CN113755339A - Phenanthrene degrading fungus in petroleum-polluted soil and preparation and application of microbial inoculum thereof - Google Patents

Abstract

The invention discloses a phenanthrene degrading fungus in petroleum-polluted soil, and preparation and application of a microbial inoculum thereof. Arthrinium gutiae LJD-2 with the deposit number: GDMCC No: 61798. the invention obtains a degrading fungus strain LJD-2 taking phenanthrene as a carbon source by domesticating and separating oil contaminated soil of Shandong, identifies the strain as Arthrium gutiae LJD-2 according to the strain morphology, physiological characteristics, ITS gene sequencing analysis and phylogenetic analysis, and has the optimal growth environmental condition of: the temperature is 28 ℃, the pH value is 6, and sodium chloride is not added; ITS gene sequencing analysis of the strain shows that the strain closest to LJD-2 is Arthrinium gutiae CBS 135835 (the similarity is 98.6%). LJD-2 can use phenanthrene as carbon source at an initial concentration of 50 mg.L^‑1After the inorganic salt culture solution is cultured for 7 days, the degradation rate of the pure bacteria can reach more than 55 percent. After the strain is prepared into the solid microbial inoculum, the degradation rate of phenanthrene can be increased to more than 70%, so that the strain has better application potential in the aspect of bioremediation of polycyclic aromatic hydrocarbon.

Description

Phenanthrene degrading fungus in petroleum-polluted soil and preparation and application of microbial inoculum thereof

The technical field is as follows:

the invention belongs to the field of degradation of organic pollutants, and particularly relates to a phenanthrene degradation fungus in petroleum-polluted soil, and preparation and application of a microbial inoculum thereof.

Background art:

with the rapid development of modern industrial processes, more and more pollutants, especially persistent organic pollutants such as Polycyclic Aromatic Hydrocarbons (PAHs), are generated in industrial operations. PAHs are ubiquitous and continuously accumulated in the environment, and national soil pollution survey reports show that the annual emission of the PAHs in China reaches 11.4 ten thousand tons, and the standard exceeding rate of the point positions of the PAHs in the soil reaches 1.4%. PAHs have the characteristics of potential carcinogenicity, teratogenicity, mutagenicity, biological accumulation and the like, and can form great harm to the ecological environment and human health, so that PAHs pollution is widely concerned by people. The polycyclic aromatic hydrocarbon in the environment is seriously overproof and causes pollution due to artificial activities of industrial and mining industry, agriculture and the like, high background value of the soil environment and the like. In addition, the main pollutants of important organic pollution places such as chemical industry park areas, peripheral soil, oil extraction areas, mining areas, sewage irrigation areas and the like mostly contain high-concentration polycyclic aromatic hydrocarbons. Phenanthrene is tricyclic aromatic hydrocarbon, has a very close relationship with carcinogenicity of PAHs, and becomes a model compound for researching the PAHs by virtue of a unique chemical structure.

The natural attenuation of toxic and harmful organic pollutants in the environment mainly depends on the metabolism of related microorganisms, and the bioremediation technology hasLow cost, good effect, no secondary pollution and the like, so the method is the most potential restoration means for restoring PAHs pollution at present. At present, few phenanthrene-degrading fungal strains have been reported, mainly including Trichoderma, Scedosporium, Fusarium, Penicillium, and Aspergillus. Since most of the microorganisms in the environment are non-culturable, many microorganisms, especially those with specific functions, cannot be isolated by pure culture. Therefore, the screening of the bacterial strain capable of effectively degrading high-concentration phenanthrene has important application value and practical significance. The mass concentration of the test is 50 mg.L^-1The phenanthrene is used as a substrate for degrading the strain, so that data support is provided for biological treatment of the polycyclic aromatic hydrocarbon.

