Gordonia cholesterol-eating YDY-4 and application thereof in degradation of n-hexadecane

Abstract

The invention discloses a Gordonia ovorans YDY-4 strain and an application of the Gordonia ovorans YDY-4 strain in degradation of n-hexadecane. The cholesterol-eating Gordonia sp. YDY-4 is named as the cholesterol-eating Gordonia sp. YDY-4, is preserved in the China Center for Type Culture Collection (CCTCC), and has the preservation number of CCTCC M 20251290, and the cholesterol-eating Gordonia sp. YDY-4 is named as the cholesterol-eating Gordonia sp. YDY-4 and is preserved in the China Center for Type Culture Collection (CCTCC). The Gordonia ovorans YDY-4 provided by the invention has the capability of degrading n-hexadecane under the condition that the pH (Potential of Hydrogen) is 7.0 to 9.0, and can grow at 28 to 32 DEG C. The Gordonia ovorans YDY-4 provided by the invention has relatively strong environmental adaptability and n-hexadecane degradation capability, and provides a basis for bioremediation of petroleum polluted environment and application of a microbial enhanced treatment technology.

Description

Technical Field

[0001] This invention relates to the field of microbiology, specifically to a highly efficient novel hexadecane-degrading bacterium—Gordonia cholesterolivorans YDY-4—and its application in the microbial degradation of hexadecane. Background Technology

[0002] With the rapid development of the global petroleum industry, a large amount of petroleum hydrocarbon pollutants continuously enter the soil and aquatic environment, posing a serious threat to ecosystems and human health. Petroleum is a complex multi-component system, mainly composed of double-chain hydrocarbons such as alkanes and cycloalkanes, aromatic compounds (benzene, toluene, ethylbenzene, xylene), polycyclic aromatic compounds (naphthalene, phenanthrene, anthracene, benzo(a)pyrene), resins, asphaltenes, and small amounts of oxygen-, sulfur-, and nitrogen-containing compounds. Due to the high hydrophobicity and chemical stability of petroleum hydrocarbons, they are difficult to remove effectively from the environment through non-biological pathways such as hydrolysis or photolysis, resulting in their long-term persistence in contaminated sites. Pollutants can cause various toxicological health problems to humans and animals, including hematologic toxicity, carcinogenicity, genotoxicity, mutagenicity, teratogenicity, cytotoxicity, neurotoxicity, immunotoxicity, nephrotoxicity, hepatotoxicity, cardiotoxicity, and ophthalmic toxicity, causing plant growth retardation, root necrosis, and decreased resistance to pests and diseases.

[0003] In petroleum hydrocarbon components, alkanes account for 50–95% of the total content. Among them, n-hexadecane (C 16 H 34 Alkylene is a major component of alkanes, and is characterized by its low water solubility (its solubility in water at 15°C is 5.21 × 10⁻⁶). -5 Petroleum pollutants (mg / L) are non-volatile and found in highly polluted oilfields, making them a representative target for petroleum pollution research. Compared with physicochemical methods, biological treatment methods have outstanding advantages such as superior performance, low cost, simple operation, and most importantly, environmental friendliness with no secondary pollution. They can degrade petroleum pollutants into non-toxic carbon dioxide and water through biological metabolism.

[0004] Numerous microorganisms possess the ability to degrade n-hexadecane, with reported bacteria including Rhodococcus, Pseudomonas, Acinetobacter, and Bacillus. However, the efficiency of *Brevibacillus nitrificans* YJ1 in degrading n-hexadecane, as described in the existing patent (CN110591972 A), is significantly limited: even with an initial concentration of 10 mL / L and an inoculum size as high as 10%, the degradation rate only reaches 66.7% after 15 days of treatment. Summary of the Invention

[0005] The purpose of this invention is to provide a cholesterol-eating bacterium Gordoniae YDY-4 and its application in the degradation of n-hexadecane.

[0006] The technical solution adopted in this invention is as follows:

[0007] (a) A strain of cholesterol-eating Gordon's bacterium YDY-4

[0008] The cholesterol-eating Gordon's bacterium YDY-4 is named *Gordonia cholesterol-eating Gordon's bacterium* YDY-4, and is deposited at the China Center for Type Culture Collection (CCTCC) on June 6, 2025, with accession number CCTCC M 20251290. The deposit address is: Wuhan University, 299 Bayi Road, Wuchang District, Wuhan, China, 430072, China.

[0009] The nucleotide sequence of the 16S rDNA of the cholesterol-eating Gordon's YDY-4 is shown in SEQ ID NO: 1.

