CN113755337B - Polycyclic aromatic hydrocarbon degrading fungi GIG-1 and GIG-2 in petroleum-polluted soil, mixed microbial inoculum thereof and application thereof - Google Patents

Abstract

The invention discloses polycyclic aromatic hydrocarbon degrading fungi GIG-1 and GIG-2 in petroleum polluted soil, a mixed microbial inoculum thereof and application thereof. The invention domesticates and separates from the petroleum polluted soil of Shandong to obtain two degrading fungus strains GIG-1 and GIG2 which take phenanthrene as carbon source. Both strains can utilize phenanthrene as a carbon source, and the initial concentration of phenanthrene is 50 mg.L^‑1After the inorganic salt culture solution is cultured for 7 days, the degradation rate of both GIG-1 and GIG2 can reach more than 40 percent. After the strains are mixed and prepared into a solid microbial inoculum for use, the degradation rate of phenanthrene can be increased to over 73 percent, so that the microbial inoculum prepared from the two strains has better application potential in the aspect of bioremediation of polycyclic aromatic hydrocarbons in petroleum-polluted fields.

Description

Polycyclic aromatic hydrocarbon degrading fungi GIG-1 and GIG-2 in petroleum-polluted soil, mixed microbial inoculum thereof and application thereof

The technical field is as follows:

the invention belongs to the field of degradation of organic pollutants, and particularly relates to polycyclic aromatic hydrocarbon degrading fungi GIG-1 and GIG-2 in petroleum-polluted soil, and a mixed microbial inoculum and application thereof.

Background art:

petroleum pollution has become one of the major environmental problems to be solved urgently in China. Since the content of hydrocarbons in petroleum can reach 95-99% of the total weight of petroleum, the treatment and research of hydrocarbon substances are generally regarded as the main tasks for treating petroleum pollution. Among hydrocarbons, Polycyclic Aromatic Hydrocarbons (PAHs) are a typical persistent organic pollutant with potential teratogenic, carcinogenic and mutagenic properties and neurotoxicity, and are a long-standing focus of research, being more toxic and more difficult to treat than aliphatic and naphthenic hydrocarbons. Phenanthrene is tricyclic aromatic hydrocarbon, has a very close relationship with carcinogenicity of PAHs, and becomes a model compound for researching biodegradation of 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 has the advantages of low cost, good effect, no secondary pollution and the like, so the method is the most potential remediation means for the current PAHs pollution remediation. 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, and phenanthrene degrading microorganisms are screened so as to provide data support for biological treatment of polycyclic aromatic hydrocarbon.

The invention content is as follows:

the first object of the present invention is to provide Cephalotrichlorophallum GIG-1 or Sceosporium dehalogii GIG-2 having polycyclic aromatic hydrocarbon-degrading ability.

Cephalotrichum dendrocephalum GIG-1 was deposited at the Guangdong province culture Collection (GDMCC) at 12.7.2021, address: building 5 of first furnance, large yard, 100, building 59, Guangdong province, Guangzhou, China, zip code: 510070, accession number: GDMCC No: 61800.

scedosporium dehalogii GIG-2, which was deposited at 12 months 7 in 2021 at the Guangdong province collection of microorganisms (GDMCC), address: building 5 of first furnance, large yard, 100, building 59, Guangdong province, Guangzhou, China, zip code: 510070, accession number: GDMCC No: 61802.

the reported fungal strains in this study were Cephalotrichum dendrophilum GIG-1 and Scedosporium dehalogii GIG-2, which were isolated from various terrestrial and aquatic habitats. At present, the research reports about the degradation of the pollutants of the two fungi are few, and the research reports about the degradation of phenanthrene by the fungi are not available at home and abroad. The research domesticates and separates two strains GIG-1 and GIG2 which take high-concentration phenanthrene as carbon sources from a petroleum-polluted site in Shandong, identifies the strains, researches the growth characteristics of the strains, and simultaneously performs the following steps of: 1 proportion is mixed to prepare the microbial inoculum, the degradation characteristic of the microbial inoculum on phenanthrene is explored, and reference is provided for bioremediation of PAHs polluted environment.

