Gordonia B7-2 and application thereof

Abstract

This invention discloses a strain of *Gordonella* B7-2 and its applications, relating to the field of microbial application technology. The *Gordonella* B7-2 strain is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20252269, with a deposit date of October 20, 2025. Research has revealed that *Gordonella* B7-2 is... Gordonia It is a potential new species and has the ability to degrade crude oil, which provides a new research direction for the development of crude oil degradation technology.

Description

Technical Field

[0001] This invention relates to the field of microbial application technology, and in particular to a strain of Gordon's B7-2 and its applications. Background Technology

[0002] Petroleum, commonly known as crude oil, is a naturally occurring liquid hydrocarbon system characterized by a complex mixture of organic compounds. Its composition typically includes saturated hydrocarbons (such as alkanes), aromatic hydrocarbons, and polar components such as asphaltenes and resins. Over the past two decades, rapid industrialization has driven significant growth in the production and consumption of petroleum products. Global crude oil demand has risen from 84 million barrels per day (2000) to 102 million barrels per day (2023). The operation of the petroleum supply chain often results in accidental hydrocarbon leaks into ecosystems during human activities, posing a serious threat to all components of these ecosystems. Therefore, removing excess petroleum hydrocarbons from the environment has become a pressing issue for society.

[0003] The development of petroleum degradation technologies is a broad and continuously evolving research field. Currently employed physical and chemical methods, including photodegradation, incineration, adsorption, separation, thermal desorption, and in-situ chemical oxidation, have been used for the degradation of petroleum hydrocarbons. However, traditional physicochemical remediation methods not only generate secondary environmental pollution byproducts but also suffer from inherent limitations such as low remediation efficiency and incomplete resource recovery. Therefore, there is an urgent need to develop an environmentally sustainable alternative. Summary of the Invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention proposes a strain of Gordon's bacterium B7-2 and its application to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A strain of Gordon's bacterium B7-2, taxonomically named Gordon a sp.B7-2, Latin name Gordon A sp., deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 20252269, deposited on October 20, 2025, at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0007] A method for culturing Gordon's bacillus B7-2 as described in claim 1, wherein the culture is carried out using any of the following culture media:

[0008] ISP1: Each 1000mL of water contains 5-10g of tryptone, 3-10g of yeast extract, and 20-30g of agar;

[0009] ISP2: Each 1000mL of water contains 4-10g of yeast extract, 10-20g of malt extract, 4-8g of glucose, and 20-30g of agar;

[0010] ISP3: Each 1000mL of water contains 20-30g of oats, 1-5mL of trace salt, and 20-30g of agar;

[0011] ISP4: Each 1000mL of water contains 10-20g of soluble starch, 1-8g of K2HPO4, 1-5g of MgSO4·7H2O, 1-3g of NaCl, 2-5g of (NH4)2SO4, 2-6g of calcium carbonate, 1-3mL of trace salts, and 20-30g of agar.

[0012] ISP5: Each 1000mL of water contains 10-20g of glycerol, 1-5g of L-aspartic acid, 1-6g of K2HPO4, 1-3mL of trace salts, and 20g of agar;

[0013] The trace salt comprises the following components: 0.1g FeSO4·7H2O, 0.1g MnCl2·4H2O, and 0.1g ZnSO4•7H2O per 100mL of water.

[0014] ISP6: Contains 15-25g peptone, 5-10g ferric peptone, 0.5-2.0g ferric citrate, 1-3g K₂HPO₄, and N per 1000mL of water. a2 S2O3 0.08-0.15g, yeast extract powder 1-3g, agar 20-30g;

[0015] ISP7: Each 1000mL of water contains 15-25g of glycerol, 0.5-3.0g of L-tyrosine, 1-5g of L-aspartic acid, 0.5-3.0g of K2HPO4, 0.5-2.0g of MgSO4·7H2O, 0.5-3.0g of NaCl, 10-20mL of FeSO4·7H2O, and 20-30g of agar;

[0016] NA: Each 1000mL of water contains 5-10g of beef extract, 5-10g of peptone, 5-10g of NaCl, and 20-30g of agar;

[0017] CA: Per 1000mL of water, there are 5-10g of sucrose, 2-3g of NaNO3, 0.5-3.0g of K2HPO4, 0.5-3.0g of MgSO4·7H2O, 0.5-2.0g of KCl, 0.01-2.50g of FeSO4·7H2O, and 20-30g of agar;

[0018] PDA: Each 1000mL of water contains 200-250g of potato, 4-10g of glucose, and 20-30g of agar.

[0019] Preferably, the optimal growth conditions for Gordon's bacterium B7-2 are: growth temperature 20-34℃, pH value 6.0-10.0, and NaCl concentration of 0% (w / v).

[0020] Preferably, the application of Gordon's B7-2 or its bacterial solution involved in this application in the degradation of crude oil.

[0021] Preferably, the application includes the following steps:

[0022] S1. Pick a single colony of Gordon's B7-2 and inoculate it into ISP2 liquid medium. Culture it on a shaker to obtain the seed culture.

[0023] S2. Inoculate the seed culture into fresh ISP2 liquid medium for expansion culture, centrifuge, discard the supernatant, resuspend the cells, and wash.

[0024] S3, Adjust the bacterial suspension concentration OD 600 =1.0 and then inoculated into crude oil culture medium for degradation.

[0025] Preferably, the inoculation amount of seed liquid in step S2 is 1-3% by volume.

[0026] Preferably, the crude oil culture medium preparation method in step S3 is as follows: add 300 mg of crude oil sterilized by ether to every 1 L of sterile crude oil base salt culture medium.

[0027] Preferably, the sterile crude oil base salt culture medium is prepared by adding 9-40g of NaCl2, 3-10g of Na2HPO4, 1-5g of KH2PO4, 1-5g of NaH2PO4, 1-6g of KNO3, 1-3g of NH4Cl, 0.7-1.5g of MgSO4·7H2O, and 1-3mL of trace element culture medium to every 1000mL of distilled water, followed by sterilization.

[0028] Preferably, the method for preparing the trace element culture medium is as follows: add 20-30 mg CaCl2, 30-40 mg FeCl3, 0.5-1.5 mg CuSO4, 0.5-2.5 mg MnSO4·H2O, and 10-20 mg ZnSO4·7H2O to every 1000 mL of distilled water, and then sterilize.

[0029] Preferably, a microbial agent for degrading crude oil has an active ingredient of at least one of the aforementioned Gordon's B7-2 or its bacterial solution.

[0030] Criteria for defining new species of prokaryotes

[0031] Molecular classification: Preliminary identification of new species is usually based on 16S rRNA gene homology. Currently, the commonly used cutoff value for 16S rRNA gene homology in new prokaryotes is 98.65%. However, due to the limited genetic information carried by the 16S rRNA gene, it has certain limitations in distinguishing closely related species and strains; therefore, many new species with high 16S rRNA gene homology are also common. The "gold standard" for prokaryotic classification is DNA-DNA hybridization (DDH), which provides a reliable basis for prokaryotic classification at the genome level, with a cutoff value of 70% for defining new species. As biological research has entered the genomics era, whole-genome data has also been applied to the classification and identification of new species. DDH has evolved from traditional biochemical experiments to digital genomic hybridization, and new calculation methods and classification standards are constantly emerging. Currently, the widely used method is average nucleotide identity (ANI), which has a close correspondence with DDH; an ANI value of 95-96% corresponds to a DDH value of 70%.

