A method for degrading TNT in soil using Bacillus thuringiensis

Abstract

The invention discloses a method for degrading TNT in soil by using Bacillus thuringiensis. The fermentation culture liquid of Bacillus thuringiensis with a deposit number of CGMCC No.1.7882 is connected to an immobilized carrier, cultured, and spray-dried to obtain immobilized Bacillus thuringiensis; the immobilized Bacillus thuringiensis is sprinkled into the TNT-containing soil to be repaired, mixed evenly, and turned over to ensure soil ventilation and microbial activity; or, the immobilized Bacillus thuringiensis is added to a slurry reactor and mixed evenly, and the TNT is degraded by reaction. The immobilized Bacillus thuringiensis provided by the invention has a simple preparation method, is easy to implement, and has a low production cost. The corn straw biochar and diatomaceous earth are mixed and used to enhance the adsorption performance of the strain. The immobilized Bacillus thuringiensis of the invention is easy to recycle, can be used multiple times, and is green and environmentally friendly.

Description

Technical Field

[0001] The invention relates to the technical field of environmental governance, and in particular to a method for degrading TNT in soil by utilizing Bacillus thuringiensis. Background Art

[0002] In recent years, energetic compounds have been widely used in various industries such as military, mining, construction, and high-energy metallurgy. The use of energetic compounds not only solves the problem of convenience, but also poses huge hidden dangers. The manufacture, transportation, and use of energetic compounds lead to soil and water pollution. TNT has many advantages such as low cost, easy casting, safe use, and good explosive performance. It has long been the largest and most commonly used monomer high explosive in the military and civil explosive fields. In various links such as its production, transportation, charging, storage, and destruction, a large amount of TNT and by-products enter the environment and cause serious pollution. TNT is toxic, carcinogenic, and teratogenic. Its harm to the environment is mainly manifested in: when humans inhale it, it can cause liver disease (toxic hepatitis), aplastic anemia, and cataracts in mild cases, and death in severe cases; when the TNT content in water reaches 1 mg / L, fish will die; when TNT enters the soil, it will react with several major compounds in the soil, increasing the concentration of organic matter in the soil, so it causes far-reaching harm to the environment. The biodegradation and transformation of TNT have been widely studied.

[0003] At present, most physical or chemical methods are used to remove TNT from the soil, such as incineration, detonation or chemical oxidation. However, the practical cost of these methods is very high and causes secondary pollution to the environment. Microbial degradation of TNT has been studied abroad since the 1940s. After continuous exploration and development, the current main work is to seek efficient degradation bacteria. Since microbial degradation of TNT utilizes the metabolic activities of microorganisms to degrade organic matter, this type of method is safe and reliable, has low operating and management costs, and has satisfactory treatment effects, and has attracted much attention from environmental researchers. Some microorganisms in nature can utilize TNT and degrade energetic compound pollution in the environment. Patent CN 106497811 B discloses a Pseudomonas aeruginosa, a bacterial agent containing the bacterium, its use in degrading TNT, and a method for degrading TNT. The Pseudomonas aeruginosa deposit number is CGMCC NO: 10842. The TNT in the TNT-contaminated sample is degraded by contacting the Pseudomonas aeruginosa with the deposit number of CGMCC NO: 10842 and / or the bacterial agent containing the Pseudomonas aeruginosa with the deposit number of CGMCC NO: 10842 with the TNT-contaminated sample. However, the conversion rate of the bacteria to completely degrade the TNT-contaminated sample in the liquid environment is relatively slow. Therefore, the search for efficient TNT-degrading bacteria and the preparation of microbial agents should continue to be explored. Summary of the invention

[0004] In view of the above prior art, the object of the present invention is to provide a method for degrading TNT in soil using Bacillus thuringiensis.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides the use of Bacillus thuringiensis in degrading soil TNT, wherein the deposit number of the Bacillus thuringiensis is CGMCC No.1.7882.

