Isolation and identification of a benzo[a]pyrene-degrading fungus Arthrinium acutiapicum DL-5 from petroleum-contaminated soil and preparation and application of its microbial inoculum

Abstract

The application discloses a benzo[a]pyrene-degrading fungus Arthrinium acutiapicum DL-5 in petroleum contaminated soil, a microbial inoculum of the Arthrinium acutiapicum DL-5 and application of the Arthrinium acutiapicum DL-5. ‑1 The benzo[a]pyrene-degrading rate of the pure Arthrinium acutiapicum DL-5 to benzo[a]pyrene can reach more than 75% after the Arthrinium acutiapicum DL-5 is cultured in an inorganic salt culture solution with an initial concentration of 25 mg / L of benzo[a]pyrene for 7 days. After the Arthrinium acutiapicum DL-5 is made into a microbial inoculum, the benzo[a]pyrene-degrading rate of the Arthrinium acutiapicum DL-5 to benzo[a]pyrene can be promoted to more than 80%, and therefore, the Arthrinium acutiapicum DL-5 and the microbial inoculum have good application potential in the bioremediation of polycyclic aromatic hydrocarbons.

Description

Technical Field

[0001] This invention belongs to the field of organic pollutant degradation, specifically involving the isolation, identification, preparation and application of Arthrinium acutiapicum DL-5, a benzo[a]pyrene-degrading fungus from petroleum-contaminated soil. Background Technology

[0002] With the rapid development of modern industrial processes, the amount of pollutants generated in industrial operations is increasing, especially persistent organic pollutants such as polycyclic aromatic hydrocarbons (PAHs). PAHs are ubiquitous in the environment and continue to accumulate. Due to their potential carcinogenic, teratogenic, mutagenic, and bioaccumulative properties, PAHs pose a significant threat to the ecological environment and human health, thus PAH pollution has received widespread attention. Human activities such as mining and agriculture, as well as high background levels in soil, often lead to severe exceedances of PAHs in the environment, causing pollution. Furthermore, high concentrations of PAHs are often found among the main pollutants in important organic pollution sites such as chemical industrial parks and surrounding soils, oilfields, mining areas, and wastewater irrigation areas. Benzo[a]pyrene, a pentacyclic aromatic hydrocarbon, is closely related to the carcinogenicity of PAHs.

[0003] The natural degradation of toxic and harmful organic pollutants in the environment mainly depends on the metabolic activity of related microorganisms. Bioremediation technology has advantages such as low cost, high efficiency, and no secondary pollution, making it the most promising remediation method for PAH pollution. Currently, there are relatively few reported benzo[a]pyrene-degrading fungal strains, mainly including *Trichoderma*, *Scedosporium*, *Fusarium*, *Penicillium*, and *Aspergillus*. Since most microorganisms in the environment are unculturable, many microorganisms, especially those with specific functions, cannot be isolated through pure culture. Therefore, screening for strains that can effectively degrade high concentrations of benzo[a]pyrene has significant application value and practical significance. This experiment used a concentration of 25 mg·L⁻¹. -1 The aim was to use benzo[a]pyrene as a substrate for degradation by the strain, in order to provide data support for the bioprocessing of polycyclic aromatic hydrocarbons. Summary of the Invention

[0004] The first objective of this invention is to provide a strain Arthriniumacutiapicum DL-5 with the ability to degrade benzo[a]pyrene, which was deposited on February 26, 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: 65989.

[0005] This study reports a strain belonging to *Arthrinium acutiapicum*, which can be isolated from various terrestrial and aquatic habitats. Currently, there are relatively few reports on the degradation of pollutants by *Arthrinium acutiapicum*, and no studies on the degradation of benzo[a]pyrene by *Arthrinium acutiapicum* have been reported domestically or internationally. In this study, a strain DL-5, which uses high concentrations of benzo[a]pyrene as a carbon source, was domesticated and isolated from an oil-contaminated site in Ningbo. The strain was identified, its growth characteristics were studied, and a bacterial agent was prepared to investigate its degradation characteristics of benzo[a]pyrene, 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 Arthrinium acutiapicum DL-5 in the degradation of benzo[a]pyrene.