[0032] Polyphasic classification refers to the use of multiple information sources, including phenotypic characteristics, chemical classification characteristics, and molecular classification (genotypic) characteristics, to study the classification and systematic evolution of microorganisms. The application of polyphasic classification heralded the arrival of taxonomic consensus and is a milestone in the development of modern bacterial classification.

[0033] (1) Phenotypic characteristics: mainly including morphological characteristics, culture characteristics and physiological and biochemical characteristics.

[0034] Morphological characteristics mainly describe colony characteristics (colony shape, size, color, luster, presence and quantity of aerial hyphae, etc.), routine staining reactions (Gram staining, flagella staining, spore staining, etc.) and cell characteristics (cell shape, size, number and growth location of flagella, spore attachment location, sporangium shape, and the way hyphae break in the substrate, etc.).

[0035] Culture characteristics mainly refer to the growth of strains on organic and synthetic media. Typically, the growth of strains on different media, colony color, mycelium in the substrate, whether soluble pigments are produced, presence and color of aerial mycelium, etc. are observed.

[0036] Physiological and biochemical characteristics mainly involve detecting the activity of various enzymes in the strain. Due to the wide variety of microorganisms and enzyme systems, physiological and biochemical experiments cover a broad range. Physiological characteristics primarily include temperature tolerance range and optimal growth temperature, pH tolerance range and optimal growth pH, ​​salt concentration tolerance range and optimal growth salt concentration, aerobic requirements, and sensitivity to antibiotics. Common biochemical reactions include enzymatic detection, carbon / nitrogen source utilization, and carbon source acid production.

[0037] (2) Chemical classification characteristics: This mainly refers to the classification and identification of the cell wall chemical composition (amino acids and sugar types), quinone components, polar lipid types, and fatty acid composition of strains using chromatographic and mass spectrometric analysis techniques. The establishment of chemical classification methods marks the deepening of microbial classification from the individual level to the cellular level.

[0038] (3) Molecular classification characteristics: This involves analyzing nucleic acids and proteins at the molecular level and classifying them based on the obtained information. Its main contents include homology analysis of 16S rRNA genes and establishment of phylogenetic trees, determination of DNA (G+C) mol% content, and DNA-DNA hybridization. Whole-genome data can provide more perspectives and reliable evidence for the identification of new species, and therefore has been applied in microbial taxonomy. Since January 2018, the authoritative journal of microbial systematics, *International Journal of Systematic and Evolutionary Microbiology*, has required authors to provide genome sequencing data when publishing new species.

[0039] Compared with the prior art, the beneficial effects of the present invention are: the present invention is the first to isolate and identify a new species of bacterium, *Gordonella*, which can be used for crude oil degradation. Gordon This strain, sp. B7-2, is taxonomically unique. Its phenotypic characteristics (morphological, cultural, and physiological / biochemical), chemotaxonomic, and molecular taxonomic features differ from existing bacterial species, making it a newly discovered strain. Gordon a sp. B7-2 exhibits excellent degradation effects on crude oil, achieving a degradation rate of 66.25% after 28 days, further enriching the crude oil degradation microbial resource library. Meanwhile... Gordon a sp.B7-2 is a naturally isolated microorganism that is non-pathogenic. Its metabolic process is mild and does not produce secondary pollutants. Compared with chemical crude oil degradation agents, it does not disrupt the ecological balance and has little impact on the indigenous microbial communities in soil and water. It conforms to the concept of green and environmentally friendly pollution control and can be used for the remediation of sensitive polluted areas with high safety requirements. Attached Figure Description

[0040] Figure 1 Colony morphology and microstructure of strain B7-2;

[0041] Figure 2 Culture characteristics of strain B7-2 on different culture media over time;

[0042] Figure 3 Temperature tolerance of strain B7-2;

[0043] Figure 4 Salinity tolerance of strain B7-2;

[0044] Figure 5 pH tolerance of strain B7-2;

[0045] Figure 6 This describes the extracellular enzyme activity characteristics of strain B7-2;

[0046] Figure 7 It serves as a carbon source available to strain B7-2;

[0047] Figure 8 It serves as a nitrogen source available to strain B7-2;

[0048] Figure 9 Analysis of quinone components in strain B7-2;

[0049] Figure 10 Whole-cell sugar analysis for strain B7-2;

[0050] Figure 11 Amino acid analysis of the cell wall of strain B7-2;

[0051] Figure 12 Fatty acid analysis for strain B7-2;

[0052] Figure 13 Phospholipid analysis of strain B7-2;

[0053] Figure 14 Phylogenetic analysis of strain B7-2 and closely related *Goldenella* species based on 16S rRNA sequences for NJ strain;

[0054] Figure 15 Phylogenetic analysis of strain B7-2 and its closely related genus *Goldenella* based on whole genome sequences, where T is the type strain;

[0055] Figure 16 KEGG functional classification diagram for strain B7-2;

[0056] Figure 17 The degradation rate of crude oil (single petroleum hydrocarbon) by strain B7-2. Detailed Implementation

[0057] To enable those skilled in the art to better understand the technical content of the present invention, the technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0058] The components of the culture media and reagents involved are shown below:

[0059] ISP1 medium: 5g tryptone, 3g yeast extract, 20g agar, 1000mL distilled water, adjust pH to 7.0-7.2;

[0060] ISP2 solid medium: 4g yeast extract powder, 10g malt extract, 4g glucose, 20g agar, 1000mL distilled water, adjust pH to 7.3;

[0061] ISP2 liquid culture medium: 4g yeast extract powder, 10g malt extract, 4g glucose, 1000mL distilled water, adjust pH to 7.3;

[0062] ISP3 medium: 20g oats, 1mL trace salt, 20g agar, 1000mL distilled water, adjust pH to 7.3;

[0063] ISP4 medium: 10g soluble starch, 1g K2HPO4, 1g MgSO4·7H2O, 1g NaCl, 2g (NH4)2SO4, 2g calcium carbonate, 1mL trace salt, 20g agar, 1000mL distilled water, adjust pH to 7.2.

