Composite strains, bacterial agents and their applications

Abstract

The present invention provides a composite bacterial strain, a microbial agent, and their use. The composite bacterial strain is composed of Gordonia C1 (CGMCC No. 25708), Cellulosus C3 (CGMCC No. 25709), and Bacillus C6 (CGMCC No. 25710). Also provided are a method for preparing a microbial agent using the composite bacterial strain and the use of the composite bacterial strain or a microbial agent containing the composite bacterial strain in the degradation of petroleum hydrocarbons, achieving efficient degradation of high-quality and high-concentration petroleum. The microbial agent prepared by the present invention has broad prospects in the field of ecological remediation of petroleum-contaminated water and soil.

Description

Technical Field

[0001] The present invention relates to the field of environmental biotechnology, and in particular to a composite bacterial strain, a bacterial agent and applications thereof. Background Art

[0002] With the increasing demand for oil, oil pollution is becoming increasingly serious. Petroleum hydrocarbons (such as polycyclic aromatic hydrocarbons) in oil-contaminated sites can be toxic, mutagenic, and carcinogenic to plants, animals, and humans, posing a serious threat to the ecological environment and human health. Petroleum hydrocarbon pollution has become a serious environmental problem. Consequently, oil pollution remediation has become a major concern, and remediation of oil-contaminated soil or water bodies is an urgent matter.

[0003] Crude oil is usually a dark brown or black viscous and flammable liquid. Crude oil is a mixture of various organic substances with very different chemical compositions. It mainly includes four material components: saturated hydrocarbons, aromatic hydrocarbons, colloids and asphaltenes. The high heterogeneity puts higher demands on the effective treatment of oil pollution. As the largest group of environmental pollutants in environmental pollution, in the past few decades, oil pollution treatment processes with physical, chemical and biological as the core have been developed. Among them, bioremediation technology is one of the preferred technologies for the remediation of crude oil contaminated soil. Compared with other strategic methods for treating oil pollution, biological treatment processes that use microorganisms to biodegrade crude oil have attracted much attention due to their environmental friendliness, high efficiency and low cost.

[0004] Ecological restoration of petroleum-contaminated sites using microorganisms as the core is an economically viable, environmentally friendly approach that produces no secondary pollutants. Currently, extensive research has been conducted on the isolation and screening of petroleum-degrading bacteria, or the complex assembly of these bacteria. However, the isolated petroleum hydrocarbon-degrading bacteria or composite bacteria are mostly used in environments with low petroleum concentrations or have low petroleum hydrocarbon degradation rates. Therefore, in order to further improve the degradation capacity of petroleum hydrocarbons and quickly and efficiently resolve the problem of high-concentration petroleum contamination, there is an urgent need to develop a bacterial strain for the remediation of petroleum-contaminated ecological environments. Summary of the Invention

[0005] In response to the above problems, the main purpose of the present invention is to provide a composite strain, a bacterial agent and its application, so as to overcome the problems of low concentration of petroleum pollution in the existing technology, single type of petroleum hydrocarbon degradation and low efficiency, and realize efficient ecological management of petroleum-contaminated environments by microorganisms.

[0006] To achieve the above object, the technical solution of the present invention is as follows:

[0007] As a first aspect of the present invention, a composite strain is provided, which is composed of Gordonia sp. Strain C1 with a preservation number of CGMCC No. 25708, Cellulosimicrobium sp. Strain C3 with a preservation number of CGMCC No. 25709, and Bacillus sp. Strain C6 with a preservation number of CGMCC No. 25710; the Gordonia C1, the Cellulosimicrobium C3, and the Bacillus sp. Strain C6 were respectively sent to the China General Microbiology Center (CGMCC) for preservation on September 15, 2022.

[0008] In some embodiments of the present invention, the composite strain is isolated from petroleum-contaminated soil.

[0009] As a second aspect of the present invention, there is provided a use of the composite strain as described above in the degradation of petroleum hydrocarbons.

