Bosea thioxidans DY-5 strain and application thereof in degradation of phenanthrene and benzopyrene

Abstract

The invention discloses a Bosea thioxidans DY-5 strain and an application of the Bosea thioxidans DY-5 strain in degradation of phenanthrene and benzopyrene. The strain Bosea thioxidans DY-5 is obtained through domestication, separation and identification from petroleum-contaminated soil of Ningbo, phenanthrene and benzopyrene can be used as carbon sources, and after the strain is cultured in an inorganic salt culture solution with the initial concentration of phenanthrene and benzopyrene being 50 mg.L <-1 > and 25 mg.L <-1 > respectively for 7 days, the degradation rate of phenanthrene and benzopyrene can reach 75% or above. Therefore, the strain has good application potential in the aspect of bioremediation of polycyclic aromatic hydrocarbon.

Description

Technical Field

[0001] This invention belongs to the field of organic pollutant degradation, specifically involving a strain of Bosea thiooxidans DY-5 and its application in the degradation of phenanthrene and benzo[a]pyrene. Background Technology

[0002] The rapid pace of modern industrialization has been accompanied by increasingly severe industrial pollution, particularly the pollution of soil environments by persistent organic pollutants such as polycyclic aromatic hydrocarbons (PAHs). PAHs possess potential carcinogenic, teratogenic, mutagenic, and bioaccumulative properties, posing significant threats to the ecological environment and human health, and have attracted widespread attention. High concentrations of PAHs are among the main pollutants found in important organic pollution sites such as chemical industrial parks and surrounding soils, oilfields, mining areas, and wastewater irrigation areas. Among these, phenanthrene (Phe) is a tricyclic aromatic hydrocarbon, and benzo[a]pyrene (BaP) is a polycyclic aromatic hydrocarbon containing a benzene ring; both possess the aforementioned typical hazardous characteristics and may cause ecological damage and human health risks.

[0003] The natural degradation of toxic and hazardous organic pollutants (PAHs) in the environment mainly relies on the metabolic processes of related microorganisms. Bioremediation technology, by enhancing this natural process, exhibits advantages such as low cost, high efficiency, and no secondary pollution, making it the most promising technical means for PAH remediation. Currently, there are relatively few reported strains that degrade phenanthrene and benzo[a]pyrene, mainly including strains from the genera *Bosea*, *Pseudomonas*, and *Rhodococcus*. Most microorganisms in the environment are unculturable, and many microorganisms, especially those with specific functions, cannot be isolated through pure culture. Therefore, screening for strains that can effectively degrade high concentrations of phenanthrene and benzo[a]pyrene has significant application value and practical significance. This experiment used a concentration of 50 mg·L⁻¹. -1 and 25 mg·L -1 The study used phenanthrene and benzo[a]pyrene as substrates for the degradation of polycyclic aromatic hydrocarbons (PAHs) to provide data support for the biological treatment of PAHs. Summary of the Invention

[0004] The first objective of this invention is to provide a strain of Boseathiooxidans DY-5 with the ability to degrade phenanthrene and / or benzo[a]pyrene, which was deposited on July 3, 2025 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC No: 66626.

[0005] The strain of this invention belongs to *Bosea thiooxidans*, a species that can be isolated from various terrestrial and aquatic habitats. Currently, there are relatively few reports on the degradation of pollutants by *Bosea thiooxidans*, and no studies on the degradation of phenanthrene and benzo[a]pyrene by *Bosea thiooxidans* have been reported domestically or internationally. This invention domesticated and isolated a strain DY-5 from petroleum-contaminated soil in Ningbo, which uses high concentrations of phenanthrene and benzo[a]pyrene as carbon sources. The strain was identified, and its growth characteristics and degradation characteristics of phenanthrene and benzo[a]pyrene were studied, providing a reference for the bioremediation of PAH-contaminated environments.

[0006] A second objective of this invention is to provide the application of the aforementioned Bosea thiooxidans DY-5 in the degradation of phenanthrene and / or benzo[a]pyrene.

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

[0008] Preferably, Bosea thiooxidans DY-5 is used in environments contaminated with phenanthrene and / or benzo[a]pyrene to degrade phenanthrene and / or benzo[a]pyrene.