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

[0008] Preferably, Arthrinium acutiapicum DL-5 is used to degrade benzo[a]pyrene in an environment contaminated with benzo[a]pyrene.

[0009] A third objective of this invention is to provide a benzo[a]pyrene degrading bacterial agent comprising Arthriniumacutiapicum DL-5 as the active ingredient.

[0010] Preferably, the preparation method of the benzo[a]pyrene degrading bacterial agent is as follows:

[0011] 1) After heating corn and water to a mass ratio of 1:5 to make a paste, add sawdust, wheat bran and sodium lignosulfonate at a mass ratio of 150:100:10:1, knead into a ball, put the ball-shaped culture medium mixture into a pelletizing machine to make spherical culture medium, sterilize and dry for later use.

[0012] 2) Prepare a bacterial suspension from the cultured Arthrinium acutiapicum DL-5;

[0013] 3) Add the bacterial solution from 2) to a 3% sodium alginate solution at a mass ratio of 1:10. Mix the spherical culture medium from 1) thoroughly with this solution. After completion, add a 4% sterile calcium chloride solution and harden the mixture to obtain spherical encapsulated fungi.

[0014] 4) Place the sealed fungal pellets into a sterile culture bag and incubate at 28°C for 3-7 days. Once the surface is covered with white mycelium, it becomes a benzo[a]pyrene degrading agent.

[0015] Preferably, in step 2), the bacterial solution is a bacterial solution with a mycelial content of 10 g / L.

[0016] The fourth object of the present invention is to provide a method for degrading benzo[a]pyrene, which involves spraying the above-mentioned Arthriniumacutiapicum DL-5 into an environment containing benzo[a]pyrene to degrade the benzo[a]pyrene.

[0017] Preferably, Arthrinium acutiapicum DL-5 is applied to an environment contaminated with benzo[a]pyrene to degrade benzo[a]pyrene.

[0018] Preferably, Arthrinium acutiapicum DL-5 is applied to petroleum-contaminated soil to degrade benzo[a]pyrene.

[0019] This invention describes the domestication and isolation of a benzo[a]pyrene-degrading strain, DL-5, from petroleum-contaminated soil in Ningbo. Based on morphological and physiological characteristics, ITS gene sequencing analysis, and phylogenetic analysis, the strain was identified as *Arthrinium acutiapicum* DL-5. The optimal growth conditions were: temperature 33℃, pH 7, and no sodium chloride added. ITS gene sequencing analysis showed that the strain most closely related to DL-5 was *Arthrinium acutiapicum* KUMCC20-0209 (99.34%). DL-5 can utilize benzo[a]pyrene as a carbon source, with an initial benzo[a]pyrene concentration of 25 mg·L⁻¹. -1 After culturing in an inorganic salt broth for 7 days, the pure strain DL-5 achieved a degradation rate of over 75% for benzo[a]pyrene. Furthermore, when DL-5 was formulated into a bacterial agent, the degradation rate of benzo[a]pyrene increased to over 80%. Therefore, this strain DL-5 and its bacterial agent show promising application potential in the bioremediation of polycyclic aromatic hydrocarbons.

[0020] Arthrinium acutiapicum DL-5 was deposited on February 26, 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: 65989. Attached Figure Description

[0021] Figure 1 These are the front and back sides of strain DL-5 grown on PDB medium for 7 days in Example 1.

[0022] Figure 2This is a phylogenetic relationship based on the ITS gene sequence of strain DL-5 and its related bacteria in Example 1. The construction method is neighbor-joining, and the expansion value is set to 1000 replicates. The figure only shows the results with an expansion value greater than 50%. The scale bar 0.01 represents the substitution rate of each nucleotide.

[0023] Figure 3 The strain DL-5 in Example 2 was grown under different culture temperatures, salinities, and pH values.

