Degradation method of benzopyrene and application thereof

Abstract

The invention provides a method for degrading benzo [a] pyrene (BaP) and application of the benzo [a] pyrene (BaP). According to the degradation method, a strain of bacillus thuringiensis PAHc-a with the preservation number of CCTCC No: M 2024700 is used for degrading BaP, and the natural degradation rates of the bacillus thuringiensis PAHc-a respectively reach 17.80%, 63.94% and 83.92% in 3 days, 6 days and 9 days. In order to further improve the degradation rate of benzopyrene, another two degradation bacteria, namely bacillus aryabhattai PAHA-e with the preservation number of CCTCC No: M 2024702 and brucella PAHB-d with the preservation number of CCTCC No: M 2024701, are simultaneously screened in the invention; the three strains are used as a composite flora, cultured and inoculated into pollutants, and the 9-day natural degradation rate of benzopyrene can be increased to 87.10%.

Description

Technical Field

[0001] The invention relates to the field of soil environmental microorganisms, and in particular to a benzopyrene degradation method and application thereof. Background Art

[0002] Benzopyrene is a condensed aromatic hydrocarbon containing a benzene ring. Depending on the condensation position of the benzene ring, benzopyrene has multiple isomers, two of which are common: benzo[a]pyrene, CAS number: 50-32-8; benzo[e]pyrene, CAS number: 192-97-2.

[0003] Benzo[a]pyrene (BaP) is widely present in the environment, including but not limited to coal tar, various types of carbon black and coal, coking, oil refining, asphalt and plastics. It is a strong carcinogen to humans and animals. It was initially discovered that it can cause skin cancer. Later, in-depth research found that it can cause cancer to various organs of the body such as the lungs, liver, esophagus, gastrointestinal tract, etc.

[0004] At present, the following methods can be used to control the pollution of benzo[a]pyrene:

[0005] (1) Adsorption method: The adsorption method uses the porosity of the adsorbent to adsorb BaP on the surface of the adsorbent. The principle is that BaP molecules are attracted by the activated carbon so that more BaP is attracted and the straight outer pores are sucked into its internal gaps. Due to the mutual attraction between molecules, the internal gaps of the activated carbon are filled.

[0006] (2) Thermal treatment: In the absence of oxygen or in the absence of oxygen, the waste containing BaP is irradiated with heat to separate the BaP, thereby achieving the purpose of removing benzene BaP.

[0007] (3) Chemical method: Use chemical oxidation to decompose BaP, thereby achieving BaP pollution control. Commonly used oxidants include O3, persulfate and Fenton's reagent.

[0008] (4) Biodegradation: Use microorganisms to convert BaP into organic compounds, and further into carbon dioxide and water. The microorganisms that degrade BaP mainly include bacteria, fungi, and algae, such as Aspergillus niger, Trichoderma, Haberella, and Penicillium. Biodegradation is the most effective technology for treating BaP pollution. Due to the unique advantages of biodegradable microorganisms, such as low cost, no secondary pollution, and large-scale application, biodegradation of BaP has attracted more and more attention.

[0009] However, the existing technologies are mainly focused on the biodegradation of medium- and low-ring PAHs, and there are few technologies for the biodegradation of high-ring BaP, and the degradation time is long and the degradation rate is low. Therefore, the field urgently needs to find strains or composite bacterial communities with better degradation effects as microbial agents to better treat BaP. Summary of the invention

[0010] In view of the shortcomings of the prior art, the object of the present invention is to provide a method for degrading benzopyrene and application thereof.

[0011] To achieve this object, the present invention adopts the following technical solutions:

[0012] In a first aspect, the present invention provides a method for degrading benzopyrene, comprising the following steps:

[0013] S1, preparing the activated degradation strain into a bacterial suspension;

[0014] S2, inoculating the bacterial suspension prepared in step S1 into the object to be treated containing benzopyrene;

[0015] Among them, the degradation strain includes strain PAHc-a (Bacillus sp), which is preserved in the China Type Culture Collection Center, the strain name is Bacillus PAHc-a, the preservation address is Wuhan University, Wuhan, China, the preservation date is April 18, 2024, and the preservation number is CCTCC No: M 2024700.

[0016] As a preferred technical solution of the present invention, the degradation strain also includes: strain PAHA-e (Priestia aryabhattai), which is preserved in the China Center for Type Culture Collection, the strain name is Bacillus aryabhattai, the preservation address is Wuhan University, Wuhan, China, the preservation date is April 18, 2024, and the preservation number is CCTCC No: M 2024702.