[0064] ISP5 medium: 10g glycerol, 1g L-aspartic acid, 1g K2HPO4, 1mL trace salt, 20g agar, 1000mL distilled water, adjust pH to 7.2;

[0065] ISP6 medium: peptone 15g, β-peptone 5g, ferric citrate 0.5g, K2HPO4 1g, N a2 0.08g S2O3, 1g yeast extract powder, 20g agar, 1000mL distilled water, adjust pH to 7.2-7.4;

[0066] ISP7 medium: 15g glycerol, 0.5g L-tyrosine, 1g L-aspartic acid, 0.5g K2HPO4, 0.5g MgSO4·7H2O, 0.5g NaCl, 10mL FeSO4·7H2O, 20g agar, 1000mL distilled water, adjust pH to 7.2-7.4;

[0067] ISP9 medium (carbon and nitrogen source basal medium): (NH4)2SO4 2.64g, KH2PO4 2.38g, K2HPO4 5.65g, MgSO4·7H2O 1g, CuSO4·5H2O (1g / L) 6.4mL, FeSO4·7H2O (1g / L) 1.1mL, MnCl·4H2O (1g / L) 7.9mL, ZnSO4·7H2O (1g / L) 1.5mL, pH 7.2-7.4, distilled water 1000mL;

[0068] NA medium: 5g beef extract, 5g peptone, 5g NaCl, 20g agar, 1000mL distilled water, adjust pH to 7.2-7.4;

[0069] CA medium: sucrose 5g, NaNO3 2g, K2HPO4 0.5g, MgSO4·7H2O 0.5g, KCl 0.5g, FeSO4·7H2O 0.01g, agar 20g, distilled water 1000mL, adjust pH to 7.2-7.4;

[0070] PDA medium: 200g potato, 4g glucose, 20g agar, 1000mL distilled water, adjust pH to 7.2;

[0071] Bennett's medium: D-glucose 10g, yeast extract 1g, beef extract 1g, casein 2g, agar 20g, pH 7.2-7.4, distilled water 1000mL. (Note: Casein should be pre-dissolved in 0.5M NaOH, heated until completely dissolved, and then the pH adjusted).

[0072] Nitrate reducing medium: MgSO4·7H2O 0.5g, K2HPO4 0.5g, KNO3 1g, sucrose 20g, NaCl 0.5g, pH 7.2-7.4, distilled water 1000mL;

[0073] Griess's reagent A: 0.5g of p-aminobenzenesulfonic acid, 150mL of dilute acetic acid (approximately 10%);

[0074] Griess's reagent B: 0.1 g α-naphthylamine, 20 mL distilled water, 150 mL dilute acetic acid (approximately 10%);

[0075] Diphenylamine reagent: Dissolve 0.5g of diphenylamine in 100mL of concentrated sulfuric acid and dilute with 20mL of distilled water;

[0076] Gelatin culture medium: 5g peptone, 20g glucose, 200g gelatin, pH 7.2-7.4, 1000mL distilled water;

[0077] Milk culture medium: 200g skim milk powder, 0.02g CaCO3, mix and boil for 30min (stirring while boiling to avoid boiling, overflowing or precipitation), centrifuge at 4000r / min for 10min, remove milk skin and precipitate, take the supernatant, repeat twice; dispense into glass test tubes, then sterilize at 100-115℃ for 10min, and sterilize intermittently twice;

[0078] Aescin hydrolysis medium: 1g aescin, 10g peptone, 5g NaCl, 0.5g ferric citrate, 20g agar, pH 7.2-7.4, 1000mL distilled water;

[0079] Starch hydrolysis agar medium: 10g soluble starch, 1g MgSO4, 0.3g K2HPO4, 1g KNO3, 2g yeast extract, 0.5g NaCl, 20g agar, pH 7.2-7.4, 1000mL distilled water;

[0080] Lugol's iodine solution: 1g iodine, 2g potassium iodide, 300mL distilled water. First, dissolve the potassium iodide in a small amount of water, then dissolve the iodine in the potassium iodide solution. After all the iodine has dissolved, bring the volume to 300mL.

[0081] CMC Na-MB medium: D-glucose 10g, yeast extract 1g, beef extract 1g, casein 2g, sodium carboxymethyl cellulose 5g, agar 20g, pH 7.2-7.4, distilled water 1000mL;

[0082] Tresner medium: 10g peptone, 0.5g ferric citrate, 20g agar, pH 7.2, 1000mL distilled water;

[0083] Liquid phenol red and urea medium: peptone 1g, NaCl 5g, glucose 1g, KH2PO4 2g, 0.2% phenol red 6mL, 30% urea 66.67mL, pH 6.8-6.9;

[0084] Lipase basal medium: 1g peptone, 5g NaCl, 0.1g CaCl·7H2O, 20g agar, pH 7.4, 1000mL distilled water;

[0085] Methyl red reagent: Weigh 0.04g of methyl red, add 60mL of 95% ethanol, dissolve, then add 40mL of distilled water and store in a brown dropper bottle;

[0086] MR / VP liquid culture medium: 5g peptone, 5g glucose, 5g K2HPO4, 1000mL distilled water;

[0087] Peptone liquid culture medium: 10g peptone, 5g NaCl, 1000mL distilled water;

[0088] Indole reagent: 2g p-dimethylaminobenzaldehyde, 190mL ethanol (95%), 40mL concentrated HCl.

[0089] Example 1: Materials and Methods

[0090] 1.1 Strains

[0091] Gordon's bacteria ( GordonGordon's strain B7-2 was collected in June 2023 from mangrove sediment samples at Qingmei Port, Sanya City, Hainan Province, China (18°13′50.9″N, 109°37′15.9″E) and isolated using the standard dilution plating method. Gordon's strain B7-2 is deposited at the China Center for Type Culture Collection (CCTCC) under accession number CCTCC NO: M 20252269.

[0092] 1.2 Identification of bacterial colony morphology and culture characteristics

[0093] The strains were inoculated into solid media including ISP1, ISP2, ISP3, ISP4, ISP-5, ISP-6, ISP-7, CA (Czapek's agar), NA (nutrient agar), and PDA (potato extract agar) using the three-zone streak method. After incubation at 28°C for 7, 14, 21, and 28 days, their growth, colony morphology and color, presence of intramural and aerial hyphae, and other culture characteristics were observed. The colony color was evaluated using the ISCC-NBS colorimetric card and recorded.

[0094] 1.3 Identification of the microscopic morphology of the strain

[0095] The morphological characteristics of mycelia and spores of the bacterial strain were observed using the petri dish inoculation method. The test bacteria were streaked onto ISP3 medium, and sterilized coverslips were inserted obliquely into the medium using forceps, with 3-4 coverslips per plate. The petri dishes with inoculated coverslips were inverted and incubated at 28℃. The coverslips were removed on days 7 and 14 for observation under an optical microscope. During microscopic examination, the coverslips were carefully removed with forceps, the culture on the reverse side was wiped off, and the bacterial side was placed on the slide to observe the morphological characteristics of aerial hyphae, spore chains, and spores. Simultaneously, the surface layer of agar containing colonies was excised, soaked overnight in 2.5% glutaraldehyde, washed twice with phosphate buffer for 15 min, and then washed twice with 30%, 50%, 70%, 85%, 95%, and 100% ethanol, 15 min for each gradient. The mixture was then freeze-dried for fixation, sporophyted with gold at 15-20 mA for 30 s, and then observed under a scanning electron microscope for spore attachment, spore chains, sporangia, and hyphal breakage.