[0007] A second aspect of the present invention provides a method for degrading TNT in soil using Bacillus thuringiensis, comprising the following steps:

[0008] (1) Carrier configuration and strain immobilization: The fermentation culture fluid of Bacillus thuringiensis with a deposit number of CGMCC No. 1.7882 was inoculated into the immobilization carrier, cultured, and spray-dried to obtain immobilized Bacillus thuringiensis;

[0009] (2) Degrading TNT in soil: Sprinkling the immobilized Bacillus thuringiensis obtained in step (1) into the TNT-containing soil to be repaired, mixing evenly, turning the soil, and ensuring soil ventilation and microbial activity; or, mixing the TNT-containing soil with water to form a slurry, adding the slurry to a slurry reactor, adding the immobilized Bacillus thuringiensis obtained in step (1) to the slurry, mixing evenly, and reacting to degrade TNT.

[0010] Preferably, in step (1), the fermentation culture liquid of Bacillus thuringiensis is obtained by the following steps: inoculating the seed culture liquid of Bacillus thuringiensis at an inoculum amount of 10.0-15.0% into the fermentation culture liquid, fermenting with ventilation and stirring at 30-35°C for 12-24h, and determining the microbial content of the fermentation culture liquid to be ≥2.0-5.0×10 8 CFU / mL.

[0011] Preferably, in step (1), the inoculation amount of the fermentation broth of Bacillus thuringiensis and the input amount of the immobilized carrier are 100 mL:(2-8) g, and the culture conditions are 30-37° C., 24-48 h, and 150-180 r / min.

[0012] Preferably, in step (1), the preparation step of the immobilized carrier is: corn straw biochar and diatomaceous earth are uniformly mixed in a weight ratio of (3-6): (4-7), sterilized at 120-130°C for 10-30 min, and dried at 60-70°C to obtain the immobilized carrier.

[0013] Preferably, in step (2), the amount of immobilized Bacillus thuringiensis sprayed is 1-2 kg / m 2 , the soil moisture content is 10-15%, and the interval between turning the soil is 5 days.

[0014] Preferably, in step (2), the TNT content in the mud is 1000 mg / kg, the amount of immobilized Bacillus thuringiensis added is 20-25% of the mud weight, and the reaction time is 7-12 days.

[0015] Beneficial effects of the present invention:

[0016] The immobilized Bacillus thuringiensis provided by the present invention has a fast degradation speed, a sustained and stable degradation effect, a high degradation rate, a simple preparation method, easy implementation, and a low production cost. The corn straw biochar and diatomaceous earth are mixed for use, thereby enhancing the adsorption performance of the strain.

[0017] The immobilized bacillus thuringiensis provided by the invention is applied to a slurry reactor and mixed with aerobic sludge, and can greatly reduce the TNT content in water and soil, and the treatment effect is stable.

[0018] The immobilized bacillus thuringiensis of the invention is easy to recycle and can be reused multiple times, which is green and environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : Degradation rate of TNT by Pseudomonas nitroreducens T1, Pseudomonas nitroreducens T2, Bacillus subtilis T3, Bacillus thuringiensis T4 and Bacillus thuringiensis T5.

[0020] Figure 2 : The degradation dynamics of Bacillus thuringiensis used in the present invention;

[0021] Figure 3 : The degradation dynamic diagram of TNT in Example 2;

[0022] Figure 4 : A dynamic diagram of the repeated degradation of TNT by Bacillus thuringiensis used in the present invention. DETAILED DESCRIPTION

[0023] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0024] As described in the background technology, based on this, the present invention provides a method for degrading TNT in soil using Bacillus thuringiensis, comprising the following steps:

[0025] (1) Preparing fermentation culture medium: inoculating Bacillus thuringiensis with a preservation number of CGMCC No. 1.7882 into LB solid medium, culturing at 30-35° C. for 10-15 h to obtain an activated strain; inoculating the activated strain of Bacillus thuringiensis into LB liquid medium, culturing at 36-37° C. for 10-15 h and shaking at 150-180 r / min to obtain a seed culture medium,

[0026] The obtained seed bacterial liquid is inoculated into the fermentation culture medium at 10.0-15.0%, the fermentation culture medium includes (NH4)2SO42.0-4.0g / L, NaH2PO41.5-3.0g / L, glucose10-15g / L, pH7.0-7.2; the fermentation is carried out by aeration and stirring, the fermentation temperature is 30-35℃, the fermentation time is 12-24h, and the microbial content in the fermentation culture medium is not less than 2.0-5.0×10 8 CFU / mL;