[0010] As a third aspect of the present invention, a bacterial agent containing the composite strain described above is provided.

[0011] In some embodiments of the present invention, the bacterial agent is prepared by the following preparation method:

[0012] Culturing the Gordonia C1, the Cellulosus C3, and the Bacillus C6 in a degradation culture system to obtain a microbial culture solution;

[0013] The microbial cultures of Gordonia C1, Cellulosus C3 and Bacillus C6 were respectively prepared at a culture concentration of OD 600 =0.6~1, the volume proportion is 50%-70%, 20%-40% and 10%-20% are mixed and compounded simultaneously.

[0014] In some embodiments of the present invention, the construction of the bacterial agent of the composite strain is mainly based on petroleum-degrading functional bacteria, supplemented by surfactant-producing strains, to achieve efficient petroleum degradation.

[0015] In some embodiments of the present invention, the degradation culture system of the above-mentioned bacterial agent includes: CH3COONa 0.2 g / L, C6H5Na3O7 0.2-2 g / L, C3H3NaO3 0.02-0.5 g / L, K2HPO4 0.5 g / L, NaNO3 0.2-0.5 g / L, NH4Cl 0.05 g / L, NaCl 10-30 g / L, CaCl2 0.1-0.3 g / L, FeC6H5O70.005 g / L, Fe(OH)30.002 g / L, yeast powder 0.1-0.4 g / L, peptone 0.2-1 g / L, trace element mixture 5 mL / L, and vitamin mixture 2 mL / L.

[0016] In some embodiments of the present invention, the trace element mixture includes MgSO4·7H2O 2 g / L, MnSO4 0.4 g / L, FeSO4·7H2O 0.2 g / L, CoCl2 0.1 g / L, ZnSO4 0.05 g / L, CuSO 4· 5H2O 0.05 g / L, H3BO3 0.02 g / L, Na2MoO4 0.01 g / L.

[0017] In some embodiments of the present invention, the vitamin mixture includes 40 mg / L of vitamin C, 20 mg / L of biotin, 10 mg / L of vitamin B9, 50 mg / L of vitamin B6, 50 mg / L of vitamin B1, 30 mg of vitamin B2, 20 mg / L of vitamin B5, and 10 mg / L of vitamin B12.

[0018] As a fourth aspect of the present invention, there is provided a use of the above-mentioned bacterial agent in the degradation of petroleum hydrocarbons.

[0019] As a fifth aspect of the present invention, there is provided a method for degrading petroleum hydrocarbons using the composite strain or bacterial agent as described above, comprising the following steps:

[0020] The composite strain or bacterial agent is inoculated into a petroleum-contaminated system to degrade petroleum hydrocarbons.

[0021] In some embodiments of the present invention, the oil-contaminated system includes a water body or soil system contaminated by oil; the oil-contaminated system is a high-concentration oil-contaminated system with an oil mass concentration of 55,000-65,000 mg / L.

[0022] In some embodiments of the present invention, the degradation of petroleum hydrocarbons is carried out by reacting at a temperature of 30-37° C. and a rotation speed of 180-200 rpm for 7-21 days.

[0023] In some embodiments of the present invention, the inoculation amount of the bacterial agent is 5-20%; the optical density value OD of the bacterial agent is 600 0.6~1. By limiting the OD of the bacterial agent 600 , inoculation amount and strain ratio can ensure the concentration of bacteria in the water or soil system contaminated by oil, and better promote the strain to degrade petroleum hydrocarbons.

[0024] In the process of realizing the present invention, the inventors discovered that Gordonia C1, Cellulosomyces C3 and Bacillus C6 are oil-degrading bacterial strains, and their cell surfaces are hydrophobic. The cells themselves have the ability to produce surfactants and degrade petroleum hydrocarbons, which laid the foundation for the subsequent application of single strains in the ecological restoration of oil-contaminated sites. In addition, among the three, Gordonia C1 has a stronger ability to degrade petroleum hydrocarbons, and Cellulosomyces C3 and Bacillus C6 have high surfactant production capabilities. Based on functional complementarity, Gordonia C1 is mainly used, and Cellulosomyces C3 and Bacillus C6 are used as auxiliary bacterial agents to achieve efficient degradation of high-quality and high-concentration oil.

