Separation and identification of fungus Nigrospora osmanthi DL-7 for degrading benzo [a] pyrene in petroleum-contaminated soil as well as preparation and application of fungicide of fungus Nigrospora osmanthi DL-7

Abstract

The invention discloses a fungus Nigrospora osmanthi DL-7 for degrading benzo [a] pyrene in petroleum contaminated soil as well as a fungicide and application of the fungus Nigrospora osmanthi DL-7. According to the invention, a degrading strain Nigrospora osmanthi DL-7 with benzo [a] pyrene as a carbon source is domesticated, separated and identified from petroleum-polluted soil in Ningbo. The DL-7 can utilize benzo [a] pyrene as a carbon source, and after the DL-7 is cultured for 7 days in an inorganic salt culture solution with the initial concentration of benzo [a] pyrene being 25 mg.L <-1 >, the degradation rate of DL-7 pure bacteria on benzo [a] pyrene can reach 85% or above. And after the strain is prepared into the microbial inoculum, the degradation rate of benzo [a] pyrene can be increased to 90% or above, so that the strain and the microbial inoculum thereof have relatively good application potential in the aspect of bioremediation of polycyclic aromatic hydrocarbons.

Description

Technical Field

[0001] The invention belongs to the field of organic pollutant degradation, and specifically relates to the isolation and identification of a benzo[a]pyrene-degrading fungus Nigrospora osmanthi DL-7 in petroleum-contaminated soil, and the preparation and application of its bacterial agent. Background Art

[0002] With the rapid development of modern industry, pollution caused by industrial wastewater is becoming increasingly serious. Various organic pollutants, such as polycyclic aromatic hydrocarbons (PAHs), persist. PAHs are a class of hydrophobic organic compounds with two or more fused rings and are ubiquitous byproducts of fossil fuels (coal, oil, etc.), waste incineration, and wood processing. PAHs have the potential to cause significant harm to the ecological environment and human health due to their carcinogenic, teratogenic, mutagenic, and bioaccumulative properties. Therefore, PAH pollution has attracted widespread attention. Factors such as human activities in industry, mining, and agriculture, as well as high background levels in the soil environment, often lead to severe excesses of PAHs in the environment and cause pollution. Benzo[a]pyrene is a pentacyclic aromatic hydrocarbon that is closely related to the carcinogenicity of PAHs. In 2020, the "List of Priority Controlled Chemicals (Second Batch)" formulated by the Ministry of Ecology and Environment and other departments listed it as a priority pollutant.

[0003] In the natural environment, organic pollutants are mainly degraded through metabolic transformation by microorganisms. Microbial remediation technology utilizes this biological process and provides the necessary conditions for microbial growth through engineering measures, thereby achieving the process of removing pollutants from the soil. Compared with physical and chemical remediation methods, microbial remediation technology has unique advantages such as low cost and environmental friendliness, and has broad prospects in the application of polycyclic aromatic hydrocarbons contaminated soil remediation. At present, 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 and obtained through pure culture. Therefore, screening strains that can effectively degrade high-concentration benzo[a]pyrene has important application value and practical significance. In this experiment, the mass concentration of 25 mg·L -1 Benzo[a]pyrene was used as a substrate for degradation by the strain, in order to provide data support for the biological treatment of polycyclic aromatic hydrocarbons. Summary of the Invention

[0004] The first object of the present invention is to provide a strain Nigrosporaosmanthi DL-7 having the ability to degrade benzo[a]pyrene, which was deposited in the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on February 26, 2025, at the 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, Postal Code: 510070, and the deposit number is: GDMCC No: 65990.

[0005] The strain reported in this study belongs to Nigrospora osmanthi, a species that can be isolated from various terrestrial and aquatic habitats. Currently, there are relatively few reports on pollutant degradation by Nigrospora osmanthi, and no studies have been reported on its ability to degrade benzo[a]pyrene (B[a]pyrene) either domestically or internationally. In this study, a strain DL-7, which is capable of using high concentrations of B[a]pyrene as a carbon source, was domesticated and isolated from a petroleum-contaminated site in Ningbo. The strain was characterized, its growth characteristics were studied, and a bacterial inoculum was prepared to investigate its B[a]pyrene degradation properties, providing a reference for bioremediation of PAH-contaminated environments.