The invention content is as follows:

the first object of the present invention is to provide Arthrinium gutiae LJD-2 having phenanthrene degradation ability, which was deposited at the Guangdong province culture Collection (GDMCC) at 7/12/2021, address: building 5 of first furnance, large yard, 100, building 59, Guangdong province, Guangzhou, China, zip code: 510070, accession number: GDMCC No: 61798.

the second purpose of the invention is to provide the application of Arthrinium gutiae LJD-2 in degrading phenanthrene.

Preferably, the degradation phenanthrene is phenanthrene in petroleum-polluted soil.

Preferably, Arthrinium gutiae LJD-2 is applied in the environment polluted by phenanthrene to degrade phenanthrene.

The third object of the present invention is to provide a phenanthrene-degrading microbial agent comprising Arthrinium gutiae LJD-2 as an active ingredient.

Preferably, the preparation method of the phenanthrene degradation microbial inoculum comprises the following steps:

a. heating corn and water according to a mass ratio of 1:5 to prepare paste, and mixing the paste according to a mass ratio of 150: adding wood dust, wheat bran and sodium lignosulfonate at a ratio of 100:10:1, kneading into a dough at a low temperature, putting the mixture of the dough-shaped culture matrixes into a pill kneading machine to prepare spherical culture matrixes, and sterilizing and drying for later use;

b. arthrinium gutiae LJD-2 is prepared into bacterial liquid.

c. Adding the bacterial liquid into a sodium alginate solution with the mass fraction of 3% according to the mass ratio of 1:10, fully mixing the spherical culture medium with the solution, adding a sterile calcium chloride solution with the mass concentration of 4% after the mixture is completely mixed, and hardening to obtain the pellet-shaped encapsulated fungi;

d. and (3) putting the encapsulated fungus pellets into a sterile culture bag, culturing in an incubator at 28 ℃ for 3-7 days until white hyphae grow on the surface, thus obtaining the phenanthrene degradation microbial inoculum.

Preferably, the bacterial liquid in the step b is bacterial liquid with hypha content of 10 g/L.

The fourth purpose of the invention is to provide a method for degrading phenanthrene, which is implemented by spraying Arthrinium gutiae LJD-2 in an environment containing phenanthrene.

Preferably, Arthronium gutiae LJD-2 is sprinkled in the environment polluted by phenanthrene to degrade the phenanthrene.

Preferably, Arthrinium gutiae LJD-2 is sprinkled in the soil polluted by petroleum to degrade phenanthrene.

The invention obtains a degrading fungus strain LJD-2 taking phenanthrene as a carbon source by domesticating and separating oil contaminated soil of Shandong, identifies the strain as Arthrium gutiae LJD-2 according to the strain morphology, physiological characteristics, ITS gene sequencing analysis and phylogenetic analysis, and has the optimal growth environmental conditions as follows: the temperature is 28 ℃, the pH value is 6, and sodium chloride is not added; ITS gene sequencing analysis of the strain shows that the strain closest to LJD-2 is Arthrinium gutiae CBS 135835 (the similarity is 98.6%). LJD-2 can use phenanthrene as carbon source at an initial concentration of 50 mg.L^-1After the inorganic salt culture solution is cultured for 7 days, the degradation rate of the pure bacteria can reach more than 55 percent. After the strain is prepared into the solid microbial inoculum, the degradation rate of phenanthrene can be increased to more than 70%, so that the strain has better application potential in the aspect of bioremediation of polycyclic aromatic hydrocarbon.

Arthrinium gutiae LJD-2 was deposited at 12 months 7/2021 at the Guangdong province culture Collection (GDMCC) address: building 5 of first furnance, large yard, 100, building 59, Guangdong province, Guangzhou, China, zip code: 510070, accession number: GDMCC No: 61798.

description of the drawings:

FIG. 1 shows the front and back surfaces of strain LJD-2 grown on PDA medium for 72 hours.

