Benzo [a] pyrene degrading fungus Z5 as well as fungicide and application thereof

Abstract

The invention provides a benzo [a] pyrene degrading fungus Z5 as well as a fungicide and application thereof, the fungus is named as Z5, is screened from a coal chemical industry polluted site and is preserved in the China General Microbiological Culture Collection Center (CGMCC), the classification name of the fungus is Aspergillus fumigasus, the preservation number is CGMCC NO.41499, the preservation time is September 6, 2024, and the fungus can take benzo [a] pyrene as a unique carbon source. Meanwhile, the invention also discloses a fungicide containing the fungus Z5, and application of the fungus Z5 and the fungicide of the fungus Z5 in treatment of polycyclic aromatic hydrocarbon pollution. Experiments prove that the degradation rate of the fungus Z5 on 10 mg / L benzo [a] pyrene within 7 days reaches 75.43%, and powerful support is provided for PAHs polluted saline alkali soil remediation and ecological protection in northwest regions.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a benzo[a]pyrene-degrading fungus Z5, a bacterial agent and an application thereof. Background Art

[0002] Polycyclic aromatic hydrocarbons (PAHs) are highly toxic organic pollutants. Due to their hydrophobicity, they are deposited in the soil, causing serious damage to the ecological environment, and therefore have a high potential for environmental pollution. PAHs can be divided into two categories according to the number of aromatic rings they contain: light molecular weight polycyclic aromatic hydrocarbons (LMW·PAHs, containing two or three aromatic rings) and high molecular weight polycyclic aromatic hydrocarbons (HMW·PAHs, containing four or more aromatic rings). As the molecular weight increases, their solubility in water decreases; the melting point and boiling point increase, and the vapor pressure decreases.

[0003] Bioremediation of HMW·PAHs-contaminated soil has become the most difficult problem in the field of HMW·PAHs remediation. Using microorganisms to deal with HMW·PAHs pollution is an ideal way, but there are currently few HMW·PAHs-degrading microbial resources. In addition, the remediation effect is limited by the toxic effect of HMW·PAHs on microorganisms, resulting in damage to the cell structure. Therefore, it is very challenging to treat HMW·PAHs pollution through microbial remediation. Therefore, screening lignin-degrading fungi with broad substrate specificity is of great significance for the remediation of HMW·PAHs-contaminated sites. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention aims to provide a benzo[a]pyrene-degrading fungus Z5 and its bacterial agent and application, wherein the fungus Z5 is a filamentous fungus that can use benzo[a]pyrene as the sole carbon source. The present invention discusses the optimal degradation conditions by changing environmental parameters, and studies its degradation mechanism and pathway by whole genome sequencing, transcriptome sequencing, spectroscopy and mass spectrometry. The research of the present invention enriches the HMW·PAHs degradation strain library, and provides strong support for the restoration and ecological protection of saline-alkali soil contaminated by PAHs in the northwest region.

[0005] The objective of the present invention is achieved through the following technical solutions:

[0006] The first aspect of the present invention provides a benzo[a]pyrene-degrading fungus, which is named Z5, screened from a coal chemical pollution site, and deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCCNO.41499, a classification name of Aspergillus fumigatus, and a deposit date of September 6, 2024. The fungus can use benzo[a]pyrene as the sole carbon source.

[0007] Furthermore, fungus Z5 was grown at 25℃~43℃, pH 6~8 and metal ion Mg 2+ , Mn 2+ Cr 2+ Well tolerated in the presence of

[0008] The second aspect of the present invention provides a bacterial agent prepared by the benzo[a]pyrene-degrading fungus described in the first aspect, wherein the bacterial agent includes a liquid bacterial agent and a solid bacterial agent.

[0009] Furthermore, the bacterial agent is prepared by the following method:

[0010] The fungus Z5 with a preservation number of CGMCC NO.41499 was inoculated into PDW liquid culture medium, cultured in a constant temperature shaking incubator at 150 r / min and 37° C. for 36 hours, and the bacterial cells were collected by centrifugation and washed with PBS buffer to obtain the bacterial agent.

[0011] The third aspect of the present invention provides the use of the fungus Z5 described in the first aspect, or the bacterial agent described in the second aspect, in the treatment of polycyclic aromatic hydrocarbon pollution.

[0012] Furthermore, the PAH pollution is high molecular weight PAH soil pollution.

[0013] Furthermore, the high molecular weight polycyclic aromatic hydrocarbon is benzo[a]pyrene.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] 1. The present invention screened a fungus Z5 that uses benzo[a]pyrene as the sole carbon source from a coal chemical pollution site. Through the analysis of the morphological characteristics and homology information of the ITS rDNA sequence of the strain Z5, it was confirmed that it belongs to the genus Aspergillus sp. in the Ascomycota phylum, specifically Aspergillus fumigatus. The strain has the characteristics of thermophilicity (the maximum growth temperature is 50°C). At the same time, after optimization of single factor conditions (temperature, pollutant concentration, shaker speed, bacterial age, inoculation amount, pH, salt concentration, co-metabolism carbon source, etc.), the degradation rate of A. fumigatus Z5 for 10 mg / L benzo[a]pyrene reached 75.43% within 7 days;

[0016] 2. The degradation rate of strain Z5 on 10 mg / kg benzo[a]pyrene contaminated soil was 27.76% within 30 days. Microbial diversity analysis showed that strain Z5 may have a certain antibacterial effect in the soil and restrict the growth of some pathogenic fungi. At the same time, strain Z5 also had a positive impact on a series of bacterial communities. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The present invention will be further described below in conjunction with the accompanying drawings and embodiments:

[0018] Figure 1 The colony photos of three fungi strains using benzo[a]pyrene as a carbon source as described in Example 1;

[0019] Figure 2 The degradation ability of the three fungi in Example 1 on benzo[a]pyrene is shown;

[0020] Figure 3 These are colony photos and microscope photos of strain Z5; A, colony photo of Z5; B, 4x microscope photo of the strain; C, 10x microscope photo of the strain; D: 40x microscope photo of the strain;

[0021] Figure 4 Phylogenetic tree analysis of ITS rRNA gene of strain Z5;

[0022] Figure 5 The effect of different conditions described in Example 2 on the growth of strain Z5 is shown; wherein A, temperature; B, pH; C, salt concentration; D: metal ions;

[0023] Figure 6 The effect of temperature and rotation speed on the degradation efficiency of benzo[a]pyrene by strain Z5 described in Example 3 is shown;

[0024] Figure 7 The tolerance of strain Z5 described in Example 3 to benzo[a]pyrene is shown;

