Separation and identification of bacteria Achromobacter deeleyi DY-7 for degrading phenanthrene and benzopyrene in petroleum-contaminated soil and application of bacteria Achromobacter deeleyi DY-7

Abstract

The invention discloses separation and identification of a bacterium Achromobacter deeleyi DY-7 for degrading phenanthrene and benzopyrene in petroleum contaminated soil and an application of the bacterium Achromobacter deeleyi DY-7. The strain Achromobactleyi DY-7 is obtained by domesticating, separating and identifying petroleum-polluted soil in Ningbo City in Zhejiang Province, phenanthrene and benzopyrene can be used as carbon sources, and after the strain is cultured in inorganic salt culture solutions with the initial concentration of phenanthrene or benzopyrene being 50 mg / L and 25 mg / L respectively for 7 days, the degradation rate of phenanthrene or benzopyrene can reach 70% or above. Therefore, the strain has good application potential in the aspect of bioremediation of polycyclic aromatic hydrocarbon.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of organic pollutant degradation, and particularly relates to separation and identification of a strain of bacteria Achromobacter deleyi DY-7 for degrading phenanthrene and benzopyrene in oil-contaminated soil and application thereof. BACKGROUND

[0002] The pollution of soil caused by the rapid development of modern industry is increasingly serious, and various persistent organic pollutants (POPs) such as polycyclic aromatic hydrocarbons (PAHs) exist in the contaminated soil. Human activities such as industrial and mining activities, agriculture and high soil environmental background value have caused serious pollution and serious over-standard of polycyclic aromatic hydrocarbons in the environment, and the main pollutants of the soil in the chemical park and the surrounding area, the oil extraction area, the mining area and the sewage irrigation area are caused by polycyclic aromatic hydrocarbons. These polycyclic aromatic hydrocarbons which exist universally and accumulate continuously in the environment have attracted widespread attention. Phenanthrene (Phe) is a kind of three-ring aromatic hydrocarbon, and it has a very close relationship with the carcinogenicity of PAHs. With its unique chemical structure, phenanthrene has become a model compound for the study of PAHs. Benzopyrene (BaP) is a planar polycyclic structure formed by the fusion of five benzene rings, and it is a strong carcinogen. It can combine with DNA in human cells to form adducts and induce gene mutations. These two types of substances have potential carcinogenic, teratogenic and mutagenic properties and biological accumulation, and can pose a major hazard to the ecological environment and human health.

[0003] The natural attenuation of toxic and harmful organic pollutants in the environment mainly depends on the metabolic action of related microorganisms. The bioremediation technology shows the advantages of low cost, good effect and no secondary pollution by strengthening this natural process, and is the most potential remediation method for phenanthrene and benzopyrene pollution repair. At present, there are few reported phenanthrene and benzopyrene degrading strains, mainly including strains of Virgibacillus, Chryseobacterium and Bacillus. Most of the microorganisms in the environment are not cultivable, and many microorganisms, especially microorganisms with specific functions, cannot be isolated by pure culture. Therefore, it has important application value and practical significance to screen strains that can effectively degrade high-concentration phenanthrene and benzopyrene. In the experiment, phenanthrene with a mass concentration of 50 mg·L -1 and benzopyrene with a mass concentration of 25 mg·L -1 were used as substrates for strain degradation, in order to provide data support for the biological treatment of polycyclic aromatic hydrocarbons. SUMMARY

[0004] The first object of the present application is to provide a strain Achromobacter deleyi DY-7 having the ability of degrading phenanthrene and / or benzopyrene, which was deposited in the Guangdong Microbial Culture Collection Center (GDMCC) on July 3, 2025, at address: 59, Building 5, 100, Martyrs' Avenue, Guangzhou, Guangdong, China, Postcode: 510070, and the deposit number is GDMCC No: 66627.

[0005] The strain of the present application belongs to Achromobacter deleyi, which can be isolated from various terrestrial and aquatic habitats. At present, there are still relatively few reports on the degradation of pollutants by Achromobacter deleyi. The present application domesticates and isolates a strain DY-7 using high-concentration phenanthrene and benzopyrene as a carbon source from petroleum-contaminated soil in Ningbo, Zhejiang Province, identifies it, and studies its growth characteristics and degradation characteristics of phenanthrene and benzopyrene, thereby providing a reference for the bioremediation of PAHs-contaminated environment.