[0096] 1.4 Identification of the physiological and biochemical characteristics of the strain

[0097] (1) Experiment on the utilization of carbon and nitrogen sources

[0098] Weigh different carbon and nitrogen sources to a final concentration of 1% (w / v), sterilize with ether, and then add to hot ISP9 medium and mix well. The bacterial strains to be tested were simultaneously inoculated onto different carbon and nitrogen source media, and the growth of the cells was observed at 7, 14, and 21 days. Carbon source media included D-galactose, mannitol, glycerol, fructose, dextrin, inositol, soluble starch, maltose, glucose, and sucrose. Nitrogen source media included peptone, soybean flour, yeast extract, ammonium sulfate, urea, and potassium nitrate.

[0099] (2) Temperature tolerance test

[0100] The test strain was streaked onto Bennett medium solid plates and incubated at 4℃, 10℃, 15℃, 20℃, 28℃, 34℃, 37℃, 40℃, 45℃, and 50℃ respectively. The results were observed after 7 days and 14 days. After initially determining the growth temperature, the test range was narrowed to obtain the optimal growth temperature.

[0101] (3) pH tolerance test

[0102] Phosphate buffers with pH values ​​of 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, and 13.0 were prepared using KH2PO4 (0.25M) and K2HPO4 (0.25M). Equal volumes of 2× Benett solid medium were then prepared for each buffer. After sterilization, the buffers with different pH values ​​were mixed together with the medium. The test strains were then inoculated into solid mediums with different pH values ​​and cultured at 28°C. The results were observed after 7 and 14 days.

[0103] (4) Salinity tolerance test

[0104] The tested strains were inoculated into Benett solid medium with NaCl concentrations of 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 15%, and 20%, respectively, and cultured at 28°C. The growth status was observed after 7 days and 14 days. After initially determining the salinity at which the strains could grow, the test range was narrowed down to obtain the optimal salinity for growth.

[0105] (5) Nitrate reduction test

[0106] Inoculate the test bacteria into nitrate-reducing medium and incubate in a static liquid. Take the culture medium from 7 days and 14 days, and add one drop each of Griess' reagent A and B. If the solution turns pink, it indicates a positive result for nitrate reduction. If no red color appears, add 1-2 drops of diphenylamine reagent. If it does not turn blue, it is a positive result; otherwise, it is a negative result.

[0107] (6) Gelatin liquefaction test

[0108] The test strain was inoculated onto the surface of gelatin culture medium tubes without puncture. Three uninoculated gelatin tubes served as blank controls. Results were observed after incubation at 28℃ for 5, 10, 20, and 30 days. Before observation, the tubes were placed in a refrigerator for 20-30 minutes to cool, then the degree of liquefaction was observed. If the gelatin curd partially or completely transformed into a flowable liquid, it was a positive reaction, indicating that the strain had produced gelatinase and the gelatin had been hydrolyzed. If it remained solid, the gelatin had not been hydrolyzed.

[0109] (7) Milk coagulation and peptone test

[0110] The test strain was inoculated into test tubes containing milk culture medium and cultured at 28°C for 5, 10, 20 and 30 days. If a clot was formed, it was considered coagulation; if it was further hydrolyzed into a liquid, it was considered peptone formation.

[0111] (8) Aescin hydrolysis test

[0112] Inoculate the bacteria to be tested onto a saescin hydrolysis medium containing 0.1% (w / v) aescin and culture for 7-14 days. Those that produce dark brown pigment are considered positive.

[0113] (9) Starch hydrolysis test

[0114] The strain was inoculated into starch hydrolysis agar medium using the three-point inoculation method and cultured at 28°C for 6-14 days. After the strain had grown well, Lugol's iodine solution was added around the colony for testing. If there was a clear, colorless ring around the colony, it was a positive result; if it turned blue-black, it was a negative result.

[0115] (10) CMC (carboxymethyl cellulase) degradation experiment

[0116] The strain was inoculated into CMC Na-MB medium using a three-point inoculation method and incubated at 28°C for 7–14 days. Staining was performed with 0.1% Congo red solution for 10 min, followed by destaining with 1 mol / L NaCl solution. Colonies showing a clear zone around their surface were considered positive for CMC degradation, while those without were considered negative.

[0117] (11) Protease experiment

[0118] Take 5g of skim milk powder (or 50mL of skim milk), and separately weigh 1.5g of agar and dissolve it in 50mL of distilled water. Sterilize the two solutions separately. After cooling to 45-50℃, mix the two solutions and pour them onto agar plates. Dry the plates by blowing them dry and inverting them overnight to allow surface moisture to evaporate. Then, use the three-point inoculation method to inoculate the bacterial strain onto the milk agar plates. Incubate at 28℃ for 3, 5, and 7 days, recording the presence and absence of a clear zone. A clear zone around the colony indicates positive protease activity; otherwise, it indicates negative activity.

[0119] (12) Catalase (H2O2 enzyme) test

[0120] Take a small loop of bacteria in the logarithmic growth phase and smear it onto a glass slide that has been dotted with 3% hydrogen peroxide. If bubbles are produced, the result is positive; otherwise, it is negative.

[0121] (13) Melanin production experiment

[0122] The test strain was inoculated onto experimental culture medium plates and cultured at 28°C for 7 days and 14 days. The presence or absence of melanin was then observed.

[0123] Experimental culture medium: composition reference ISP-6 medium.

[0124] (14) Experiment on hydrogen sulfide generation

[0125] The test strain was inoculated onto Tresner medium slant and incubated at 28°C for 7 days and 14 days. If it turned black, it was considered a positive result.

[0126] (15) Urease test

[0127] The experimental strain was inoculated into test tubes with liquid phenol red urea medium and cultured at 28°C and 180 rpm for 2-14 days. The results were then observed. If negative, the culture was continued for another 5-7 days. A pink color in the medium indicated a positive result, while no color change indicated a negative result.

[0128] (16) Lipase experiment (Tween-20, Tween-60, Tween-80)

[0129] Tween-20, Tween-60, and Tween-80 were sterilized at 121℃ for 20 min. Lipase basal medium was prepared, cooled to 50℃, and sterilized Tween-20, Tween-60, and Tween-80 were added to a final concentration of 1%. Plates were then poured. The strains were streaked onto the plates and incubated at 28℃ for 7-14 days, with daily observation. A faint halo around the bacterial cells indicates a positive result; the absence of a halo indicates a negative result.

[0130] (17) Methyl red test (MR test)

[0131] Inoculate the strain into MR / VP liquid medium, set up 3 replicates and 1 blank, and incubate at 28℃ and 180rpm for 2-14 days. Then add 1 drop of methyl red reagent to each culture. If it turns red, it is positive and yellow is negative.

[0132] (18) Volt-Phase experiment (VP experiment)

[0133] The strain was inoculated into MR / VP liquid medium, with 3 replicates and 1 blank. Incubation was carried out at 28°C and 180 rpm for 2-14 days. After the bacteria had grown well, an equal volume of the culture medium was mixed with 40% NaOH, and a small amount of creatine was added. A positive result was indicated if the culture medium turned red after 10 minutes. Sometimes, a longer incubation period is required for the red reaction to appear.