The second purpose of the invention is to provide the application of Cephalotrichum dendrocephalum GIG-1 or Sceosporium dehalogii GIG-2 in degrading polycyclic aromatic hydrocarbon.

Preferably, the polycyclic aromatic hydrocarbon is degraded polycyclic aromatic hydrocarbon in soil or sewage.

Preferably, the polycyclic aromatic hydrocarbon is phenanthrene.

The third object of the present invention is to provide a polycyclic aromatic hydrocarbon-degrading bacterial agent comprising, as an active ingredient, a cephalothin dendrimer GIG-1, a scorosporium dehalogii GIG-2 or a mixed bacterial agent thereof.

The preparation method of the mixed 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. the cultured fungal solutions of Cephalotrichum dendrophilum GIG-1 and Sceosporium dehalogii GIG-2 were cultured in the following ratio of 1:1, mixing the materials in a mass ratio to prepare bacterial liquid with the content of 10 g/L;

c. adding the bacterial liquid into a 3% sodium alginate solution according to the volume ratio of 1:10, fully mixing the spherical culture medium and the bacterial liquid, adding a sterile calcium chloride solution with the mass concentration of 4% by mass after the mixture is finished, and hardening to obtain pills of 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, and thus obtaining the mixed microbial inoculum of the strains GIG-1 and GIG 2.

The fourth purpose of the invention is to provide a method for degrading polycyclic aromatic hydrocarbon, which is to sprinkle the Cephalotrichum dendrophilum GIG-1, Sceosporium dehalogii GIG-2 or the mixed microbial inoculum thereof in the environment containing polycyclic aromatic hydrocarbon to degrade the polycyclic aromatic hydrocarbon.

Preferably, phenanthrene is degraded by spraying the phenanthrene contaminated environment with Cephalotrichum dendrophilum GIG-1, Sceosporium dehalogii GIG-2, or a mixture thereof.

The invention domesticates and separates from certain oil contaminated soil of Shandong to obtain two degrading fungus strains GIG-1 and GIG2 which take phenanthrene as carbon source, according to the strain morphology, physiological characteristics, ITS gene sequencing analysis and phylogenetic analysis, the strains are identified as Cephalotrichum dendrophilum GIG-1 and Sceosporium deologii GIG2, and the optimal environmental conditions for growth are as follows: the temperature is 28 ℃, and the pH value is 6; ITS gene sequencing analysis result of the strain shows that the strain Cephalotrichum dendrocephalum LZ16-03(MT898686.1) is most similar to GIG-1 (similarity is 100%); the most similar strain of GIG-2 was Scedosporium dehalogii DTO 401-F6(MT316369.1) (similarity 100%). Both strains can utilize phenanthrene as a carbon source, and the initial concentration of phenanthrene is 50 mg.L^-1After the inorganic salt culture solution is cultured for 7 days, the degradation rate of both GIG-1 and GIG2 can reach more than 40 percent. After the strains are mixed and prepared into a solid microbial inoculum for use, the degradation rate of phenanthrene can be increased to over 73 percent, so that the microbial inoculum prepared from the two strains has better application potential in the aspect of bioremediation of polycyclic aromatic hydrocarbons in petroleum-polluted fields.

Cephalichum dendrocephalum GIG-1, deposited at the Guangdong province collection of microorganisms (GDMCC) at 7/12/2021, address: building 5 of first furios middle way 100 large yard 59, Guangdong province, Guangzhou, zip code: 510070, accession number: GDMCC No: 61800.

scedosporium dehalogii GIG-2, deposited at 12 months 7 of 2021 at Guangdong province collection of microorganisms (GDMCC), address: building 5 of first furnance, large yard, 100, building 59, Guangdong province, Guangzhou, China, zip code: 510070, accession number: GDMCC No: 61802.

description of the drawings:

FIG. 1 is front and back sides of GIG-1(a) and GIG-2(b) grown on PDA medium for 72 hours.

FIG. 2 is an ITS gene sequence-based phylogenetic relationship between GIG-1(a) and GIG-2(b) and related bacteria, constructed by the neighbor joining method, in which the setting of the bootstrap value is repeated 1000 times, and only the results of the bootstrap value of more than 50% are shown, and scales 0.01 and 0202 represent the substitution rate per nucleotide.