[0027] (2) Carrier configuration and strain immobilization: corn straw biochar and diatomaceous earth are mixed uniformly in a weight ratio of (3-6): (4-7), sterilized at 120-130° C. for 10-30 min, and dried at 60-70° C. to obtain an immobilized carrier; the fermentation culture obtained in step (1) is inoculated into the immobilized carrier, the inoculation amount (ml) of the fermentation culture and the amount (g) of the immobilized carrier are 100:(2-8), the culture conditions are 30-37° C., 24-48 h, 150-180 r / min, and spray dried to obtain immobilized Bacillus thuringiensis;

[0028] (3) Degradation of TNT in soil: The immobilized Bacillus thuringiensis obtained in step (2) was added at a rate of 1-2 kg / m 2 The amount of the TNT is sprinkled into the soil to be repaired containing 1000 mg / kg TNT, mixed evenly, and the soil is turned over every 5 days. The soil moisture content is 10-15% to ensure soil ventilation and microbial activity; alternatively, the TNT-containing soil is mixed with water to form a slurry and added to a slurry reactor. The TNT content in the slurry is 1000 mg / kg. 20-25% of the weight of the slurry is added to the slurry and mixed evenly. The reaction takes 7-12 days to degrade the TNT.

[0029] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0030] The test materials used in the examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels.

[0031] Screening experiments

[0032] The nitroreduced Pseudomonas T1 used in the following experiments was purchased from the China General Microbiological Culture Collection Center with the accession number: CGMCC No.1.8737; the nitroreduced Pseudomonas T3 used has the accession number: CGMCC No.1.4928; the Bacillus subtilis T3 used has the accession number: CGMCC No.1.15792; the Bacillus thuringiensis T4 used has the accession number: CGMCC No.1.7882; and the Bacillus thuringiensis T5 used has the accession number: CGMCC No.1.15822.

[0033] Pseudomonas nitroreducens T1, Pseudomonas nitroreducens T2, Bacillus subtilis T3, Bacillus thuringiensis T4 and Bacillus thuringiensis T5 were activated in LB liquid medium and cultured at 150r / min and 37℃ for 12 hours to obtain seed bacterial liquid. These five bacteria were added to 1 / 5 LB medium containing 100mg / L TNT and cultured at 150r / min and 37℃ for 12 hours. The strain screening results are shown in Figure 1. Figure 1 As shown, by measuring the TNT content, it is known that the degradation rate of nitroreducing Pseudomonas T1 is 62.95%, the degradation rate of nitroreducing Pseudomonas T2 is 47.2%, the degradation rate of Bacillus subtilis T3 is 61.56%, the degradation rate of Bacillus thuringiensis T4 is 100%, and the degradation rate of Bacillus thuringiensis T5 is 54.22%. Among them, Bacillus thuringiensis T4 has the best degradation effect, and Bacillus thuringiensis with a preservation number of CGMCC No.1.7882 is selected to remove TNT in soil.

[0034] The Bacillus thuringiensis with the deposit number CGMCC No.1.7882 was cultured in 1 / 5LB liquid medium containing 100 mg / L TNT, with an initial OD600 of the bacterial solution = 0.05, and was cultured at 150 rpm and 37°C for 4 hours. The degradation dynamics are shown in Figure 2. Figure 2 shown.

[0035] Example 1

[0036] (1) preparing a fermentation culture medium: inoculating Bacillus thuringiensis with a deposit number of CGMCC No. 1.7882 into LB solid medium, and culturing at 35° C. for 12 h to obtain an activated strain of Bacillus thuringiensis;

[0037] The activated strain of Bacillus thuringiensis was inoculated into LB liquid culture medium with an inoculation amount of 2.0% by volume, and cultured under shaking conditions of 150 r / min and 35° C. for 12 hours to obtain a seed bacterial solution of Bacillus thuringiensis.

[0038] The seed liquid of Bacillus thuringiensis was inoculated into the fermentation medium at an inoculum volume fraction of 10.0%, the fermentation medium included (NH4)2SO4 3.0g / L, NaH2PO4 2.0g / L, glucose 15g / L, pH 7.0; the fermentation was carried out at 35°C with aeration and stirring for 24h, and the microbial content in the fermentation medium was determined to be not less than 5.0×10 8 CFU / mL.