[0025] Figure 8 The effect of the bacterial age, bacterial dosage and pH on the degradation efficiency of benzo[a]pyrene by strain Z5 described in Example 3 is shown;

[0026] Fig. 9 The tolerance of strain Z5 described in Example 3 to NaCl, different heavy metal ions and PAHs of different molecular weights is shown;

[0027] Fig.10 The degradation of benzo[a]pyrene by strain Z5 described in Example 3 under the optimal conditions is shown;

[0028] Fig.11 The effect of the cometabolic carbon source described in Example 3 on the degradation efficiency of benzo[a]pyrene is shown;

[0029] Fig.12 The color change of the plate for screening the lignin peroxidase system of the degrading bacteria in Example 3 is shown; wherein A is laccase; B is manganese peroxidase; C is lignin peroxidase;

[0030] Fig.13The quantitative results of the intracellular and extracellular enzyme activities of the degradation bacteria in Example 3 are shown; wherein A, laccase; B, manganese peroxidase; C, cytochrome P450 enzyme system; D, the degradation efficiency of Aspergillus fumigatus, intracellular enzymes, and extracellular enzymes on benzo[a]pyrene;

[0031] Fig.14 FT-IR graph of the functional group changes during the degradation of benzo[a]pyrene;

[0032] Fig.15 The degradation of benzo[a]pyrene contaminated soil was simulated as described in Example 4; wherein, A, the effect of the amount of strain added on the degradation rate; B, the degradation trend of benzo[a]pyrene by the degrading bacteria within 30 days;

[0033] Fig.16 The colonization ability of strain Z5 in Example 4 and its effect on indigenous microorganisms in soil are shown; the dominant species and relative division of (A) fungi and (B) bacteria at the phylum classification level, and the results are presented as means. (C) Clustering results of abundance similarity between fungi and (D) bacteria species. The color changes reflect the similarities and differences in community composition of different groups at each taxonomic level;

[0034] Note: All statistical analyses in the present invention were performed using t-test: *, p<0.5; **, p<0.01; ***, p<0.001; ***, p<0.0001. DETAILED DESCRIPTION

[0035] Example 1: Enrichment of benzo[a]pyrene-degrading fungus Z5

[0036] In this example, soil samples were collected from a coal chemical site in northern Shaanxi (soil samples of about 6 cm on the surface were peeled off, 10 cm to 15 cm of soil samples were collected with a sterile shovel, transferred into sterile plastic bags, sealed and stored in a low-temperature environment), and fungi with benzo[a]pyrene degradation function were screened out. The specific method is:

[0037] (1) Screening and domestication of degradation bacteria

[0038] 5 g of each of the five soil samples collected were suspended in 45 mL of sterilized distilled water. The suspension was then placed on a shaker at 30°C and 150 r / min overnight and allowed to settle for 1 hour. Next, 2 mL of the supernatant was inoculated into Bushnell Haas (BH) broth containing 10 mg / L benzo[a]pyrene and cultured for 5 days at 30°C and 150 r / min. After that, 5 mL of the stock solution was taken from the shake flask again and added to the MSM medium containing 10 mg / L benzo[a]pyrene and cultured under the same conditions until obvious mycelial ball formation was observed.

[0039] (2) Isolation of degrading bacteria

[0040] The MSM medium enriched for 5 days was serially diluted (10 -1 -10 -4 ) were used for fungal isolation. 100 μL of each dilution was applied to a PDA plate containing erythromycin (0.0015 g / 100 mL) and incubated at 30 °C for 3 days. The morphology of the strains was observed and the isolates were further subcultured until a single colony appeared.

[0041] (3) Preservation of strains

[0042] The bacterial solution was thoroughly mixed with glycerol (40%) and stored in a -80°C refrigerator.

[0043] Strain domestication results:

[0044] like Figure 1 As shown in the figure, three fungi were screened in the MSM culture system with benzo[a]pyrene as the carbon source, named Z1, Z3, and Z5. After being purified into single colonies on PDA plates, their degradation abilities to benzo[a]pyrene were measured.

[0045] The results of the test on the degradation ability of the strain to benzo[a]pyrene:

[0046] The degradation ability of benzo[a]pyrene (10 mg / L) was tested on the three selected fungi Z1, Z3 and Z5. Figure 2 As shown. Within 7 days, under the condition of 30℃, the degradation rate of strain Z1 was 16.4%, the degradation rate of strain Z3 was 24.7%, and the degradation rate of strain Z5 was 45.1%. Statistical analysis showed that the three strains were significantly different from each other in terms of degradation rate, among which strain Z5 had a very significant difference relative to Z1 (p<0.001), and strain Z5 had a very significant difference relative to strain Z3 (p<0.01). Compared with strain Z5, strains Z1 and Z3 grow slowly, metabolize slowly, and have lower benzo[a]pyrene degradation ability. In summary, strain Z5 showed strong degradation ability and growth advantage, so we chose strain Z5 as the starting strain for benzo[a]pyrene degradation in this application.

[0047] Morphological characteristics of the strain:

[0048] Strain Z5 grew rapidly and appeared velvety. It was dark green on the PDA plate in the early growth stage and became darker in color in the later growth stage. Figure 3 A). Under the microscope, complex branching hyphae can be observed ( Figure 3 Middle B) and round black spores ( Figure 3 Middle C), the conidial heads are loose and radial, and the apical capsule is approximately spherical ( Figure 3 D), preliminarily diagnosed as mold.

[0049] Genome extraction and ITS rDNA sequence amplification of strain Z5:

[0050] The genome of fungus Z5 was detected by agarose gel electrophoresis. After PCR amplification of ITS rDNA sequence, the length obtained was about 568bp. The sequence was imported into the NCBI database for similar sequence comparison to determine the species of strain Z5.

[0051] Phylogenetic tree construction of strains:

[0052] After obtaining the ITS rDNA sequence of strain Z5, we entered it into the NCBI BLAST database for homologous sequence retrieval. We selected 18 strains belonging to four different genera and strain Z5 from the Genebank database, analyzed the sequences using MEGAX, cut them to achieve maximum homology, constructed a phylogenetic tree using the neighbor-joining method, and performed 1000 bootstrap tests to verify the reliability of the evolutionary tree ( Figure 4 ). The results showed that the sequence similarity between strain Z5 and Aspergillus.fumigatus (NR121481.1) was 99.47%, and the bootstrap value was 100%, indicating the closest evolutionary relationship. Strain Z5 had the most distant evolutionary relationship with A.niger, A.tubingensis, and A.terreus. Therefore, based on the morphological characteristics of strain Z5 and the homology information of the ITS rDNA sequence, strain Z5 belongs to Aspergillus fumigatus of the genus Aspergillus sp. in the phylum Ascomycola. Therefore, strain Z5 was named Aspergillus fumigatus Z5.