[0009] A third objective of this invention is to provide a phenanthrene and / or benzo[a]pyrene degrading bacterial agent comprising the aforementioned Boseathiooxidans DY-5 as an active ingredient.

[0010] A fourth objective of this invention is to provide a method for degrading phenanthrene and / or benzo[a]pyrene by sprinkling the aforementioned Boseathiooxidans DY-5 in an environment containing phenanthrene and / or benzo[a]pyrene to degrade the phenanthrene and / or benzo[a]pyrene.

[0011] Preferably, the Bosea thiooxidans DY-5 is sprayed in an environment contaminated with phenanthrene and / or benzo[a]pyrene to degrade phenanthrene and / or benzo[a]pyrene.

[0012] Preferably, the Bosea thiooxidans DY-5 is applied to petroleum-contaminated soil to degrade phenanthrene and / or benzo[a]pyrene.

[0013] This invention describes the domestication and isolation of a biodegrading strain, DY-5, from petroleum-contaminated soil in Ningbo, which utilizes phenanthrene and benzo[a]pyrene as carbon sources. Based on morphological analysis, 16S rDNA sequencing, and phylogenetic analysis, the strain was identified as *Boseathiooxidans* DY-5. The optimal growth conditions for this strain were: temperature 28℃, pH 7, and no sodium chloride added. 16S rDNA sequencing analysis showed that the strain most closely related to DY-5 was *Bosea thiooxidans* strain BI-42 (99.48%). DY-5 can utilize phenanthrene and benzo[a]pyrene as carbon sources, with initial concentrations of phenanthrene and benzo[a]pyrene of 50 mg·L⁻¹. -1 and 25 mg·L -1 After culturing in an inorganic salt medium for 7 days, the degradation rates of phenanthrene and benzo[a]pyrene both reached over 75%. Therefore, this strain has good application potential in the bioremediation of polycyclic aromatic hydrocarbons.

[0014] Bosea thiooxidans DY-5 was deposited on July 3, 2025 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC No: 66626. Attached Figure Description

[0015] Figure 1 These are the front and back sides of strain DY-5 grown on LB solid medium for 72 hours in Example 1.

[0016] Figure 2 This is a phylogenetic relationship based on the 16S rRNA gene sequence of strain DY-5 and its related bacteria in Example 1. The construction method is neighbor-joining, the expansion value is set to 1000 replicates, and the scale bar 0.5 represents the substitution rate of each nucleotide.

[0017] Figure 3 The strain DY-5 in Example 2 was grown under different culture temperatures, salinities, and pH conditions.

[0018] Figure 4 This refers to the degradation efficiency of strain DY-5 in Example 3 in an inorganic salt medium containing high concentrations of phenanthrene and benzo[a]pyrene (initial concentrations of phenanthrene and benzo[a]pyrene were 50 mg·L⁻¹). -1 and 25 mg·L -1 ). Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention 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 Bosea thiooxidans DY-5

[0021] 1. Materials and Methods

[0022] 1.1 Sample Source

[0023] Soil samples were collected from near an oil-polluted plant in Ningbo. The samples were acclimatized for a long period of time using high concentrations of phenanthrene and benzo[a]pyrene as carbon sources. Through multiple screenings and purifications, highly efficient phenanthrene and benzo[a]pyrene degrading bacteria were obtained.

[0024] 1.2 Culture medium

[0025] 1.2.1 Inorganic Salt Culture Medium

[0026] Inorganic salt culture medium is used for the enrichment culture of microorganisms in samples and for the degradation experiments of phenanthrene and benzo[a]pyrene under pure bacterial conditions. The formulation of this culture medium is shown in Table 1 (containing phenanthrene and benzo[a]pyrene solution). The preparation method is to add each component to the solvent water, mix evenly, and sterilize.

[0027] Table 1. Inorganic Salt Culture Medium Formulation

[0028]

[0029]

[0030] 1.2.2 Nutrient Culture Medium

[0031] Nutrient media are used for the isolation, purification, preservation, and activation of bacteria and other routine microbial cultures. The types and components of the liquid nutrient media used in this experiment are shown in Table 2. If a solid medium is required, simply add 1.5-2% agar powder to the existing medium formula. Unless otherwise specified, the pH of the medium should be adjusted to 7. The nutrient media is prepared by adding all components to a solvent of water, mixing thoroughly, and then sterilizing.