FIG. 3 is a graph showing that GIG-1(a) and GIG-2(b) were grown under different temperature, pH conditions;

FIG. 4 is a graph showing the degradation efficiency of GIG-1 and GIG-2 and their mixed Microbial Inoculum (MIX) in the inorganic salt medium 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 Cephalotrichum dendrophilum GIG-1 and Sceosporium dehalogii GIG-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 pure bacteria conditions. 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 bacteria. 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 bacterial growth, respectively at concentration of 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. Using 100 mg.L^-1And (4) carrying out strain domestication on an inorganic salt culture medium with phenanthrene as a carbon source, wherein 7d is a domestication period. Inoculating 10% of the inoculum size by volume to a fresh medium with the same culture system at 100 mg.L^-1The enrichment process is repeated three times in an inorganic salt culture medium with phenanthrene as a carbon source.

The fourth generation enrichment culture samples obtained above were spread and isolated by dilution plating, and the samples were isolated using nutrient medium (PDA). 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 on a nutrient medium plate. 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. 2 strains GIG-1 and GIG-2 with high-efficiency degradation performance to phenanthrene are 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

As shown in FIG. 1, after activation of GIG-1, colonies with a diameter of 2-3mm, irregular edges, a rounded shape, and upward growth of white villous hyphae were formed after 72 hours of growth on PDA plates under aerobic conditions at 28 ℃. The strain is an obligate aerobic strain. GIG-2 is cultured for 72h at 28 deg.C under aerobic condition to form colony with diameter of 4-7mm, gray to light brown, irregular round edge, and upward growth of gray villous hypha, and reddish brown back for secreting laccase. The strain is an obligate aerobic strain.

B. Characteristics of molecular biology

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 because of ITS high degree of conservation, specificity and appropriate sequence length.

The Polymerase Chain Reaction (PCR) is mainly used for amplifying different gene segments, 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 desired gene, preparing gel block with 0.75-1% agarose and nucleic acid stain GelRed, adding PCR product and various length piecesAnd (3) placing the DNA marker (maker) in the section into an electrophoresis apparatus, filling TBE (Tris boric acid) buffer solution into the electrophoresis apparatus, taking out the electrophoresis apparatus after the electrophoresis apparatus works for 20min under a certain voltage, and placing the electrophoresis apparatus under an ultraviolet lamp of 300nm for observation to confirm that the PCR product amplification reaction is successful. Then, the 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 fungal ITS gene sequence obtained by sequencing is uploaded to NCBI, and the website can compare the submitted sequence with the ITS gene sequence of a typical strain of a recognized species to obtain the similarity information among the sequences. According to the result analysis of the sequence comparison, the corresponding typical strain can be selected as the model strain of the experimental isolated strain, and meanwhile, the ITS gene sequence of the model strain can be obtained, and phylogenetic analysis is constructed to prove that the model strain and the experimental isolated strain have difference, so that the isolated strain is identified. Construction of phylogenetic tree the phylogenetic tree is constructed by using the MEGA 5.05 program, usually by using an adjacency method, a minimum evolution method and a maximum reduction method, wherein the adjacency method is most commonly used, and the self-expansion value is usually set to be calculated repeatedly for 1000 times.

The ITS gene alignment shows that the gene similarity of the strain GIG-1 (the ITS sequence is shown as SEQ ID NO. 1) and Cephalotrichum dendrocephalum LZ16-03(MT898686.1) is 100%. From the above results, it was found that the fungus GIG-1 isolated in this experiment is of the species Cephalotrichum dendrocephalum. The gene similarity of GIG-2 (the ITS sequence is shown in SEQ ID NO. 2) and Scedosporium dehalogii DTO 401-F6(MT316369.1) is 100%. The isolated fungus GIG-2 was described as a species of Scedosporium dehalogii.