[0053] Example 2: Study on the growth characteristics of fungus Z5

[0054] In this example, the strain Z5 obtained by screening in Example 1 was subjected to growth specialization research, and the specific method included:

[0055] First, the moisture content of strain Z5 was determined, and the temperature (20℃, 25℃, 30℃, 37℃, 43℃, 50℃), initial pH (4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0), NaCl concentration (0g / L, 2g / L, 4g / L, 6g / L, 8g / L, 10g / L) and metal ion (Mg) content in PDW culture medium were investigated. 2+ , Mn 2+ , Fe 2+ , Cu 2+ Cr 2+ 、Co 2+ and Pb 2+, three different concentrations of each ion were selected (10, 20, 30 mg / L) on the growth of Z5. All treatment groups were cultured on a shaker at 170 r / min for 48 h, and each treatment was repeated three times.

[0056] The method for determining the moisture content of the bacteria is as follows: first filter the bacteria through a double layer of gauze, then wash with distilled water several times to ensure that they are clean. Then, weigh a certain amount of bacteria, heat them in a drying oven at 105°C to constant weight, and finally calculate the moisture content.

[0057] The method for determining the bacterial growth amount is as follows: first filter the bacterial solution through a double layer of gauze, then wash it with distilled water for several times, collect the wet bacterial balls after filtration and weigh them, and calculate the wet weight to obtain the bacterial growth amount.

[0058] result:

[0059] (1) After measurement, the moisture content of the Z5 bacterial pellet was 93.78%.

[0060] (2) Effect of temperature on strain growth

[0061] Temperature has a greater impact on strain Z5, such as Figure 5 As shown in A, the biomass of strain Z5 is 30.64 g / L at 20°C. As the temperature rises, the biomass of strain Z5 also continues to rise. When the temperature is 43°C, the biomass of strain Z5 reaches a maximum of 289.76 g / L. When the temperature reaches 50°C, the biomass of strain Z5 drops sharply to 187 g / L. Therefore, it is preliminarily judged that strain Z5 is a thermophilic fungus. Considering that high temperature will cause the culture medium to evaporate and affect the growth of the strain, 37°C is selected as the temperature for subsequent experiments.

[0062] (3) Effect of pH on strain growth

[0063] pH has a certain effect on the growth of strain Z5. Figure 5 As shown in B, when pH ≦ 6, the biomass of the strain is less than 175 g / L; when pH = 7, the biomass of the strain is 231.22 g / L; when pH ≧ 8, the biomass of the strain is less than 150 g / L. Therefore, it shows that strain Z5 is suitable for growth under neutral conditions, and acidic and alkaline conditions pose a certain threat to the growth of the strain.

[0064] (4) Effect of salt concentration on strain growth

[0065] Using NaCl as the experimental salt, the results are as follows Figure 5As shown in Figure C, when no NaCl was added to the culture system, the biomass of strain Z5 reached a peak of 227.64 g / L. When the NaCl concentration was 2 g / L, the biomass of Z5 dropped to 164.82 g / L. As the NaCl concentration continued to rise, the growth of strain Z5 was inhibited and the biomass dropped sharply. When the NaCl concentration was 10 g / L, the biomass of Z5 dropped to the lowest value of 47.74 g / L. Therefore, the addition of NaCl to the culture system would affect the growth of strain Z5.

[0066] (5) Effects of metal ions on strain growth

[0067] Different metal ions can promote or inhibit the growth of strain Z5, such as Figure 5 As shown in D. When the concentration of metal ions is 10 mg / L, Mg 2+ , Mn 2+ Cr 2+ and Fe 2+ It significantly promoted the growth of strain Z5 and increased its biomass. When the concentration of metal ions was 20 mg / L, Mg 2+ , Mn 2+ Cr 2+ It significantly promoted the growth of strain Z5 and increased its biomass. When the concentration of metal ions was 30 mg / L, only Mg 2+ and Cr 2+ It significantly promoted the growth of strain Z5. 2+ , Pb 2+ and Co 2+ When the concentration was greater than 10 mg / L, it had a significant inhibitory effect on the growth of strain Z5. 2+ When the concentration is greater than 20 mg / L, it has a strong toxic effect on strain Z5, causing strain Z5 to be unable to grow. 2+ When the concentration was greater than 20 mg / L, it had a significant inhibitory effect on the growth of strain Z5. In summary, the effects of metal ions on strain Z5 varied depending on the type and concentration. When the metal ion concentration was less than 30 mg / L, Mg was selected. 2+ and Mn 2+ It is better to promote the growth of strain Z5.

[0068] Example 3: Study on the degradation characteristics of benzo[a]pyrene by fungus Z5

[0069] This example studies the optimization of degradation conditions for strain Z5, which has a good benzo[a]pyrene degradation effect, including conditions such as temperature, pH, salt concentration, bacterial age and pollutant concentration, and measures the intracellular and extracellular enzyme activities of strain Z5. In addition, this example also measures the types of PAHs degraded by strain Z5, providing more microbial resources for the restoration of PAHs pollution. Finally, the changes in functional groups and intermediate degradation products during the degradation of benzo[a]pyrene were studied by Fourier transform infrared spectroscopy (FT-IR) and gas chromatography-mass spectrometry (GC-MS), providing more basic data for the pathway of benzo[a]pyrene degradation by strain Z5.

[0070] 1. Study results on the degradation performance of strain Z5 on benzo[a]pyrene

[0071] (1) Effect of temperature on degradation ability

[0072] like Figure 6 The results showed that temperature had a significant effect on the degradation of benzo[a]pyrene. From 20 to 30°C, the degradation rate increased with the increase of temperature, and the degradation efficiency was significantly improved (p<0.01). When the temperature was 37°C, the degradation rate reached a peak of 54.62%. However, as the temperature continued to rise, the degradation rate showed a downward trend. At 43°C, the degradation rate was only 43.78%, and the degradation efficiency decreased significantly (p<0.05). This shows that high temperature will reduce the enzyme activity and make the microorganism lack the power to degrade. Therefore, 37°C is the optimal temperature for strain Z5 to degrade benzo[a]pyrene.