[0039] (2) Carrier configuration and strain immobilization: 6 parts of corn straw biochar and 4 parts of diatomaceous earth were mixed evenly, sterilized at 121°C and 0.105 MPa high-pressure steam for 20 min, and then dried at 65°C to prepare the immobilized carrier.

[0040] The fermentation liquid of Bacillus thuringiensis was inoculated into the immobilized carrier, the inoculation amount of the fermentation liquid of Bacillus thuringiensis and the input amount of the immobilized carrier were 100mL:8g, and the mixture was cultured at 150rpm / min and 37°C for 48h, and the immobilized Bacillus thuringiensis was obtained by spray drying.

[0041] (3) Degradation of TNT: The immobilized Bacillus thuringiensis obtained in step (2) was added to the mixture at a rate of 2 kg / m 2 Sprinkle the amount into the soil to be repaired containing 1000 mg / kg TNT, mix evenly, maintain the soil moisture content at 10%, turn the soil over every 5 days to ensure soil ventilation and microbial activity, and incubate in a biochemical incubator.

[0042] Soil samples were taken at 30 days, 40 days, and 50 days to determine the TNT content. The specific steps are as follows:

[0043] Take 1g of soil and add 2mL of acetonitrile to mix, ultrasonically extract for 60min, centrifuge at 8000rpm for 5min, filter the supernatant through a 0.22μm microporous filter membrane and transfer it into a chromatographic bottle, and use high performance liquid chromatography to determine the TNT content. The liquid chromatography parameters are as follows: mobile phase of water: methanol = 50:50, flow rate of 0.7mL / min, and column temperature of 33℃.

[0044] It was determined that the TNT degradation rate was 59.6% at 30 days, 85.6% at 40 days, and 99.8% at 50 days.

[0045] Example 2

[0046] (1) preparing a fermentation culture medium: inoculating Bacillus thuringiensis with a deposit number of CGMCC No. 1.7882 into LB solid medium, and culturing at 35° C. for 12 h to obtain an activated strain of Bacillus thuringiensis;

[0047] The activated strain of Bacillus thuringiensis was inoculated into LB liquid culture medium with an inoculation amount of 2.0% by volume, and cultured at 150 r / min and 35° C. for 12 hours to obtain a seed bacterial solution of Bacillus thuringiensis.

[0048] The seed culture of Bacillus thuringiensis was inoculated into the fermentation medium at an inoculum volume fraction of 15.0%. The fermentation medium included (NH4)2SO4 3.0 g / L, NaH2PO4 2.0 g / L, glucose 15 g / L, and pH 7.0. The fermentation was carried out under aeration and stirring at 35°C for 24 hours. The microbial content in the fermentation medium was determined to be not less than 5.0×10 8 CFU / mL.

[0049] (2) Carrier configuration and strain immobilization: 6 parts of corn straw biochar and 4 parts of diatomaceous earth were mixed evenly, sterilized at 121°C and 0.105 MPa high-pressure steam for 20 min, and then dried at 65°C to prepare the immobilized carrier.

[0050] The fermentation culture fluid of Bacillus thuringiensis was inoculated into the immobilized carrier, the inoculation amount of the fermentation culture fluid of Bacillus thuringiensis and the input amount of the immobilized carrier was 100mL:8g, and the culture was carried out at 150rpm / min and 37°C for 48h, and the immobilized Bacillus thuringiensis was obtained by spray drying.

[0051] (3) Degradation of TNT: The soil containing TNT is mixed with water to obtain a slurry with a TNT content of 1000 mg / kg. The slurry is added to a slurry reactor, and immobilized Bacillus thuringiensis is added to the slurry at a weight percentage of 20%, mixed evenly, and aerated to react.