The ITS gene sequences of the two strains and the ITS gene sequences with higher similarity are used for making a phylogenetic tree, so that the homology results between the ITS genes and the ITS genes with higher similarity are obtained. A phylogenetic tree constructed by the orthotopic grafting method is shown in FIG. 2. At present, there are few reports about the application of the strain in the environmental field. Therefore, the obtained efficient phenanthrene degrading bacteria have important theoretical and practical significance for treatment and deep remediation of polycyclic aromatic hydrocarbons in petroleum-polluted soil.

From the above results, it can be derived:

the isolated strain GIG-1 in this experiment was named Cephalotrichum dendrocephalum GIG-1, which was deposited at the Guangdong province culture Collection (GDMCC) at 12.7.7.2021, address: building 5 of first furnance, large yard, 100, building 59, Guangdong province, Guangzhou, China, zip code: 510070, accession number: GDMCC No: 61800.

the strain GIG-2 isolated in this experiment was named Scedosporium dehalogii GIG-2, which was deposited at 12 months 7 in 2021 at the Guangdong province culture Collection (GDMCC) with the address: building 5 of first furnance, large yard, 100, building 59, Guangdong province, Guangzhou, China, zip code: 510070, accession number: GDMCC No: 61802.

example 2: growth conditions

A. Measurement of growth temperature:

preparing a liquid PDA nutrient medium required by the growth of the strain, and taking the prepared liquid PDA nutrient medium into a sterilization pot for sterilization. Inoculating 0.1ml of activated strain into 10ml of PDA culture medium (experimental group), taking the culture medium without fungus inoculation as a control (control group), putting the culture medium into different temperatures for culturing for 7 days, repeating the control group and the experimental group corresponding to each temperature for three times, observing the growth condition of the fungus every day, pouring the culture medium into a centrifuge tube for weighing after 7 days, centrifuging for 30min at 5000 revolutions, pouring out supernatant, putting into a 60-DEG oven, drying to constant weight, and weighing to calculate the dry weight of fungus hyphae. The test temperatures were as follows: 18 ℃, 23 ℃, 28 ℃, 33 ℃, 35 ℃ and 38 ℃.

B. Measurement of growth pH:

preparing a liquid nutrient medium required by the growth of the strain, and adjusting the pH of a culture solution by using a buffer system with the pH of 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. Inoculating activated strain 0.1ml into 10ml culture medium, repeating three times per pH, culturing at optimum temperature for new strain growth for 7 days with culture medium without fungus as control, and pouring culture medium after 7 daysCentrifuging for 30min after centrifuging the tube at 5000 rpm, pouring out the supernatant, putting into a 60-DEG oven, drying to constant weight, and weighing to calculate the dry weight of the fungal hyphae. The pH tested was as follows: 4.0, 5.0, 6.0, 7.0, 8.0, 9.0.

As shown in FIG. 3, in the nutrient medium, GIG-1 and GIG-2 were able to grow at a temperature of 18 to 38 ℃ and the optimum growth temperatures were both the enrichment temperature of 28 ℃; they can grow under the pH condition of 4.0-9.0, and the optimum growth pH is 6.0.

Example 3: 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 (3) mixing the cultured GIG-1 and GIG2 fungus liquid according to the ratio of 1:1 mass ratio to prepare bacterial liquid with the content of 10 g/L.

3. Adding the bacterial liquid into a sodium alginate solution with the mass fraction of 3% according to the volume 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 mixed microbial inoculum of GIG-1 and GIG 2.

Secondly, respectively inoculating the strains GIG-1 and GIG-2 which are activated and cultured for 7 days and the mixed microbial inoculum according to the inoculum size of 10 percent of the mass ratio to the initial strain containing 50 mg.L^-1In the inorganic salt medium of phenanthrene (example 1), the temperature is 28 ℃ for 7d with shaking, and 3 times of parallel experiments are carried out. The treatment without adding pure bacteria was a control treatment.

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: and (3) determining the content of PAHs in each processed sample by adopting an Agilent 7890 gas chromatograph-5975 mass spectrometer. The column used was an Agilent DB 5-MS capillary column (column length 30m, inner diameter 0.25mm, film thickness 0.25 μm). An Agilent 7890 gas chromatograph-5975 mass spectrometer is used for measuring the content of PAHs. The separation analysis was carried out using an Agilent DB 5-MS (column length 30m, inner diameter 0.25mm, film thickness 0.25 μm) capillary chromatography column. 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 microorganism cell concentration is measured by a drying and weighing method.