FIG. 2 shows the phylogenetic relationship of strain LJD-2 and ITS related bacteria based on ITS gene sequences, which is constructed by the neighbor joining method, and the setting of the auto-expansion value is repeated 1000 times, only the result of the auto-expansion value greater than 50% is shown in the figure, and the scale bar 0.01 represents the substitution rate of each nucleotide.

FIG. 3 shows the growth of strain LJD-2 under different temperature, pH and salinity tolerance conditions.

FIG. 4 shows the degradation efficiency of strains LJD-2 and LJD-2 inoculum (LGD-2-agent) in medium with inorganic salts of phenanthrene (initial concentration 50 mg. multidot.L)^-1)。

The specific implementation mode is as follows:

the following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Example 1: isolation and characterization of Arthrinium gutiae LJD-2

1. Materials and methods

1.1 sample sources

Taking a soil sample from a certain petroleum-polluted site in Shandong, carrying out long-term domestication by taking high-concentration phenanthrene as a carbon source, and obtaining the efficient phenanthrene degrading bacteria through multiple screening, separation and purification.

1.2 culture Medium

1.2.1 inorganic salt Medium

The inorganic salt culture medium is used for enrichment culture of microorganisms in a sample, and phenanthrene degradation experiments under the conditions of pure bacteria and microbial agents. The medium formulation is shown in table 1. The preparation method comprises adding the above materials into solvent water, mixing, and sterilizing.

TABLE 1 inorganic salt media formulation

1.2.2 nutrient Medium

The nutrient medium is used for culturing conventional microorganisms such as separation, purification, preservation, activation and the like of fungi. The types and compositions of the liquid nutrient media used in this experiment are shown in Table 2. If a solid culture medium needs to be prepared in an experiment, only agar powder with the mass fraction of 1.5-2% needs to be added on the basis of the original culture medium formula. If the culture conditions of the strains are not specified, the pH of the culture medium is adjusted to 6. The preparation method comprises adding the above materials into solvent water, mixing, and sterilizing.

TABLE 2 Potatto-Dextrose broth (PDA) composition

1.3 acclimatization, screening and isolation of strains

Adding the collected contaminated soil into inorganic salt culture medium, adding streptomycin sulfate and penicillin (concentration is 100ug/ml) to inhibit fungus growth, and adding 100 mg.L^-1The phenanthrene is used as a degradation substrate and is placed in an incubator at 28 ℃ and subjected to shake culture in the dark. 10% of the inoculum size was transferred to fresh 100 mg.L with the same culture system^-1And (3) in an inorganic salt culture medium with phenanthrene as a carbon source, repeating the enrichment process, wherein 7d is an acclimatization period, and repeating the process for three times.

And (3) coating and separating the fourth generation enrichment culture sample obtained by the method of dilution plate, and separating the sample by using nutrient medium. Culturing the coated sample at 28 deg.C for about 48 hr to form obvious single colony on the surface of the culture medium, selecting different single colonies according to the characteristics of colony, such as morphology, size, color, and hypha, streaking, purifying, and culturing. If single colonies of different characteristics are still observed on the streaked plates, they are streaked again until only single colonies of the same characteristics are observed on the same plate. 1 strain LJD-2 with high degradation performance to phenanthrene is obtained by screening in the experiment. Selecting purified single colony to culture in corresponding solid test tube nutrient medium, sealing with sterilized liquid paraffin, and storing at-4 deg.C for a long period.

A. Morphological characteristics

The strain LJD-2 is a fungus separated from petroleum-contaminated soil in Shandong, and can form a colony with a diameter of 8mm and a round shape with upward growth of white villous hyphae after being activated and growing on a PDA plate for 72 hours under the aerobic condition at 28 ℃ (FIG. 1). The strain is an obligate aerobic strain.

