongying, Bohai Sea in July 2023. 1,2-dichloropropane degradation bacteria agent is added to make the initial bacterial count of the system about 6×10 7 cfu / mL, water quality conditions: pH7.9, COD about 1.1mg / L, ammonia nitrogen about 0.2mg / L, salt content 32.987g / kg; 1,2-dichloropropane was added to a concentration of 200mg / L, the treated water volume was 1L, and aeration treatment was performed at room temperature to make the dissolved oxygen above 2mg / L. After 24h, the 1,2-dichloropropane content in the water was <1mg / L, indicating that the bacteria has a certain salt tolerance, and can achieve efficient and rapid treatment when there is a certain amount of 1,2-dichloropropane in seawater.

[0070] 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 Brevibacillus ruderi ( Brevibacillus reuszeri )YJY23-07, characterized by: Its deposit number is CGMCC NO. 26981, and it was deposited in the General Microbiology Center of China Culture Collection Administration on March 31, 2023.

2. A method for preparing a bacterial agent, characterized in that: The preparation is carried out using the Brevibacillus ruderi described in claim 1, and the specific steps are as follows: 1) Activation of bacteria: Move the test tube bacteria of Brevibacillus russii to room temperature for activation for 4h-8h; 2) Seed solution preparation: The activated test tube strains were inoculated into the corresponding liquid culture medium, 1 test tube strain was inoculated into 50-100 mL beef extract peptone medium, and the seed solution was obtained by shaking culture for 12-18 hours. The culture conditions were: temperature 32-37°C, shaking speed 100-200 r / min; 3) Fermentation: Inoculate the seed liquid of Brevibacillus russii in a volume percentage of 1%-5% into the corresponding fermentation medium for liquid deep fermentation to obtain a fermentation liquid; the culture conditions are: temperature 32-37° C., rotation speed 100-200 r / min, and culture time 12-18 h; The amount of bacteria in the fermentation liquid was 0.5×10 8 -8×10 9 cfu / mL; 4) Drying and crushing: The fermentation liquid obtained by the above fermentation is adsorbed, dried and crushed to obtain a single bacterial agent with an effective live bacterial count of more than 10 billion / g.

3. The method for preparing a microbial agent according to claim 2, characterized in that: The beef extract peptone medium in step (2) has a formula of: 5% beef extract, 10% peptone, 5% NaCl, pH 7.0, and the balance is water; The fermentation medium formula in step (3) is as follows: by weight percentage, soybean cake powder 1-3%, glucose 1-3%, corn steep liquor dry powder 1-3%, peptone 0.1-0.5%, magnesium sulfate 0.01-0.05%, sodium chloride 0-0.5%, ammonium sulfate 0.1-0.6%, potassium dihydrogen phosphate 0.01-0.1%, calcium carbonate 0.1-1%, pH 7.0-8.0, and the balance is water.

4. The use of Brevibacillus ruderi according to claim 1, characterized in that: The application includes biochemical treatment of seawater or saline-alkali land containing 1,2-dichloropropane or wastewater containing 1,2-dichloropropane.

Images