[0017] Preferably, the degradation strain also includes: strain PAHB-d (Brucella sp.), which is preserved in the China Center for Type Culture Collection, the strain name is Brucella, the preservation address is Wuhan University, Wuhan, China, the preservation date is April 18, 2024, and the preservation number is CCTCC No: M 2024701.

[0018] As a preferred technical solution of the present invention, the bacterial suspension OD prepared in step S1 is 600 It is 0.6-1.2, for example, it can be 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 or 1.2, etc.

[0019] Preferably, the volume ratio of the bacterial suspension of strain PAHc-a and strain PAHA-e is 1:(0.8-1.2), for example, it can be 1:0.8, 1:0.85, 1:0.9, 1:1, 1:1.05, 1:1.1, 1:1.5, 1:1.2, etc.

[0020] Preferably, the volume ratio of the bacterial suspension of strain PAHc-a and strain PAHB-d is 1:(0.8-1.2), for example, it can be 1:0.8, 1:0.85, 1:0.9, 1:1, 1:1.05, 1:1.1, 1:1.5, 1:1.2, etc.

[0021] In a second aspect, the present invention provides a microbial agent for degrading benzopyrene, comprising: strain PAHc-a (Bacillus sp.), with a deposit number of CCTCC No: M 2024700.

[0022] As a preferred technical solution of the present invention, the microbial agent also includes: strain PAHA-e (Priestia aryabhattai), with a preservation number of CCTCC No: M 2024702; and strain PAHB-d (Brucella sp.), with a preservation number of CCTCC No: M 2024701.

[0023] Preferably, during use, the inoculation ratio of the three strains can be set according to 1:(0.8-1.2):(0.8-1.2), for example, it can be 1:1:1, 1:1:0.8, 1:1:1.2, 1:0.8:1, 1:1.2:1, 1:0.8:0.8, 1:1.2:1.2, 1:0.9:1.1, 1:0.95:1.15, 1:1.1:0.9, etc.

[0024] As a preferred technical solution of the present invention, the preparation method of the microbial agent is:

[0025] Inoculating the bacterial cells directly into a liquid culture solution, culturing to a logarithmic growth phase, centrifuging, concentrating, and washing the bacterial cells to obtain the microbial agent;

[0026] Alternatively, the microorganisms are compounded according to the formula amount and inoculated into a liquid culture solution, cultured to the logarithmic growth phase, centrifuged, concentrated, and washed to obtain the microbial agent;

[0027] Alternatively, the bacterial cells are directly inoculated into a liquid culture solution, compounded according to a formula, and then inoculated into the liquid culture solution, cultured to a logarithmic growth phase, centrifuged, concentrated, and washed to obtain the microbial agent.

[0028] Therefore, when used for pollutant treatment, the strain or composite bacterial community can be cultured and then directly inoculated into the soil or water source to degrade benzopyrene and other polycyclic aromatic hydrocarbons in the treated material.

[0029] In a third aspect, the present invention further provides the use of the degradation method as described in the first aspect or the microbial agent as described in the second aspect in remediating soil or water contaminated by benzopyrene.

[0030] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) The present invention provides a method for degrading benzo[a]pyrene, wherein the method uses the strain to degrade BaP, and the natural degradation rates thereof reach 17.80%, 63.94% and 83.92% at 3 days, 6 days and 9 days, respectively;

[0033] (2) On this basis, considering that benzopyrene is mostly present in pollutants such as soil or water sources contaminated by complex aromatic hydrocarbons, in order to further improve the degradation rate of benzopyrene and other polycyclic aromatic hydrocarbon pollutants, the present invention simultaneously screened two other degrading bacteria, namely Bacillus aggregatis PAHA-e with a preservation number of CCTCC No: M 2024702 and Brucella sp. PAHB-d with a preservation number of CCTCC No: M 2024701; the strains do not have an antagonistic relationship with the strain PAHc-a and will not inhibit each other's growth under the same environmental conditions.

[0034] The three strains are used as a composite bacterial community and inoculated into pollutants after cultivation, and the natural degradation rates of benzopyrene can be increased to 23.16%, 86.42% and 87.10% at 3 days, 6 days and 9 days respectively.

[0035] (3) In addition, the composite bacterial community can also be used to degrade composite aromatic hydrocarbon pollutants. Its degradation targets cover low, medium and high polycyclic aromatic hydrocarbons. The 9-day natural degradation rates of phenanthrene, pyrene and benzopyrene can reach 73.14%, 71.34% and 87.10%, respectively. Therefore, the composite bacterial community is simple to use, has a more comprehensive degradation range, a higher degradation efficiency, a wide range of use, and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the growth curve of benzopyrene-degrading bacteria Ca.

