CN117903990A

CN117903990A - Bacillus belicus and application thereof

Info

Publication number: CN117903990A
Application number: CN202410099993.6A
Authority: CN
Inventors: 张颖; 张�林; 曲建华; 徐洋; 刘芮忻; 林春颖; 魏书奇; 李梓玮; 孟繁松; 王蕾; 张博
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2024-01-24
Filing date: 2024-01-24
Publication date: 2024-04-19
Anticipated expiration: 2044-01-24
Also published as: CN117903990B

Abstract

Bacillus belicus and application thereof, and relates to the technical field of ecological restoration of environmental pollution. The invention aims to solve the problem that the existing microorganism has low degradation efficiency on BaA in the environment. Bacillus belicus is Bacillus belicus (Bacillus velezensis) DNX24, and is preserved in China center for type culture Collection, wherein the preservation address is China, university of Wuhan, and the preservation date is 2023, 12 months and 25 days, and the preservation number is CCTCCNO: m20232676. An application of bacillus beleiensis (Bacillus velezensis) DNX24 in degrading polycyclic aromatic hydrocarbon pollutants. The invention can obtain bacillus belicus and application thereof.

Description

Bacillus belicus and application thereof

Technical Field

The invention relates to the technical field of ecological restoration of environmental pollution, in particular to bacillus belicus and application thereof.

Background

Benzo [ a ] anthracene (BaA) is a world-recognized strong oncogenic Polycyclic Aromatic Hydrocarbon (PAHs) pollutant with a 4-ring structure, and has the characteristics of high stability, difficult degradation, strong toxicity, three-induced (oncogenic, teratogenic, mutagenic) effect and the like. Therefore BaA was early blacklisted by the U.S. environmental protection agency to control toxic organic pollutants preferentially. It often exists in petroleum pollutants, enters the soil through the leakage of petroleum exploitation and transportation processes, the irrigation of petroleum polluted water, the sedimentation of atmospheric fly ash and the like, is difficult to degrade in natural environment, is easy to accumulate in the environment such as the soil and causes soil pollution, and is therefore attracting attention of more researchers.

BaA enters soil, atmosphere and water body environments through various ways, is widely detected in different environment media and organism tissues, and threatens the safety of an ecological system and the health of a human body through migration transformation and biological chain transfer. Numerous studies have demonstrated that BaA, due to its neurotoxicity, has an interfering effect on the endocrine system, and at the same time has reproductive developmental toxicity, immunological toxicity and oncogenic toxicity, which pose a great threat to human health and the ecological environment. Accordingly, there is a constant need for intensive research into degradation techniques.

For BaA degradation, the methods mainly adopted in China at present are a chemical oxidation method and a biological method, and in the research process of various degradation technologies, biodegradation is considered as one of the most promising means for BaA degradation at present, and is an effective technical means for pollution repair and exposure risk reduction. Microbial degradation is an important degradation way of organic pollutants in the environment, and is widely paid attention to because of low cost, high sustainability and obvious degradation effect, and no secondary pollution to the environment.

BaA aerobic microorganisms degrade more efficiently and rapidly and more thoroughly than anaerobic microorganisms, however, among the microorganisms obtained at present, aerobic microorganisms capable of degrading BaA are not more and the degradation effect is not remarkable. Therefore, searching for a high-efficiency degradation strain aiming at BaA is a hot spot problem of environmental treatment and pollution repair research on BaA pollution.

Disclosure of Invention

The invention aims to solve the problem of low degradation efficiency of BaA in the environment caused by the existing microorganism, and provides bacillus belicus and application thereof.

Bacillus belicus is Bacillus belicus (Bacillus velezensis) DNX24, and is preserved in China center for type culture Collection, wherein the preservation address is China, university of Wuhan, and the preservation date is 2023, 12 months and 25 days, and the preservation number is CCTCC NO: m20232676.

An application of bacillus beleiensis (Bacillus velezensis) DNX24 in degrading polycyclic aromatic hydrocarbon pollutants.

The principle of the invention is as follows:

And (3) carrying out gradient domestication on the concentration of the pollutants: the targeting environment matrix BaA is added into the bacterial culture medium to enable the bacillus berryis DNX24 to adapt to and rely on BaA, so that the bacillus berryis DNX can also show better growth trend and degradation characteristic in BaA polluted environment.

