Bacillus cereus CY-1 and its application and degradation agent

Abstract

The present invention relates to the field of microbial technology, and more particularly to a strain of Bacillus cereus CY-1 and its application and degradation agent. The Bacillus cereus CY-1 was deposited in the China Center for Type Culture Collection on April 30, 2024, with a deposit number of CCTCC NO:M2024859. The degradation rate of Bacillus cereus CY-1 provided by the present invention for 200mg / L of chlorobenzene in 24h was 95.75%, the degradation rate for 700mg / L of chlorobenzene in 48h was 61.3%, and the degradation rate for 1000mg / L of chlorobenzene in 48h was 50.2%. The strain has strong tolerance to chlorobenzene and is well adapted to the environment. The temperature when used to repair contaminated sites can be 20-40°C, the pH can be 4-9, and it can be used to degrade chlorobenzene, benzene, toluene and o-dichlorobenzene.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, in particular to a Bacillus cereus CY-1 strain and its application and degradation agent. Background Art

[0002] Chlorobenzene, a benzene ring compound containing chlorine atoms, is widely used in industrial production as an organic solvent, raw material, and intermediate in the manufacture of chemical products such as dyes, pesticides, and pharmaceuticals. Chlorobenzene production and use can result in emissions and leaks, leading to chlorobenzene pollution. Chlorobenzene is highly toxic and bioaccumulative. Long-term or high-concentration exposure to chlorobenzene can cause serious harm to the human body, including damage to the central nervous system, liver, and kidneys. Furthermore, chlorobenzene and its metabolites are considered to be potentially carcinogenic to humans.

[0003] Currently, physical and chemical methods for treating chlorobenzene are difficult to achieve effective results in practice due to high costs, high energy consumption, and the risk of secondary pollution. Microbial remediation has attracted widespread attention due to its low cost, wide applicability, lack of secondary pollution, and excellent treatment results. By screening microorganisms capable of degrading chlorobenzene and studying their degradation efficiency, the remediation potential of degrading bacteria in natural environments was enhanced. This research not only provides a reference for identifying potential degrading bacteria at other contaminated sites, but also provides a theoretical basis for environmental remediation technologies such as bioremediation of chlorobenzene contamination in groundwater, providing scientific support for alleviating chlorobenzene pollution. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides a Bacillus cereus CY-1 and its application and degradation agent. The Bacillus cereus CY-1 provided by the present invention can degrade aromatic hydrocarbons and has a high degradation effect on chlorobenzene.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a Bacillus cereus CY-1. The Bacillus cereus CY-1 was deposited in the China Center for Type Culture Collection on April 30, 2024, with a deposit number of CCTCC NO: M2024859.

[0007] The present invention also provides the use of the Bacillus cereus CY-1 described in the above technical solution in the degradation of aromatic hydrocarbons.

[0008] The present invention also provides an aromatic hydrocarbon degradation agent, which contains the Bacillus cereus CY-1 described in the above technical solution.

[0009] Preferably, the content of Bacillus cereus CY-1 in the aromatic hydrocarbon degrading agent is 3.3×107 CFU / mL.

[0010] The present invention also provides the use of the aromatic hydrocarbon degradation agent described in the above technical solution in degrading aromatic hydrocarbons.

[0011] Preferably, the aromatic hydrocarbons include one or more of benzene, toluene, chlorobenzene and o-dichlorobenzene.

[0012] Beneficial effects of the present invention:

[0013] 1. The strain of the present invention was screened from leachate from a waste treatment plant and has strong environmental adaptability and chlorobenzene tolerance. It can effectively degrade chlorobenzene at temperatures between 20-40°C and pH between 4-9, and can efficiently degrade up to 1000 mg / L of chlorobenzene.