[0073] (2) Effect of shaking table speed on degradation capacity

[0074] The results of the speed optimization of strain Z5 are as follows Figure 6 As shown in the figure, when the speed is 130r / min, the degradation rate is 39.04%. As the speed increases, the degradation efficiency of benzo[a]pyrene also increases significantly (p<0.01). When the speed is 170r / min, the degradation rate reaches a peak of 64.66%. After that, as the speed continues to increase, the degradation rate shows a downward trend. When the speed is 190r / min, there is a significant downward trend compared with the degradation rate at 170r / min (p<0.01). Therefore, the optimal degradation speed of strain Z5 is 170r / min.

[0075] (3) Benzo[a]pyrene concentration tolerance

[0076] The appropriate concentration of benzo[a]pyrene can enable the strain to fully exert its activity. The results of benzo[a]pyrene tolerance of strain Z5 are shown in Figure 7As shown. When benzo[a]pyrene concentration is 5mg / L, degradation rate reaches peak value 74.83%, and with the increase of benzo[a]pyrene concentration, degradation efficiency is in a downward trend, and with the degradation efficiency significant difference (p<0.01) at 5mg / L. When benzo[a]pyrene concentration increases to 40mg / L, degradation rate reaches minimum value 23.38%. This may be due to the increase of benzo[a]pyrene concentration, the resistance of cell colony to toxicity weakens, causing strain vigor to decline. However, when benzo[a]pyrene concentration is 2mg / L, degradation efficiency does not reach peak value, which may be due to insufficient carbon source, so that bacterial growth and development are limited, affecting its degradation to benzo[a]pyrene. Therefore, in order to ensure sufficient carbon source in the system and no obvious restriction on the growth of bacterial body, it is 10mg / L to select benzo[a]pyrene concentration in the present embodiment.

[0077] (4) Effect of bacterial age on degradation capacity

[0078] The bacterial age can directly affect the activity and metabolism of the strain. The effect of the bacterial age of strain Z5 on the degradation of benzo[a]pyrene is shown in Figure 8 As shown. The degradation rate of benzo[a]pyrene by the strain cultured at 37℃ for 12h was 49.74%; the degradation rate of the strain cultured for 24h increased to 61.53%, which was significantly different from the strain cultured for 12h (p<0.01); when the culture time increased to 36h, the degradation rate decreased to 41.01%, which was significantly different from the strain cultured for 12h (p<0.01). With the extension of the culture time of the strain, the degradation rate of benzo[a]pyrene continued to decrease, and when the culture time increased to 72h, the degradation rate decreased to 28.12%. Therefore, after strain Z5 was cultured at 37℃ for 24h, the strain had strong activity and the highest degradation rate of 10mg / L benzo[a]pyrene.

[0079] (5) Effect of bacterial dosage on degradation capacity

[0080] The appropriate amount of bacterial cells added can adsorb and degrade more pollutants and improve the degradation efficiency. The effect of the addition amount of strain Z5 on the degradation of benzo[a]pyrene is shown in the figure. Figure 8As shown. When the initial dosage was set to 4g / L, the degradation rate of benzo[a]pyrene was 26.48%. At this time, the degradation rate was low. It may be that the bacterial community was toxic and collapsed due to the lack of bacterial cells, and lost its growth vitality. The degradation rate increased with the increase of the bacterial cell dosage. When the bacterial cell dosage reached 40g / L, the degradation rate of benzo[a]pyrene reached a peak of 63.44%. Compared with the dosage of 30g / L, the degradation efficiency was significantly improved (p<0.001). When the bacterial cell dosage was further increased to 50g / L, the degradation rate of benzo[a]pyrene did not increase, but showed a slight downward trend, and the degradation rate reached 62.93%. This may be because the excessive amount of bacterial cells added caused the bacterial community to compete for the limited nutrients in the culture system, limiting the growth of the bacteria, thereby keeping the degradation rate at a stable level. Therefore, the optimum dosage of strain Z5 is 40 g / L, at which the degradation rate of benzo[a]pyrene is the highest.

[0081] (6) Effect of pH on degradation ability

[0082] Microorganisms are usually sensitive to acidic and alkaline conditions, and most strains have the strongest biological activity under neutral conditions. However, for PAHs-contaminated sites such as oil pollution and coal chemical site pollution, the site environment is alkaline, so optimizing the optimal pH of the strain is the key to improving the remediation efficiency. The pH optimization results of strain Z5 are shown in Figure 2. Figure 8 As shown in the results, when the pH was 5.0-7.0, the degradation rate was maintained at about 30.00%-60.00%. When the pH was 4.0, the degradation rate was the lowest (9.73%). When the pH was 8.0, the degradation rate was the highest (67.43%), which was significantly different from the degradation efficiency at pH = 7.0 (p < 0.01). As the pH increased, the degradation rate gradually decreased. The degradation rate at pH = 9.0 was 52.46%, which was significantly different from the degradation efficiency at pH = 7.0 (P < 0.01). This is different from the growth pH of strain Z5 in PDW liquid medium. Z5 has the largest biomass in PDW liquid medium with a pH of 7.0, while the optimal pH in MSM medium is alkaline. It is speculated that this is because benzo[a]pyrene produces some acidic substances during the degradation process, which neutralizes the alkaline environment. Therefore, pH = 8.0 was selected as the optimal degradation condition for strain Z5 in subsequent experiments.

[0083] (7) Salt concentration tolerance

[0084] Most PAHs-contaminated soils are highly saline and alkaline. Therefore, it is necessary to investigate the tolerance of strain Z5 to NaCl. The results of the tolerance of strain Z5 to NaCl in MSM medium are shown in Figure 2. Fig. 9As shown. When no NaCl was added, the degradation rate of strain Z5 was the highest, reaching 65.72%. When 2g / L NaCl was added, the degradation rate of strain Z5 decreased to 55.36%, which was significantly different from the degradation rate when no NaCl was added (p<0.05). When the NaCl concentration increased to 4g / L, the degradation rate of strain Z5 decreased slightly compared with the addition of 2g / L NaCl. As the NaCl concentration continued to increase, the degradation rate of strain Z5 continued to decrease, which was consistent with Figure 5 The growth trends of strains in C in PDW medium with different NaCl concentrations are consistent, indicating that high salt concentration is not conducive to the growth of strain Z5. Because the high salt concentration environment makes it impossible for the strain cells to reach an isotonic environment, some cells will die. Therefore, this application does not change the NaCl concentration in the MSM medium.