Maximum contaminant concentration acclimation: the bacillus belicus DNX24 treated by high concentration stress shows the advantage of strong removal BaA, which shows that the bacillus belicus DNX24 has strong tolerance characteristics to high-ring polycyclic aromatic hydrocarbon (BaA).

Domestication of strains on solid inorganic salt medium: due to the high concentration pollution pressure of the external environment, single bacillus bailii is easy to have the problem of poor stress impact resistance; thus, bacterial colony degradation BaA of bacillus belicus DNX24 was enriched by acclimatization of the strain on solid inorganic salt medium.

In the gradient domestication process, along with the gradual increase of BaA concentration, the colony number in the solid culture medium gradually decreases, and along with the gradual stabilization stage of domestication, the single bacillus baileyi DNX24 colony with BaA as the only carbon source is finally obtained, and the bacillus baileyi DNX24 colony not only can resist the stress of high concentration BaA, but also can show stronger BaA degradation capability.

The invention has the beneficial effects that:

(1) With the deep understanding and research of bacillus belicus, more and more researchers find that the strain of the genus bacillus plays a great potential in the degradation of various pollutants, and the strain has better degradation capability on high molecular weight PAHs (PAHs with more than three rings) and can grow by taking benzo [ a ] anthracene as a unique carbon source.

The bacillus belicus DNX24 is subjected to a pollutant concentration gradient domestication method, a maximum pollutant concentration domestication method and domestication of a strain on a solid inorganic salt culture medium, and the result shows that the strain has a prominent removal effect under the stress of high concentration BaA with BaA as the sole carbon source compared with the stress of low concentration, and has good tolerance to the stress of high concentration BaA; meanwhile, the strain has high degradation efficiency for BaA, the required condition is mild, and the strain can be well degraded at room temperature. Because of the cytotoxicity and high loop number of BaA, most of the reported microbial tolerant substrate concentrations that degrade BaA are not high, the strain shows great potential for use, especially in highly contaminated soil or water. The degradation rate of Bacillus bailii DNX24 in BaA days with the concentration of 25mg/kg in soil is as high as 73.0%, and the Bacillus bailii DNX24 has extremely strong degradation capability.

(2) Under the direct domestication condition, the strain survival rate is extremely limited even after multiple times of expansion culture, which shows that BaA with high concentration pollutes the environment and has obvious inhibition effect on the growth and propagation of bacillus belicus. Therefore, in order to enhance the tolerance of bacillus belicus to the high-concentration polycyclic aromatic hydrocarbon BaA environment and improve the capability of removing BaA of microorganisms in the high-concentration polycyclic aromatic hydrocarbon BaA environment, the invention adopts a gradient domestication method to screen benzo [ a ] anthracene degrading bacteria. For normal strains in the BaA-containing culture system, the higher the BaA concentration is, the longer the delay period is, which shows that the unaccounted strain is sensitive to external BaA stress pressure response. For the domesticated strain, the growth condition is optimally 50mg/L BaA of the domesticated strain under stress, namely Bacillus bailii DNX24.

The invention can obtain bacillus belicus and application thereof.

Drawings

FIG. 1 is a phylogenetic tree of Bacillus bailii DNX24 of the present invention (D represents DNX 24);

FIG. 2 shows the degradation rate of Bacillus bailii DNX24 of the present invention on BaA days in water;

FIG. 3 shows the degradation rate of Bacillus bailii DNX24 of the present invention in soil for BaA days.

Detailed Description

The first embodiment is as follows: the bacillus beleiensis is bacillus beleiensis (Bacillus velezensis) DNX24, and is preserved in China center for type culture Collection, wherein the preservation address is China, university of Wuhan, and the preservation date is 2023, 12 months and 25 days, and the preservation number is CCTCC NO: m20232676.

The second embodiment is as follows: the application of the bacillus belicus (Bacillus velezensis) DNX24 in degrading polycyclic aromatic hydrocarbon pollutants is provided.

And a third specific embodiment: the second difference between this embodiment and the specific embodiment is that: the bacillus belicus (Bacillus velezensis) DNX24 is used for degrading polycyclic aromatic hydrocarbon pollutants in water or soil.

The other steps are the same as those of the second embodiment.

The specific embodiment IV is as follows: the difference between this embodiment and the second or third embodiment is that: the polycyclic aromatic hydrocarbon pollutant is benzo [ a ] anthracene.