[0024] Figure 4 This refers to the degradation efficiency of strain DL-5 and DL-5 inoculum in inorganic salt medium containing benzo[a]pyrene in Example 3 (initial concentration 25 mg·L⁻¹). -1 ). Detailed Implementation

[0025] 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.

[0026] Example 1: Isolation and Identification of Arthrinium acutiapicum DL-5

[0027] 1. Materials and Methods

[0028] 1.1 Sample Source

[0029] Soil samples were obtained from an oil-contaminated site in Ningbo. The samples were acclimatized for a long period of time using high concentrations of benzo[a]pyrene as a carbon source. Through multiple screenings and purifications, highly efficient benzo[a]pyrene-degrading strains were obtained.

[0030] 1.2 Culture medium

[0031] 1.2.1 Inorganic Salt Culture Medium

[0032] Inorganic salt culture medium is used for the enrichment culture of microorganisms in samples, pure bacteria, and benzo[a]pyrene degradation experiments under bacterial agent conditions. The formula of this culture medium is shown in Table 1 (containing benzo[a]pyrene solution). The preparation method is to add each component to the solvent water, mix evenly, and sterilize.

[0033] Table 1. Inorganic Salt Culture Medium Formulation

[0034]

[0035]

[0036] 1.2.2 Nutrient Culture Medium

[0037] Nutrient media are used for the isolation, purification, preservation, and activation of fungi and other routine microbial cultures. The types and components of the 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.

[0038] Table 2. Composition of Potato-Dextrose Broth Medium (PDB)

[0039]

[0040] 1.3 Domestication, screening and isolation of strains

[0041] The collected contaminated soil was added to the aforementioned inorganic salt culture medium, along with streptomycin sulfate and penicillin (concentration of 100 ug / ml) to inhibit bacterial growth, at a concentration of 50 mg·L⁻¹. -1 Benzo[a]pyrene was used as the degradation substrate, and the culture was carried out in a 28°C incubator in the dark with shaking. The strain was acclimatized using an inorganic salt medium with benzo[a]pyrene as the carbon source, with each acclimatization cycle lasting 7 days. A 10% inoculum was transferred to a fresh inorganic salt medium with the same culture system, and the above enrichment process was repeated three times.

[0042] The fourth-generation enriched culture samples obtained above were separated by dilution plating using a nutrient medium. The samples were then 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 hyphae, 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. In this experiment, a strain DL-5 with highly efficient degradation capabilities for benzo[a]pyrene was screened. The purified single colonies were picked and cultured in appropriate solid test tubes of nutrient medium, sealed with sterilized liquid paraffin, and stored at -4℃ for long-term preservation.

[0043] 1.4 Identification of strains

[0044] The strain DL-5 was identified based on its morphological and molecular biological characteristics.

[0045] 1.4.1 Morphological characteristics

[0046] DL-5 is a fungus isolated from petroleum-contaminated soil in Ningbo. After activation, under aerobic conditions at 28℃, it grows on PDB plates for 7 days, forming colonies with a diameter of 12.5 mm, consisting of white, round, white, fluffy mycelia growing upwards. This fungus is an obligate aerobic bacterium. Figure 1 ).

[0047] 1.4.2 Molecular biological characteristics

[0048] Molecular biological characterization mainly includes sequencing and phylogenetic tree construction. Before sequencing and constructing the phylogenetic tree, fungal DNA needs to be extracted (the rapid fungal genomic DNA extraction kit used in the experiment was from Sangon Biotech (Shanghai) Co., Ltd.). For fungal taxonomic studies, it is usually necessary to amplify the ITS gene and construct a phylogenetic tree. The amplified gene is a segment of DNA encoded by rRNA in eukaryotes. Due to its high conservation, specificity, and suitable sequence length, it is commonly used for the detection and identification of fungi.

[0049] Polymerase chain reaction (PCR) is mainly used to amplify different gene fragments. PCR requires different primers (ITS1: 5'-TCCGTAGGTGAACCTGCGG-3'; ITS4: 5'-TCCTCCGCTTATTGATATGC-3'). 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: 95℃ pre-denaturation for 3 min, 95℃ for 45 s, 56℃ annealing for 30 s, 72℃ extension for 45 s, 30 cycles. Final extension at 72℃ for 10 min. Store at 4℃ after the reaction. 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 operated at a specific voltage for 20 minutes, then removed and observed under a 300 nm UV lamp to confirm successful PCR amplification. The successfully amplified PCR product was then sequenced using the same primers as the amplification primers.