[0006] The second object of the present application is to provide the use of the above-mentioned Achromobacter deleyi DY-7 in degrading phenanthrene and / or benzopyrene.

[0007] Preferably, the degradation of phenanthrene and / or benzopyrene is the degradation of phenanthrene and / or benzopyrene in petroleum-contaminated soil.

[0008] Preferably, the Achromobacter deleyi DY-7 is applied to the environment contaminated by phenanthrene and / or benzopyrene to degrade phenanthrene and / or benzopyrene.

[0009] The third object of the present application is to provide a phenanthrene and / or benzopyrene-degrading microbial agent, which comprises the above-mentioned Achromobacter deleyi DY-7 as an active ingredient.

[0010] The fourth object of the present application is to provide a method for degrading phenanthrene and / or benzopyrene, which sprays the above-mentioned Achromobacter deleyi DY-7 in an environment containing phenanthrene and / or benzopyrene to degrade phenanthrene and / or benzopyrene.

[0011] Preferably, the Achromobacter deleyi DY-7 is sprayed in the environment contaminated by phenanthrene and / or benzopyrene to degrade phenanthrene and / or benzopyrene.

[0012] Preferably, the Achromobacter deleyi DY-7 is sprayed in the petroleum-contaminated soil to degrade phenanthrene and / or benzopyrene.

[0013] The application obtains a strain DY-7 which degrades phenanthrene and benzopyrene as carbon source from the oil-polluted soil in Ningbo, Zhejiang Province. According to the strain morphology, 16S rDNA gene sequencing analysis and phylogenetic analysis, the strain is identified as Achromobacter deleyi DY-7. The optimal environmental conditions for the growth of DY-7 are as follows: the temperature is 28℃, the pH value is 7, and no sodium chloride is added. The 16S rDNA gene sequencing analysis result of the strain shows that the strain most similar to DY-7 is Achromobacter deleyi strain LMG 3458 (GenBank accession number NR 152014.1, similarity 99.94%). DY-7 can utilize phenanthrene and benzopyrene as carbon source, and the degradation rates of phenanthrene and benzopyrene can both reach more than 70% after the strain is cultured in inorganic salt culture solution with the initial concentrations of phenanthrene and benzopyrene being 50mg·L -1 and 25mg·L -1 respectively for 7 days. Therefore, the strain has good application potential in the bioremediation of polycyclic aromatic hydrocarbons.

[0014] Achromobacter deleyi DY-7, which was preserved in Guangdong Microbial Culture Collection Center (GDMCC) on July 3, 2025, is located at No. 59 Building, 5th Floor, Guangzhou Institute of Military Personnel, 100 Middle Martyrs Road, Guangzhou, Guangdong, China, with a postal code of 510070, and a preservation number of GDMCC No: 66627. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 is the front and back of the strain DY-7 in Example 1 grown on LB solid medium for 36 hours.

[0016] Figure 2 is the phylogenetic relationship of the strain DY-7 in Example 1 and its related bacteria based on 16s rRNA gene sequence, the construction method is neighbor-joining method, the self-exhibition value is set to repeat 1000 times, and the scale of 0.002 represents the replacement rate of each nucleotide.

[0017] Figure 3 is the growth of the strain DY-7 in Example 2 under different culture temperatures, salinity and pH conditions.

[0018] Figure 4 is the degradation efficiency of the strain DY-7 in Example 3 in inorganic salt culture medium with high concentration of phenanthrene and benzopyrene (the initial concentrations of phenanthrene and benzopyrene are 50mg·L -1 and 25mg·L -1 respectively). DETAILED DESCRIPTION

[0019] In order to make the objectives, technical solutions and advantages of the present application clearer, further detailed description will be made to the present application in combination with the accompanying drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and not used to limit the present application. In addition, the technical features involved in each embodiment of the present application described below can be combined with each other as long as they do not conflict with each other.

[0020] Example 1 Isolation and identification of Achromobacter deleyi DY-7

[0021] 1. Materials and methods

[0022] 1.1 Sample source

[0023] The soil sample was collected from a petroleum contaminated soil in Ningbo, Zhejiang Province, and long-term acclimated with high concentration of phenanthrene and benzopyrene as carbon source, respectively. Through multiple screening and purification, high-efficiency phenanthrene and benzopyrene-degrading bacteria were obtained.