B. Molecular biological characteristics of Strain LJD-2

The molecular biological characteristic identification mainly comprises sequencing and the construction of a phylogenetic tree. Before sequencing and constructing phylogenetic tree, DNA of fungus needs to be extracted (the rapid extraction kit of the fungus genome DNA used in the experiment is from the company of biological engineering (Shanghai)). In order to study the taxonomy of fungi, it is usually necessary to amplify ITS gene, which is a piece of DNA in the eukaryotic coding rRNA component, and construct phylogenetic tree, which is usually used for detecting and identifying fungi due to ITS high degree of conservation, specificity and appropriate sequence length.

The Polymerase Chain Reaction (PCR) is mainly used for amplifying different gene fragments, the PCR requires different primers (ITS1: 5'-TCCGTAGGTGAACCTGCGG-3'; ITS4: 5'-TCCTCCGCTTATTGATATGC-3'), and the PCR amplification reaction system: 10 XBuffer 2.5. mu.l, Mg²⁺1.5. mu.l (25mmol/l), 0.3. mu.l dNTP (25mmol/l), 0.5. mu.l forward primer (10mmol/l), 0.5. mu.l reverse primer (10mmol/l), Taq enzyme: 0.25. mu.l, 0.1. mu.l of DNA set template, 19.35. mu.l of deionized water. PCR amplification reaction conditions: pre-denaturation at 95 ℃ for 3min, annealing at 95 ℃ for 45s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 45s, for 30 cycles. Extending for 10min at 72 ℃, and storing at 4 ℃ after the reaction is finished. Amplifying the needed gene, adding 0.75-1% agarose and nucleic acid stain GelRed to prepare a gel block, adding PCR products and DNA markers (marker) containing fragments of various lengths into the gel block, placing the gel block into an electrophoresis apparatus, filling TBE (Tris boric acid) buffer solution into the electrophoresis apparatus, taking out the gel block after the electrophoresis apparatus works for 20min under a certain voltage, and placing the gel block under an ultraviolet lamp of 300nm for observation to confirm that the PCR product amplification reaction is successful. Then theThe pcr product successfully amplified is sent to Huada Gene science and technology Limited for sequencing, and the sequencing primer is the same as the amplification primer.

The sequencing obtained fungal ITS gene sequence is uploaded to EzTaxon-e (http:// EzTaxon-e. ezbiocloud. net), the website compares the submitted sequence with the ITS gene sequence of a recognized typical strain to obtain the similarity information between the sequences, the corresponding typical strain can be selected as the model bacteria of the experimental isolate strain according to the analysis of the sequence comparison result, meanwhile, the ITS gene sequence of the model bacteria can be obtained, phylogenetic analysis is constructed to prove that the model bacteria and the experimental isolate strain have difference so as to identify the isolated strain, the phylogenetic tree is constructed by utilizing MEGA 5.05 program, the adjacency method, the minimum evolution method and the maximum reduction method are usually adopted to construct the evolutionary tree, wherein the most common adjacency method is the adjacency method, and the self-development value is usually set to repeat 1000 times of calculation.

An ITS gene sequence with the length of 623bp obtained by PCR and gene sequencing has a nucleotide sequence shown in SEQ ID NO. 1. The ITS gene alignment shows that the similarity of the LJD-2 strain and Arthrinium gutiae CBS 135835 gene is 98.5%.

And (3) making a phylogenetic tree by using the ITS gene sequence of the LJD-2 and the ITS gene sequence with higher similarity, thereby obtaining a homology result between the ITS gene of the LJD-2 and the ITS gene with higher similarity. A phylogenetic tree constructed by the orthotopic grafting method is shown in FIG. 2.