[0032] Table 2. Composition of Luria-Bertani medium (LB)

[0033]

[0034] 1.3 Domestication, screening and isolation of strains

[0035] The collected contaminated soil was added to the enrichment medium (the above-mentioned inorganic salt medium) at a concentration of 50 mg·L⁻¹. -1 25 mg·L -1 Phenanthrene and benzo[a]pyrene were used as substrates for degradation and cultured in a 28°C incubator in the dark with shaking. The strain was acclimatized using an inorganic salt medium with phenanthrene and benzo[a]pyrene as carbon sources, with each acclimatization cycle lasting 7 days. A 10% inoculum was then transferred to a fresh enrichment medium with the same culture system, and the enrichment process was repeated three times.

[0036] The fourth-generation enriched culture samples obtained above were separated by dilution plating using a nutrient medium. The plated samples were incubated at the original culture temperature. After approximately 48 hours, distinct single colonies formed on the surface of the medium. Several single colonies with different characteristics were selected based on their morphology, size, color, and transparency, and then streaked onto nutrient medium plates for purification. If single colonies with different characteristics were still observed on the purified plates, they were streaked again until only single colonies with the same characteristics were observed on the same plate. A strain, DY-5, exhibiting high degradation performance against both phenanthrene and benzo[a]pyrene, was screened in the experiment. The purified single colonies were picked and cultured in the appropriate liquid nutrient medium until the logarithmic growth phase. The bacterial culture was then mixed with sterile glycerol and aliquoted into sterile 2ml cryovials (glycerol concentration 15%), and stored at -80℃ for long-term preservation.

[0037] 1.4 Identification of strains

[0038] The strain DY-5 was identified based on its morphological characteristics and molecular biological properties.

[0039] 1.4.1 Morphological characteristics

[0040] DY-5 is a bacterium isolated from petroleum-contaminated soil in Ningbo. After activation, under aerobic conditions at 28°C, it can form white, smooth, slightly convex, opaque, non-spore-forming, and non-motile colonies with a diameter of about 1.8 mm after 72 hours of growth on LB agar plates. Figure 1 ).

[0041] 1.4.2 Molecular biological characteristics

[0042] Molecular biological characterization mainly involves sequencing and phylogenetic tree construction. Before sequencing and constructing the phylogenetic tree, bacterial DNA needs to be extracted (the rapid bacterial genomic DNA extraction kit used in the experiment was from Beijing Adley Biotechnology Co., Ltd.). For bacterial taxonomic studies, it is usually necessary to amplify the 16S rRNA gene and construct a phylogenetic tree. The amplified gene is a segment of DNA encoding rRNA in prokaryotes, and due to its high conservation, specificity, and suitable sequence length, it is commonly used for bacterial detection and identification.

[0043] Polymerase chain reaction (PCR) is mainly used to amplify different gene fragments. PCR requires different primers (27F and 1492R). The PCR amplification reaction system consists of: 10× buffer 2.5 μl, Mg 2+ 1.5 μl of 25 mmol / L primer, 0.3 μl of dNTP (25 mmol / L), 0.5 μl of forward primer (10 mmol / L), 0.5 μl of reverse primer (10 mmol / L), 0.25 μl of Taq enzyme, 0.1 μl of DNA template, and 19.35 μl of deionized water. PCR amplification conditions: denaturation at 95℃, annealing at 55℃, and extension at 72℃, repeated 30 times, with a final extension at 72℃ for 10 min. After the PCR reaction, the sample was stored at 4℃. After amplifying the desired gene, a gel block was prepared using 0.75-1% agarose and the nucleic acid staining agent GelRed. The PCR product and DNA markers containing fragments of various lengths were added to the gel block, which was then placed in an electrophoresis apparatus filled with TBE (Tris borate) buffer. The apparatus was run at a specific voltage for 20 minutes, then removed and observed under a 300nm UV lamp to confirm successful PCR amplification. The successfully amplified PCR product was then sent to BGI Genomics Co., Ltd. for sequencing, using the same primers as the amplification primers.