[0085] (8) Heavy metal ion tolerance

[0086] Some metal ions can play an important role as cofactors in the metabolism of microorganisms. The effects of metal ions on the degradation efficiency of strain Z5 are as follows: Fig. 9 When the metal ion concentration is 10 mg / L, Mg 2+ (p<0.01), Mn 2+ (p<0.05), Cr 2+ (p<0.001), Fe 2+ (p<0.001) had a significant promoting effect on the degradation of benzo[a]pyrene. 2+ 、Co 2+ and Pb 2+ It has a significant inhibitory effect on the degradation of benzo[a]pyrene; when the metal ion concentration is 20 mg / L, only Fe 2+ It has a promoting effect on the degradation of benzo[a]pyrene, while other metal ions inhibit the degradation of benzo[a]pyrene; when the metal ion concentration is 30 mg / L, all metal ions inhibit the degradation of benzo[a]pyrene. Therefore, the auxiliary effect of metal ions on microorganisms varies depending on the type and concentration. In this example, when the metal ion concentration is 10 mg / L, Mg 2+ , Mn 2+ Cr 2 + , Fe 2+ It is a beneficial metal ion for strain Z5, which promotes the growth and metabolism of the strain. 2+ When the concentration was increased to 20 mg / L, it could still promote the growth and metabolism of the strain, which was consistent with the effect of metal ions on strain Z5 in PDW medium. In subsequent application experiments, Fe 2+ To promote the degradation of PAHs.

[0087] (9) Tolerance to different PAHs

[0088] In order to study whether strain Z5 has a broad spectrum of PAHs degradation, we selected three-ring phenanthrene and anthracene, four-ring fluoranthene and pyrene, and five-ring benzopyrene, and dissolved them in 50 mL of MSM medium at a final concentration of 50 mg / L. Within 7 days, we found that strain Z5 had different degradation of PAHs of different molecular weights. Fig. 9 As shown in the data, the degradation rate of strain Z5 for benzo[a]pyrene was 15.49%, for fluoranthene was 47.73%, for pyrene was 51.25%, for anthracene was 66.59%, and for phenanthrene was 69.02%. Statistical analysis showed that the degradation of PAHs with less than four rings by strain Z5 was significantly different from that of PAHs with five rings (p<0.0001), which may be caused by the difference in molecular weight. With the increase of molecular weight, PAHs are less susceptible to the attack of fungal intracellular and extracellular enzymes, and their toxicity to cells is also enhanced.

[0089] Therefore, strain Z5 can degrade PAHs with five rings or less, and the smaller the molecular weight, the higher the degradation efficiency, and it has the ability to degrade a broad spectrum of PAHs.

[0090] (10) Degradation results of benzo[a]pyrene under optimal conditions

[0091] According to the results of single factor optimization, this example selected 2 g of cocci (wet weight) activated at 37 ° C for 24 h to be added to 50 mL of MSM medium containing 10 mg / L benzo[a]pyrene, and FeSO4 was added to make Fe 2+ The concentration was 10 mg / L. After that, the culture was incubated at 37°C in a shaker (170 r / min) and pH 8 for 7 days. The residual amount of benzo[a]pyrene was measured every 24 hours without adding any metal ions or other carbon sources. The results are as follows: Fig.10 shown.

[0092] In the early stage of degradation, the degradation rate of benzo[a]pyrene by strain Z5 was less than 10%, which may be because benzo[a]pyrene is a high molecular weight PAHs, which has a greater toxic effect on cells, so the strain needs a certain amount of adaptation time. With the extension of culture time, the degradation rate of benzo[a]pyrene gradually increased, and the degradation rate increased sharply on the 4th day, reaching 73.81% on the 6th day. Compared with the 5th day, the degradation rate was significantly different (p<0.001), and the activity and metabolic capacity of the strain reached a peak at this time. At the end of the degradation, the degradation trend gradually slowed down and stabilized, and the final degradation rate reached 75.43%.

[0093] 2. Research results of co-metabolism carbon source bacteria

[0094] Different carbon sources are used by microorganisms in different ways, so the growth activity and metabolic efficiency they exhibit vary greatly. Although strain Z5 in this application can grow with benzo[a]pyrene as the only carbon source, benzo[a]pyrene is a harmful carbon source and has a certain toxic effect on the strain. In addition, the strain has a certain adaptation period to benzo[a]pyrene, which greatly delays the degradation efficiency. Therefore, it is necessary to add an appropriate proportion of harmless carbon sources and low molecular weight carbon sources to the degradation system to assist the growth and metabolism of microorganisms. The effect of co-metabolism carbon sources on strain Z5 is as follows. Fig.11 As shown. Compared with the control group with benzo[a]pyrene as the only carbon source, after adding different co-metabolism carbon sources, the carbon sources that have a significant promoting effect on the degradation efficiency are glucose (p<0.01), lactose (p<0.05), sucrose (p<0.01), starch (p<0.001), salicylic acid (p<0.0001), phthalic acid (p<0.01), and phenol (p<0.0001). Peptone and yeast powder have no significant promoting effect on the degradation of benzo[a]pyrene. Compared with the control group, salicylic acid and phenol have a very significant promoting effect on the degradation of benzo[a]pyrene (p<0.0001), which may be because salicylic acid and phenol are intermediates in the degradation of benzo[a]pyrene.

[0095] The salicylic acid and phenol added in this example are most likely intermediate products produced during the degradation of benzo[a]pyrene, thereby reducing the reaction energy barrier of benzo[a]pyrene and activating the corresponding enzyme system. Phenol, as a low molecular weight aromatic hydrocarbon compound, can also play a similar auxiliary role, thereby providing sufficient energy for strain Z5 and improving the degradation efficiency.

[0096] 3. Research results of enzymatic reaction of benzopyrene-degrading bacteria

[0097] (1) Qualitative results of lignin peroxidase system

[0098] Different enzymes were qualitatively screened based on the color changes of the lignin peroxidase screening plate. The laccase produced by strain Z5 oxidized guaiacol to iron red ( Fig.12 A), the manganese peroxidase produced oxidizes the phenol red in the culture medium into yellow ( Fig.12 However, there is no reddish brown area on the lignin peroxidase screening plate ( Fig.12 Middle C), indicating that strain Z5 does not produce lignin peroxidase.

[0099] (2) Quantitative results of lignin peroxidase system

[0100] Qualitative analysis showed that strain Z5 could produce laccase and manganese peroxidase. We further conducted quantitative analysis on laccase and manganese peroxidase. Fig.13As shown in Figure A, the production of laccase reached a peak of 26.78 U / L on the 4th day, and then showed a downward trend, and there was a significant difference between the enzyme activities on the 3rd and 4th days (p<0.01). Fig.13 As shown in B, the production of manganese peroxidase reached a peak of 11.18 U / L on the 5th day, and then gradually decreased. There was a significant difference between the enzyme activity on the 4th day and the 5th day (p<0.05).