[0134] (19) Tryptophan decomposition (indole production) test

[0135] The bacterial strain was inoculated into peptone liquid medium, with 5 replicates and corresponding controls, and incubated at 28°C. After 1, 3, 5, 7, and 9 days of incubation, indole reagent (3-5 mm high) was slowly added along the tube wall to the surface of the culture medium. A red color at the interface indicates a positive reaction. If the color is not obvious, 4-5 drops of diethyl ether can be added to the peptone culture medium, shaken to disperse the ether, and allowed to stand for 2-3 minutes until the ether floats to the surface before adding indole reagent.

[0136] (20) Citrate test

[0137] Inoculate the bacterial strain into citrate slant agar. Set up three replicates and corresponding controls, incubate at 28°C for 3-7 days, and observe for growth and color change of the test bacteria. A blue color indicates a positive reaction, while a green color indicates a negative reaction.

[0138] (21) Iron Carrier Capacity Test (CAS)

[0139] Single colonies of the test strain were picked and inoculated onto modified CAS medium plates and incubated at 28°C in the dark for 7 days. Strains that produce an orange-yellow halo around the colony are capable of producing siderophores. The larger the halo, the stronger the siderophore-producing ability. Each strain was replicated three times.

[0140] (22) Determination of the ability to produce β-1,3-glucanase

[0141] Single colonies of the biocontrol strain were inoculated onto β-1,3-glucanase agar plates and incubated at 28°C in the dark for 7 days. If a clear halo appeared around the colony, it indicated that the strain could produce β-1,3-glucanase, and was positive for β-1,3-glucanase degradation; otherwise, it was negative. Each strain was repeated three times.

[0142] 1.5 Identification of strains by chemical classification

[0143] (1) Amino acid analysis of cell wall

[0144] The determination was performed using thin-layer chromatography (TLC) of microcrystalline cellulose.

[0145] (2) Whole-cell glycogen analysis

[0146] The determination was performed using thin-layer chromatography (TLC) of microcrystalline cellulose.

[0147] (3) Detection of fatty acid components in whole cells

[0148] Fatty acids were analyzed and determined using the whole-cell fatty acid methyl esterification-GC-MS qualitative and quantitative analysis method.

[0149] (4) Quinone component analysis

[0150] The extraction and analysis of methylnaphthoquinone were mainly carried out in accordance with the method of Collins (Collins et al., 1977).

[0151] (5) Phospholipid analysis

[0152] Phospholipid analysis mainly employs thin-plate chromatography (TLC) (Minnikin, 1984; Ruan Jisheng et al., 1990).

[0153] 1.6 Molecular identification of strains

[0154] (1) Extraction of genomic DNA

[0155] Gordon's bacteria Gordon A strain of sp. B7-2 was streaked onto ISP2 solid medium and cultured at 28°C for 7 days. Subsequently, a single colony from the solid medium was picked and inoculated into 50 mL of ISP2 liquid medium, and cultured at 28°C with shaking at 180 rpm for approximately 5 days to obtain a seed culture. Next, 2 mL of the seed culture was transferred to 200 mL of fresh ISP2 liquid medium and cultured a second time under the same conditions (28°C, 180 rpm) for another 7 days. After the culture was completed, the culture was centrifuged at 4°C, 8,000 rpm for 10 min, and washed with sterile water to obtain young bacterial cells. Genomic DNA of strain B7-2 was extracted using a bacterial genomic DNA extraction kit (magnetic bead method) (Beijing Tiangen Biotech Co., Ltd.), following the manufacturer's instructions. The extracted genomic DNA was purified and quantified, and high-quality DNA was selected for subsequent studies.

[0156] (2) Phylogenetic analysis based on 16S rRNA gene sequence

[0157] The 16S rRNA gene sequence was amplified and sequenced. After sequencing, the gene sequences were compared with those of the model strains with high similarity. Multiple sequence alignment was performed using Clustal W in MEGA 7.0 software. Neighbour-Joining was used for cluster analysis, and a phylogenetic tree was constructed. The phylogenetic matrix was estimated based on the Kimura-2-parameter model. Bootstrap analysis was performed after 1000 repeated samplings to evaluate the stability of the phylogenetic tree topology.

[0158] (3) Genome sequencing and assembly

[0159] We commissioned Meiji Biopharmaceutical Technology Co., Ltd. (Shanghai, China) to perform whole-genome sequencing on the PacBio Sequel II and Illumina HiSeq 2000 platforms. They provided PacBio sequencing data and Illumina sequencing data with a coverage depth of no less than 100 times that of the genome to ensure a more complete and accurate assembly, ultimately assembling a complete genome chromosome.

[0160] (4) Phylogenetic analysis based on whole genome sequence

[0161] use Gordon A phylogenetic tree was constructed from the whole genome sequence of sp. B7-2, and the ANI and dDDH values ​​of B7-2 and closely related strains were calculated.

[0162] 1.7 Genome annotation of strain B7-2

[0163] Genome structure annotation can be performed using the free online platform of MajorBio Cloud Platform (http: / / cloud.majorbio.com).

[0164] 1.8 Validation of the petroleum degradation performance of strain B7-2

[0165] Aseptic crude oil basic salt culture medium: Add 29g NaCl, 3g Na2HPO4, 1g KH2PO4, 1g NaH2PO4, 1g KNO3, 1g NH4Cl, 0.7g MgSO4·7H2O, and 1mL trace element culture medium to 1000mL of distilled water, and sterilize at 121℃ for 20min. The formula for the trace element culture medium is: Add 20mg CaCl2, 30mg FeCl3, 0.5mg CuSO4, 0.5mg MnSO4·H2O, and 10mg ZnSO4·7H2O to 1000mL of distilled water, and sterilize.

[0166] Crude oil culture medium: Add 300 mg of crude oil sterilized by ether to 1 L of sterile crude oil base salt culture medium.

[0167] To study Gordon's bacteria Gordon To investigate the biodegradability of *Sp. B7-2* on petroleum, following the method of Yang et al., *Sp. B7-2* was inoculated into a crude oil culture medium with crude oil as the sole carbon source. After cultivation, its degradation rate on crude oil was determined. Single colonies of *Sp. B7-2* were inoculated into ISP2 liquid medium and cultured at 28°C and 180 rpm for 3 days to obtain a seed culture. This seed culture was then inoculated into ISP2 liquid medium at a 1% inoculation rate for 7 days. After centrifugation at 8000 rpm for 20 minutes at 4°C, the supernatant was discarded, and the bacterial cells were resuspended in sterile water. The cells were washed twice to remove residual culture medium. Finally, the bacterial suspension concentration was adjusted to OD500 with sterile water. 600 =1.0. Then, a 2% (v / v) bacterial suspension was inoculated into 100 mL of crude oil culture medium, while the control group was directly inoculated with 2% (v / v) sterilized liquid ISP2 culture medium. Three parallel experiments were set up, and the absorbance of the remaining crude oil was measured using a spectrophotometer after culturing at 28℃ and 180 rpm for 7 days, 14 days, 21 days, and 28 days, respectively.