[0010] The characteristics of the cholesterol-eating Gordon's bacterium YDY-4 are: colonies are white or pale yellow, round, opaque, and have a smooth surface. Scanning electron microscopy reveals that the morphology of this bacterium is that of a bacillus.

[0011] The *Gordonella oryzae* strain YDY-4 is an aerobic bacterium that can reproduce under pure culture conditions using n-hexadecane as the sole carbon source, producing intermediate products such as aldol acids, and ultimately generating CO2 and H2O. Within a pH range of 8.0–10.0, with an initial n-hexadecane content ranging from 0.1% to 1.0% (v / v), this bacterium demonstrates the ability to degrade n-hexadecane, exhibiting strong environmental adaptability.

[0012] (II) A bacterial solution

[0013] The active ingredient in the bacterial solution includes the aforementioned cholesterol-eating Gordon's bacterium YDY-4.

[0014] (III) A method for preparing bacterial culture

[0015] The preparation method specifically involves culturing the cholesterol-eating Gordon's bacterium YDY-4 in LB medium to obtain a bacterial culture.

[0016] The LB medium comprises deionized water and the following components at the following concentrations: NaCl 10.0 g / L, tryptone 10.0 g / L, yeast extract 5.0 g / L, and pH 7.0.

[0017] (iv) Application of a cholesterol Gordon's bacterium YDY-4, the above-mentioned bacterial solution, or the bacterial solution obtained by the above preparation method in the degradation of n-hexadecane.

[0018] (V) A method for degrading n-hexadecane

[0019] Specifically, the bacterial agent containing *Gordonella cholerae* YDY-4, the above-mentioned bacterial solution, or the bacterial solution obtained by the above preparation method is inoculated into an environment containing n-hexadecane to be degraded, and the n-hexadecane is degraded under conditions of 28–32°C and pH = 8.0–10.0.

[0020] Preferably, hexadecane is degraded at 30°C and pH 9.0.

[0021] Specifically, in an environment containing n-hexadecane to be degraded, the OD at the time of bacterial inoculation is... 600 The inoculum size is 0.8–1.5, and the volumetric inoculum size is 1%–5%, preferably 1.5%–5%, and most preferably 2%.

[0022] Preferably, in the environment containing the desired degradation of n-hexadecane, the concentration of n-hexadecane is 0.1% to 1.0% (v / v).

[0023] More preferably, the concentration of n-hexadecane is 0.1% to 0.5% (v / v).

[0024] Most preferably, the concentration of n-hexadecane is 0.5% (v / v).

[0025] Preferably, the environment is wastewater and / or contaminated soil.

[0026] The beneficial effects of the present invention are as follows:

[0027] 1. The *Gordonella oryzae* strain YDY-4 provided by this invention can reproduce under pure culture conditions using n-hexadecane as the sole carbon source, producing intermediate products such as aldol acids, and ultimately generating CO2 and H2O. Within a pH range of 8.0–10.0 and an initial n-hexadecane content of 0.1%–1.0% (v / v), this bacterium exhibits the ability to degrade n-hexadecane, demonstrating strong environmental adaptability.

[0028] 2. By adjusting the BH medium with hexadecane as the sole carbon source to 9.0 and the initial concentration of hexadecane to 0.5% (v / v), and the culture temperature to 30℃, the removal rate of cholesterol-eating Gordon's bacteria YDY-4 reached 99.01% after 15 days. Attached Figure Description

[0029] Figure 1 A streak plate diagram of *Gordonella salina* YDY-4, which is provided by the present invention;

[0030] Figure 2 A scanning electron microscope image of *Gordonella oryzae* YDY-4, which is provided by this invention;

[0031] Figure 3 Phylogenetic tree diagram of Cholesterol-eating Gordon's YDY-4 provided by the present invention;

[0032] Figure 4 The growth curve of cholesterol-eating Gordon's bacterium YDY-4 in LB medium provided by the present invention;

[0033] Figure 5 Comparison of the degradation performance of hexadecane by Gordon's bacteria YDY-4 at different pH levels;

[0034] Figure 6 Comparison of the degradation performance of cholesterol-eating Gordon's YDY-4 under different initial n-hexadecane concentrations;

[0035] Figure 7 Comparison of the degradation performance of hexadecane by Gordon's bacteria YDY-4 with different inoculum amounts. Detailed Implementation

[0036] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0037] This invention provides a cholesterol-eating bacterium, Gordon's YDY-4.