[0014] The chlorobenzene-degrading bacteria provided by the present invention are screened from landfill leachate and are highly effective in degrading chlorobenzene. They also have a significant impact on benzene, toluene, and o-dichlorobenzene, playing an important role in the remediation of chlorobenzene-contaminated soil and water. This not only provides a reference for identifying potential degrading bacteria in other contaminated sites but also provides a theoretical basis for environmental remediation technologies such as bioremediation of chlorobenzene contamination in groundwater.

[0015] 3. The Bacillus cereus CY-1 provided by the present invention has a degradation rate of 95.75% for 200 mg / L of chlorobenzene in 24 hours, a degradation rate of 78.15% for 500 mg / L of chlorobenzene in 48 hours, and a degradation rate of 61.3% for 700 mg / L of chlorobenzene in 48 hours. This strain has strong tolerance to chlorobenzene and good environmental adaptability. When used to remediate contaminated sites, the temperature can be 20-40°C and the pH can be 4-9. It can be used to degrade chlorobenzene, benzene, toluene, and o-dichlorobenzene. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0017] Figure 1 The colony morphology of Bacillus cereus CY-1 provided by the present invention, wherein A is Gram staining and B is colony morphology;

[0018] Figure 2 The degradation rate results of the strain of the present invention under different pH conditions;

[0019] Figure 3 The results of the degradation rate of different substrates by the strain of the present invention are shown in FIG.

[0020] Figure 4 The degradation effect of the strain of the present invention on chlorobenzene at different concentrations;

[0021] Figure 5 A diagram showing the degradation effect of the strain of the present invention on chlorobenzene in actual groundwater.

[0022] Biological Deposit Description

[0023] Bacillus cereus CY-1, also known as Bacillus cereus in Latin, was deposited in the China Center for Type Culture Collection, Wuhan University, Wuhan, China on April 30, 2024, with the deposit number CCTCC NO: M2024859. DETAILED DESCRIPTION

[0024] The present invention provides a strain of Bacillus cereus CY-1, which was deposited with the China Center for Type Culture Collection on April 30, 2024, with a deposit number of CCTCC NO: M2024859. In the present invention, the Bacillus cereus CY-1 is a Gram-positive bacterium that forms round or nearly round, soft, pigment-free, slightly shiny, white, candle-like colonies with a diameter of 5-7 mm on a common agar plate medium (Luria-Bertani medium) at 37° C. for 24 hours.

[0025] The present invention also provides the use of the Bacillus cereus CY-1 described in the above technical solution in the degradation of aromatic hydrocarbons. In the present invention, the aromatic hydrocarbons preferably include one or more of benzene, toluene, chlorobenzene and o-dichlorobenzene.

[0026] The present invention also provides an aromatic hydrocarbon degrading agent, comprising the Bacillus cereus CY-1 described in the above technical solution. In the present invention, the bacterial content of Bacillus cereus CY-1 in the aromatic hydrocarbon degrading agent is preferably 3.3×10 7 The present invention has no particular limitation on the preparation method of the aromatic hydrocarbon degradation agent, and those skilled in the art can prepare the agent using conventional microbial agent preparation methods.

[0027] The present invention also provides the use of the aromatic hydrocarbon degradation agent described in the above technical solution in the degradation of aromatic hydrocarbons. In the present invention, the aromatic hydrocarbons preferably include one or more of benzene, toluene, chlorobenzene and o-dichlorobenzene.

[0028] In order to further illustrate the present invention, the present invention is described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0029] Example 1

[0030] Screening of strains:

[0031] (1) Chlorobenzene-degrading bacteria were obtained by collecting samples of leachate from a waste treatment plant through enrichment, screening, acclimation, isolation, and purification. Five milliliters of the collected leachate were added to an inorganic salt culture medium containing chlorobenzene as the sole carbon source. After constant temperature and shaking in a shaking incubator at 30°C and 150 rpm for five days, the chlorobenzene concentration was increased and subcultured for five cycles of acclimation.