[0024] 1.2 Culture medium

[0025] 1.2.1 Inorganic salt medium

[0026] The inorganic salt medium was used for enrichment culture of microorganisms in the sample and phenanthrene and benzopyrene degradation experiments under pure bacterial conditions. The medium formula is shown in Table 1 (containing phenanthrene or benzopyrene solution), and the preparation method is to add each component into solvent water, mix uniformly, and sterilize to obtain.

[0027] Table 1 Formula of inorganic salt medium

[0028]

[0029] 1.2.2 Nutrient medium

[0030] The nutrient medium was used for the isolation, purification, preservation and activation of bacteria and the culture of conventional microorganisms. The types and components of the liquid nutrient medium used in this experiment are shown in Table 2. If solid medium is needed for the experiment, only 1.5-2% agar powder needs to be added to the original medium formula. If there is no special instruction for the culture conditions of the strain, the pH of the medium is adjusted to 7. The preparation method of the nutrient medium is to add each component into solvent water, mix uniformly, and sterilize to obtain.

[0031] Table 2 Composition of Luria-Bertani medium (LB)

[0032]

[0033] 1.3 Acclimation, screening and isolation of strains

[0034] The collected contaminated soil was added to the enrichment medium (the above-mentioned inorganic salt medium), and phenanthrene and benzopyrene were used as degradation substrates at concentrations of 50 mg·L -1 and 25 mg·L -1 , respectively, and placed in a 28°C incubator for culture in the dark. The inorganic salt medium with phenanthrene or benzopyrene as the carbon source was used for strain acclimation, and 7 days were taken as one acclimation cycle. A 10% inoculum was transferred to fresh enrichment medium with the same culture system, and the above-mentioned enrichment process was repeated three times.

[0035] The fourth-generation enrichment culture sample obtained above was subjected to coating separation by the dilution plating method, and the sample was separated with a nutrient medium. The coated sample was placed in the original culture temperature condition for culture, and after about 48 hours, obvious single colonies were formed on the surface of the culture medium. According to the characteristics of the colony size, color, transparency, etc., several single colonies with different characteristics were picked and streaked on a nutrient medium plate for culture. If different characteristic single colonies can still be observed on the streaked plate, the streaking separation is performed again until only single colonies with the same characteristics can be observed on the same plate. In the experiment, one strain DY-7 with high-efficiency degradation performance for phenanthrene and benzopyrene was screened. The purified single colony was picked and cultured in the corresponding liquid nutrient medium to the logarithmic phase, and the bacterial liquid was mixed with sterile glycerol to a concentration of 25% and then was divided into sterile 2-ml cryotubes for long-term storage at -80°C.

[0036] 1.4 Identification of the strain

[0037] The strain DY-7 was identified according to its morphological characteristics and molecular biological properties.

[0038] 1.4.1 Morphological characteristics

[0039] DY-7 is a bacterium isolated from a petroleum-contaminated soil in Ningbo City, Zhejiang Province. After activation, it can form white, round, smooth-surfaced, slightly convex, opaque, non-spore, and non-flagellated colonies with a diameter of 0.5-1.5 mm on a plate prepared from LB medium at 28°C in the presence of oxygen after 36 hours of growth. Figure 1

[0040] 1.4.2 Molecular biological properties

[0041] ​Molecular biological characteristics identification mainly includes sequencing and phylogenetic tree construction. Before sequencing and phylogenetic tree construction, bacterial DNA needs to be extracted (bacterial genomic DNA rapid extraction kit used in the experiment is from Beijing Aidley Biotechnology Co., Ltd.). In order to study the taxonomy of bacteria, it is usually necessary to amplify 16S rRNA gene and construct phylogenetic tree, and the amplified gene is a piece of DNA in the part of rRNA encoding in prokaryotes, which is usually used for detection and identification of bacteria due to its high conservation, specificity and suitable sequence length.