The other steps are the same as those of the second or third embodiment.

Fifth embodiment: the second to fourth embodiments differ from the present embodiment in that: before degrading benzo [ a ] anthracene, bacillus belicus (Bacillus velezensis) DNX24 is inoculated on a solid culture medium for culture.

Other steps are the same as those of the second to fourth embodiments.

Specific embodiment six: the present embodiment differs from the second to fifth embodiments in that: the concentration of benzo [ a ] anthracene in the solid culture medium is 5-100 mg/L.

Other steps are the same as those of the second to fifth embodiments.

Seventh embodiment: the present embodiment differs from the second to sixth embodiments in that: the solid medium consisted of 3g ammonium sulfate, 0.5g potassium dihydrogen phosphate, 0.5g disodium hydrogen phosphate, 0.3g magnesium sulfate and l mL trace element solution, and ph=7.0.

Other steps are the same as those of the second to sixth embodiments.

Eighth embodiment: the present embodiment differs from the second to seventh embodiments in that: the preparation method of the microelement solution comprises the following steps: 0.3g of ferric trichloride, 0.3g of ferrous sulfate heptahydrate, 0.15g of manganese sulfate hydrate, 0.14g of zinc sulfate and 0.2g of cobalt chloride were added to distilled water, and the volume was set to 1000mL.

The other steps are the same as those of the second to seventh embodiments.

Detailed description nine: the present embodiment differs from the second to eighth embodiments in that: the culture condition is that the culture is carried out for 6 to 8 days at 30 ℃.

Other steps are the same as those of embodiments two to eight.

Detailed description ten: the present embodiment differs from one of the second to ninth embodiments in that: bacillus belicus (Bacillus velezensis) DNX24 was degraded for 7 days in water containing 5mg/L benzo [ a ] anthracene and for 15 days in soil containing 25mg/kg benzo [ a ] anthracene.

The other steps are the same as those of the second to ninth embodiments.

The following examples are used to verify the benefits of the present invention:

Example 1:

1. sample source: collecting a soil sample polluted by a bearing factory BaA in Harbin city of Heilongjiang province;

A large amount of harmful substances such as BaA contained in a bearing factory are harmful to all surrounding environment media, and are important pollution sources, so that the human health and life safety are seriously endangered. Collecting soil from a bearing factory (0-20 cm), removing impurities in the sample, placing the sample in a sterile bag, collecting the sample by a sterile sampling bag, and rapidly taking the sample back to a laboratory by using a sample refrigerator.

2. Main experimental reagent and culture medium components:

the main experimental reagents include: peptone, sodium chloride, agar, yeast powder, baA, etc., all of which are purchased in the Allatin reagent market.

Acclimatization medium (g/L): preparing BaA stock solution with acetone at a concentration of 500mg/L, taking a certain amount of BaA stock solution, placing into a sterilized conical flask, and adding sterilized inorganic salt liquid culture medium/solid culture medium after acetone is volatilized completely.

Inorganic salt liquid medium/solid medium (g/L): 3g of ammonium sulfate, 0.5g of monopotassium phosphate, 0.5g of disodium hydrogen phosphate, 0.3g of magnesium sulfate and l mL of microelement solution, wherein the pH=7.0, the volume is fixed to 1000mL by ddH ₂ O, and the sterilization is carried out for 20min at 121 ℃ for later use. Wherein, the preparation of the microelement solution comprises the following steps: ferric trichloride 0.3g, ferrous sulfate heptahydrate 0.3g, manganese sulfate hydrate 0.15g, zinc sulfate 0.14g and cobalt chloride 0.2g, and the volume is fixed to 1000mL, and the solution is filtered and sterilized for standby. The solid culture medium is added with 20g of agar based on inorganic salt liquid culture medium, the culture medium is poured into a conical flask, and the culture medium is sterilized for 20min at the high temperature of 121 ℃ for standby.

LB enriched medium formulation (liquid): 10g of peptone, 5g of yeast powder, 10g of sodium chloride and 1000mL of distilled water, and sterilizing at 121 ℃ for 20min.

LB enriched medium formulation (solids): 10g of peptone, 5g of yeast powder, 10g of sodium chloride, 1000mL of distilled water and 20g of agar, and sterilizing at 121 ℃ for 20min.