[0044] The bacterial 16S rRNA gene sequence obtained from sequencing was uploaded to EzTaxon-e (http: / / eztaxon-e.ezbiocloud.net / ). This website compares the submitted sequence with the 16S rRNA gene sequences of typical strains of recognized species to obtain sequence similarity information. Based on the sequence alignment results, the corresponding typical strain can be selected as the model strain for this experiment. The 16S rRNA gene sequence of the model strain can also be obtained, and a phylogenetic analysis can be constructed to demonstrate the differences between the model strain and the experimental isolate, thereby identifying the isolated strain. The phylogenetic tree is constructed using the MEGA 5.05 program, typically employing the neighbor-joining method, minimum evolution method, and maximum parsimony method. The neighbor-joining method is the most commonly used, and the bootstrap value is usually set to 1000 iterations.

[0045] A 1363 bp 16S rRNA gene sequence was obtained through PCR and gene sequencing. Comparison of the 16S rRNA gene revealed a 99.48% genetic similarity between this strain and *Bosea thiooxidans* strain BI-42 (GenBank accession number NR114668.1). Based on these results, the bacteria DY-5 isolated in this experiment can be identified as *Bosea thiooxidans*.

[0046] A phylogenetic tree was constructed using the 16S rRNA gene sequence of DY-5 and 16S rRNA gene sequences with high similarity to it, thereby obtaining the homology results between the 16S rRNA gene of DY-5 and 16S rRNA genes with high similarity. The phylogenetic tree constructed using the neighbor-joining method is shown below. Figure 2 Currently, there are few reports on the application of this strain in the environmental field. Therefore, obtaining highly efficient phenanthrene and benzo[a]pyrene degrading bacteria is of significant theoretical and practical importance for the treatment and deep remediation of PAHs contaminated soils containing phenanthrene and benzo[a]pyrene.

[0047] The 16S rRNA gene sequence of DY-5 is shown in SEQ ID NO.1, specifically as follows:

[0048] GTCGCCTGCCTCCTTGCGGTTAGCGCGACGCCTTCGGGTAAACCCAACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATGCTGATCCACGATTACTAGCGATTCCACCTTCATGCACTCGAGTTGCAGAGTGCAATCTGAACTGAGACGGCTTTTTGGGATTAGCTCGAGGTCGCCCTTTCGCTGCCCATTGTCACCGCCATTGTAGCACGTGTGTAGCCCAGCCTGTAAGGGCCATGAGGACTTGACGTCATCCCCACCTTCCTCGCGGCTTATCACCGGCAGTCCCCCTAGAGTTCCCAACTGAATGATGGCAACTAGGGGCGAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTGTTCCGGCCAGCCGAACTGAAGAAAGGCATCTCTGCCGATCAAACCGGACATGTCAAAAGCTGGTAAGGTTCTGCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTTAATCTTGCGACCGTACTCCCCAGGCGGAATGCTTAAAGCGTTAGCTGCGCCACTGAAGAGCAAGCTCCCCAACGGCTGGCATTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTCAGCGTCAGTTTCGGACCAGTTGGCCGCCTTCGCCACTGGTGTTCTTGCGAATATCTACGAAT TTCACCTCTACACTCGCAGTTCCACCAACCTCTTCCGAACTCAAGACTCCCAGTATCGAAGGCAATTCCAGGGTTGAGCCCTGGGCTTTCACCCCCGACTTAAGAGTCCGCCTACGCGCCCTTTACGCCCAGTGATTCCGAGCAACGCTAGCCCCCTTCGTATTACCGCGGCTGCTGGCACGAAGTTAGCCGGGGCT TATTCTTCCGGTACAGTCATTATCTTCCCGGACAAAAGAGCTTTACAACCCTAAGGCCTTCATCACTCACGCGGCATGGCTGGATCAGGCTTGCGCCCATTGTCCAATATTCCCCACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCTGATCATCCTCTCAGACCAGCTACTGATCGTC GCCTTGGTGAGCCATTACCTCACCAACTAGCTAATCAGACGCGGGCCGATCTATCGGCGATAAATCTTTCCCCGAAGGGCGTATCCGGTATTAGTCCAAGTTTCCCTGAATTATTCCGAACCGAAAGGTACGTTCCCACGTGTTACTCACCCGTCTGCCACTAGCACCGAAGTGCCCGTTCGACTTGCAGGTAAGC

[0049] The isolated strain DY-5 was named *Bosea thiooxidans* DY-5 and deposited on July 3, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC No. 66626.