From the above results, it was found that the fungus LJD-2 isolated in this experiment is Arthrinium gutiae, which has accession number NR 154698.1 in GenBank. Named Arthrinium gutiae LJD-2, deposited at the Guangdong province Collection of microorganisms (GDMCC) at 12 months 7 in 2021, address: building 5 of first furnance, large yard, 100, building 59, Guangdong province, Guangzhou, China, zip code: 510070, accession number: GDMCC No: 61798.

example 2: growth conditions of Arthrinium gutiae LJD-2

A. Measurement of growth temperature:

a liquid nutrient medium (PDA medium in example 1) required for growth of the strain was prepared, and the prepared liquid nutrient medium was sterilized in a sterilizer. The activated strain Arthrinium gutiae LJD-20.1 ml is inoculated into 10ml PDA culture medium (experimental group), the PDA culture medium without fungus inoculation is used as a control group, the culture medium is placed at different temperatures for 7 days, the control group and each experimental group corresponding to the temperature are repeated three times, the growth condition of the fungus is observed every day, after 7 days, the culture medium is poured into a weighing centrifuge tube, then the culture medium is centrifuged for 30min by 5000 turns, after the supernatant is poured out, the culture medium is placed into a 60-DEG oven to be dried to constant weight, and the dry weight of the fungus hyphae is weighed and calculated. The test temperatures were as follows: 18 ℃, 23 ℃, 28 ℃, 33 ℃ and 38 ℃.

B. Measurement of growth pH:

preparing a liquid nutrient medium (PDA medium in example 1) required for strain growth, and adjusting the pH of the culture solution with the following buffer system, wherein the pH is 4.0-5.0, 0.1mol/l sodium citrate and 0.1mol/l citric acid; pH 6.0-8.0, 0.1mol/l NaOH and 0.1mol/l KH₂PO₄；pH 9.0，0.1mol/l NaHCO₃And 0.1mol/l Na₂CO₃. The activated Arthronium gutiae LJD-20.1 ml strain is inoculated into 10ml PDA culture media (experimental group) with different pH values, each pH value is repeated three times, the culture media without fungi inoculation are used as a control, the culture media are placed into the optimum temperature for new fungi growth for 7d, the growth condition of the fungi is observed every day, after 7 days, the culture media are poured into a weighed centrifuge tube, then the centrifuge tube is rotated 5000 times for centrifugation for 30min, after the supernatant is poured out, the culture media are placed into a 60-DEG oven to be dried to constant weight, and the dry weight of the fungal hyphae is weighed and calculated. The pH tested was as follows: 4.0, 5.0, 6.0, 7.0, 8.0, 9.0.

C. Salt concentration tolerance: liquid nutrient medium (PDA medium of example 1) required for growth of the strain was prepared, and the salt concentration of the medium was adjusted. Inoculating activated Arthrinium gutiae LJD-20.1 ml of strain into 10ml of PDA culture medium (experimental group) with different salt concentrations, repeating three times for each salt concentration, using culture medium without inoculation as a control, placing the culture medium at the optimum temperature for new bacterium growth for culturing for 7d, pouring the culture medium into a centrifuge tube for weighing after 7 days, centrifuging for 30min at 5000 revolutions, pouring out supernatant, placing into a 60-DEG oven for drying to constant weight, weighing and calculating the dry weight of fungal hyphae to obtain the salt concentration range which can be tolerated by the new bacterium. The salt concentrations tested were as follows: the mass fraction is 0%, 2%, 4%, 6%, 8%, 10%.

As shown in FIG. 3, in the PDA culture medium, LJD-2 can grow at 23-38 ℃, and the optimal growth temperature is 28 ℃ of the enrichment temperature of the strain; the strain can grow under the pH condition of 4.0-9.0, and the optimal growth pH is 6.0; the bacteria have weak salt tolerance, can grow under the condition that the salt concentration is 0-6%, and grow best under the condition of no salt.

Example 4: experiment on degradation of phenanthrene

Preparation of microbial inoculum

1. Heating corn and water according to a mass ratio of 1:5 to prepare paste, and mixing the paste according to a mass ratio of 150: adding wood dust (200 mesh sieve), wheat bran and sodium lignosulfonate into the mixture at a ratio of 100:10:1, and kneading the mixture into a dough below the temperature. Placing the mixture of the bulk culture medium into a pill rolling machine to obtain spherical culture medium with diameter of 8mm, sterilizing, and oven drying.