[0101] (3) Quantitative results of cytochrome P450 enzyme system

[0102] Cytochrome P450 enzymes are important intracellular enzymes of lignin-degrading fungi. Therefore, we conducted a quantitative analysis of the cytochrome P450 enzymes of strain Z5. The changes in cytochrome P450 enzymes within 7 days were as follows: Fig.13 As shown in C, the cytochrome P450 enzyme reached a peak of 44.53 U / L on the 5th day, and the growth trend from the 3rd day to the 4th day was significantly different (p<0.01), and then gradually decreased.

[0103] (4) Results of enzymatic degradation of benzopyrene

[0104] The degradation of benzo[a]pyrene by the complete bacteria, extracellular enzymes and intracellular enzymes of strain Z5 within 120 h is shown in the figure. Fig.13 As shown in D. In the first 72 hours, the degradation trend of benzo[a]pyrene by bacteria, extracellular enzymes and intracellular enzymes was in an upward state, and the degradation efficiency of intact bacteria was always higher than that of extracellular enzymes and intracellular enzymes, which may be the result of the adsorption of bacteria. After 72 hours, the degradation trend of bacteria, extracellular enzymes and intracellular enzymes gradually stabilized, but the degradation efficiency of intracellular enzymes was finally higher than that of extracellular enzymes. Therefore, we preliminarily inferred that the mode of action of strain Z5 in degrading benzo[a]pyrene mainly depends on cytochrome P450 enzymes.

[0105] 4. Functional group changes during benzo[a]pyrene degradation

[0106] This example uses Fourier transform infrared spectroscopy to identify the changes in specific organic functional groups during the degradation of benzo[a]pyrene. The functional group changes of benzo[a]pyrene (red) sampled and tested after 3 days of degradation in MSM culture medium are as follows: Fig.14 Compared with the control group, benzo[a]pyrene showed a decrease in the peak at 3313 cm after 3 days of degradation. -1 and 2360cm -1 An absorption peak appeared, 3313cm -1 The 2360cm-2 is caused by the vibration of the -OH group, indicating that benzo[a]pyrene undergoes an oxygenation process under the action of intracellular and extracellular enzymes of strain Z5, generating benzo[a]pyrene derivatives with -OH groups. -1It is caused by the stretching vibration of the -NH2 group, indicating that some hydrogen atoms of benzopyrene are replaced by -NH2 groups, possibly forming benzo[a]pyreneamine. The secondary metabolites of the strain itself also contain -NH2 groups, which cannot be ignored. 1707cm -1 The absorption peaks at 1357cm are caused by the stretching of C=O of carboxyl, C=O in quinone, ketone and ester groups, indicating that acid, quinone, ketone and ester are generated during the degradation of benzo[a]pyrene, which are intermediate products generated by the action of enzymes inside and outside the cell of strain Z5. -1 The absorption peaks near 1224-1000cm indicate the presence of carboxyl groups, which is a signal for the presence of small molecule compounds. -1 The absorption peaks appearing at 1000-500cm are caused by the deformation and stretching of CH bonds, CC bonds, and CO bonds. CH bonds appear in aliphatic hydrocarbons, aromatic hydrocarbons, alcohols, ethers, etc. The vibration of CC bonds in benzene rings appears in this region, and the stretching vibration of CO bonds in alcohols and ethers also appears in this region. -1 The absorption peaks at 3 and 4 were slightly stronger than those without strain Z5, indicating that a large number of aromatic hydrocarbon compounds appeared in the fermentation broth after 3 days of degradation. The absorption peaks in these two regions indicated that small molecular organic compounds in the fermentation broth were also degraded, further indicating that benzo[a]pyrene was degraded into compounds with smaller molecular weight under the action of strain Z5.

[0107] Benzo[a]pyrene was degraded in MSM medium for 7 days (blue) and the functional group changes of samples were tested. Fig.14 The absorption peak of benzo[a]pyrene after 7 days of degradation has no significant change in type compared with the absorption peak after 3 days of degradation, but has a significant change in absorption intensity. -1 The absorption peak of the -OH group at 2360 cm-1 was significantly enhanced. Combined with the fact that the degradation rate reached a peak on the 5th day under the above optimal conditions, it can be seen that the oxygenation reaction of strain Z5 reached its strongest on the 5th day. -1 The absorption peak of the -NH2 group at 1707 cm was also enhanced, indicating that the reaction of hydrogen atoms being replaced by -NH2 groups was enhanced, and it was more likely that the secondary metabolites containing amino groups of strain Z5 reached the peak. -1 The absorption peak intensities of C=O at carboxyl, C=O in quinone, ketone and ester groups also increased, indicating that the concentrations of acid, quinone, ketone and ester in the fermentation broth increased. -1 The absorption peak near 1224-1000cm is slightly enhanced, that is, the C=O and -OH of the carboxyl group increase, indicating that the carboxyl group in the fermentation broth has increased. -1 The absorption peak intensity of CH bond, CC bond and CO bond at 1000-500cm does not change much, and these functional groups mostly appear in low molecular weight compounds, which can be further degraded into the tricarboxylic acid cycle, so the absorption peak intensity of this band does not change much.-1 The enhanced absorption peak intensity of the side chain aromatic ring indicated that the concentration of small molecule aromatic rings generated during the degradation of benzo[a]pyrene was higher than that on the third day, which means that the degradation ability of strain Z5 was most active at this time.

[0108] In summary, benzo[a]pyrene was degraded under the action of the enzyme secreted by strain Z5. The absorption peaks of -OH group and -NH2 group were significantly enhanced under the action of oxygen. In the further degradation process, the absorption peak intensity of acid, quinone, ketone and ester was enhanced. The absorption peak intensity of the generated carboxyl group and aromatic ring was also enhanced. This is the process of transformation from macromolecular compounds to small molecular compounds.