[0032] 2) Take 1 ml of the bacterial solution obtained after 5 cycles of acclimation, streak it onto an inorganic salt solid medium, and incubate it in a 30°C constant temperature incubator. Select the strain growing on the inorganic salt solid medium and continue to purify it in LB medium. Transfer the selected single bacteria to an inorganic salt medium containing chlorobenzene as the sole carbon source to verify their ability to degrade chlorobenzene. If the chlorobenzene concentration decreases and the bacteria grow, then the chlorobenzene-degrading bacteria are obtained.

[0033] 3) By comparing the 16s rDNA sequence of the strain using BLAST on NCBI, it was found that the strain was highly homologous to Bacillus cereus, and it was speculated that the strain was a Bacillus cereus. It was named Bacillus cereus CY-1 and deposited in the China Center for Type Culture Collection on April 30, 2024, with the deposit number CCTCC NO: M2024859.

[0034] The inorganic salt culture medium of step 1) is composed of: 1.0 g / L (NH4)2SO4, 0.5 g / L KH2PO4, 1.5 g / L K2HPO4, 1.0 g / L NaCl, 0.1 g / LMgSO4; 2 ml of trace elements are added to each liter of culture medium; the trace elements are: 0.3 g H3BO3, 0.1 g ZnCl, 0.1 g Na2MoO4.2H2O, 0.75 g NiCl2.6H2O, 1.0 g MnCl2.4H2O, 0.1 g CuCl2.2H2O, 1.5 g CoCl2.6H2O, 0.02 g Na2SeO3, and 0.1 g Al2(SO4)3.18H2O, and the volume is adjusted to 1 L.

[0035] The chlorobenzene concentration in step 1) was adjusted to 300 mg / L, 400 mg / L, 500 mg / L, 600 mg / L and 700 mg / L. The degradation rate was measured every 24 hours. Figure 4 In 48 hours, the degradation rates of 300 mg / L chlorobenzene were 96.85%, 400 mg / L chlorobenzene was 92.5%, 500 mg / L chlorobenzene was 78.15%, 600 mg / L chlorobenzene was 70.25%, and 700 mg / L chlorobenzene was 61.3%.

[0036] During the entire domestication process, the tools used are 100mL serum bottles, sealed with aluminum caps and stoppers, and the liquid added is generally 20mL. Fresh bacterial liquid is taken and sent to Universal Bio (Anhui) Co., Ltd. for sequencing. The BLAST alignment sequence has 100% homology with Bacillus cereus.

[0037] Example 2

[0038] Degradation characteristics of chlorobenzene-degrading bacteria:

[0039] Chlorobenzene-degrading bacteria (Bacillus cereus CY-1) were cultured in Luria-Bertani medium (Tryptone 10 g / L, Yeast extract 5 g / L, Sodium chloride (NaCl) 10 g / L) for 24 h, centrifuged at 8000 rpm for 10 min, washed twice with the inorganic salt medium in Example 1, washed to remove the carbon source, and resuspended in the inorganic salt medium. 20 ml of the inorganic salt medium with chlorobenzene as the sole carbon source was added to a 100 ml serum bottle, and then 0.2 ml of the resuspended bacterial solution was added. The OD of the bacterial solution was 0. 600 The final OD value of the culture medium is 10. 600 The value is 0.1 (i.e. the bacterial content is 3.3×10 7 CFU / mL) and a chlorobenzene concentration of 200 mg / L. Measure absorbance at 600 nm, setting the initial absorbance to 0.1. Seal the serum vial with a stopper and aluminum cap and incubate in a constant-temperature shaking incubator at 30°C and 150 rpm. Measure the chlorobenzene concentration after 24 hours. Change the initial chlorobenzene concentration and pH value. After 24 hours, take samples and measure the chlorobenzene concentration and absorbance at 600 nm.