[0025] As can be seen from the above, the composite strain, bacterial agent and application thereof provided by the present invention have at least the following beneficial effects:

[0026] The present invention prepares a microbial agent by compounding Gordonia C1, Cellulosus C3 and Bacillus C6 in a certain proportion based on functional complementarity. The microbial agent is prepared mainly with Gordonia C1 having a petroleum degradation function, and Cellulosus C3 and Bacillus C6 having high surfactant production capabilities as auxiliary agents. The hydrophobic effect of Gordonia C1 promotes the degradation of petroleum hydrocarbons by the microbial agent, and the emulsification effect of the high surfactant production capabilities of Cellulosus C3 and Bacillus C6 enhances the hydrophilicity of the cells, promotes the growth of the strains in the microbial agent, and achieves efficient degradation of petroleum hydrocarbons in water bodies and soil systems contaminated by high-mass concentrations (55,000-65,000 mg / L) of petroleum while ensuring the high activity of the strains in the microbial agent. The strain culture cost is low, and the prepared microbial agent has broad prospects in the field of ecological restoration of petroleum-contaminated water bodies and soils.

[0027] By using the bacterial agent provided by the present invention, the total petroleum hydrocarbon degradation rate in a high-concentration (60,000 mg / L) petroleum degradation system is greater than 80%. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0029] Figure 1 is a 16S rDNA gene phylogenetic tree constructed based on the Maximum likelihood method provided in an embodiment of the present invention;

[0030] Figure 2 is the total petroleum hydrocarbon degradation rate of a single strain provided by an embodiment of the present invention after 14 days of biodegradation in a petroleum degradation medium with a petroleum mass concentration of 60,000 mg / L;

[0031] Figure 3 This is a gas chromatography-mass spectrometry analysis peak graph of the blank group provided in an embodiment of the present invention after 14 days of biodegradation of the bacterial agent in a petroleum degradation medium with a petroleum mass concentration of 60,000 mg / L;

[0032] Figure 4 This is a gas chromatography-mass spectrometry analysis peak diagram of the experimental group provided in the embodiment of the present invention after 14 days of biodegradation of the bacterial agent in a petroleum degradation medium with a petroleum mass concentration of 60,000 mg / L;

[0033] Figure 5 It is the total petroleum hydrocarbon degradation rate after 7 days and 14 days of bacterial agent biodegradation in a petroleum degradation culture medium with a petroleum mass concentration of 60,000 mg / L provided by the embodiment of the present invention. DETAILED DESCRIPTION

[0034] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0035] The following examples are intended to illustrate the present invention and are not intended to limit the present invention. The experimental methods in the following examples are all routine experimental procedures unless otherwise specified. All instruments, consumables, and reagents in the following examples are commercially available unless otherwise specified. Three biological replicates were performed for each quantitative experiment in the following examples.

[0036] The Gordonia sp. Strain C1, Cellulosimicrobium sp. Strain C3, and Bacillus sp. Strain C6 provided by the present invention have been deposited in the China General Microbiology Center (CGMCC) of the China Culture Collection Administration. The address of the CGMCC is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with deposit numbers CGMCC No. 25708, CGMCC No. 25709, and CGMCC No. 25710, respectively.

[0037] The raw materials and main equipment used in each embodiment of the present invention are as follows:

[0038] Degradation medium: CH₃COONa 0.2 g / L, C₆H₅Na₃Oₐ 1 g / L, C₃H₃NaO₃ 0.02 g / L, K₂HPO₄ 0.5 g / L, NaNO₃ 0.2 g / L, NH₄Cl 0.05 g / L, NaCl 20 g / L, CaCl₂ 0.2 g / L, FeC₆H₅Oₐ 0.005 g / L, Fe(OH)₃ 0.002 g / L, yeast extract 0.15 g / L, peptone 0.4 g / L, natural pH. Sterilize at 115°C for 20 min. After cooling, add 5 mL / L of trace element mixture and 2 mL / L of vitamin mixture.