The strains GIG-1, GIG-2 and the microbial inoculum can degrade phenanthrene by GC-MS determination and analysis, and the degradation rate can reach more than 40 percent after the strains are cultured in an inorganic salt culture solution containing phenanthrene with the concentration of 50mg/L for 7 days (figure 4). The degradation rates of the strains GIG-1 and GIG-2 are 53.8% and 42.9% respectively, and the degradation efficiency of the microbial inoculum prepared by mixing the strains is 73.1%.

And (4) conclusion:

1. two phenanthrene degrading bacteria GIG-1 and GIG-2 which can grow by taking phenanthrene as a carbon source are obtained by enrichment and separation from petroleum polluted soil in Shandong, and are mixed to prepare the solid microbial inoculum.

2. Both strains are obligate aerobic bacteria. According to the analysis of molecular biology means, the fungus GIG-1 separated in the experiment is Cephalotrichum dendrophilum, GIG-2 is Sceosporium dehalogii, and the evolutionary tree 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 conditions for both strains were 28 ℃ and pH 6.0. All of them can utilize PAH phenanthrene as carbon source, and after the phenanthrene is cultured in inorganic salt culture solution whose initial concentration is 50 mg.L-1 for 7 days, the degradation rate of GIG-1 and GIG2 can be up to above 40%. After the bacillus subtilis and the bacillus subtilis are mixed and prepared into a solid microbial inoculum for use, the degradation rate of phenanthrene can be increased to more than 73%, and in conclusion, GIG-1 and GIG2 are two strains capable of degrading phenanthrene, the adaptability to polycyclic aromatic hydrocarbon is strong, and the microbial inoculum prepared from the two strains has good application potential in the bioremediation aspect of polycyclic aromatic hydrocarbon in petroleum-polluted sites.

Sequence listing

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Claims (9)

1. Cephalotrichum dendrocephalum GIG-1 with a deposit number of: GDMCC No: 61800.
2. scedosporium dehalogii GIG-2 with accession number: GDMCC No: 61802.
3. 3. use of the cephalothin dendrimer GIG-1 of claim 1 or the Scedosporium dehalogii GIG-2 of claim 2 or a mixture thereof for degrading polycyclic aromatic hydrocarbons.
4. 4. The use of claim 3, wherein said polycyclic aromatic hydrocarbon is a polycyclic aromatic hydrocarbon in soil or sewage.
5. 5. Use according to claim 3, wherein the polycyclic aromatic hydrocarbon is phenanthrene.
6. 6. A polycyclic aromatic hydrocarbon-degrading bacterial agent comprising, as an active ingredient, the Cephalotrichum dendrophilum GIG-1 of claim 1, the Scedosporium dehalogii GIG-2 of claim 2, or a mixture thereof.
7. 7. The polycyclic aromatic hydrocarbon degrading bacterial agent according to claim 6, wherein the preparation method of the mixed bacterial agent 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. the cultured fungal solutions of Cephalotrichum dendrophilum GIG-1 and Sceosporium dehalogii GIG-2 were cultured in the following ratio of 1:1, mixing the materials in a mass ratio to prepare bacterial liquid with the content of 10 g/L;

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

   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, and thus obtaining the mixed microbial inoculum of the strains GIG-1 and GIG 2.
8. 8. A method for degrading a polycyclic aromatic hydrocarbon, which comprises spraying the Cephalotrichum dendrophilum GIG-1 of claim 1, the Scedosporium dehalogii GIG-2 of claim 2 or a mixed microbial preparation thereof into an environment containing the polycyclic aromatic hydrocarbon to degrade the polycyclic aromatic hydrocarbon.
9. 9. The method of claim 8, wherein phenanthrene is degraded by spraying a cephalosporium dendrophilum GIG-1, a scordosporium dehalogii GIG-2, or a mixture thereof into an environment contaminated with phenanthrene.

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