[0109] This embodiment aims to improve the degradation efficiency of benzo[a]pyrene by strain Z5 by changing environmental parameters. After optimizing single factor conditions (temperature, pollutant concentration, shaker speed, bacterial age, inoculation amount, pH, salt concentration, co-metabolism carbon source, etc.), strain Z5 achieved a degradation rate of 75.43% for 10 mg / L benzo[a]pyrene within 7 days. Tony et al. studied the degradation efficiency of 10 mg / L benzo[a]pyrene by Pleurotuseryngii F032 under static and stirring conditions. The results showed that within 20 days, static culture removed 59% of benzo[a]pyrene, while stirring culture removed the highest amount (73%). In this embodiment, strain Z5 has a strong advantage in the repair cycle and can greatly shorten the repair cycle. NorAsyikin et al. optimized the degradation conditions of benzo[a]pyrene by a strain of Aspergillus brasiliensis (A.brasiliensis) by response surface methodology. The results showed that the optimal pH value for the growth of Aspergillus brasiliensis and the degradation of benzo[a]pyrene was 6, the maximum tolerance concentration of benzo[a]pyrene was 40mg / L, and when surviving under the optimal conditions, the maximum growth amount was 0.254g / L, and the maximum degradation rate of benzo[a]pyrene was 44.14%. This shows that different strains have optimal conditions for the degradation of polycyclic aromatic hydrocarbons, wherein the influence of pH value on the degradation efficiency is relatively large, and most microorganisms prefer to degrade polycyclic aromatic hydrocarbons under neutral pH conditions, because acidic conditions have a destructive effect on microorganisms. However, the optimal pH value of strain Z5 in the present embodiment is 8, which may be due to the generation of acidic groups in the degradation process, forcing the pH value of the culture solution to be neutralized to neutral conditions, avoiding the damage caused to the strain by excessively acidic environments.

[0110] When the metal ion concentration is 10 mg / L, Mg 2+ , Mn 2+ Cr 2+ , Fe 2+It is a beneficial metal ion for strain Z5, which promotes the growth and metabolism of the strain. In addition, strain Z5 can degrade PAHs with five rings or less, and the smaller the molecular weight, the higher the degradation efficiency, and has the ability to degrade a wide spectrum of PAHs. Some metal ions such as Fe 2+ , Mn 2+ 、Co 2+ It has been proven that these metal ions can promote the microbial degradation activity of PAHs. These metal ions may participate in the microbial degradation enzyme system as coenzymes or enzyme active centers, thereby accelerating the degradation rate of PAHs. On the other hand, high concentrations of certain metal ions may inhibit the growth and metabolic activity of microorganisms, thereby slowing down the degradation rate of PAHs. This inhibitory effect may directly affect the physiological activity of microbial cells or inhibit the activity of related degradation enzymes. The sensitivity of some microorganisms to specific metal ions may result in their growth being restricted in an environment containing the metal ions, thereby affecting the overall degradation process. Therefore, in the actual environmental governance and bioremediation process, the influence of metal ions needs to be comprehensively considered to optimize the efficiency and effectiveness of microbial degradation.

[0111] The biodegradation of PAHs by microorganisms depends on the action of intracellular or extracellular enzymes, and fungi mainly rely on the lignin peroxidase system and the cytochrome P450 enzyme system. This example shows that strain Z5 mainly degrades benzo[a]pyrene through the cytochrome P450 enzyme system through the enzymatic degradation reaction and the appearance of dibutyl phthalate, pyrene and benzo[a]pyrene-1,6-diquinone in the degradation products. In fact, [7,10- 14 C BaP] as substrate, by measuring 14 The CO2 release was used to verify whether cytochrome P450 was involved in the oxidation of benzo[a]pyrene. The release amount decreased by 66% after 10 days of cultivation, indicating that this enzyme is involved in the degradation of benzo[a]pyrene.

[0112] Example 4: Degradation of simulated benzo[a]pyrene contaminated soil and analysis of microbial diversity

[0113] In order to verify the remediation efficiency of strain Z5 in the environment, this example simulated benzo[a]pyrene contaminated soil and analyzed the soil microbial diversity. The PAHs-free soil used in this example was collected from a campus garden (Xi'an, China).

[0114] Soil pretreatment method: Put 50g of uncontaminated soil into several 100mL Erlenmeyer flasks, seal them, and sterilize them three times at 121℃. After drying, add acetone-soluble benzo[a]pyrene solution to the non-sterile soil to make the benzo[a]pyrene content in the soil reach 10mg / kg. Then place the soil at room temperature in the dark for more than 2 weeks.

[0115] Soil experiment system: The pretreated soil was tested according to the treatment shown in Table 1. In order to achieve the best degradation effect, it is necessary to determine the optimal addition amount of the strain, set the gradient addition amount of 1g, 2g, 3g, 4g, and 5g, cover with sealing film and place in a 37°C constant temperature incubator, and measure the degradation rate after 30 days. In addition, to ensure the normal growth of the strain, the MSM inorganic salt medium was added for the first water supplement, and then sterile water was used for water supplementation, so that the moisture content of the soil system was about 30%. Samples were taken at 5, 10, 15, 20, 25, and 30 days to detect the residual amount of benzo[a]pyrene.

[0116] Table 1 Experimental design and treatment methods

[0117]

[0118] 1. Results of soil physical and chemical properties test

[0119] After the collected soil without PAHs pollution was sieved (<1mm), 5g was taken to determine the physical and chemical properties of the soil, and the content of organic matter, hydrolyzable nitrogen, available phosphorus, available potassium, pH and conductivity in the soil were detected respectively. After measurement, the physical and chemical properties of the soil collected from the campus are shown in Table 2. The content of organic matter in the soil is 26.8g / kg, the content of hydrolyzable nitrogen is 73mg / kg, the content of available phosphorus is 33.4mg / kg, the content of available potassium is 437mg / kg, the content of N is 1.25g / kg, the conductivity is 19.2S / m, and the pH is 8.32, which is a weakly alkaline soil.

[0120] Table 2 Results of soil physical and chemical properties

[0121]

[0122] 2. Determination results of residual benzopyrene in soil

[0123] In order to detect the degradation ability of strain Z5 on PAHs in soil, this example simulated soil contaminated with benzo[a]pyrene. When the inoculum size of the strain was 3 g ( Fig.15 In A), the degradation rate of benzo[a]pyrene in soil was 31.23%. As the inoculation amount increased, the degradation rate tended to decrease. This may be because too many strains would compete for limited nutritional resources, resulting in a slow growth rate of each strain, thus affecting the overall rate of the degradation reaction. Therefore, it is more appropriate to inoculate 3g of strain Z5 in every 50g of soil. The degradation of non-sterile contaminated soil by strain Z5 within 30 days is shown in Figure 1. Fig.15As shown in Figure B, in the first 20 days, the degradation rate of benzo[a]pyrene continued to rise, and the final degradation rate was 27.76%. As time went on, the degradation rate of benzo[a]pyrene stopped rising, and the limited nutrients in the soil could not provide a lasting energy source for the strain.