[0006] The second object of the present invention is to provide the use of the above-mentioned Nigrospora osmanthi DL-7 in the degradation of benzo[a]pyrene.

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

[0008] Preferably, Nigrospora osmanthi DL-7 is used in a benzo[a]pyrene-contaminated environment to degrade benzo[a]pyrene.

[0009] The third object of the present invention is to provide a benzo[a]pyrene-degrading bacterial agent comprising the above-mentioned Nigrosporaosmanthi DL-7 as an active ingredient.

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

[0011] 1) Corn and water are heated in a mass ratio of 1:5 to form a paste, sawdust, wheat bran, and sodium lignin sulfonate are added in a mass ratio of 150:100:10:1, and the mixture is kneaded into a ball. The ball-shaped culture matrix mixture is placed in a pelletizer to produce a spherical culture matrix, which is sterilized and dried for later use;

[0012] 2) preparing a bacterial suspension of the cultured Nigrospora osmanthi DL-7;

[0013] 3) adding the bacterial solution from 2) to a 3% sodium alginate solution at a mass ratio of 1:10, thoroughly mixing the spherical culture matrix from 1) with the solution, and then adding a 4% sterile calcium chloride solution to harden the mixture to obtain pellets of encapsulated fungi;

[0014] 4) The encapsulated fungal pellets are placed in a sterile culture bag and cultured in a 28°C incubator for 3-7 days. When the surface is covered with white hyphae, the benzo[a]pyrene-degrading bacterial agent is obtained.

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

[0016] The fourth object of the present invention is to provide a method for degrading benzo[a]pyrene, which comprises spraying the above-mentioned Nigrosporaosmanthi DL-7 into an environment containing benzo[a]pyrene to degrade benzo[a]pyrene.

[0017] Preferably, Nigrospora osmanthi DL-7 is sprayed into an environment contaminated with benzo[a]pyrene to degrade benzo[a]pyrene.

[0018] Preferably, Nigrospora osmanthi DL-7 is sprayed on petroleum-contaminated soil to degrade benzo[a]pyrene.

[0019] The present invention domesticates and isolates a benzo[a]pyrene-degrading strain DL-7 from petroleum-contaminated soil in Ningbo. The strain is identified as Nigrospora osmanthi DL-7 based on its morphological and physiological characteristics, ITS gene sequencing analysis, and phylogenetic analysis. The optimal environmental conditions for its growth are: a temperature of 28°C, a pH of 7, and no sodium chloride addition. ITS gene sequencing analysis of the strain shows that the strains most similar to DL-7 are Nigrospora osmanthiisolate 39A-1X and Nigrospora osmanthi CGMCC 3.18126 (100%). DL-7 is able to utilize benzo[a]pyrene as a carbon source and can degrade benzo[a]pyrene at an initial benzo[a]pyrene concentration of 25 mg / L. -1 After seven days of cultivation in an inorganic salt-rich medium, the DL-7 strain achieved a benzo[a]pyrene degradation rate exceeding 85%. When DL-7 was formulated into a bacterial inoculant, the degradation rate was increased to nearly 90%. Therefore, this strain and its inoculant have great potential for bioremediation of polycyclic aromatic hydrocarbons.

[0020] Nigrospora osmanthi DL-7 was deposited on February 26, 2025 in Guangdong Provincial Microbiological Culture Collection Center (GDMCC), address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, Postal Code: 510070, with the deposit number: GDMCC No: 65990. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0022] Figure 2 This is the phylogenetic relationship of strain DL-7 and its related bacteria based on ITS gene sequences in Example 1. The construction method was the neighbor-joining method, and the bootstrap value was set to 1000 repetitions. Only the results with a bootstrap value greater than 50% are shown in the figure. The scale of 0.01 represents the substitution rate of each nucleotide.

[0023] Figure 3 The strain DL-7 in Example 2 was grown under different culture temperatures, salinities, and pH values.

[0024] Figure 4 The degradation efficiency of strain DL-7 and DL-7 bacterial agent in Example 3 in inorganic salt medium containing benzo[a]pyrene (initial concentration 25 mg·L -1 ). DETAILED DESCRIPTION

[0025] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is 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 for the purpose of explaining the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0026] Example 1 Isolation and Identification of Nigrospora osmanthi DL-7

[0027] 1 Materials and Methods

[0028] 1.1 Sample Source

[0029] Soil samples were obtained from a petroleum-contaminated site in Ningbo. They were acclimated for a long time using high concentrations of benzo[a]pyrene as a carbon source. Through multiple screening, separation and purification, an efficient benzo[a]pyrene-degrading strain was obtained.