[0168] Crude oil standard curve determination: A petroleum standard solution (5000 mg / L) was prepared using petroleum ether as the solvent and serially diluted to obtain a series of calibration solutions with concentrations of 0 mg / L, 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, and 500 mg / L. A full-wavelength scan of the petroleum-petroleum ether standard solution was performed to determine the appropriate detection wavelength, and the concentration of petroleum was determined using a UV-Vis spectrophotometer at the maximum absorption wavelength of 600 nm. A standard curve was plotted with the concentration of the petroleum standard solution on the x-axis and absorbance on the y-axis.

[0169] Sample pretreatment: The bacterial culture broth infused with crude oil was centrifuged at 9000 rpm for 20 min to remove cell biomass. The supernatant was collected and the residual crude oil was extracted with petroleum ether using a separatory funnel. The extract was transferred to a 100 mL volumetric flask and brought to volume.

[0170] Take 3 mL of the extract and measure its absorbance (OD) at 600 nm using a quartz cuvette. 600 The crude oil residue in the supernatant was calculated based on the standard curve. All experiments were performed in triplicate, and the average value was taken.

[0171]

[0172] Where C0 represents the initial content of petroleum (mg / L), Ct This indicates the residual content of petroleum at time t (mg / L).

[0173] Example 2: Classification characteristics of strain B7-2

[0174] Gordon The cells of sp. B7-2 are short rod-shaped, Gram-positive, and the colonies are orange, indicating that it is a strict aerobic bacterium. Figure 1 This strain can grow on ISP1, ISP2, ISP3, ISP4, ISP5, ISP6, ISP7, NA, CA, and PDA media, but does not produce diffusible pigments, including melanin-like pigments. Figure 2 The growth temperature range is 15–37°C (optimal 20–34°C). Figure 3 The NaCl tolerance concentration is 0-10% (w / v) (optimal is no addition). Figure 4 The pH tolerance range is 4.0-12.0 (optimal pH is 6.0-10.0). Figure 5 Catalase activity was positive, and nitrate reduction activity was weakly positive. It can degrade esculin and sodium carboxymethyl cellulose, but cannot degrade gelatin, casein, starch, or dextran. It can produce siderophores (…). Figure 6 Available carbon sources include: D-galactose, D-mannose, D-fructose, D-trehalose, D-maltose, D-glucose, L-arabinose, L-rhamnose, mannitol, glycerol, olive oil, dextrin, inositol, soluble starch, maltose, glucose, ethanol, sucrose, and n-hexadecane. Growth is vigorous when glucose is used as a carbon source. Potassium acetate is not available. Figure 7 Available nitrogen sources include: D-glutamic acid, L-alanine, L-leucine, L-proline, peptone, soybean meal, yeast extract, diammonium hydrogen phosphate, ammonium sulfate, and urea; potassium nitrate and soybean meal are not available. Figure 8 The main methylnaphthoquinone is MK-9(H2) (100%). Figure 9 Whole-cell glycosides include arabinose and galactose. Figure 10 The cell wall amino acid components include alanine, glycine, and meso-diaminopimelic acid (meso-DAP). Figure 11 ( ), is the cell wall of type IV actinomycetes. Its main fatty acid component is C. 16:0 Summary Feature 3 (C) 16:1 ω7c / C 16:1 ω6c), 10-methylC 18:0 (tuberculous stearic acid), C 18:1 ω9c, C 17:0 and C 18:0 ( Figure 12The main polar lipids include: diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside, two unidentified phospholipids, one unknown aminoglycolipid, and five unidentified lipids. Figure 13 ).

[0175] Example 3: Phylogenetic analysis of strain B7-2

[0176] 3.1 Phylogenetic analysis based on 16S rRNA

[0177] The 16S rRNA sequence of strain B7-2 was compared with that of closely related strains to... Streptomyces griseoincarnatus LMG 19316T (AJ781321) was an outgroup, and a phylogenetic tree of NJ was constructed using MEGA11.0. The results are as follows: Figure 14 As shown, strain B7-2 and Gordonia polyisoprenivorans NBRC 16320 (98.52%) Gordonia oryzae RS15-1S (98.15%) clustered into a single branch, with its maximum similarity to closely related strains all below 99%, suggesting that it may be a... Gordonia It is a potential new species.

[0178] 3.2 Phylogenetic analysis based on whole genome sequence

[0179] Furthermore, a phylogenetic tree based on the whole genome sequence of strain B7-2 and closely related strains was constructed online, and the ANI and dDDH values ​​of B7-2 and closely related strains were calculated. The results are as follows: Figure 15 As shown, strain B7-2 forms a separate branch, which is closely related to... Gordonia The ANI values ​​of the strains ranged from 74.31 to 77.63, and the dDDH values ​​ranged from 20.1 to 23.0, both below the critical values ​​for prokaryotic classification and identification (ANI value <95%-96% and dDDH value <70%). However, *Gordonella glutinis*, which has the most similar 16S rRNA gene sequence, was the most similar. Gordonia polyisoprenivorans The ANI value of NBRC 16320 (98.52%) and strain B7-2 was 75.90, and the dDDH value was 21.2; *Gordonella rice* Gordonia oryzae The ANI value of RS15-1S (98.15%) and strain B7-2 was 75.83, and the dDDH value was 20.7, which further confirms that B7-2 is the most likely candidate strain. Gordonia It is a new strain.

[0180] 3.3 Differential characteristics of strain B7-2 and closely related species of Gordon's bacterium

[0181] 3.3.1 Growth and Colony Morphology Characteristics in Different Culture Media

[0182] Based on molecular sequence characteristics, strain B7-2 and Gordonia oryzae RS15-1S and Gordonia polyisoprenivorans NBRC 16320 showed the highest similarity. Further comparisons were made between strain B7-2 and the two strains mentioned above, examining their growth and colony morphology on ISP2-7 medium at 28°C for 7 days. The results are shown in Table 1. The culture characteristics of B7-2 are from this study. Gordonia polyisoprenivorans NBRC 16320 and Gordonia rice The culture characteristics of RS15-1S were derived from the studies of Linos et al. (Linos A, Steinbüchel A, Spröer C, Kroppenstedt RM. Gordonia polyisoprenivorans sp. nov., a rubber-degradingactinomycete isolated from an automobile tyre. Int J Syst Bacteriol 1999;49:1785–1791.) and Muangham et al. (Muangham S, Lipun K, Thamchaipenet A, Matsumoto A and Duangmal K. Gordonia oryzae sp. nov., isolated from rice plant stems (Oryza sativa L.). Int J Syst Evol Microbiol 2019;69:1621-1627). The results indicate that strain B7-2... T Growth and colony morphology characteristics on different culture media can be compared with G.oryzae RS15-1S T and G. polyisoprenivorans NBRC 16320 T There are differences. Specifically, B7-2 T and G. polyisoprenivorans NBRC16320 T It grows well on ISP2 agar medium, while G.oryzae RS15-1S T Then growth is weaker; G. polyisoprenivorans NBRC 16320 T Growth is moderate in ISP5, while in B7-2 T and G.oryzae RS15-1S TGrowth is weaker; B7-2 T It grows well on ISP6, while G.oryzae RS15-1S T Growth is weak.