[0050] The fungal ITS gene sequence obtained from sequencing was uploaded to EzTaxon-e (http: / / eztaxon-e.ezbiocloud.net / ). This website compares the submitted sequence with the ITS 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. Simultaneously, the ITS gene sequence of the model strain can be obtained, and phylogenetic analysis can be constructed to demonstrate the differences between the model strain and the experimental isolate, thereby identifying the isolated strain. Phylogenetic trees are 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, with the bootstrap value usually set to 1000 iterations.

[0051] A 603 bp ITS gene sequence was obtained through PCR and gene sequencing. ITS gene alignment revealed a 99.34% genetic similarity between this strain and *Arthrinium acutiapicum* KUMCC 20-0209 (GenBank accession number MT946342.1). Based on these results, the fungus DL-5 isolated in this experiment can be identified as *Arthrinium acutiapicum*.

[0052] A phylogenetic tree was constructed using the ITS gene sequence of DL-5 and highly similar ITS gene sequences to obtain the homology results between the DL-5 ITS gene and its highly similar ITS gene. 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 benzo[a]pyrene degrading bacteria is of great theoretical and practical significance for the treatment and deep remediation of benzo[a]pyrene-contaminated water and soil and PAH pollution.

[0053] The ITS gene sequence of DL-5 is shown in SEQ ID NO.1, specifically:

[0054] .

[0055] Based on the above results, the strain DL-5 isolated in this experiment can be identified as *Arthrinium acutiapicum*, and is named *Arthrinium acutiapicum* DL-5. It was deposited on February 26, 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. 65989.

[0056] Example 2: Growth conditions of Arthrinium acutiapicum DL-5

[0057] Measurement of growth temperature:

[0058] Prepare the nutrient medium required for the growth of the strain (Example 1), and sterilize it in an autoclave after preparation. Inoculate the activated strain *Arthrinium acutiapicum* DL-5 into the nutrient medium (experimental group), and use uninoculated nutrient medium as a control (control group). Incubate the medium at different temperatures for 7 days. There are three replicates for both the control group and the experimental group at each temperature. Observe the fungal growth daily. After 7 days, pour the medium into a weighing centrifuge tube, centrifuge at 4500 rpm for 30 minutes, discard the supernatant, and dry in a 60°C oven until constant weight. Weigh and calculate the dry weight of the fungal mycelium. The test temperatures are as follows: 18°C, 23°C, 28°C, 33°C, and 38°C.

[0059] Determination of pH for growth:

[0060] Prepare the nutrient medium required for the growth of the strain (Example 1). Adjust the pH of the culture medium using 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, 0.1 mol / L NaHCO3 and 0.1 mol / L Na2CO3. Inoculate the fungus *Arthrinium acutiapicum* DL-5 into the culture medium, performing three replicates for each pH. Use an uninoculated nutrient medium as a control. Incubate the medium at the optimal temperature for new fungal growth for 7 days, observing the fungal growth daily. After 7 days, pour the medium into a weighing centrifuge tube, centrifuge at 4500 rpm for 30 min, discard the supernatant, and dry in a 60°C oven until constant weight. Weigh and calculate the dry weight of the fungal mycelium. The pH values ​​tested were as follows: 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0.

[0061] Salt concentration tolerance:

[0062] Prepare the nutrient medium required for the growth of the strain (Example 1) and adjust the salt concentration of the medium. Inoculate the activated new strain *Arthrinium acutiapicum* DL-5 into the sterilized medium. Perform three replicates for each salt concentration. Use uninoculated nutrient medium as a control. Incubate the medium at the optimal temperature for new strain growth for 7 days. After 7 days, pour the medium into a weighed centrifuge tube and centrifuge at 4500 rpm for 30 min. Discard the supernatant and dry in a 60°C oven until constant weight. Weigh the tube and calculate the dry weight of the fungal mycelium to obtain the optimal salt concentration range that the new strain can tolerate. The tested salt concentrations are as follows: 0%, 2%, 4%, 6%, 8%, and 10% (mass fraction).