[0042] Polymerase chain reaction (PCR) is mainly used to amplify different gene fragments. Different primers (27F and 1492R) are needed for PCR. The system of PCR amplification reaction is as follows: 10x buffer 2.5 μl, Mg 2+ (25 mmol / l) 1.5 μl, dNTP (25 mmol / l) 0.3 μl, forward primer (10 mmol / l) 0.5 μl, reverse primer (10 mmol / l) 0.5 μl, Taq enzyme: 0.25 μl, DNA group template 0.1 μl, deionized water 19.35 μl. The PCR amplification reaction conditions are as follows: denaturation at 95°C, annealing at 55°C, and extension at 72°C, which is cycled for 30 times, extension at 72°C for 10 min, and preservation at 4°C after the end of PCR reaction. After amplifying the required gene, 0.75-1% agarose is added and nucleic acid staining agent GelRed is prepared into a gel block. The PCR product and DNA marker containing various length fragments are added to the gel block and placed in an electrophoresis instrument. TBE (Tris borate) buffer is loaded into the electrophoresis instrument, and the electrophoresis instrument is worked at a certain voltage for 20 min, then taken out and placed under a 300 nm ultraviolet lamp for observation to determine whether the PCR product amplification reaction is successful. Then the successfully amplified PCR product is sent to Huada Gene Technology Co., Ltd. for sequencing, and the sequencing primer is the same as the amplification primer.

[0043] The sequencing of bacterial 16s rRNA gene sequence was uploaded to EzTaxon-e (http: / / eztaxon-e.ezbiocloud.net / ), which compared the submitted sequence with the 16S rRNA gene sequence of the typical strain of the recognized species to obtain the similarity information between the sequences. According to the results of sequence alignment analysis, the corresponding typical strain was selected as the model strain of the isolated strain in the experiment, and the 16S rRNA gene sequence of the model strain was obtained, and the phylogenetic analysis was constructed to prove that the model strain and the isolated strain had differences, so as to identify the isolated strain. The phylogenetic tree was constructed by MEGA 5.05 program, usually using the neighbor-joining method, the minimum evolution method and the maximum parsimony method to construct the phylogenetic tree, among which the most commonly used was the neighbor-joining method, and the self-exhibition value was usually set to repeat 1000 times.

[0044] A 1404bp 16S rRNA gene sequence was obtained by PCR and gene sequencing. By 16S rRNA gene alignment, it was found that the strain had a gene similarity of 99.94% with Achromobacter deleyi strain LMG 3458 (GenBank accession number NR 152014.1). From the above results, it can be concluded that the bacteria DY-7 isolated in the experiment is Achromobacter deleyi.

[0045] The 16S rRNA gene sequence of DY-7 and the 16S rRNA gene sequence with high similarity were used to make a phylogenetic tree, so as to obtain the homology results between the 16S rRNA gene of DY-7 and the 16S rRNA gene with high similarity. The phylogenetic tree constructed by the neighbor-joining method is shown in Figure 2 At present, there are few reports on the application of this strain in the field of environment. Therefore, it is of great theoretical and practical significance to obtain high-efficiency phenanthrene and benzopyrene degrading bacteria for the treatment and deep repair of soil contaminated with phenanthrene and benzopyrene and PAHs pollution.

[0046] The 16S rRNA gene sequence of DY-7 is shown in SEQ ID NO. 1, which is specifically as follows:

[0047]

[0048] The isolated strain DY-7 is named Achromobacter deleyi DY-7, which is preserved in Guangdong Microbial Culture Collection Center (GDMCC) on July 3, 2025, at address: 59, 5th Floor, Building 100, Xianlie Middle Road, Guangzhou, Guangdong, China, Postcode: 510070, with preservation number: GDMCC No: 66627.

[0049] Example 2 Growth conditions of Achromobacter deleyi DY-7

[0050] Determination of growth temperature:

[0051] Prepare the liquid nutrient medium required for the growth of the strain (Example 1), and sterilize it with a sterilization pot after preparation. The activated strain Achromobacter deleyi DY-7 is inoculated into the medium (experimental group), and the medium without bacterial inoculation is used as a control (control group). The medium is cultured at different temperatures for 12 h. The control group and each temperature corresponding experimental group have three repeats, and the growth of bacteria is observed. When the results are difficult to distinguish with the naked eye, the visible-ultraviolet spectrophotometer is used to measure the absorbance of the medium at wavelength λ = 600 nm, and finally the growth temperature and the optimum growth temperature range of the new bacteria are obtained. The test temperature is as follows: 13℃, 18℃, 23℃, 28℃, 33℃, 38℃.