[0050] Example 2: Growth conditions of Bosea thiooxidans DY-5

[0051] Measurement of growth temperature:

[0052] Prepare the liquid nutrient medium required for the growth of the bacterial strain (Example 1), and sterilize it in an autoclave after preparation. Inoculate the activated strain *Bosea thiooxidans* DY-5 into the medium (experimental group), and use uninoculated medium as a control (control group). Incubate the medium at different temperatures for 12 hours. There are three replicates for both the control group and the experimental group at each temperature. Observe the bacterial growth. When results are difficult to distinguish with the naked eye, measure the absorbance of the medium at a wavelength of λ = 600 nm using a visible-ultraviolet spectrophotometer. Finally, determine the optimal growth temperature range and the temperature at which the new bacteria can grow. The test temperatures are as follows: 13℃, 18℃, 23℃, 28℃, 33℃, and 38℃.

[0053] Determination of pH for growth:

[0054] Prepare the liquid nutrient medium required for the growth of the bacterial strain (Example 1). Adjust the pH of the culture medium using the following buffer systems: 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. Inoculate the bacteria *Bosea thiooxidans* DY-5 into the culture medium, performing three replicates for each pH. Use uninoculated medium as a control. Incubate the medium at the optimal temperature for new bacterial growth for 12 hours and observe the bacterial growth. When results are difficult to distinguish with the naked eye, measure the absorbance of the medium at a wavelength of λ = 600 nm using a visible-ultraviolet spectrophotometer. Finally, determine the pH at which the new bacteria can grow and the optimal pH range for growth. The pH values ​​tested were as follows: 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0.

[0055] Salt concentration tolerance:

[0056] Prepare the liquid nutrient medium required for the growth of the bacterial strain (Example 1) and adjust the salt concentration of the medium. Inoculate the activated strain *Bosea thiooxidans* DY-5 into the sterilized medium, performing three replicates for each salt concentration. Use uninoculated medium as a control. Incubate the medium under the optimal conditions for new bacterial growth for 12 hours and observe the bacterial growth. When the growth is difficult to distinguish with the naked eye, measure the absorbance of the medium at a wavelength of λ = 600 nm using a visible-ultraviolet spectrophotometer to determine the range of salt concentrations that the new bacteria can tolerate. The tested salt concentrations are as follows: 0%, 1%, 2%, 3%, 4%, and 5% (mass fraction).

[0057] The results of growth condition measurements are as follows Figure 3 As shown, in nutrient broth medium, DY-5 can grow at temperatures ranging from 13 to 38°C, with the optimal growth temperature being the enrichment temperature of 28°C. The bacterium can grow at pH values ​​ranging from 4.0 to 9.0, with the optimal growth pH being 7.0. The bacterium has weak salt tolerance, growing at salt concentrations from 0% to 3%, and exhibits the best growth under salt-free conditions. Based on these experimental results, the optimal growth conditions for the strain were determined to be a temperature of 28°C, pH 7.0, and no sodium chloride addition. Degradation experiments of strain DY-5 in high concentrations of phenanthrene and benzo[a]pyrene were conducted under these conditions.

[0058] Example 3: Degradation experiment of phenanthrene and benzo[a]pyrene in Bosea thiooxidans DY-5

[0059] The activated strain *Bosea thiooxidans* DY-5 was inoculated at a volume ratio of 10% into an inorganic salt culture medium containing an initial phenanthrene concentration of 50 mg / L or an initial benzo[a]pyrene concentration of 25 mg / L (refer to Example 1, but without the addition of NaCl). The medium was incubated for 7 days in the dark with shaking at 28°C and pH 7.0. The treatment without the addition of strain DY-5 served as the control group.