2. And preparing the cultured LJD-2 fungi into a bacterial liquid with the hypha content of 10 g/L.

3. Adding the bacterial liquid into a sodium alginate solution with the mass fraction of 3% according to the mass ratio of 1:10, fully mixing the spherical culture medium and the solution, adding a sterile calcium chloride solution with the mass concentration of 4% after the mixture is finished, and hardening for 20min to obtain the pellet-shaped encapsulated fungi.

4. And (3) putting the encapsulated fungus pellets into a sterile culture bag, culturing in an incubator at 28 ℃ for 3-7 days until white hyphae grow on the surface, and thus obtaining the LJD-2 microbial inoculum.

Secondly, respectively inoculating the strain LJD-2 or LJD-2 microbial inoculum after 7 days of activated culture to a strain containing 50 mg.L initially according to the inoculation amount of 10 percent^-1The phenanthrene was cultured in an inorganic salt medium (example 1, NaCl removed, pH 6.0) at 28 ℃ for 7 days with shaking, and 3 parallel experiments were performed. The control treatment was carried out without adding any of the pure Arthronium gutiae LJD-2 and LJD-2 agents.

Taking each processed sample for chemical analysis, and the specific steps are as follows: (1) sample pretreatment: adding dichloromethane into each culture sample for extraction, simultaneously adding 5 mu L of recovery rate indicator (phenanthrene-d 10) with the concentration of 200mg/L, fully shaking, and transferring into a separating funnel for standing. And collecting organic phases after layering, putting the lower layer liquid back to a shake flask, repeatedly extracting with dichloromethane of the same volume, combining the extracts, transferring the combined extracts to a flat-bottomed flask containing a proper amount of activated copper sheets for rotary evaporation, concentrating to about 2mL, adding a small amount of n-hexane (about 5mL), carrying out rotary evaporation to 2mL, repeatedly washing for three times, and replacing the organic solvent with the n-hexane. The concentrate after the displacement was purified by means of a glass-packed column (diameter: about 9 mm). The column packing was 3cm 3% deactivated neutral alumina, 3cm 3% deactivated silica gel and 1cm anhydrous sodium sulfate from bottom to top. Activating the column with an appropriate amount of n-hexane, rinsing the packed column with 15mL of mixed n-hexane/dichloromethane (volume ratio of 1:1), collecting about 15mL of eluate in a brown reagent bottle, blowing nitrogen to concentrate the eluate to about 0.5mL, transferring the eluate to a 1.5mL cell bottle, and freezing and storing the eluate. 5 mul internal standard hexamethylbenzene was added before the machine measurement, and the concentration was 200 mg/L. (2) Analyzing by an instrument: an Agilent 7890 gas chromatograph-5975 mass spectrometer is used for measuring the content of PAHs. The separation and analysis were carried out by means of an Agilent DB 5-MS (column length 30m, inner diameter 0.25mm, film thickness 0.25 μm) capillary chromatography column at a flow rate of 1.2 ml/min. The data obtained were processed using an agilent chromatography workstation and phenanthrene quantification was performed using a 6-point calibration curve and an internal standard method.

The strain LJD-2 and the microbial inoculum can degrade phenanthrene by GC-MS determination and analysis, and the degradation rate can reach more than 55% after the strain is cultured in an inorganic salt culture solution containing phenanthrene with the concentration of 50mg/L for 7 days (figure 4). The degradation rate of the strain LJD-2 is 55.3%, the degradation efficiency of the microbial inoculum is 73.5%, and the degradation efficiency is increased by 18.2% in a same ratio, so that the strain LJD-2 is a powerful bacterium capable of degrading phenanthrene, and the effect is better after the strain is prepared into the microbial inoculum.