[0030] 1.2 Culture medium

[0031] 1.2.1 Inorganic salt culture medium

[0032] Inorganic salt culture medium is used for enrichment of microorganisms in samples and for benzo[a]pyrene degradation experiments under pure bacterial and inoculum conditions. The medium formula is shown in Table 1 (including benzo[a]pyrene solution). Preparation is by adding all ingredients to a water solvent, mixing thoroughly, and sterilizing.

[0033] Table 1 Inorganic salt culture medium formula

[0034]

[0035]

[0036] 1.2.2 Nutrient medium

[0037] Nutrient media are used for the isolation, purification, preservation, and activation of fungi, as well as for the cultivation of common microorganisms. The types and compositions 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 original medium formula. Unless otherwise specified, the pH of the medium is adjusted to 7. Nutrient media is prepared by adding all ingredients to a water solvent, mixing thoroughly, and sterilizing.

[0038] Table 2 Potato-Dextrose broth medium (PDB) composition

[0039]

[0040] 1.3 Domestication, screening and isolation of strains

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

[0042] The fourth-generation enrichment culture sample obtained above was spread and separated using the dilution plating method, and the sample was separated using a nutrient medium. The spread sample was incubated at the original culture temperature. After approximately 48 hours, distinct single colonies formed on the surface of the culture medium. Several distinct single colonies were selected based on their morphology, size, color, hyphae, and other characteristics. These colonies were streaked and purified on nutrient medium plates and cultured. If single colonies with different characteristics were still observed on the streak-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-7 with high benzo[a]pyrene degradation performance was screened and obtained. The purified single colonies were cultured in the corresponding solid test tube nutrient medium, sealed with sterilized liquid paraffin, and stored at -4°C for long-term storage.

[0043] 1.4 Identification of strains

[0044] The strain DL-7 was identified based on its morphological characteristics and molecular biological properties.

[0045] 1.4.1 Morphological characteristics

[0046] DL-7 is a fungus isolated from oil-contaminated soil in Ningbo. After activation, it can form white, round, and upward-growing white fuzzy hyphae with a diameter of 13.5 mm after growing on PDB plates under aerobic conditions at 28°C for 7 days. This fungus is an obligate aerobe ( Figure 1 ).

[0047] 1.4.2 Molecular biological characteristics

[0048] Molecular biological characterization primarily involves sequencing and phylogenetic tree construction. Before sequencing and phylogenetic tree construction, fungal DNA must be extracted (the rapid fungal genomic DNA extraction kit used in the experiment was from Sangon Biotech (Shanghai) Co., Ltd.). To study fungal taxonomy, it is often necessary to amplify the ITS gene and construct a phylogenetic tree. The amplified gene is a segment of DNA that encodes rRNA in eukaryotes. Due to its high conservation, specificity, and suitable sequence length, it is often used for fungal detection and identification.

[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 is: 10× buffer 2.5μl, Mg 2+1.5 μl of dNTP (25 mmol / l), 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 reaction conditions included 30 cycles of initial denaturation at 95°C for 3 min, followed by 30 cycles of 95°C for 45 s, annealing at 56°C for 30 s, and extension at 72°C for 45 s. The reaction was then extended at 72°C for 10 min. The reaction was then stored at 4°C. After amplifying the desired gene, a gel block is prepared using 0.75-1% agarose and the nucleic acid stain GelRed. The PCR product and DNA markers of various lengths are added to the gel block and placed in an electrophoresis apparatus filled with TBE (Tris boric acid) buffer. The apparatus is operated at a constant voltage for 20 minutes, then removed and observed under a 300nm UV lamp to confirm successful PCR amplification. Successful PCR products are then sequenced using the same sequencing primers as the amplification primers.