3. Main experimental instrument:

Electronic balance, beaker, glass rod, 200ml volumetric flask, conical flask, triangle flask, YXQ-100A vertical pressure steam sterilizer, test tube, inoculating loop, alcohol lamp, SW-CJ-2D ultra-clean bench, SPX-250B-Z biochemical incubator, BSD-YX2200 vertical intelligent precision shaking table, centrifuge tube and pipette.

4. The experimental method comprises the following steps:

4.1, concentration gradient domestication method of pollutant:

The concentration of BaA mg/L, 10mg/L, 25mg/L, 50mg/L and 100mg/L in the bacterial domestication system is sequentially increased, 7 days is one period of domestication, and five periods of domestication are taken as a whole.

4.2 Maximum contaminant concentration acclimation method:

The concentration of the bacterial domestication system BaA is 100mg/L, and the domestication is carried out every 7 days for five periods, so that a flora which can survive or grow in a growth environment with BaA as the sole carbon source is obtained.

4.3 Domestication of strains on solid mineral salts Medium:

The obtained degradation strain is transferred to a solid culture medium with BaA mg/L concentration for culture by a plate streaking method, and is placed in a constant temperature incubator for culture at 30 ℃ for 7 days. After 7 days, colonies with different forms and vigorous growth are picked by a fungus inoculation ring, and transferred to a sterilized solid culture medium with the concentration of BaA mg/L to continue the separation and purification by flat plate streaking. Inoculating the single colony to fresh and sterilized solid culture medium every 7 days until BaA concentration is increased to 100mg/L and colony morphology is not changed, and finishing further domestication process of degrading bacteria in inorganic salt solid culture medium to obtain pure BaA degrading strain.

4.4 Gradient dilution coating method:

Absorbing 1mL of bacterial liquid in the strain screening culture medium of the last period degradation, diluting the bacterial liquid into liquid with a gradient of 10 ^-1～10^-7 according to a 10-fold dilution method, taking 30 mu L of liquid to be coated on a yeast extract solid culture medium, uniformly coating the liquid by a sterilization coating rod, and culturing in a constant-temperature incubator at 30 ℃.

4.5 Isolation and purification of bacteria:

The cultured gradient dilution coated plate is placed in an ultra-clean workbench to finish the bacterial separation and purification work. After single colonies are grown, screening is carried out according to morphological characteristics of each colony, different types of strains are separated by observing the shape, diameter, color, viscosity and the like of the strains, three-area lineation is carried out on an inorganic salt solid flat-plate culture medium by dipping a strain body through a sterilized inoculating loop, and after the three-area flat-plate is cultured for 24 hours in a constant-temperature incubator, the single colonies grown in the three areas are picked out on the inorganic salt flat-plate culture medium to carry out one-area lineation. After a section line plate grows out the strain, observing whether the strain is a single type of pure strain under a microscope, and if not, repeatedly carrying out line drawing purification until a single colony is screened to obtain pure bacteria.

4.6 Determination of degradation Capacity of Strain:

500mg/L BaA of acetone stock solution was added to a conical flask in 500. Mu.L, and after n-hexane had volatilized under aseptic conditions, 50mL of LB liquid medium was added, and after BaA had been sufficiently dissolved, the final concentration of BaA in the reaction system was 5mg/L. Inoculating the bacteria to be detected into an conical flask filled with LB liquid under the aseptic condition, and fully and uniformly mixing; and then placing the degradation reaction system in a constant temperature shaking incubator at 30 ℃ for 7d at 150r/min, measuring BaA content in the system, calculating the degradation rate of the strain, screening out the strain with highest BaA degradation rate as a target strain, and naming the target strain as DNX24.

4.7 Identification of strains:

4.7.1 morphological identification of strains:

strain DNX24 was grown on LB plates for two days, morphological observations and gram staining, the results are shown in table 1, table 1 being the morphological identification of the target strain;

TABLE 1

Color of

Diameter of

Texture of

Shape and shape

Edge of the sheet

Surface of the body

Gram staining

Yellow colour

5mm

Viscous and thick

Rod-shaped

Irregular shape

Fold

Positive and negative

4.7.2 Physiological and biochemical identification of strains:

Carrying out physiological and biochemical identification on the strain DNX24, wherein the results are shown in table 2, and table 2 is the physiological and biochemical identification of the target strain;

TABLE 2

VP determination	Oxidase enzyme	Contact enzyme	Amylase enzyme	Methyl red
					+	+	+	+	-

Physiological and biochemical identification results: VP test positive, oxidase reaction positive, contact enzyme reaction positive, starch hydrolysis positive and methyl red reaction negative.