[0038] The name of the cholesterol-eating Gordon's bacterium YDY-4 is Gordon's cholesterol-eating bacterium (Gordonia cholesterolsterolivorans) YDY-4. It is deposited at the China Center for Type Culture Collection (CCTCC) on June 6, 2025, with accession number CCTCC M 20251290. The deposit address is: Wuhan University, 299 Bayi Road, Wuchang District, Wuhan, China, 430072.

[0039] The nucleotide sequence of the 16S rDNA of *Gordonella cholesterolophila* YDY-4 is shown in SEQ ID NO: 1.

[0040] The characteristics of Gordon's cholesterol-eating bacteria YDY-4 are: colonies are white or pale yellow, round, opaque, and have a smooth surface. Under a scanning electron microscope, the morphology of this bacterium is that of a bacillus.

[0041] The cholesterol-eating Gordon's bacterium strain YDY-4 is an aerobic bacterium that can reproduce under pure culture conditions using n-hexadecane as the sole carbon source, producing intermediate products such as aldol acids, and ultimately generating CO2 and H2O. Within a pH range of 8.0–10.0, with an initial n-hexadecane content ranging from 0.1% to 1.0% (v / v), this bacterium demonstrates the ability to degrade n-hexadecane, exhibiting strong environmental adaptability.

[0042] The present invention also provides a bacterial suspension. The active ingredient of the bacterial suspension includes the above-mentioned cholesterol-eating Gordon's bacterium YDY-4.

[0043] The present invention also provides a method for preparing bacterial suspension. Specifically, Gordon's cholesterol-eating bacteria YDY-4 is cultured in LB medium to obtain bacterial suspension.

[0044] Preferably, the LB medium comprises deionized water and components with the following concentrations: NaCl 10.0 g / L, tryptone 10.0 g / L, yeast extract 5.0 g / L, and pH 7.0.

[0045] The present invention also provides the application of Cholesterol Gordon's YDY-4, the above-mentioned bacterial solution, or the bacterial solution obtained by the above preparation method in the degradation of n-hexadecane.

[0046] The present invention also provides a method for degrading n-hexadecane. Specifically, the method involves inoculating an environment containing n-hexadecane to be degraded with a bacterial agent containing *Gordonella cholesterol-eating* YDY-4, the above-mentioned bacterial solution, or a bacterial solution obtained by the above preparation method, and degrading the n-hexadecane under conditions of 28–32°C and pH = 8.0–10.0.

[0047] Preferably, hexadecane is degraded at 30°C and pH 9.0.

[0048] Among them, the OD at inoculation of the bacterial culture in an environment containing n-hexadecane to be degraded... 600 The inoculum size is 0.8–1.5, and the volumetric inoculum size is 1%–5%, preferably 1.5%–5%, and most preferably 2%.

[0049] Preferably, in an environment containing the desired degradation of n-hexadecane, the concentration of n-hexadecane is 0.1% to 1.0% (v / v).

[0050] More preferably, the concentration of n-hexadecane is 0.1% to 0.5% (v / v).

[0051] Most preferably, the concentration of n-hexadecane is 0.5% (v / v).

[0052] The environment refers to wastewater and / or contaminated soil.

[0053] Specific embodiments of the present invention are as follows:

[0054] The culture medium formulation used in this embodiment of the invention is as follows:

[0055] LB medium (g / L): NaCl 10.0, tryptone 10.0, yeast extract 5.0, pH 7.0, autoclaved at 121℃ for 20 min.

[0056] Inorganic salt culture medium (g / L): Na2HPO4·12H2O 5.0, KH2PO4 1.0, NH4Cl 1.0, MgSO4·7H2O 0.2, CaCl2 0.05, trace elements 1mL, n-hexadecane 1% (v / v), autoclaved at 121℃ for 20min.

[0057] Trace elements (g / L): FeSO4·7H2O 0.05, H3BO3 0.014, MnSO4·4H2O 0.10, Na2MoO4·2H2O 0.02, CoCl2·6H2O 0.02, folic acid 0.00089, D-pantothenic acid (vitamin B5) 0.0035, vitamin B2 0.0023, niacin 0.0023, biotin (vitamin H) 0.0023.

[0058] Bushnell-Haas (BH) medium (g / L): MgSO4·7H2O 0.2, CaCl2 0.02, KH2PO4 1.0, K2HPO4 1.0, (NH4)2SO4 1.0, FeCl3 0.05, n-hexadecane 1% (v / v), autoclaved at 121℃ for 20 min.

[0059] Example 1

[0060] This embodiment describes the isolation, purification, and identification of Gordonia cholesterolivorans YDY-4.