[0050] The fungal ITS gene sequences obtained by sequencing were uploaded to EzTaxon-e (http: / / eztaxon-e.ezbiocloud.net / ). This website will compare the submitted sequences with the ITS gene sequences of representative strains of recognized species to obtain similarity information between the sequences. Based on the sequence alignment results, the corresponding representative strain can be selected as the model strain for the experimental isolate. The ITS gene sequence of the model strain can also be obtained to construct a phylogenetic analysis to demonstrate the differences between the model strain and the experimental isolate, thereby identifying the isolate. Phylogenetic trees are constructed using the MEGA 5.05 program. The neighbor-joining method, minimum evolution method, and maximum parsimony method are commonly used to construct phylogenetic trees. The neighbor-joining method is the most commonly used, and the bootstrap value is often set to 1000 replicates.

[0051] A 553-bp ITS gene sequence was obtained through PCR and gene sequencing. Comparison of the ITS gene revealed that the strain shared 100% similarity with Nigrospora osmanthi isolate 39A-1X and Nigrospora osmanthi CGMCC3.18126 (GenBank accession numbers MT556397.1 and NR 153474.1, respectively). These results confirm that the fungus DL-7 isolated in this experiment is a species of Nigrospora osmanthi.

[0052] The phylogenetic tree was constructed using the ITS gene sequence of DL-7 and its ITS gene sequence with high similarity, thereby obtaining the homology results between the ITS gene of DL-7 and its ITS gene with high similarity. The phylogenetic tree constructed using the neighbor-joining method is shown in 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 has important theoretical and practical significance for the treatment and deep remediation of benzo[a]pyrene-contaminated water and soil and PAHs pollution.

[0053] The ITS gene sequence of DL-7 is shown in SEQ ID NO. 1, specifically:

[0054] .

[0055] Based on the above results, it can be concluded that the strain DL-7 isolated in this experiment is the species Nigrospora osmanthi. It was named Nigrospora osmanthi DL-7 and was deposited on February 26, 2025, at the Guangdong Provincial Microbiological Culture Collection Center (GDMCC), Building 59, 5th Floor, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, Postal Code: 510070, with the deposit number: GDMCC No: 65990.

[0056] Example 2 Growth conditions of Nigrospora osmanthi DL-7

[0057] Determination of growth temperature:

[0058] The nutrient medium required for strain growth was prepared (Example 1), and after preparation, it was sterilized in an autoclave. The activated strain Nigrospora osmanthi DL-7 was accessed into the nutrient medium (experimental group), and a culture medium not inoculated with fungi was used as a control (control group). The culture medium was cultured at different temperatures for 7 days. The control group and the experimental group corresponding to each temperature were repeated three times. The growth of the fungus was observed every day. After 7 days, the culture medium was poured into a weighed centrifuge tube and centrifuged at 4500 rpm for 30 minutes. After discarding the supernatant, the tube was placed in a 60-degree oven and dried to constant weight. The dry weight of the fungal mycelium was calculated by weighing. The test temperatures were as follows: 23°C, 28°C, 33°C, 35°C, and 38°C.

[0059] Determination of growth pH:

[0060] A nutrient medium (Example 1) was prepared for strain growth, and the pH of the culture medium was adjusted 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; and pH 9.0, 0.1 mol / l NaHCO3 and 0.1 mol / l Na2CO3. The fungus Nigrospora osmanthi DL-7 was inoculated into the culture medium, with three replicates performed at each pH. A nutrient medium without fungus inoculation served as a control. The culture medium was incubated at the optimal temperature for new fungal growth for 7 days, with fungal growth observed daily. After 7 days, the culture medium was poured into a weighed centrifuge tube and centrifuged at 4500 rpm for 30 minutes. The supernatant was discarded, and the tube was dried in a 60°C oven to a constant weight. The dry weight of the fungal mycelium was then calculated by weighing. The pH tested was as follows: 4.0, 5.0, 6.0, 7.0, 8.0, 9.0.

[0061] Salt concentration tolerance:

[0062] The nutrient medium required for strain growth was configured (Example 1) and the salt concentration of the medium was adjusted. The activated new bacterium Nigrospora osmanthi DL-7 was inoculated into a sterilized culture medium. Three replicates were performed for each salt concentration. A nutrient medium without inoculation was used as a control. The culture medium was placed at the optimal temperature for the growth of the new bacterium and cultured for 7 days. After 7 days, the culture medium was poured into a weighed centrifuge tube and centrifuged at 4500 rpm for 30 minutes. After discarding the supernatant, the tube was placed in a 60-degree oven and dried to constant weight. The dry weight of the fungal hyphae was calculated by weighing to obtain the salt concentration range that the new bacterium can tolerate. The test salt concentrations were as follows: mass fraction 0%, 2%, 4%, 6%, 8%, 10%.