[0063] The results are as follows Figure 3As shown, DL-5 can grow in nutrient broth medium at temperatures ranging from 18 to 38°C, with the optimal growth temperature being the enrichment temperature of 33°C. The bacterium can grow at pH levels ranging from 4.0 to 9.0, with the optimal growth pH being 7.0. The bacterium has weak salt tolerance, exhibiting the best growth under salt-free conditions. It can grow at salt concentrations of 0% to 6%, but its growth decreases significantly with increasing salinity, and it hardly grows at 8%.

[0064] Example 3: Degradation experiment of benzo[a]pyrene by Arthrinium acutiapicum DL-5 and its bacterial agent.

[0065] 1. The preparation steps of the microbial agent are as follows:

[0066] 1) Heat corn and water at a mass ratio of 1:5 to make a paste. Then add sawdust (passed through a 200-mesh sieve), wheat bran and sodium lignosulfonate at a mass ratio of 150:100:10:1. Knead the mixture into a ball and place the ball-forming culture medium mixture into a pelletizing machine to obtain spherical culture medium with a diameter of 8mm. Sterilize and dry the mixture for later use.

[0067] 2) Prepare a bacterial solution with a mycelial content of 10 g / L from the cultured DL-5 fungus.

[0068] 3) Add the bacterial solution to a 3% sodium alginate solution at a mass ratio of 1:10. Mix the above spherical culture medium thoroughly with this solution. After completion, add a 4% sterile calcium chloride solution and harden for 20 minutes to obtain spherical encapsulated fungi.

[0069] 4) Place the sealed fungal pellets into a sterile culture bag and incubate at 28°C for 3-7 days. Once the surface is covered with white mycelium, it is DL-5 fungal agent.

[0070] 2. Degradation experiment of benzo[a]pyrene

[0071] After 7 days of activation culture, strain DL-5 or DL-5 agent (DL-5-agent) was inoculated into a solution containing an initial concentration of 25 mg / L. -1 In an inorganic salt culture medium for benzo[a]pyrene (Example 1), the culture was shaken and repeated three times for each treatment. The culture conditions were: temperature 33°C, pH 7.0, and no NaCl added. The inoculum of pure DL-5 and DL-5 inoculum was 10% by weight. The treatment without pure bacteria or inoculum was used as a control.

[0072] 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 (deuterated PAHs) with a concentration of 200 mg / L was added. After shaking thoroughly, the sample was 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% activated neutral alumina, 3 cm of 3% activated 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). The eluent of about 15 mL was collected in a brown reagent bottle and concentrated to about 0.5 mL by nitrogen blowing. Finally, it was transferred to a 1.5 mL cell culture flask and frozen for storage. Before the instrumental analysis, 5 μL of hexamethylbenzene, with a concentration of 200 mg / L, was added. (2) Instrumental analysis: The content of PAHs in each treatment sample was determined by Agilent 7890 gas chromatograph-5975 mass spectrometer. The chromatographic column used was an Agilent DB 5-MS capillary column (30 m in length, 0.25 mm in inner diameter, and 0.25 μm in membrane thickness). The obtained data were processed by an Agilent chromatography workstation. The quantification of benzo[a]pyrene was performed using a 6-point calibration curve and the internal standard method. The concentration of microbial cells was determined by drying and weighing.

[0073] The results showed that the degradation rate of benzo[a]pyrene itself was 24.2% after 7 days. GC-MS analysis confirmed that both strain DL-5 and the bacterial agent could degrade benzo[a]pyrene, and the degradation rate reached over 80% after 7 days of cultivation in an inorganic salt culture medium containing 25 mg / L benzo[a]pyrene. Figure 4 The pure strain DL-5 showed a degradation rate of 82.5% for benzo[a]pyrene, while the bacterial agent showed a degradation efficiency of 5.7% higher than that of the pure strain. This indicates that strain DL-5 is a potent bacterium capable of degrading benzo[a]pyrene, and its effectiveness is even better when prepared as a bacterial agent.