[0060] The samples were used for chemical analysis. The specific steps are as follows: (1) Sample pretreatment: Dichloromethane was added to each culture sample for extraction. At the same time, 5 μL of recovery indicator with a concentration of 200 mg / L was added (for phenanthrene and benzo[a]pyrene treated samples, deuterated polycyclic aromatic hydrocarbons were added). After thorough shaking, the samples were transferred to a separatory funnel and allowed to stand. After separation, the organic phase was collected. The lower liquid was returned to the shake flask and extracted again with an equal volume of dichloromethane. The extracts were combined and transferred to a flat-bottomed flask containing an appropriate amount of activated copper sheet for rotary evaporation. The solution was concentrated to about 2 mL. A small amount of n-hexane (about 5 mL) was added and the solution was rotary evaporated to 2 mL. The washing was repeated three times to replace the organic solvent with n-hexane. The concentrated solution after replacement was purified with a glass packed column (about 9 mm in diameter). The column packing from bottom to top consisted of 3 cm of 3% deactivated neutral alumina, 3 cm of 3% deactivated silica gel, and 1 cm of anhydrous sodium sulfate. The column was activated with an appropriate amount of n-hexane, and the packed column was rinsed with a 15 mL mixture of n-hexane / dichloromethane (volume ratio 1:1). Approximately 15 mL of eluent was collected in a brown reagent bottle and concentrated to approximately 0.5 mL by nitrogen blowing. The eluent was then transferred to a 1.5 mL cell culture flask and frozen. Before analysis, 5 μL of hexamethylbenzene (200 mg / L) was added as an internal standard. (2) Instrumental analysis: The PAH content in each treated sample was determined using an Agilent 7890 gas chromatograph coupled with a 5975 mass spectrometer. The chromatographic column used was an Agilent DB 5-MS capillary column (30 m long, 0.25 mm inner diameter, 0.25 μm membrane thickness). The obtained data were processed using an Agilent chromatography workstation. Quantification of phenanthrene and benzo[a]pyrene was performed using a 6-point calibration curve and the internal standard method. The concentration of microbial cells was determined by photoelectric turbidimetry, expressed as OD, which is the optical density value of ultraviolet light transmitted through the measured bacterial culture sample at a wavelength of 600 nm.

[0061] GC-MS analysis showed that strain DY-5 could degrade phenanthrene and benzo[a]pyrene, and the degradation rate reached over 75% after 7 days of culture in inorganic salt solutions containing 50 mg / L and 25 mg / L of phenanthrene and benzo[a]pyrene. Figure 4 This indicates that strain DY-5 is a strain that can degrade both phenanthrene and benzo[a]pyrene, and has a strong tolerance to both compounds.

[0062] in conclusion

[0063] 1) This invention obtained a phenanthrene and benzo[a]pyrene degrading bacterium, DY-5, from petroleum-contaminated soil in Ningbo, which can grow using phenanthrene and benzo[a]pyrene as carbon sources, respectively.

[0064] 2) This strain DY-5 can form colonies that are approximately 1.8 mm in diameter, white, smooth, slightly convex, opaque, non-spore-forming, and non-flagellated. Based on molecular biological analysis, the bacteria DY-5 isolated in this experiment was identified as *Bosea thiooxidans*, and its phylogenetic tree was constructed. Currently, there are few reports on the applications of this strain, especially studies on its degradation of phenanthrene and benzo[a]pyrene.

[0065] 3) The optimal growth conditions for strain DY-5 are a temperature of 28℃, pH 7.0, and no NaCl addition. DY-5 can utilize phenanthrene and benzo[a]pyrene as carbon sources, with initial concentrations of phenanthrene and benzo[a]pyrene of 50 mg·L⁻¹. -1 and 25 mg·L -1 After culturing in an inorganic salt medium for 7 days, the degradation rates of phenanthrene and benzo[a]pyrene both reached over 75%. In summary, DY-5 is a strain capable of degrading phenanthrene and benzo[a]pyrene and exhibiting strong tolerance to these substances. It also demonstrates strong adaptability to polycyclic aromatic hydrocarbons and shows good application potential in bioremediation.

Claims

1. Bosea thiooxidans DY-5, accession number: GDMCC No: 66626.

2. The use of the Bosea thiooxidans DY-5 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 Bosea thiooxidans DY-5 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 Boseathiooxidans DY-5 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 Boseathiooxidans DY-5 as described in 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 Bosea thiooxidans DY-5 into environments 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 Bosea thiooxidans DY-5 to petroleum-contaminated soil to degrade phenanthrene and / or benzo[a]pyrene.

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