[0039] The trace element mixture contains the following substances: MgSO4·7H2O 2 g / L, MnSO4 0.4 g / L, FeSO4·7H2O0.2 g / L, CoCl2 0.1 g / L, ZnSO4 0.05 g / L, CuSO 4· 5H2O 0.05 g / L, H3BO3 0.02 g / L, Na2MoO40.01 g / L.

[0040] The vitamin mixture contains the following: vitamin C 40 mg / L, biotin 20 mg / L, vitamin B9 10 mg / L, vitamin B6 50 mg / L, vitamin B1 50 mg / L, vitamin B2 30 mg, vitamin B5 20 mg / L, and vitamin B12 10 mg / L. Filter sterilize, aliquot for later use, and store at -20°C.

[0041] Petroleum degradation medium: Add 60,000 mg / L petroleum to the degradation medium and sterilize at 115°C for 45 min.

[0042] Gas chromatography-mass spectrometry (GC-MS-QP2010 Ultra, Japan) and multi-function microplate reader (molecular devices SpectraMax i3x, USA) were used.

[0043] Example 1

[0044] This example is a molecular biological identification of a single strain:

[0045] Single colonies of the three bacterial strains isolated and purified in our laboratory were transferred to degradation culture medium and cultured at 37°C and 150-200 rpm for 3-5 days. The bacteria were collected and the bacterial genome was extracted. PCR amplification was performed using the universal primers 27F and 1492R for the 16S rDNA gene, and the PCR products were sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for full-length 16S rDNA gene sequencing.

[0046] The 16S rDNA gene sequences of the above strains were subjected to Blast analysis on NCBI, and the 16S rDNA gene sequences of related strains were selected to construct a phylogenetic tree based on the Maximum likelihood method using PhyML 3.0 (e.g. Figure 1 The strains were shown in Figure 2 ), named Gordonia sp. Strain C1, Cellulosimicrobium sp. Strain C3 and Bacillus sp. Strain C6 respectively.

[0047] Example 2

[0048] This example characterizes the functional traits of Gordonia C1, Cellulosus C3, and Bacillus C6. :

[0049] 1. Enrichment Culture of Single Strains of Gordonia C1, Cellulosus C3, and Bacillus C6

[0050] Gordonia C1, Cellulosus C3, and Bacillus C6, all isolated and identified in our laboratory, were inoculated into degradation medium at 5%-20% inoculum and cultured at 37°C and 200 rpm for 4 days. The cells and supernatant were obtained by centrifugation (10,000 × g, 3 min, 16°C). The cells were washed three times with urea phosphate buffer and resuspended in urea phosphate buffer. The OD value of the suspension was measured using a multifunctional microplate reader. 600 , and adjust the bacterial suspension OD 600 About 0.6.

[0051] 2. Functional Characterization of Gordonia C1, Cellulosus C3, and Bacillus C6

[0052] The functional traits of a single strain mainly include cell surface hydrophobicity, emulsification properties and petroleum degradation potential.

[0053] 2.1 Cell surface hydrophobicity

[0054] The cell surface hydrophobicity of the bacteria was measured by MATH test, and the results are shown in Table 1. As can be seen from Table 1, the cell surface hydrophobicity of Gordonia C1 is the highest, and it has the ability to quickly adsorb to the surface of petroleum and potentially degrade petroleum hydrocarbons, followed by Cellulosomyces C3.

[0055] Table 1 Cell surface hydrophobicity (CSH%) of different strains

[0056]

[0057] 2.2 Ability to produce surfactants

[0058] Using paraffin oil as a crude oil substitute, the surfactant production ability of bacteria was evaluated by the emulsification index, and the results are shown in Table 2. As can be seen from Table 2, compared with the other two bacterial strains, Bacillus C6 has the highest emulsification index and has a high ability to produce surfactants.