And (4) conclusion:

1.1 strain of phenanthrene degrading bacteria LJD-2 which can grow by taking phenanthrene as a carbon source is obtained by enrichment and separation from petroleum polluted soil in Shandong, and the solid microbial inoculum is prepared.

2. The strain can form a colony with the diameter of about 8mm and growing upwards of white, round and white villous hyphae. The strain is an obligate aerobic strain. According to the analysis of a molecular biology method, the fungus LJD-2 separated in the experiment can be obtained as Arthrinium gutiae strain, and a clade thereof is drawn. At present, few reports are made on the application of the strain, and particularly, the research on degrading phenanthrene by using the strain is not reported.

3. The optimal growth condition of the strain LJD-2 is to determine that the optimal growth condition of the strain is temperature of 28 ℃, pH 6.0 and NaCl is not added. LJD-2 can be used as carbon source and degrade phenanthrene whose initial concentration is 50 mg.L^-1After the inorganic salt culture solution is cultured for 7 days, the degradation rate of the inorganic salt culture solution can reach 55.3 percent. And the solid microbial inoculum prepared from LJD-2 has better effect of explaining phenanthrene, and reaches 73.5 percent. In conclusion, LJD-2 is a strain capable of degrading phenanthrene, has strong adaptability to polycyclic aromatic hydrocarbon, and the prepared microbial inoculum has better degradation effect and better application potential in the aspect of bioremediation.

Sequence listing

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Claims (10)

1. Arthrinium gutiae LJD-2 with the deposit number: GDMCC No: 61798.
2. 2. use of Arthrinium gutiae LJD-2 of claim 1 for degrading phenanthrene.
3. 3. The use according to claim 2, wherein the degrading phenanthrene is degrading phenanthrene in petroleum contaminated soil.
4. 4. The use according to claim 2, wherein Arthrinium gutiae LJD-2 is used in a phenanthrene contaminated environment to degrade phenanthrene.
5. 5. A phenanthrene-degrading bacterial agent comprising Arthrinium gutiae LJD-2 of claim 1 as an active ingredient.
6. 6. The phenanthrene degradation microbial inoculum according to claim 5, wherein the preparation method of the phenanthrene degradation microbial inoculum comprises the following steps:

   a. heating corn and water according to a mass ratio of 1:5 to prepare paste, and mixing the paste according to a mass ratio of 150: adding wood dust, wheat bran and lignin sodium lignosulfonate at a ratio of 100:10:1, kneading into a dough at a position below zero, putting the mixture of the dough-shaped culture matrixes into a pill kneading machine to prepare spherical culture matrixes, and sterilizing and drying for later use;

   b. arthrinium gutiae LJD-2 is prepared into bacterial liquid.

   c. Adding the bacterial liquid into a sodium alginate solution with the mass fraction of 3% according to the mass ratio of 1:10, fully mixing the spherical culture medium with the solution, adding a sterile calcium chloride solution with the mass concentration of 4% after the mixture is completely mixed, and hardening to obtain the pellet-shaped encapsulated fungi;

   d. and (3) putting the encapsulated fungus pellets into a sterile culture bag, culturing in an incubator at 28 ℃ for 3-7 days until white hyphae grow on the surface, thus obtaining the phenanthrene degradation microbial inoculum.
7. 7. The phenanthrene degradation bacterial agent according to claim 6, wherein the bacterial liquid in the step b is 10g/L bacterial liquid.
8. 8. A method for degrading phenanthrene, comprising spraying Arthrinium gutiae LJD-2 of claim 1 into an environment containing phenanthrene to degrade the phenanthrene.
9. 9. The method of claim 8, wherein the degradation of phenanthrene is carried out by spraying Arthrinium gutiae LJD-2 into a phenanthrene contaminated environment.
10. 10. The method of claim 9, wherein the degradation of phenanthrene is carried out by spraying Arthrinium gutiae LJD-2 on the petroleum contaminated soil.

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