4.7.3 Identification of Strain 16S rDNA:

And adopting a colony PCR method, and directly taking single colony lysate as a template to carry out PCR. Universal primers for PCR amplification of 16S rDNA (genomic PCR amplification-primer sequence: 27F:5 '-AGAGTTTGATCCTGGGCTCAG-3';

1492R:5'-GGTTACCTTGTTACGACTT-3', PCR reaction system: ddH 2O-10. Mu.L, 27F-30.5. Mu.L, 1492R-0.5. Mu. L, taq-12.5. Mu. L, DNA-1.5. Mu.L, and 25. Mu.L total volume, and mixing. Gel electrophoresis conditions: u=120v, a=120 ma, t=20 min; the PCR reaction conditions are shown in Table 3). Primers and sequencing work were done by Hangzhou Info technologies, inc.

Table 3 identifies PCR reaction conditions for strain 16S rDNA:

TABLE 3 Table 3

The sequencing result is compared with the target sequence and the reference sequence by CLUSTAL W in MEGA7.0 software Alignment, and the parameters are default values. The phylogenetic tree is constructed as shown in FIG. 1.

The results show that the Bacillus belicus DNX24 provided by the present invention has up to 99.7% homology with Bacillus belicus, and thus it is confirmed that the strain DNX24 of the present invention belongs to the genus Bacillus belicus, and NCBI accession No. SUB14003740 of ITS ITS.

5. Degradation test:

5.1BaA polluted water:

(1) Bacillus belicus DNX24 was inoculated in 20mL of liquid LB medium, and shake cultured at 30℃overnight for activation.

(2) Centrifuging the cultured fungus solution at 8000r/min for 2min, discarding supernatant, re-suspending with sterile water, centrifuging at 8000r/min for 2min again, discarding supernatant, and re-suspending with sterile water. The bacterial liquid OD ₆₀₀ obtained by resuspension was adjusted to 1.0, transferred to a 250mL triangular flask containing 50mL MSM medium with BaA as a single carbon source according to an inoculum size of 5% (V/V), shake cultured at 30 ℃ in a 150r/min shaker for 7d, sampled at 1 st, 3 rd, 5 th and 7d respectively, and each treatment was repeated for 3 groups.

(3) Respectively adding an equal volume of n-hexane organic solvent for extraction, oscillating and extracting for 30min by a shaking table, pouring into a separating funnel, standing for 15min, transferring the lower organic phase into a 50mL centrifuge tube after liquid is layered, pouring the upper aqueous phase into a triangular flask again, and repeating the extraction for 1 time.

(4) The total 100mL of the extract obtained by 2 times of extraction was centrifuged, and the residual water in the upper layer was sucked dry by a pipette.

(5) The extract obtained 2 times was evaporated to dryness under reduced pressure in a water bath at 40℃using a rotary evaporator, and to the dried round-bottomed flask was added 50mL of chromatographically pure acetonitrile to sufficiently dissolve BaA.

(6) 1ML of the obtained BaA acetonitrile solution was filtered off impurities with a 0.22 μm organic filter to prepare a sample to be tested.

5.2BaA contaminated soil:

(2) Centrifuging the cultured fungus solution at 8000r/min for 2min, discarding supernatant, re-suspending with sterile water, centrifuging at 8000r/min for 2min again, discarding supernatant, and re-suspending with sterile water. The bacterial liquid OD ₆₀₀ obtained by resuspension was adjusted to 1.0, transferred to BaA soil at a concentration of 25mg/kg according to an inoculum size of 5% (V/V), shake cultured at 30℃for 15d with a shaking table at 150r/min, sampled at 1 st, 3 rd, 5 th, 7 th, 9 th, 11 th, 13 th and 15d, respectively, and each treatment was repeated for 3 th groups.

(3) Centrifuging, and extracting supernatant in the same manner as 5.1. Respectively adding equal volumes of n-hexane into soil samples: acetone=1: 1, carrying out ultrasonic extraction for 2 hours, centrifuging, taking supernatant, blowing nitrogen, and then using acetonitrile to fix the volume to 10mL.

(4) 1ML of the obtained BaA acetonitrile solution was filtered off impurities with a 0.22 μm organic filter to prepare a sample to be tested.