[0059] Table 2 Emulsification index of supernatants from different strains

[0060]

[0061] 2.3 Petroleum degradation potential

[0062] Gordonia C1, Cellulosus C3 and Bacillus C6 were inoculated into the petroleum degradation medium at a 10% inoculum rate, respectively, and cultured at 37 °C and 200 rpm for 14 days.

[0063] The oil residues in the fermentation broth after biodegradation were extracted by ultrasonic extraction, and the total petroleum hydrocarbon degradation rate of a single strain was evaluated by weight method. Figure 2 As shown in the figure, the total petroleum hydrocarbon degradation rate of Gordonia C1 was 43.86%, the total petroleum hydrocarbon degradation rate of Cellulosus C3 was 20.91%, and the total petroleum hydrocarbon degradation rate of Bacillus C6 was 13.81%. Therefore, Gordonia C1 has a strong ability to remediate petroleum pollution.

[0064] Example 3

[0065] This example is about the bacterial preparation obtained by compounding the strains and the functional testing of the bacterial preparations. :

[0066] 1. Compounding of strains to obtain bacterial agents

[0067] The strains are compounded in a manner that Gordonia C1 is the main strain, and Cellulosus C3 and Bacillus C6 are the auxiliary strains.

[0068] Gordonia C1, Cellulosus C3 and Bacillus were inoculated into the degradation culture medium at an inoculum amount of 5%-20%, and cultured separately at a temperature of 30-37°C and a rotation speed of 150-200 rpm for 3-5 days.

[0069] Use degradation medium to adjust the microbial cell suspension to its OD 600 = 1, Gordonia C1, Cellulosus C3 and Bacillus C6 were simultaneously mixed at a ratio of 60%, 30% and 10% respectively to prepare a bacterial agent.

[0070] 2. Bacterial agent function test

[0071] The microbial agent was inoculated into a petroleum degradation medium with a petroleum mass concentration of 60,000 mg / L at an inoculum size of 10%, and a petroleum degradation medium with a petroleum mass concentration of 60,000 mg / L without the microbial agent was used as a blank. The culture was carried out at a temperature of 37°C and a rotation speed of 180-200 rpm for 14 days.

[0072] The petroleum residues in the fermentation broth after 14 days of biodegradation were extracted with n-hexane, and the remaining petroleum hydrocarbons in the experimental group and the blank group were analyzed by GC-MS. Figure 3 and Figure 4 As shown in the GC-MS analysis peak diagram, the peak height of the blank group is one order of magnitude higher than that of the experimental group, indicating that the bacterial agent has a significant effect on C 10 -C 40 The degradation efficiency of aliphatic hydrocarbons is good, and the statistical calculation shows that C 10 -C 40 The degradation rate of petroleum hydrocarbons is 92.90%.

[0073] The degradation rate of total petroleum hydrocarbons by the inoculum was evaluated by gravimetric method on the 7th and 14th days. Figure 5 As shown in the figure, the total petroleum hydrocarbon degradation rate was 57.16% on the 7th day and reached 81.96% on the 14th day. These results indicate that the bacterial agent has a higher petroleum hydrocarbon degradation rate than the strain, suggesting that this bacterial agent can play a greater role in the remediation of petroleum-contaminated soil or water.

[0074] Example 4

[0075] This example is to evaluate the petroleum degradation performance of bacterial agents obtained by compounding strains in different compounding ratios :

[0076] The implementation method is as described in Example 3, and the compounding ratio of the strains is adjusted, and Gordonia C1, Cellulosus C3 and Bacillus C6 are simultaneously mixed at a ratio of 50%, 30% and 20%, respectively, to prepare a bacterial agent.

[0077] Example 5

[0078] This example is to evaluate the petroleum degradation performance of bacterial agents obtained by compounding strains in different compounding ratios :

[0079] The implementation method is as described in Example 3, and the compounding ratio of the strains is adjusted, and Gordonia C1, Cellulosus C3 and Bacillus C6 are simultaneously mixed at a ratio of 70%, 20% and 10%, respectively, to prepare a bacterial agent.