[0124] 3. Analysis of soil microbial diversity

[0125] In order to determine the colonization of strain Z5 in the soil and its effect on indigenous microorganisms, 5 g of soil was taken from treatment B (non-sterile soil + free bacteria) on the 20th day and the non-sterile soil stored at low temperature was used for After DNA was extracted by the Spin Kit, it was quickly frozen in liquid nitrogen for 10 minutes and mailed to Shanghai Meiji Biomedical Technology Co., Ltd. with dry ice for environmental PacBio microbial diversity analysis, with 3 replicates for each treatment. The extracted DNA was amplified by PCR, and after fluorescence quantification, it was constructed with PacBio library and sequenced on the machine. The data after the machine was obtained by the ccs module of SMRTLinkv11.0 analysis software to obtain the hifi reads (or ccs reads) sequence file, which is in fastq format. After distinguishing the samples by barcode, the correct sequencing sequence was identified according to the sequencing primer information, and the sequence direction was corrected and the primer sequence was removed, and then OTU clustering and species taxonomy analysis were performed, and statistical analysis of community structure was performed based on taxonomic information.

[0126] Microbial diversity was assessed at the OUT level. After clustering the sequencing sequences at a threshold of 97%, the average OUT of fungi in the control group (CK) was 78, and the average OUT of bacteria was 609. The average OUT of fungi in the treatment group (B) was 40, and the average OUT of bacteria was 660.

[0127] The changes in fungal communities were monitored by sequencing ITS rRNA gene amplicon. The fungi in the soil were analyzed at the microbial phylum level and the results showed that ( Fig.16 In the middle A), the bacterial communities with higher abundance in the control group were Ascomycota, Mortierellomycota, unclassified Fungi, Basidiomycola, Chylridiomycola, Rozellomycota, Blasocladiomycota, and Aphelidiomycota. After adding strain Z5, the abundance of Ascomycota in the soil increased, and the abundance of the remaining bacterial communities decreased, preliminarily indicating that strain Z5 can colonize in the soil.

[0128] In order to gain a deeper understanding of the differences in soil fungi before and after the addition of strain Z5, the heat map method was used to conduct an in-depth analysis of the dominant microbial groups in the soil. The results showed that ( Fig.16 In the middle C), the species with relatively high abundance in the control group were A. fumigatus, N. rubicola, G. circinata, and unclassified Fusicolla. Within 30 days after the addition of strain Z5 (A. fumigatus Z5), the abundance of A. fumigatus in the soil increased, the abundance of N. rubicola changed slightly, while the abundance of G. circinata, unclassified Fusicolla, and other fungi decreased significantly, indicating that strain Z5 has a strong colonization ability in the soil. In addition, it also inhibited the growth of G. circinata, unclassified Fusicolla, and other fungi in the soil.

[0129] The changes in bacterial communities were monitored by 16S rRNA gene amplicon sequencing, and the bacteria in the soil were analyzed at the microbial phylum level. The results showed that ( Fig.16 In the figure (B), the five most abundant bacterial groups are Proteobacteria, Firmicutes, Bacteroidota, Actinobacteria, and Acidobacteria. The addition of strain Z5 increased the abundance of Proteobacteria and Bacteroidota. The heat map method shows the dominant bacterial populations in the soil in detail ( Fig.16 In Figure D), strain Z5 promoted the growth of 12 bacteria, such as Bordetella, Xenophilus, and Devosia, and antagonized the growth of 10 bacteria, such as Bacillus, Symbiobacterium, and Niallia.

[0130] This example aims to simulate the biodegradation process of benzo[a]pyrene contaminated soil to evaluate the effect of strain Z5 in practical applications. The experimental results of inoculating different amounts of strains showed that the most suitable amount of bacteria was 3g of bacterial balls inoculated in 50g of soil. In the 30-day experiment, the degradation rate of the strain for 10mg / kg benzo[a]pyrene in the soil was 27.76%. As time went on, the degradation rate of benzo[a]pyrene gradually stabilized, indicating that the strain was able to use benzo[a]pyrene in the soil as a carbon source.

[0131] The results of the study on microbial diversity in soil revealed the complex ecological effects after adding strain Z5. Previous studies have shown that the basic properties of soil (pH value, particle composition, organic matter, total nitrogen, total phosphorus and available phosphorus) significantly affect the composition and function of the entire microbial community. In addition, the addition of exogenous microorganisms also has a significant effect on the composition of the microbial community. In the study of this embodiment, in addition to the increase in the abundance of Aspergillus fumigatus (A.fumigatus), the abundance of Neosporum (N.rubicola) is relatively stable, and the abundance of G.circinata, unclassified Fusicolla and other fungi have shown a significant downward trend. This shows that strain Z5 may have a certain antibacterial effect in the soil, which limits the growth of certain pathogenic fungi. At the same time, strain Z5 also had a positive impact on a series of bacterial communities, especially the growth-promoting effect on Proteobacteria, Firmicutes, Bacteroidota, Actinobacteria, and Acidobacteria.

[0132] Finally, it should be noted that the above is only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred arrangement scheme, a person skilled in the art should understand that the technical solution of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A benzo[a]pyrene-degrading fungus, characterized in that: The fungus was named Z5, screened from a coal chemical contaminated site, and deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration. Its classification name was Aspergillus fumigatus, the preservation number was CGMCC NO.41499, and the preservation time was September 6, 2024. The fungus can use benzo[a]pyrene as the sole carbon source.

2. The benzo[a]pyrene-degrading fungus according to claim 1, characterized in that Fungus Z5 at 25℃~43℃, pH 6~8 and metal ion Mg 2+ , Mn 2+ Cr 2+ Well tolerated in the presence of 3. A bacterial agent prepared by the benzo[a]pyrene-degrading fungus as claimed in claim 1 or 2, characterized in that: The bacterial agent includes liquid bacterial agent and solid bacterial agent.

4. The bacterial agent according to claim 3, characterized in that The bacterial agent is prepared by the following method: The fungus Z5 with a preservation number of CGMCC NO.41499 was inoculated into PDW liquid culture medium, cultured in a constant temperature shaking incubator at 150 r / min and 37° C. for 36 hours, and the bacterial cells were collected by centrifugation and washed with PBS buffer to obtain the bacterial agent.

5. Use of the fungus Z5 described in claim 1 or 2, or the bacterial agent described in claim 3 or 4 in the treatment of polycyclic aromatic hydrocarbon pollution.

6. The use according to claim 5, characterized in that: The PAH pollution is high molecular weight PAH soil pollution.

7. The use according to claim 6, characterized in that: The high molecular weight polycyclic aromatic hydrocarbon is benzo[a]pyrene.

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