[0037] Figure 2 It is a bar graph of the degradation rate of BaP in the culture medium.

[0038] Figure 3 Figure 1 is the growth curve of three phenanthrene-degrading bacteria (Aa, Ac and Ae), wherein Figure I represents strain Aa, Figure II represents strain Ac, and Figure III represents strain Ae.

[0039] Figure 4Figure 1 shows the growth curves of three pyrene-degrading bacteria (Ba, Bb and Bd), where Figure I represents strain Ba, Figure II represents strain Bb, and Figure III represents strain Bd.

[0040] Figure 5 This is a physical picture of the colony morphology of the composite strain.

[0041] Figure 6 This is a bar graph showing the degradation rate of the composite bacterial community ABC-eba on a mixture of Phe, Pyr and BaP. DETAILED DESCRIPTION

[0042] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simple examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0043] In the following examples, unless otherwise specified, all reagents and consumables used were purchased from conventional reagent manufacturers in the field; unless otherwise specified, all experimental methods and technical means used were conventional methods and means in the field.

[0044] Example 1 Screening of efficient degradation bacteria

[0045] An appropriate amount of soil was collected from farmland soil heavily contaminated by PAHs around chemical companies and brought back to the laboratory. Stones and plant debris were removed and the soil was passed through a 10-mesh sieve. After freeze-drying, the soil was stored in a brown glass bottle and refrigerated in a 4°C refrigerator for later use.

[0046] Take 10.0 g of the test soil sample in a 250 mL conical flask, maintain 65% of the field water capacity, and culture it at 25°C for 7 days to restore the activity of soil microorganisms.

[0047] Weigh 1.0 g of the above soil sample or 5 mL of soil suspension and add it into a conical flask containing 50 mL of inorganic salt liquid culture medium, then add benzo[a]pyrene (BaP) to make the BaP concentration in the culture medium 2.0 mg·L -1 , at 30℃, 180r·min -1 The cells were cultured in a shaker in the dark for 7 days.

[0048] 5 mL of the above culture medium was taken and inoculated into new inorganic salt culture medium (BaP concentration was 5.0 mg·L -1 ), the culture conditions and time remain unchanged and the above operation is repeated.

[0049] The BaP concentrations were 2.0, 5.0, 10.0 and 20.0 mg·L -1 , in order to complete the domestication of the bacterial flora in the soil sample.

[0050] Take the last bacterial culture solution, use phosphate buffered saline (PBS) as diluent, and dilute it to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 and 10 -7 times.

[0051] Take 0.1 mL of the diluted bacterial culture solution and spread it evenly on a tryptone yeast powder solid culture medium plate, culture it at 30°C for 24 to 72 hours, and observe the growth of the colonies.

[0052] The well-grown colonies were selected and separated and purified multiple times on tryptone yeast powder solid culture medium plates, and finally the obtained bacterial colonies were preserved on slants.

[0053] Through enrichment and acclimatization as well as separation and purification at different gradient concentrations, a benzopyrene-degrading bacterium was finally screened and numbered Ca.

[0054] Figure 1 This is the growth curve of benzopyrene-degrading bacteria Ca. The lag phase of the strain is 6 hours. During this period, it is the adaptation stage of the strain. The cells do not divide and the bacterial solution is relatively clear. The logarithmic growth phase is 6 to 12 hours. During this stage, the cells adapt to the new growth environment and begin to replicate, and then the number of cells grows rapidly exponentially, and the bacterial solution gradually becomes turbid. As time goes on, cells begin to die due to lack of nutrients and increase of harmful substances in the culture medium. This stage is called the decay phase, which is manifested as a decrease in biomass on the growth curve. The decay phase of strain Ca occurs at 12 hours.

[0055] According to the growth curve, the lag phase of strain Ca is shorter, indicating that the strain has strong adaptability.

[0056] The screened strains were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing, and the obtained 16S rDNA sequences were uploaded to the National Center for Biotechnology Information (NCBI) database of the United States, and then BLAST comparison was performed to obtain the strain sequence with the highest similarity to the strain.

[0057] The 16S rDNA sequence of strain Ca has the highest similarity with Bacillus thuringiensis, with a homology of 100%. Strain Ca was deposited under the deposit name Bacillus PAHc-a.