[0061] 1. Isolation and purification of Gordonia cholesterolivorans YDY-4

[0062] Activated sludge was collected from the production wastewater of an oil refinery in Changzhou, Jiangsu Province, China. After settling for 24 hours, 5 mL of the supernatant was inoculated into 100 mL of inorganic salt medium with n-hexadecane as the sole carbon source. After culturing at 30°C and 160 rpm for 7 days with shaking, 5 mL of the suspension was inoculated into 100 mL of fresh inorganic salt medium and cultured at 30°C and 160 rpm for 7 days with shaking. This process was repeated 3–5 times. A certain amount of the bacterial suspension was then serially diluted to obtain bacterial suspensions of different concentrations. The obtained bacterial suspensions were purified by repeated streak plating on inorganic salt solid selective medium (e.g., ...). Figure 1 As shown in the figure, a single colony, namely the hexadecane-degrading strain, was obtained and denoted as strain YDY-4.

[0063] 2. Identification of strain YDY-4

[0064] Based on 16S rRNA sequence analysis and identification, strain YDY-4 was identified as *Gordonia cholesterolivorans*. The specific steps are as follows:

[0065] DNA from strain YDY-3 was extracted and purified using the Gentra Puregene Handbook (Qiagen) kit (Zhejiang Tianke Biotechnology Co., Ltd.) and stored at 4℃. The purified DNA was then amplified by PCR using universal primers F27 and 1492R. The primer sequences are as follows:

[0066] F27: 5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO: 2;

[0067] 1492R: 5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO: 3.

[0068] The PCR reaction system (25 μL) consisted of: 1 μL template DNA, 1 μL each of primer F27 and primer 1492R, 12.5 μL Phanta MaxMaster Mix (including PCR buffer, Taq DNA polymerase, and dNTPs), and 9.5 μL sterile deionized water.

[0069] The PCR reaction program was set as follows: pre-denaturation at 95℃ for 1 min; followed by denaturation at 95℃ for 10 s, annealing at 50℃ for 30 s, extension at 72℃ for 30 s, for 30 cycles; then extension at 72℃ for 4 min; and finally hold at 4℃ for 10 min. The PCR products were sequenced (Zhejiang Tianke), and the sequencing results are shown in SEQ ID NO: 1.

[0070] The 16S rDNA sequence of strain YDY-4 was uploaded to GenBank for homology comparison. The results showed that it belongs to the genus *Gordonia*, and has the highest homology (100%) with *Gordonia cholesterolivorans*. Figure 2 This is a bioelectron micrograph of the strain. Figure 3 This is a phylogenetic tree diagram of the strain. To further confirm the reliability of the identification results, after strain identification, strain YDY-4 was finally determined to belong to *Gordonia cholesterolivorans*. Therefore, strain YDY-4 is named *Gordonia cholesterolivorans* YDY-4, and is deposited at the China Center for Type Culture Collection (CCTCC), accession number: CCTCC M 20251290, deposit date: June 6, 2025, address: Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, China, postcode: 430072.

[0071] Example 2

[0072] This embodiment describes the preparation of Gordonia cholesterolivorans YDY-4 bacterial culture and seed culture.

[0073] The preparation method of *Gordonella cholerae* YDY-4 bacterial suspension is as follows: the screened degrading strain is inoculated into a 250mL glass bottle containing 100mL LB medium, and cultured with shaking at 30℃ and 160rpm until the logarithmic growth phase, obtaining the OD of the bacterial suspension. 600 The value was 1.0–1.5. The growth curve of *Gordonella cholesterolophila* YDY-4 in LB medium is shown in the figure. Figure 4 As shown.

[0074] The preparation method of *Gordonella cholerae* YDY-4 seed culture is as follows: Centrifuge the bacterial culture cultured to the logarithmic growth phase at 8000 rpm for 10 min, remove the supernatant, and collect the bacterial cells. Wash the bacterial cells with an appropriate amount of phosphate buffer, centrifuge at 8000 rpm for 10 min, and remove the supernatant. Repeat the above operation 2-3 times. Finally, add an appropriate amount of phosphate buffer to resuspend the bacterial cells to OD0.05. 600 A bacterial suspension with a value of 1.0 was used as the seed culture.

[0075] Example 3

[0076] This embodiment tested the hexadecane degradation performance of Gordonia cholesterolivorans YDY-4.

[0077] 1. Investigate the degradation performance of the strain under different pH conditions.