5.3 Determination of BaA degradation Rate:

(1) And detecting the obtained sample to be detected by using High Performance Liquid Chromatography (HPLC), and calculating the concentration according to a standard curve of the peak area and the concentration which are measured in advance.

(2) The high performance liquid chromatography detection is carried out, the model of the high performance liquid chromatography instrument is Agilent 1260, the chromatographic column is 120EC-C18 chromatographic column 4.6X150 mm, the mobile phase is 100% acetonitrile, the mobile phase rate is set to be 1mL/min, the sample loading amount is 20 mu L each time, and the detection wavelength is 254nm.

5.4 Degradation effects:

5.4.1 the degradation rate of the Bacillus bailii DNX24 on BaA days with the concentration of 5mg/L in water body is up to 60.1 percent, and the degradation rate data piece is shown in figure 2. FIG. 2 shows the BaA content of water over time during biodegradation. From the results, baA degradation rate was gradually increased with time, which indicates that the content of benzo [ a ] anthracene remained in the culture medium was gradually decreased with the growth of DNX24, and the highest degradation rate of benzo [ a ] anthracene in the polluted water body reached 60.1% after 7 days.

5.4.2 The Bacillus bailii DNX24 has high degradation rate of 73.0% in BaA days with the concentration of 25mg/kg in soil and extremely strong degradation capability. Degradation rate data is shown in fig. 3, and the change of BaA content in soil with time in the biodegradation process is shown in fig. 3. From this, it can be seen that BaA degradation rate gradually increased with time, which means that with the growth of DNX24, the content of benzo [ a ] anthracene remained in the contaminated soil gradually decreases, and after 15 days, the benzo [ a ] anthracene degradation rate in the contaminated soil reaches the highest 73.0%.

Investigation of degradation rate:

according to the invention, the bearing black soil field with rich organic matter content is sampled and screened, and three-step domestication is utilized to enrich bacillus beijerinus DNX24 which can adapt to extreme adversity, namely high concentration BaA pollution stress, so that the strain has strong BaA degradation capability.

Claims

1. The bacillus beleiensis is bacillus beleiensis (Bacillus velezensis) DNX24, and is preserved in China center for type culture Collection, wherein the preservation address is China, university of Wuhan, and the preservation date is 2023, 12 months and 25 days, and the preservation number is CCTCC NO: m20232676.

2. The use of bacillus beleideri according to claim 1, characterized in that said bacillus beliederi (Bacillus velezensis) DNX24 is used for degrading polycyclic aromatic hydrocarbon contaminants.

3. The use of bacillus beleidersonii according to claim 2, wherein said bacillus belsonii (Bacillus velezensis) DNX24 is used to degrade polycyclic aromatic hydrocarbon contaminants in water or soil.

4. The use of bacillus belgium according to claim 3, wherein the polyaromatic hydrocarbon contaminant is benzo [ a ] anthracene.

5. Use of a bacillus beleiensis according to claim 2, 3 or 4, characterized in that before degrading benzo [ a ] anthracene, said bacillus beleiensis (Bacillus velezensis) DNX24 is inoculated onto a solid medium for cultivation.

6. The use of Bacillus belicus according to claim 5, wherein the concentration of benzo [ a ] anthracene in the solid medium is 5-100 mg/L.

7. The use of Bacillus belicus according to claim 5, wherein the solid medium consists of 3g ammonium sulphate, 0.5g potassium dihydrogen phosphate, 0.5g disodium hydrogen phosphate, 0.3g magnesium sulphate and l mL trace element solution, and the pH=7.0.

8. The use of bacillus beljalis according to claim 7, characterized in that the preparation of the trace element solution comprises the following steps: 0.3g of ferric trichloride, 0.3g of ferrous sulfate heptahydrate, 0.15g of manganese sulfate hydrate, 0.14g of zinc sulfate and 0.2g of cobalt chloride were added to distilled water, and the volume was set to 1000mL.

9. The use of Bacillus belicus according to claim 5, wherein the culture conditions are at 30℃for 6 to 8 days.

10. The use of bacillus beleiensis according to claim 2, 3 or 4, wherein said bacillus beleiensis (Bacillus velezensis) DNX24 degrades water containing benzo [ a ] anthracene at a concentration of 5mg/L for 7 days and soil containing benzo [ a ] anthracene at a concentration of 25mg/kg for 15 days.