[0052] On the 12th and 22nd day, mud samples were taken to determine their TNT content. The specific steps are as follows:

[0053] Take 2mL of mud in a centrifuge tube, centrifuge at 8000rpm for 5min, take 500 microliters of supernatant, add an equal amount of acetonitrile for extraction, filter the extract through a 0.22μm microporous filter membrane and transfer it into a chromatographic bottle; add 2mL of acetonitrile to the precipitate and mix well, ultrasonically extract for 60min, centrifuge at 8000rpm for 5min, filter the supernatant through a 0.22μm microporous filter membrane and transfer it into a chromatographic bottle, use high performance liquid chromatography to determine the TNT content, the liquid chromatography parameters are mobile phase water: methanol = 50:50, flow rate is 0.7mL / min, column temperature is 33℃.

[0054] It was determined that after 12 days of aeration, the TNT degradation rate was 94.2%. The degradation dynamics are as follows: Figure 3 As shown; after 22 days of aeration reaction, the TNT degradation rate reached a maximum value of 98.4%.

[0055] Repeated degradation experiments

[0056] In order to determine the sustained reusability of the immobilized Bacillus thuringiensis of the present invention, the immobilized Bacillus thuringiensis ( Figure 4 The fixed bacteria in the TNT soil were used to simulate the degradation experiment of TNT contaminated soil (100 mg / LTNT). After each cycle, the immobilized carrier particles of the immobilized Bacillus were washed three times with sterile distilled water and added to the TNT soil again for reaction. Figure 4 The TNT degradation rate was determined by using Biochar in the test tube as a control. The operation was repeated 5 times, and the TNT degradation rate was still as high as 93.4%. Figure 4 shown.

[0057] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. The application of Bacillus thuringiensis in degrading soil TNT is characterized in that: The deposit number of the Bacillus thuringiensis is CGMCC No.1.7882.

2. A method for degrading TNT in soil using Bacillus thuringiensis, characterized in that: The following steps are involved: (1) Carrier configuration and strain immobilization: The fermentation culture fluid of Bacillus thuringiensis with a deposit number of CGMCC No. 1.7882 was inoculated into the immobilization carrier, cultured, and spray-dried to obtain immobilized Bacillus thuringiensis; (2) Degrading TNT in soil: Sprinkling the immobilized Bacillus thuringiensis obtained in step (1) into the TNT-containing soil to be repaired, mixing evenly, turning the soil, and ensuring soil ventilation and microbial activity; or, mixing the TNT-containing soil with water to form a slurry, adding the slurry to a slurry reactor, adding the immobilized Bacillus thuringiensis obtained in step (1) to the slurry, mixing evenly, and reacting to degrade TNT.

3. The method for degrading TNT in soil using Bacillus thuringiensis according to claim 2, characterized in that: In step (1), the fermentation culture liquid of Bacillus thuringiensis is obtained by the following steps: inoculating the seed culture liquid of Bacillus thuringiensis at an inoculum amount of 10.0-15.0% into the fermentation culture liquid, fermenting with ventilation and stirring at 30-35°C for 12-24h, and determining the microbial content of the fermentation culture liquid to be ≥2.0-5.0×10 8 CFU / mL.

4. The method for degrading TNT in soil using Bacillus thuringiensis according to claim 2, characterized in that: In step (1), the inoculation amount of the fermentation liquid of Bacillus thuringiensis and the input amount of the immobilized carrier are 100mL:(2-8)g, and the culture conditions are 30-37°C, 24-48h, and 150-180r / min.

5. The method for degrading TNT in soil using Bacillus thuringiensis according to claim 2, characterized in that: In step (1), the preparation step of the immobilized carrier is: corn straw biochar and diatomaceous earth are mixed evenly in a weight ratio of (3-6): (4-7), sterilized at 120-130° C. for 10-30 min, and dried at 60-70° C. to obtain the immobilized carrier.

6. The method for degrading TNT in soil using Bacillus thuringiensis according to claim 2, characterized in that: In step (2), the amount of immobilized Bacillus thuringiensis sprayed is 1-2 kg / m 2 , the soil moisture content is 10-15%, and the interval between turning the soil is 5 days.

7. The method for degrading TNT in soil using Bacillus thuringiensis according to claim 2, characterized in that: In step (2), the TNT content in the mud is 1000 mg / kg, the amount of immobilized Bacillus thuringiensis added is 20-25% of the mud weight, and the reaction time is 7-12 days.

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