[0058] Example 2 Degradation characteristics of strain PAHc-a on BaP

[0059] (1) Preparation of bacterial suspension

[0060] The selected optimal BaP-degrading bacteria were inoculated into the sterilized tryptone yeast powder liquid culture medium and placed in an oscillating incubator at 30°C and 180 r / min. -1 After culturing for 24 h, the culture solution was divided into 100 mL sterile centrifuge tubes and centrifuged at 4000 r / min at room temperature. -1 Centrifuge for 10 minutes, collect the wet bacteria at the bottom of the tube, then wash repeatedly with sterilized saline (at least 3 times), and then prepare it into a bacterial suspension with saline to make its OD 600 is 1.0.

[0061] (2) Vaccination

[0062] According to the inoculum volume of 5%, the bacterial suspension was inoculated to 100 mL of BaP with a concentration of 50 mg·L -1 Sterile tryptone yeast powder liquid medium was used at 30℃ and 180r·min -1 The shaking culture was carried out for 3, 6 and 9 days under the conditions;

[0063] At the same time, a tryptone yeast powder liquid culture medium without bacteria was set as a reference and three experimental replicates. Samples were taken for analysis at 3d, 6d and 9d of culture to determine the degradation rate of BaP by a single strain.

[0064] (3) Calculation of degradation rate

[0065] like Figure 2 As shown, with the extension of culture time, the degradation rate of BaP in the culture medium increased significantly.

[0066] The natural degradation rates of BaP were 0.63%, 2.15% and 4.31% on the 3rd, 6th and 9th days, respectively. After adding strain PAHc-a, the degradation rates increased to 17.80%, 63.94% and 83.92% on the 3rd, 6th and 9th days, respectively.

[0067] This shows that strain PAHc-a can significantly degrade benzopyrene in the culture medium.

[0068] Example 3 Screening of composite flora

[0069] Screening was performed from the same farmland soil as in Example 1, and the screening steps were referred to Example 1;

[0070] The difference is that phenanthrene (Phe) or pyrene (Pyr) drugs are added to the culture medium to make the concentration of Phe or Pyr in the culture medium 2.0 mg·L -1 .

[0071] Finally, three phenanthrene-degrading bacteria and three pyrene-degrading bacteria were screened out and numbered Aa, Ac, Ae, Ba, Bb and Bd, respectively.

[0072] Figure 3 The following are the growth curves of three phenanthrene-degrading bacteria (Aa, Ac and Ae), where Figure I represents strain Aa, Figure II represents strain Ac, and Figure III represents strain Ae. It can be seen from the figure that the lag phases of strains Aa, Ac and Ae in liquid culture medium are 6h, 10h and 4h respectively, the logarithmic growth phases are 6-20h, 10-22h and 4-12h respectively, and the decay phases appear at 20h, 22h and 12h.

[0073] According to the growth curve, strain Ae has the shortest lag phase, indicating that the strain has stronger adaptability than the other two strains and can be used as one of the candidate strains for the composite flora. The 16S rDNA sequence of strain Ae is most similar to Bacilus aryabhattai (Bacillus aryabhattai), with a homology of 100%; strain Ae is preserved and the preservation name is Bacillus PAHA-e.

[0074] Figure 4 Figure 1 is the growth curve of three pyrene-degrading bacteria (Ba, Bb and Bd), where Figure I represents strain Ba, Figure II represents strain Bb, and Figure III represents strain Bd. It can be seen from the figure that the lag phase of strains Ba, Bb and Bd are 12h, 10h and 8h respectively, the logarithmic growth phase is 12-24h, 10-24h and 8-20h respectively, and the decay phase occurs at 24h, 24h and 10h.

[0075] According to the growth curve, strain Bd has the shortest lag phase, indicating that the strain has stronger adaptability than the other two strains and can be used as one of the candidate strains for the composite flora. The 16S rDNA sequence of strain Bd has the highest similarity with Brucella sp, with a homology of 100%; strain Bd is preserved and the preservation name is Brucella PAHB-d.

[0076] Antagonism between strains: refers to the process of inoculating two or more different strains into the culture medium of the same culture dish. During the growth of the strains, different strains resist each other's invasion, and a dark antagonism line visible to the naked eye will be formed at the junction of the strains. If no antagonism line appears in the antagonism experiment, it indicates that there is no competition between the strains.

[0077] Strains Ae, Bd and Ca were randomly combined and streaked on solid medium for 3 days. Figure 5As shown, the growth state is good and the growth momentum is similar, indicating that there is no antagonistic relationship between the strains, that is, the three strains will not inhibit each other's growth under the same environmental conditions.

[0078] Example 4 Degradation of benzopyrene pollutants by composite bacterial flora ABC-eba

[0079] The selected best Phe, Pyr and BaP degradation bacteria were made into OD 600 The mixture was mixed evenly in a volume ratio of 1:1:1 to construct a composite bacterial community ABC-EBA solution.