[0040] Depend on Figure 2 It can be seen that the degradation rate of Bacillus cereus CY-1 is the fastest at pH = 7, reaching 95.75% within 24 hours, which has certain advantages. The degradation rate is 58.9% at pH = 4, 75.65% at pH = 5, 88.1% at pH = 6, 95.75% at pH = 7, 94.25% at pH = 8, and 82.4% at pH = 9.

[0041] Example 3

[0042] Study on the broad spectrum of substrates of chlorobenzene-degrading bacteria (Bacillus cereus CY-1):

[0043] Chlorobenzene-degrading bacteria (Bacillus cereus CY-1) were cultured in Luria-Bertani medium for 24 h, centrifuged at 8000 rpm for 10 min, washed twice with the inorganic salt medium in Example 1, and the carbon source was washed away. The culture was resuspended in the inorganic salt medium to obtain a resuspended bacterial solution. 20 ml of inorganic salt medium was added to a 100 ml serum bottle, with benzene, toluene, and o-dichlorobenzene as the sole carbon source (final concentration 50 mg / L), and 0.2 ml of the resuspended bacterial solution was added. The OD value of the bacterial solution was 0. 600 The value is 10, the final OD of the culture medium 600 The value is 0.1 (i.e. the bacterial content is 3.3×10 7 The serum bottles were sealed with a cap. The serum bottles were placed in a constant temperature shaking incubator at 30°C, 150 rpm, and pH 7.0. The concentrations of benzene, toluene, and o-dichlorobenzene were measured every 24 hours.

[0044] Depend on Figure 3 It can be seen that Bacillus cereus CY-1 can effectively degrade benzene, toluene and o-dichlorobenzene, indicating that chlorobenzene-degrading bacteria have a broad spectrum of substrates.

[0045] Table 1 Degradation effect

[0046] Time (d) Benzene degradation rate / % Toluene degradation rate / % Ethylbenzene degradation rate / % Degradation rate of o-dichlorobenzene / % 0 0 0 0 0 1 9.75 32.5 25.5 8.2 2 17.4 56 38.25 17.95 3 23 74 56.7 33.4 4 28.25 83.35 75.5 40.1 5 28.5 87.2 82.1 41.5

[0047] Example 4

[0048] Degradation of high concentrations of chlorobenzene in groundwater by Bacillus cereus CY-1:

[0049] Bacillus cereus CY-1 was grown overnight in Luria-Bertani medium. The washed strain was resuspended in an inorganic salt medium without a carbon source and the bacterial suspension (OD 600 Add a solution (value of 0.1) to 20 mL of chlorobenzene-contaminated groundwater, add chlorobenzene to 1000 mg / L, and degrade at 150 rpm and 20°C. The initial absorbance at 600 nm is 0.1. Samples are taken at intervals to determine the residual chlorobenzene concentration.

[0050] Figure 5 It showed that chlorobenzene was rapidly degraded within the first 72 hours, with a degradation rate of about 65.3%.

[0051] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A strain of Bacillus cereus ( Bacillus cereus )CY-1, characterized in that The Bacillus cereus CY-1 was deposited in the China Center for Type Culture Collection on April 30, 2024, with the deposit number being CCTCC NO: M2024859.

2. Use of the Bacillus cereus CY-1 according to claim 1 in the degradation of aromatic hydrocarbons; The aromatic hydrocarbon is one or more of benzene, toluene, chlorobenzene and o-dichlorobenzene.

3. An aromatic hydrocarbon degradation agent, characterized in that Contains the Bacillus cereus CY-1 according to claim 1.

4. The aromatic hydrocarbon degradation agent according to claim 3, characterized in that The bacterial content of Bacillus cereus CY-1 in the aromatic hydrocarbon degrading agent is 3.3×10 7 CFU / mL.

5. Use of the aromatic hydrocarbon degradation agent according to claim 3 or 4 in the degradation of aromatic hydrocarbons; The aromatic hydrocarbon is one or more of benzene, toluene, chlorobenzene and o-dichlorobenzene.

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