[0063] The results are as follows Figure 3 As shown, in nutrient broth culture medium, DL-7 can grow at a temperature of 18-38°C, and the optimal growth temperature is the enrichment temperature of the bacteria, 28°C; the bacteria can grow at a pH of 4.0-9.0, with the optimal growth pH being 7.0, but the growth rate decreases significantly with increasing alkalinity; the bacteria has a weak salt tolerance and grows best in the absence of salt. It can grow under conditions of salt concentrations of 0% to 4%, but the growth rate decreases significantly with increasing salinity, and almost no growth occurs at 6%.

[0064] Example 2 Benzo[a]pyrene degradation experiment of Nigrospora osmanthi DL-7

[0065] 1. Preparation of the microbial agent, the steps are as follows:

[0066] 1) Corn and water were heated in a mass ratio of 1:5 to form a paste. Sawdust (passed through a 200-mesh sieve), wheat bran, and lignin sodium lignin sulfonate were added in a mass ratio of 150:100:10:1. The mixture was kneaded into a ball. The ball was placed in a pelletizer to produce 8 mm diameter balls. The balls were sterilized and dried for later use.

[0067] 2) The cultured DL-7 fungus was prepared into a bacterial liquid with a mycelium content of 10 g / L.

[0068] 3) The bacterial liquid was added to a 3% sodium alginate solution at a mass ratio of 1:10, and the spherical culture matrix was thoroughly mixed with the solution. After completion, a 4% sterile calcium chloride solution was added and hardened for 20 minutes to obtain pellets of encapsulated fungi.

[0069] 4) Place the encapsulated fungal pellets into a sterile culture bag and culture in a 28°C incubator for 3-7 days. When the surface is covered with white hyphae, it is the DL-7 fungal agent.

[0070] 2. Benzo[a]pyrene degradation experiment

[0071] After 7 days of activation culture, the strain DL-7 or DL-7 agent (DL-7-agent) was inoculated into a medium containing an initial concentration of 25 mg·L -1 The cells were shaken in an inorganic salt medium containing benzo[a]pyrene (Example 1), with three replicates per treatment, at 28°C, pH 7.0, and without the addition of NaCl. The inoculum size of both the pure DL-7 strain and the DL-7 inoculum was 10% by weight. The treatment without the pure strain or inoculum served as the control.

[0072] Each treated sample was taken for chemical analysis. The specific steps are as follows: (1) Sample pretreatment: Each culture sample was extracted with dichloromethane and 5 μL of a recovery indicator (deuterated-PAHs) at a concentration of 200 mg / L was added. After thorough shaking, the sample was transferred to a separatory funnel and allowed to stand. After separation, the organic phase was collected, the lower layer 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 sheets for rotary evaporation and concentrated to about 2 mL. A small amount of n-hexane (about 5 mL) was added and rotary evaporated to 2 mL. The washing was repeated three times and the organic solvent was replaced with n-hexane. The concentrated solution after replacement was purified using a glass filled column (about 9 mm in diameter). The column fillings were 3 cm 3% deactivated neutral alumina, 3 cm 3% deactivated silica gel and 1 cm anhydrous sodium sulfate from bottom to top. The column was activated with an appropriate amount of n-hexane, and the packed column was eluted with 15 mL of a mixed reagent of n-hexane / dichloromethane (volume ratio of 1:1). About 15 mL of the eluate was collected in a brown reagent bottle, concentrated to about 0.5 mL by nitrogen blowing, and finally transferred to a 1.5 mL cell bottle and frozen for storage. Before the determination on the machine, 5 μL of the internal standard hexamethylbenzene was added, and its concentration was 200 mg / L. (2) Instrumental analysis: The PAHs content in each treated sample was determined using an Agilent 7890 gas chromatograph-5975 mass spectrometer. The chromatographic column used was an Agilent DB 5-MS capillary column (column length 30 m, inner diameter 0.25 mm, film thickness 0.25 μm). The data obtained were processed using an Agilent chromatography workstation, and 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 using the drying and weighing method.