[0183] Table 1 Comparison of culture characteristics of strain B7-2 and closely related species of the genus *Goldenella*

[0184]

[0185] 3.3.2 Physiological and Biochemical Characteristics

[0186] Strain B7-2 T The similarities and differences in the physiological and biochemical characteristics of strain B7-2 and its closely related genus *Gordonella* are shown in Table 2. T It can grow in environments with a pH range of 10.0-12.0, and grows vigorously in an environment with a pH of 10.0. G.oryzae RS15-1S T It cannot grow in an environment with a pH of 10.0; strain G.oryzae RS15-1S T Salt tolerance up to 13%, B7-2 T Its salt tolerance is only 10%; at 40°C, G. polyisoprenivorans NBRC 16320 T It can grow, while strain B7-2 T and G.oryzae RS15-1S T Then it cannot grow. Strain B7-2 T It can hydrolyze aescin and sodium carboxymethyl cellulose, and has nitrate reducing properties, but G.oryzae RS15-1S T I do not have this ability. G.oryzae RS15-1S T It can produce hydrogen sulfide, while B7-2 T No, it is not possible. (B7-2) T It can utilize D-maltose, L-arabinose, L-rhamnose, D-galactose, D-mannose, mannitol, and inositol as the sole carbon source for growth, while G.oryzae RS15-1S T No, G. polyisoprenivorans NBRC 16320 T Only L-rhamnose, D-galactose, mannitol, and inositol can be used; D-maltose and D-mannose cannot. B7-2 T It can be grown using L-alanine, L-leucine, and L-proline as the sole nitrogen source, while G. polyisoprenivorans NBRC 16320 T L-leucine cannot be utilized. G.oryzae RS15-1ST It can grow using potassium nitrate as the sole nitrogen source, while B7-2 T Then it is not possible.

[0187] This indicates that strain B7-2 T It differs from closely related species in the genus *Gordonella* in physiological and biochemical characteristics, suggesting that strain B7-2... T It is a new species of the genus *Gordonella*.

[0188] Table 2. Strain B7-2 T Comparative analysis of physiological and biochemical characteristics with closely related strains of Gordon's disease

[0189]

[0190] Note: "+" indicates that the physiological and biochemical test result is "positive", "-" indicates negative, and "ND" indicates not measured.

[0191] 3.3.3 Characteristics of Chemical Taxonomy

[0192] Strain B7-2 T The methylnaphthoquinone is MK-9(H2) (100%), while G.oryzae RS15-1S T The methylnaphthoquinone components are MK-9(H2) and MK-8(H2). G. polyisoprenivorans NBRC 16320 T Like the B7-2, only the MK-9 (H2) was produced. (B7-2) T The whole-cell glycosides contain arabinose and galactose, while G.oryzae RS15-1S T The total glycogen content of the cell consists of arabinose, galactose, mannose, and ribose. G. polyisoprenivorans NBRC 16320 T It consists of glucose and arabinose. B7-2 T The cell wall amino acid composition contains alanine, glycine, and meso-diaminopimelic acid (meso-DAP), which is characteristic of type IV actinomycete cell walls. The strain... G.oryzae RS15-1S T and G. polyisoprenivorans NBRC 16320 T meso-DAP was then used as the sole amino acid. (Strain B7-2) T The main polar lipids include diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside, two unidentified phospholipids, one unknown aminoglycolipid, and five unidentified lipids. G.oryzae RS15-1S TThe polar lipids include diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol mannoside, an unidentified polar lipid, and two unidentified phospholipids. G. polyisoprenivorans NBRC 16320 T Polar lipids consist of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside, and some unidentified glycolipids. (Strain B7-2) T and G.oryzae RS15-1S T and G. polyisoprenivorans NBRC 16320 T The differences in cellular fatty acid composition are shown in Table 3.

[0193] Table 3. Comparative analysis of cellular fatty acids between strain B7-2 and its close relative, *Gordonella* strain.

[0194]

[0195] Example 4: Genome profile of strain B7-2

[0196] Gordon's B7-2 ( Gordon The total genome length of sp. B7-2 is 5,393,941 bp. The genome profile is shown in Table 4.

[0197] Table 4. Overview of the whole genome

[0198]

[0199] Example 5: Functional annotation of strain B7-2

[0200] 5.1 Gene Annotation

[0201] Gordon a sp. B7-2 annotated a total of 4887 coding sequences across the entire genome, with a total length of 4879074 bp, representing 90.45% of the total genome. Among these coding genes, 3313 genes provided GO information, 3821 genes provided COG information, 2240 genes provided KEGG information, 4813 genes provided NR information, and 3400 genes provided Swiss-Prot information.

[0202] 5.2 Annotation of the GO (GeneOntology) database

[0203] GO database annotation includes three aspects: biological processes (bp), cellular components (cc), and molecular functions (mf). The statistical results for B7-2 in the GO database across these three categories are as follows: GO classification identifies 3313 genes across 42 functional categories, representing 67.79% of all genes. Among these, 1494 genes are associated with bp, 1250 with cc, and 2748 with mf. Based on GO gene annotations and enzyme activities, further analysis revealed core genes associated with petroleum degradation, including: protocatechuic acid 3,4-dioxygenase α / β subunits (genes 4336, 4337), biphenyl-2,3-diol 1,2-dioxygenase (gene 0804), 4-hydroxy-2-oxovalerate aldolase (genes 0791, 2627, 4366), alkanol monooxygenase (gene 1604), acetaldehyde dehydrogenase (acetylation) (genes 0790, 2625, 2626, 4367), and acetyl-CoAC-acyltransferase (genes 1860, 4089, 1401). This indicates that B7-2 does not function independently in petroleum degradation but rather participates synergistically in multiple metabolic pathways, forming a complete degradation network.

[0204] Aromatic hydrocarbon degradation pathway: gene0804 (biphenyl-2,3-diol dioxygenase) → gene4336 / 4337 (protocatechuic acid dioxygenase) → gene0791 / 2627 / 4366 (4-hydroxy-2-oxovalerate aldolase) → gene0790 / 2625, etc. (acetaldehyde dehydrogenase) → enters the tricarboxylic acid cycle.

[0205] Alkane degradation pathway: gene1604 (alkanol monooxygenase) → gene0138 (aldehyde dehydrogenase) → gene1860 / 4089, etc. (acetyl-CoA C-acyltransferase) → fatty acid β-oxidation → entering the tricarboxylic acid cycle.

[0206] 5.3 KEGG (Kyoto Encyclopedia of Genes and Genomes) Database Annotations

[0207] KEGG functional classifications such as Figure 16As shown, strain B7-2 had 4053 genes annotated. Enrichment analysis revealed 2341 genes annotated in metabolic pathways. Of these, 244 genes (10.42%) were related to carbohydrate metabolism, and 124 genes (5.30%) were related to xenobiotic degradation and metabolism. A total of 119 genes were annotated in cellular processes, with the largest number of annotated genes found in prokaryotes. Metabolic pathways annotated for xenobiotic degradation and metabolism included benzoic acid degradation (ko00362), xylene degradation (ko00622), chlorocyclohexane and chlorobenzene degradation (ko00625), styrene degradation (ko00643), and chloroalkyl and chloroolefin degradation (ko00361).