[0052] Determination of growth pH:

[0053] Prepare the liquid nutrient medium required for the growth of the strain (Example 1), and adjust the pH of the culture solution with the following buffer system: pH 4.0-5.0, 0.1 mol / l sodium citrate and 0.1 mol / l citric acid; pH 6.0-8.0, 0.1 mol / l NaOH and 0.1 mol / l KH2PO4; pH 9.0-10.0, 0.1 mol / l NaHCO3 and 0.1 mol / l Na2CO3; pH 11.0, 0.1 mol / l NaOH and 0.05 mol / l Na2HPO4. The strain Achromobacter deleyi DY-7 is inoculated into the medium, and each pH is repeated three times. The medium without bacterial inoculation is used as a control. The medium is placed in the optimum temperature for the growth of the new bacteria and cultured for 12 h. The growth of bacteria is observed. When the results are difficult to distinguish with the naked eye, the visible-ultraviolet spectrophotometer is used to measure the absorbance of the medium at wavelength λ = 600 nm, and finally the growth pH and the optimum growth pH range of the new bacteria are obtained. The tested pH is as follows: 4.0, 5.0, 6.0, 7.0, 8.0, 9.0.

[0054] Salt concentration tolerance:

[0055] The liquid nutrient medium required for the growth of the strain (Example 1) was prepared and the salt concentration of the medium was adjusted. The activated strain Achromobacter deleyi DY-7 was inoculated into the sterilized medium, three replicates were made for each salt concentration, and the medium without inoculation was used as a control. The medium was incubated for 12 h under the optimal salinity for the growth of the strain, and the growth of the bacteria was observed. When it was difficult to distinguish with the naked eye, the visible-ultraviolet spectrophotometer was used to measure the absorbance of the medium at a wavelength of λ = 600 nm, and the salt concentration range that the strain could tolerate was determined. The salt concentrations tested were as follows: mass fraction 0%, 2%, 4%, 6%, 8%, and 10%.

[0056] The results of the growth condition determination are shown in Table 1. Figure 3 As shown in Table 1, in the nutrient broth medium, DY-7 could grow at a temperature of 13-38°C, and the optimal growth temperature was the enrichment temperature of the strain, which was 28°C. The strain could grow at a pH of 5.0-9.0, and the optimal growth pH was 7.0. The salt tolerance of the strain was weak, and it could grow at a salt concentration of 0% to 6%, but it grew best without the addition of sodium chloride, and its growth was greatly inhibited when the salt concentration increased. According to the above experimental results, the optimal growth conditions for the strain were determined as follows: temperature 28°C, pH 7.0, and no addition of NaCl. The growth and degradation experiments of strain DY-7 in high-concentration phenanthrene and benzopyrene were all carried out under these conditions.

[0057] Example 3 Degradation of phenanthrene and benzopyrene by Achromobacter deleyi DY-7

[0058] The activated strain Achromobacter deleyi DY-7 was inoculated into the inorganic salt culture solution (see Reference Example 1, but without the addition of NaCl) containing an initial phenanthrene concentration of 50 mg / L or an initial benzopyrene concentration of 25 mg / L at an inoculation volume of 10%, and was cultured in the dark for 7 days at a temperature of 28°C and a pH of 7.0. The treatment without the addition of strain DY-7 was used as a control.

[0059] The treated samples were taken for chemical analysis, and the specific steps were as follows: (1) sample pretreatment: each culture sample was added into dichloromethane extraction, and 5 μL of recovery indicator with a concentration of 200 mg / L was added (for phenanthrene and benzopyrene treated samples, deuterated polycyclic aromatic hydrocarbons were added), and after shaking, it was transferred into a separatory funnel and stood. After layering, the organic phase was collected, the lower liquid was put back into the flask and was repeatedly extracted with an equal volume of dichloromethane, the extraction liquid was combined and was transferred into a flat-bottomed flask containing a proper amount of activated copper sheet for rotary evaporation, and was concentrated to about 2 mL, a small amount of n-hexane (about 5 mL) was added, and rotary evaporation was repeated three times until the organic solvent was replaced with n-hexane. The concentrated liquid after replacement was purified by a glass packed column (about 9 mm in diameter). The column packing was 3 cm of 3% deactivated neutral alumina, 3 cm of 3% deactivated silica gel and 1 cm of anhydrous sodium sulfate from bottom to top. The column was activated with a proper amount of n-hexane, 15 mL of n-hexane / dichloromethane (1:1 by volume) mixed reagent was used to elute the packed column, and about 15 mL of eluent was collected in a brown reagent bottle, and was concentrated to about 0.5 mL by nitrogen blowing, and finally was transferred into a 1.5 mL cell bottle and was stored in the freezer. 5 μL of internal standard hexamethylbenzene with a concentration of 200 mg / L was added before instrument analysis. (2) Instrument analysis: the content of PAHs in each treated sample was determined by using Agilent 7890 gas chromatograph-5975 mass spectrometer. The chromatographic column used was Agilent DB 5-MS capillary chromatographic column (30 m in length, 0.25 mm in diameter and 0.25 μm in film thickness). The obtained data was processed by using Agilent chromatographic workstation, and the quantification of phenanthrene and benzopyrene was performed by using 6-point calibration curve and internal standard method. The concentration of microbial cells was determined by using photoelectric turbidimetry, and OD was used to represent the optical density value of the determined bacterial liquid sample at a wavelength of 600 nm.