[0073] The results showed that the degradation rate of benzo[a]pyrene itself was 24.2% in 7 days. According to GC-MS analysis, both strain DL-7 and the bacterial agent were able to degrade benzo[a]pyrene, and after culturing in an inorganic salt culture medium containing 25 mg / L benzo[a]pyrene for 7 days, the degradation rate reached over 85%. Figure 4 The degradation rate of benzo[a]pyrene by pure DL-7 strain was 86.4%, while that by the bacterial agent was 88.8%, indicating that strain DL-7 is a potent bacterium capable of degrading benzo[a]pyrene, and that the degradation capacity of the bacterium was not affected after the bacterial agent was prepared.

[0074] in conclusion

[0075] 1) A benzo[a]pyrene-degrading bacterium strain DL-7, which can grow using benzo[a]pyrene as a carbon source, was enriched and isolated from petroleum-contaminated soil in Ningbo and prepared into a solid bacterial agent.

[0076] 2) The strain DL-7 forms colonies with white, round, upward-growing, fuzzy hyphae approximately 13.5 mm in diameter. It is an obligate aerobe. Molecular biological analysis indicates that the fungus DL-7 isolated in this experiment is a strain of Nigrospora osmanthi, and a phylogenetic tree has been constructed. Currently, there are few reports on the application of this strain, particularly its use in the degradation of benzo[a]pyrene.

[0077] 3) The optimal growth conditions for strain DL-7 were 28°C, pH 7.0, and no NaCl. DL-7 was able to use benzo[a]pyrene as a carbon source and degrade it. When the initial benzo[a]pyrene concentration was 25 mg·L -1 After 7 days of cultivation in an inorganic salt culture medium, the degradation rate reached 86.4%. Furthermore, the solid inoculant prepared from DL-7 showed even better degradation of benzo[a]pyrene, reaching 88.8%. In summary, DL-7 is a strain capable of degrading benzo[a]pyrene, has strong adaptability to polycyclic aromatic hydrocarbons, and the inoculant prepared from it exhibits excellent degradation efficiency, suggesting promising potential for bioremediation.

Claims

1. Nigrospora osmanthi DL-7, preservation number: GDMCC No: 65990.

2. Use of Nigrospora osmanthi DL-7 according to claim 1 in the degradation of benzo[a]pyrene.

3. The application according to claim 2, wherein The degradation of benzo[a]pyrene is the degradation of benzo[a]pyrene in petroleum-contaminated soil.

4. The application according to claim 2, wherein It is to apply Nigrospora osmanthi DL-7 in an environment contaminated with benzo[a]pyrene for the degradation of benzo[a]pyrene.

5. A benzo[a]pyrene-degrading microbial agent, characterized in that, It contains Nigrospora osmanthi DL-7 according to claim 1 as an active ingredient.

6. The benzo[a]pyrene-degrading bacterial agent according to claim 5, wherein The preparation method of the benzo[a]pyrene-degrading microbial agent is as follows: 1) After heating corn and water in a mass ratio of 1:5 to make a paste, add sawdust, wheat bran, and sodium lignosulfonate in a mass ratio of 150:100:10, knead into a ball, and put the spherical culture medium mixture into a pill-making machine to obtain spherical culture medium, sterilize and dry for later use; 2) Prepare a bacterial solution of Nigrospora osmanthi DL-7; 3) Add the bacterial solution in 2) to a 3% sodium alginate solution in a mass ratio of 1:10, fully mix the spherical culture medium in 1) with this solution, and after completion, add a 4% sterile calcium chloride solution for hardening treatment to obtain pill-shaped encapsulated fungi; 4) Put the encapsulated fungal pellets into a sterile culture bag, and culture them in an incubator at 28°C for 3-7 days. When the surface is covered with white hyphae, it is the benzo[a]pyrene-degrading microbial 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 mycelium content of 10 g / L.

8. A method for degrading benzo[a]pyrene, characterized in that, Sprinkle Nigrospora osmanthi DL-7 according to claim 1 on an environment containing benzo[a]pyrene for the degradation of benzo[a]pyrene.

9. The method according to claim 8, characterized in that, It is to sprinkle Nigrospora osmanthi DL-7 on an environment contaminated with benzo[a]pyrene for the degradation of benzo[a]pyrene.

10. The method according to claim 8, characterized in that, It is to sprinkle Nigrospora osmanthi DL-7 on petroleum-contaminated soil for the degradation of benzo[a]pyrene.

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