[0080] The composite bacterial flora ABC-eba solution was added to 100 mL of BaP at a concentration of 50 mg·L at a 5% inoculum concentration. -1 Sterile tryptone yeast powder liquid medium was used at 30℃ and 180r·min -1 The cells were cultured under shaking conditions for 3, 6 and 9 days. A tryptone yeast powder liquid culture medium without bacteria was set as a reference and three experimental replicates. Samples were taken for analysis at 3, 6 and 9 days of culture to detect the degradation rate of BaP pollutant by the composite bacterial community ABC-eba.

[0081] The results show that the degradation rate of BaP pollutants by the composite bacterial community ABC-eba is higher than the degradation efficiency of a single strain.

[0082] Example 5 Degradation of polycyclic aromatic hydrocarbons by composite bacterial flora ABC-eba

[0083] The only difference compared with Example 4 is that the composite bacterial flora ABC-eba solution is added to 100 mL of Phe, Pyr and BaP at a concentration of 50 mg·L -1 The degradation rate of BaP pollutant by the composite bacterial community ABC-eba was detected in sterile tryptone yeast powder liquid culture medium.

[0084] Figure 6 This is the degradation of a mixture of Phe, Pyr and BaP by the composite bacterial community ABC-eba. As can be seen from the figure, with the extension of time, the degradation ability of the composite bacterial community ABC-eba on mixed pollutants has improved to varying degrees.

[0085] Among them, the degradation rates of BaP by strain Ca and the composite bacterial community are shown in Table 1. The degradation rates of BaP at 3d, 6d and 9d were 23.16%, 86.42% and 87.10%, which were 3.18% higher than that of a single strain.

[0086] Table 1

[0087]

[0088]

[0089] In addition, the composite bacterial community ABC-eba also showed degradation rates of 37.84%, 66.04% and 73.14% for Phe during the test period, among which the 9-day degradation rate was 5.68% higher than when the composite strain was not added; the degradation rates of Pyr were 36.96%, 52.50% and 71.34%; among which the 9-day degradation rate was 21.8% higher than when the composite strain was not added.

[0090] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for degrading benzopyrene, characterized in that: include: S1, preparing the activated degradation strain into a bacterial suspension; S2, inoculating the bacterial suspension prepared in step S1 into the object to be treated containing benzopyrene; Wherein, the degradation strain includes strain PAHc-a (Bacillus sp.), and the preservation number is CCTCC No: M2024700.

2. The degradation method according to claim 1, characterized in that: The degradation strain also includes: strain PAHA-e (Priestia aryabhattai), with the preservation number being CCTCC No: M 2024702.

3. The degradation method according to claim 1, characterized in that: The degradation strain also includes: strain PAHB-d (Brucella sp.), with a preservation number of CCTCC No: M 2024701.

4. The degradation method according to claim 1, characterized in that: OD of bacterial suspension prepared in step S1 600 It is 0.6-1.

2.

5. The degradation method according to claim 2 or 3, characterized in that: The bacterial suspension volume ratio of the strain PAHc-a to the strain PAHA-e is 1:(0.8-1.2); The bacterial suspension volume ratio of the strain PAHc-a to the strain PAHB-d is 1:(0.8-1.2).

6. A microbial agent for degrading benzopyrene, characterized in that: The microbial agent comprises: strain PAHc-a (Bacillus sp.), with a preservation number of CCTCC No: M 2024700.

7. The microbial agent according to claim 6, characterized in that: The microbial agent also includes: strain PAHA-e (Priestia aryabhattai), with a deposit number of CCTCC No: M 2024702; And, strain PAHB-d (Brucella sp.), deposited with CCTCC No: M 2024701.

8. The microbial agent according to claim 6 or 7, characterized in that: The preparation method of the microbial agent is as follows: Inoculating the bacterial cells directly into a liquid culture solution, culturing to a logarithmic growth phase, centrifuging, concentrating, and washing the bacterial cells to obtain the microbial agent; Alternatively, the microorganisms are compounded according to the formula amount and inoculated into a liquid culture solution, cultured to the logarithmic growth phase, centrifuged, concentrated, and washed to obtain the microbial agent; Alternatively, the bacterial cells are directly inoculated into a liquid culture solution, compounded according to a formula, and then inoculated into the liquid culture solution, cultured to a logarithmic growth phase, centrifuged, concentrated, and washed to obtain the microbial agent.

9. Use of the degradation method according to any one of claims 1 to 8 or the microbial agent according to any one of claims 6 to 8 in remediation of soil or water contaminated by benzopyrene.

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