[0060] According to the determination and analysis by GC-MS, it was found that the strain DY-7 could degrade phenanthrene and benzopyrene ( Figure 4 ), and after being cultured in inorganic salt culture solution containing 50 mg / L of phenanthrene and 25 mg / L of benzopyrene for 7 days, the degradation rates could reach more than 70%, and were 83.1% (phenanthrene) and 70.8% (benzopyrene) respectively. It was proved that the strain DY-7 was a strain which could degrade phenanthrene and benzopyrene and had strong tolerance to the two compounds.

[0061] Conclusion

[0062] 1) The present application enriches and separates a phenanthrene and benzopyrene degrading strain DY-7 which can grow with phenanthrene and benzopyrene as carbon sources from a petroleum contaminated soil in Ningbo City, Zhejiang Province.

[0063] 2) The strain DY-7 can form white, round, smooth surface, slightly convex, opaque, non-spore, non-flagellum colony with diameter of 0.5-1.5mm. According to the analysis of molecular biology, the bacteria DY-7 isolated in this experiment is Achromobacter deleyi strain, and its phylogenetic tree is drawn. At present, the application of the strain is rarely reported, especially the research of degrading phenanthrene and benzopyrene has not been reported.

[0064] 3) The optimal growth conditions of the strain DY-7 are temperature 28℃, pH 7.0, and no NaCl added. DY-7 can use phenanthrene and benzopyrene as carbon source. After 7 days of culture in inorganic salt medium with initial concentration of phenanthrene 50mg / L and benzopyrene 25mg / L, the degradation rate of phenanthrene or benzopyrene can reach more than 70%. In summary, DY-7 is a strain that can degrade phenanthrene and benzopyrene and has strong tolerance to phenanthrene and benzopyrene, and has strong adaptability to polycyclic aromatic hydrocarbons, which has good application potential in bioremediation.

Claims

1. Achromobacterdeleyi DY-7, accession number: GDMCC No: 66627.

2. The use of Achromobacterdeleyi DY-7 as described in claim 1 in the degradation of phenanthrene and / or benzo[a]pyrene.

3. The application according to claim 2, characterized in that, The degradation of phenanthrene and / or benzo[a]pyrene refers to the degradation of phenanthrene and / or benzo[a]pyrene in petroleum-contaminated soil.

4. The application according to claim 2, characterized in that, The Achromobacterdeleyi DY-7 is used to degrade phenanthrene and / or benzo[a]pyrene in environments contaminated with phenanthrene and / or benzo[a]pyrene.

5. A phenanthrene and / or benzo[a]pyrene degrading bacterial agent, characterized in that, It contains Achromobacterdeleyi DY-7 as described in claim 1 as an active ingredient.

6. A method for degrading phenanthrene and / or benzo[a]pyrene, characterized in that, The method involves spraying the Achromobacterdeleyi DY-7 according to claim 1 into an environment containing phenanthrene and / or benzo[a]pyrene to degrade phenanthrene and / or benzo[a]pyrene.

7. The method according to claim 6, characterized in that, The method involves spraying Achromobacterdeleyi DY-7 into an environment contaminated with phenanthrene and / or benzo[a]pyrene to degrade phenanthrene and / or benzo[a]pyrene.

8. The method according to claim 6, characterized in that, The method involves applying Achromobacterdeleyi DY-7 to petroleum-contaminated soil to degrade phenanthrene and / or benzo[a]pyrene.

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