[0080] Example 6

[0081] This example is to evaluate the petroleum degradation performance of bacterial agents obtained by compounding strains in different compounding ratios :

[0082] The implementation method is as described in Example 3, and the compounding ratio of the strains is adjusted, and Gordonia C1, Cellulosus C3 and Bacillus C6 are simultaneously mixed at a ratio of 50%, 40% and 10%, respectively, to prepare a bacterial agent.

[0083] The bacterial agents prepared in Examples 4, 5, and 6 were inoculated into a petroleum degradation medium with a petroleum mass concentration of 60,000 mg / L at an inoculum size of 10%. The petroleum degradation performance of the bacterial agents was evaluated using the gravimetric method. The details are shown in Table 3.

[0084] Table 3 Total petroleum hydrocarbon degradation rate of bacterial agents with different ratios

[0085]

[0086] By analyzing the total petroleum hydrocarbon degradation rate of different strain ratios, increasing the proportion of petroleum-degrading bacteria (Gordonella C1) helps to quickly achieve petroleum degradation.

[0087] Example 7

[0088] This example evaluates the petroleum degradation performance of different inoculation amounts of the microbial agent:

[0089] The implementation method is as described in Example 3. The inoculum size of the bacterial agent is adjusted, and Gordonia C1, Cellulosus C3, and Bacillus C6 are simultaneously mixed at a ratio of 60%, 30%, and 10%, respectively, to prepare a bacterial agent. The inoculum size of 5% of the bacterial agent is inoculated into a petroleum degradation medium with a petroleum mass concentration of 60,000 mg / L.

[0090] Example 8

[0091] This example evaluates the petroleum degradation performance of different inoculation amounts of the microbial agent:

[0092] The difference between this embodiment and Example 7 is that the inoculation is performed at an inoculum amount of 20% of the bacterial agent.

[0093] Example 9

[0094] This example evaluates the petroleum degradation performance of different inoculation amounts of the microbial agent:

[0095] The implementation method is as described in Example 3. The inoculum size of the bacterial agent is adjusted, and Gordonia C1, Cellulosus C3, and Bacillus C6 are simultaneously mixed at a ratio of 70%, 20%, and 10%, respectively, to prepare a bacterial agent. The inoculum size of 5% of the bacterial agent is inoculated into a petroleum degradation medium with a petroleum mass concentration of 60,000 mg / L.

[0096] Example 10

[0097] This example evaluates the petroleum degradation performance of different inoculation amounts of the microbial agent:

[0098] The difference between this embodiment and Example 9 is that the inoculation is performed at an inoculum amount of 20% of the bacterial agent.

[0099] Example 11

[0100] This example evaluates the petroleum degradation performance of different inoculation amounts of the microbial agent:

[0101] The implementation method is as described in Example 3. The inoculum size of the bacterial agent is adjusted, and Gordonia C1, Cellulosus C3, and Bacillus C6 are simultaneously mixed at a ratio of 50%, 30%, and 20%, respectively, to prepare a bacterial agent. The inoculum size of 5% of the bacterial agent is inoculated into a petroleum degradation medium with a petroleum mass concentration of 60,000 mg / L.

[0102] Example 12

[0103] This example evaluates the petroleum degradation performance of different inoculation amounts of the microbial agent:

[0104] The difference between this embodiment and Example 11 is that the inoculation is carried out at an inoculum amount of 20% of the bacterial agent.

[0105] The petroleum degradation performance of the bacterial agents prepared in Examples 7 to 12 was evaluated using the gravimetric method, and the details are shown in Table 4.

[0106] Table 4 Total petroleum hydrocarbon degradation rate with different inoculation amounts of bacterial agents

[0107]

[0108] Through the analysis of the total petroleum hydrocarbon degradation rate with different inoculum dosages, it was found that increasing the inoculum dosage of the microbial agent is helpful to achieve rapid petroleum degradation.