[0208] The metabolic pathways annotated for xenobiotic degradation and metabolism are shown in Table 5. Statistical analysis indicates that a significant number of genes involved in the metabolism of petroleum pollutants were annotated for xenobiotic degradation and metabolism, suggesting that this strain has the potential to degrade petroleum pollutants.

[0209] Table 5. Partial biodegradation and metabolic regulatory pathways and related genes in the B7-2 genome.

[0210]

[0211] This indicates that the strain may have a high efficiency in degrading aromatic hydrocarbons, and is particularly adept at degrading various monocyclic and polycyclic aromatic hydrocarbons such as benzoic acid, toluene, xylene, ethylbenzene, naphthalene, and styrene through the catechol and protocatechuic acid pathways.

[0212] Example 6: Analysis of the petroleum degradation characteristics of strain B7-2

[0213] Will Gordon a sp. B7-2 was inoculated into a crude oil medium containing 300 mg / L crude oil as the sole carbon source and cultured in shake flasks. The concentration of residual crude oil was measured to assess the degradation characteristics of the strain on crude oil. The results are as follows: Figure 17 As shown, after 7 days of cultivation, the crude oil concentration in the culture system decreased from 300 mg / L to 192.03 ± 42.02 mg / L, with a degradation rate of 35.98% ± 13.99%. With the extension of cultivation time to 14 days, 21 days, and 28 days, the degradation rate increased to 42.41 ± 16.16, 60.08 ± 10.28, and 64.33 ± 12.00, respectively. The results indicate that the degradation efficiency of B7-2 is positively correlated with cultivation time (R0). 2The degradation kinetics of the crude oil (>0.98) can be divided into two phases: the adaptation phase (0-14 days), characterized by a slow degradation rate (1.71% per day), as the bacteria need to adapt to the hydrophobic substrate; and the exponential phase (14-28 days), where degradation accelerates (2.32% per day), indicating that the enzyme system is fully induced, driving the ring-opening of aromatic hydrocarbons. This dynamic process clearly shows that strain B7-2 maintained vigorous metabolic activity throughout the experimental period, and that the degradation of crude oil was a continuous and stable process. Even in the later stages of cultivation (21-28 days), the degradation rate continued to increase steadily, indicating that the strain can effectively adapt to a growth environment where crude oil is the main carbon source and continuously exert its degradation effect.

[0214] The above description is only a preferred embodiment of the present invention and is 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 within the protection scope of the present invention.

Claims

1. A strain of Gordon's bacteria ( Gordoni (a sp.) B7-2, characterized in that, It is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M 20252269, and the deposit date is October 20, 2025.

2. A method for culturing Gordon's bacterium B7-2 as described in claim 1, characterized in that, Cultured using any of the following culture media: ISP1: Each 1000mL of water contains 5-10g of tryptone, 3-10g of yeast extract, and 20-30g of agar; ISP2: Each 1000mL of water contains 4-10g of yeast extract, 10-20g of malt extract, 4-8g of glucose, and 20-30g of agar; ISP3: Each 1000mL of water contains 20-30g of oats, 1-5mL of trace salt, and 20-30g of agar; ISP4: Each 1000mL of water contains 10-20g of soluble starch, 1-8g of K2HPO4, 1-5g of MgSO4·7H2O, 1-3g of NaCl, 2-5g of (NH4)2SO4, 2-6g of calcium carbonate, 1-3mL of trace salts, and 20-30g of agar. ISP5: Each 1000mL of water contains 10-20g of glycerol, 1-5g of L-aspartic acid, 1-6g of K2HPO4, 1-3mL of trace salts, and 20g of agar; ISP6: Each 1000mL of water contains 15-25g of peptone, 5-10g of acetic acid peptone, 0.5-2.0g of ferric citrate, 1-3g of K2HPO4, 0.08-0.15g of Na2S2O3, 1-3g of yeast extract powder, and 20-30g of agar; ISP7: Each 1000mL of water contains 15-25g of glycerol, 0.5-3.0g of L-tyrosine, 1-5g of L-aspartic acid, 0.5-3.0g of K2HPO4, 0.5-2.0g of MgSO4·7H2O, 0.5-3.0g of NaCl, 10-20mL of FeSO4·7H2O, and 20-30g of agar; NA: Each 1000mL of water contains 5-10g of beef extract, 5-10g of peptone, 5-10g of NaCl, and 20-30g of agar; CA: Per 1000mL of water, there are 5-10g of sucrose, 2-3g of NaNO3, 0.5-3.0g of K2HPO4, 0.5-3.0g of MgSO4·7H2O, 0.5-2.0g of KCl, 0.01-2.50g of FeSO4·7H2O, and 20-30g of agar; PDA: Each 1000mL of water contains 200-250g of potato, 4-10g of glucose, and 20-30g of agar; The trace salt consists of the following components: 0.1g FeSO4·7H2O, 0.1g MnCl2·4H2O, and 0.1g ZnSO4•7H2O per 100mL of water.

3. A method for culturing Gordon's bacterium B7-2 as described in claim 1, characterized in that, Gordonella B7-2 strain was inoculated onto Bennett medium and cultured. The Bennett medium consisted of: 10g D-glucose, 1g yeast extract, 1g beef extract, 2g casein, 20g agar, pH 7.2-7.4, and 1000mL distilled water. The growth conditions for Gordonella B7-2 were: growth temperature 20-34℃, and NaCl concentration 0% (w / v).

4. The application of Gordon's B7-2 as described in claim 1 in the degradation of crude oil.

5. The application as described in claim 4, characterized in that, Includes the following steps: S1. Pick a single colony of Gordon's B7-2 and inoculate it into ISP2 liquid medium. Culture it on a shaker to obtain the seed culture. S2. Inoculate the seed culture into fresh ISP2 liquid medium for expansion culture, centrifuge, discard the supernatant, resuspend the cells, and wash. S3, Adjust the bacterial suspension concentration OD 600 =1.0 and then inoculated into crude oil culture medium for degradation.

6. The application as described in claim 5, characterized in that, In step S2, the inoculation amount of seed liquid is 1-3% by volume.

7. The application as described in claim 5, characterized in that, The method for preparing the crude oil culture medium in step S3 is as follows: 300 mg of crude oil sterilized with ether is added to every 1 L of sterile crude oil base salt culture medium; the method for preparing the sterile crude oil base salt culture medium is as follows: 29-40 g of NaCl, 3-10 g of Na2HPO4, 1-5 g of KH2PO4, 1-5 g of NaH2PO4, 1-6 g of KNO3, 1-3 g of NH4Cl, 0.7-1.5 g of MgSO4·7H2O, and 1-3 mL of trace element culture medium are added to every 1000 mL of distilled water and then sterilized; the method for preparing the trace element culture medium is as follows: 20-30 mg of CaCl2, 30-40 mg of FeCl3, 0.5-1.5 mg of CuSO4, 0.5-2.5 mg of MnSO4·H2O, and 10-20 mg of ZnSO4·7H2O are added to every 1000 mL of distilled water and then sterilized.

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