[0074] in conclusion:

[0075] 1) A benzo[a]pyrene degrading bacterium DL-5, which can grow with benzo[a]pyrene as a carbon source, was enriched and isolated from petroleum-contaminated soil in Ningbo and made into a solid bacterial agent.

[0076] 2) This strain DL-5 forms colonies with white, round, white, fluffy hyphae growing upwards, approximately 12.5 mm in diameter. It is an obligate aerobic bacterium. Based on molecular biological analysis, the fungus DL-5 isolated in this experiment was identified as *Arthrinium acutiapicum*, and its phylogenetic tree was constructed. Currently, there are few reports on the applications of this strain, especially studies on its degradation of benzo[a]pyrene.

[0077] 3) The optimal growth conditions for strain DL-5 are a temperature of 33℃, pH 7.0, and no NaCl added. DL-5 can use benzo[a]pyrene as a carbon source and degrade it, with an initial benzo[a]pyrene concentration of 25 mg·L⁻¹. -1 After culturing in an inorganic salt culture medium for 7 days, the degradation rate reached 82.5%. Furthermore, the solid bacterial agent prepared from DL-5 showed even better degradation of benzo[a]pyrene, reaching 88.2%. In summary, DL-5 is a strain capable of degrading benzo[a]pyrene, exhibits strong adaptability to polycyclic aromatic hydrocarbons, and its prepared bacterial agent demonstrates superior degradation performance, showing promising application potential in bioremediation.

Claims

1. Arthrinium acutiapicum DL-5, accession number: GDMCC No: 65989.

2. The claim 1 Arthrinium acutiapicum Application of DL-5 in the degradation of benzo[a]pyrene.

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

4. The application according to claim 2, characterized in that, It is Arthrinium acutiapicum DL-5 is used to degrade benzo[a]pyrene in environments contaminated with benzo[a]pyrene.

5. A benzo[a]pyrene degrading bacterial agent, characterized in that, Includes the claims 1 Arthrinium acutiapicum DL-5 is the active ingredient.

6. The benzo[a]pyrene degrading bacterial agent according to claim 5, characterized in that, The preparation method of the benzo[a]pyrene degrading bacterial agent is as follows: 1) After heating corn and water to a mass ratio of 1:5 to make a paste, add sawdust, wheat bran and sodium lignosulfonate at a mass ratio of 150:100:10:1, knead into a ball, put the ball-shaped culture medium mixture into a pelletizing machine to make spherical culture medium, sterilize and dry for later use. 2) Arthrinium acutiapicum DL-5 was prepared into a bacterial solution; 3) Add the bacterial solution from 2) to a 3% sodium alginate solution at a mass ratio of 1:

10. Mix the spherical culture medium from 1) thoroughly with this solution. After completion, add a 4% sterile calcium chloride solution and harden the mixture to obtain spherical encapsulated fungi. 4) Place the sealed fungal pellets into a sterile culture bag and incubate at 28°C for 3-7 days. Once the surface is covered with white mycelium, it becomes a benzo[a]pyrene degrading agent.

7. The benzo[a]pyrene degrading bacterial agent according to claim 6, characterized in that, In step 2), the bacterial solution is a bacterial solution with a mycelial content of 10 g / L.

8. A method for degrading benzo[a]pyrene, characterized in that, The claim 1 Arthrinium acutiapicum DL-5 is applied to environments containing benzo[a]pyrene to degrade benzo[a]pyrene.

9. The method according to claim 8, characterized in that, It is Arthrinium acutiapicum DL-5 was applied to environments contaminated with benzo[a]pyrene to degrade benzo[a]pyrene.

10. The method according to claim 8, characterized in that, It is Arthrinium acutiapicum DL-5 was applied to petroleum-contaminated soil to degrade benzo[a]pyrene.

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