[0109] In summary, through the above relevant embodiments and comparative examples, the total petroleum hydrocarbon degradation rate data analysis of different strain combination ratios and different inoculation amounts of the bacterial agent was carried out. The bacterial agent constructed by mainly using petroleum-degrading functional bacteria (Gordonella C1) and supplemented with surfactant-producing bacteria (Cellulospora C3 and Bacillus C6) has excellent petroleum degradation performance. For the degradation culture system of 60,000 mg / L, the total petroleum hydrocarbon degradation rate is above 80%.

[0110] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A composite strain, characterized in that The composite strain consists of Gordonia sp. Strain C1 with a preservation number of CGMCC No. 25708, Cellulosimicrobium sp. Strain C3 with a preservation number of CGMCC No. 25709, and Bacillus sp. Strain C6 with a preservation number of CGMCC No. 25710.

2. Use of the composite strain as claimed in claim 1 in the degradation of petroleum hydrocarbons.

3. A bacterial agent containing the composite strain according to claim 1.

4. The microbial agent according to claim 3, characterized in that The bacterial agent is prepared by the following preparation method: Culturing the Gordonia C1, the Cellulosus C3, and the Bacillus C6 in a degradation culture system to obtain a microbial culture solution; The microbial cultures of Gordonia C1, Cellulosus C3 and Bacillus C6 were respectively prepared at a culture concentration of OD 600 =0.6~1, the volume proportion is 50%-70%, 20%-40% and 10%-20% are mixed and compounded simultaneously.

5. The microbial agent according to claim 4, characterized in that The degradation culture system comprises: CH3COONa 0.2 g / L, C6H5Na3O7 0.2-2 g / L, C3H3NaO3 0.02-0.5 g / L, K2HPO4 0.5 g / L, NaNO3 0.2-0.5 g / L, NH4Cl 0.05 g / L, NaCl 10-30 g / L, CaCl2 0.1-0.3 g / L, FeC6H5O7 0.005 g / L, Fe(OH)3 0.002 g / L, yeast powder 0.1-0.4 g / L, peptone 0.2-1 g / L, trace element mixture 5 mL / L, and vitamin mixture 2 mL / L; The trace element mixed solution includes MgSO4·7H2O 2 g / L, MnSO4 0.4 g / L, FeSO4·7H2O 0.2 g / L, CoCl2 0.1 g / L, ZnSO4 0.05 g / L, CuSO 4· 5H2O 0.05 g / L, H3BO3 0.02 g / L, Na2MoO4 0.01 g / L; The vitamin mixture includes 40 mg / L of vitamin C, 20 mg / L of biotin, 10 mg / L of vitamin B9, 50 mg / L of vitamin B6, 50 mg / L of vitamin B1, 30 mg of vitamin B2, 20 mg / L of vitamin B5, and 10 mg / L of vitamin B12.

6. Use of the bacterial agent according to any one of claims 3 to 5 in the degradation of petroleum hydrocarbons.

7. A method for degrading petroleum hydrocarbons using the composite strain according to claim 1 or the bacterial agent according to any one of claims 3 to 5, characterized in that: The method comprises the following steps: The composite strain or bacterial agent is inoculated into a petroleum-contaminated system to degrade petroleum hydrocarbons.

8. The method according to claim 7, characterized in that The petroleum pollution system is selected from a water body or soil system contaminated by petroleum; the petroleum pollution system is a high-concentration petroleum pollution system with a petroleum mass concentration of 55,000 to 65,000 mg / L.

9. The method according to claim 7 or 8, characterized in that The degradation of petroleum hydrocarbons is carried out by reacting at a temperature of 30-37° C. and a rotation speed of 180-200 rpm for 7-21 days.

10. The method according to claim 7 or 8, characterized in that The inoculation amount of the bacterial agent is 5-20%; the optical density value OD of the bacterial agent is 600 It is 0.6~1.

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