[0078] Experiments were conducted on the degradation of n-hexadecane by *Gordonella oryzae* YDY-4 under different initial pH conditions. It was found that pH 9.0 was the optimal pH, at which the degradation rate was highest. The specific implementation steps are as follows:

[0079] Seven different pH values ​​were selected: 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0. The pH of the BH medium was adjusted using 1 mol / L HCl and 1 mol / L NaOH. After high-temperature sterilization, 1% (v / v) of n-hexadecane was added to obtain BH mediums with different pH values ​​using n-hexadecane as the sole carbon source.

[0080] The seed culture was inoculated at a volume of 1% (v / v) into BH medium with different pH values ​​and hexadecane as the sole carbon source, with each experiment repeated three times. After culturing with shaking at 160 rpm for 15 days, samples were taken, and the concentration of residual hexadecane in the strain was determined by gas chromatography-mass spectrometry.

[0081] The degradation rate of n-hexadecane by strain YDY-4 after 15 days of culture under different pH conditions is as follows: Figure 5 As shown. Figure 5 It was found that the optimal pH range for the growth of strain YDY-4 is 8.0–10.0. The highest degradation rate of strain YDY-4 (72.63%) was observed at pH 9.0. When the pH was below 9.0, the degradation rate of n-hexadecane was 59.43% (pH 8). When the pH was above 9.0, the degradation rate of n-hexadecane decreased to 62.9% (pH 10).

[0082] Furthermore, the pH value of strain YDY-4 decreased after cultivation when the pH range was 4.0–10.0, indicating that acidic products were generated during the degradation of n-hexadecane by strain YDY-4.

[0083] Subsequent experimental strains were cultured at a pH of 9.0.

[0084] 2. Investigate the degradation performance of the strain under different initial n-hexadecane concentrations.

[0085] Five different initial concentrations of n-hexadecane were selected: 0.1%, 0.5%, 1%, 2%, and 5% (v / v). The pH of the BH medium was adjusted to 9.0, and after autoclaving, n-hexadecane was added. This resulted in BH medium with n-hexadecane as the sole carbon source, at initial concentrations of 0.1%, 0.5%, 1%, 2%, and 5% (v / v).

[0086] Seed culture was inoculated at a volume of 1% (v / v) into BH medium with n-hexadecane as the sole carbon source. Each experiment was repeated three times. After inoculation, the culture system was incubated at 30℃ and 160 rpm with shaking for 15 days, and the concentration of residual n-hexadecane was measured. The degradation rate of n-hexadecane by the strain under different initial n-hexadecane concentrations is shown in the figure below. Figure 6 As shown.

[0087] Depend on Figure 6 It was found that after 15 days of cultivation, the degradation rate of n-hexadecane in strain YDY-4 gradually decreased with increasing initial concentrations from 0.1% to 5% (v / v). Specifically, the degradation rates reached 98.36% and 98.55% at initial concentrations of 0.1% and 0.5%, respectively. However, the degradation efficiency plummeted to 5.48% at an initial concentration of 5%. This indicates that initial concentrations of n-hexadecane significantly inhibited the metabolic activity of the strain, possibly due to the toxic effects of high initial concentrations, which disrupted cellular physiological functions. Based on this, subsequent experiments selected 0.5% (v / v) n-hexadecane as the optimized concentration parameter.

[0088] 3. Investigate the degradation performance of the bacterial strains under different inoculum amounts.

[0089] The pH of BH medium was adjusted to 9.0, and after high-temperature sterilization, an appropriate amount of n-hexadecane was added to bring the initial concentration of n-hexadecane to 0.5% (v / v). Seed culture was added to BH medium with n-hexadecane as the sole carbon source at inoculum levels of 0.5%, 1%, 1.5%, 2%, and 5%, respectively. An uninoculated degrading bacteria control group was used. Each experiment was repeated three times. After inoculation, the culture system was incubated at 30℃ and 160 rpm with shaking for 15 days, and the remaining concentration of n-hexadecane was measured. The degradation rate of n-hexadecane at different inoculum levels is shown in the figure below. Figure 7 As shown.

[0090] From Figure 7 It was observed that the degradation rate of n-hexadecane, measured after 15 days, initially increased with increasing inoculum size, reaching its highest level of 99.01% at an inoculum size of 2%. However, when the inoculum size reached 5%, the degradation rate of n-hexadecane decreased to 98.26%. This may be due to factors such as substrate competition between strains and the oxygen content in the culture medium, which slightly reduced the growth and degradation capabilities of the strains.

[0091] In summary, by adjusting the BH medium with n-hexadecane as the sole carbon source to pH 9.0, the initial concentration of n-hexadecane to 0.5% (v / v), the inoculum size to 2%, and the culture temperature to 30°C, this strain achieved a removal rate of 99.01% after 15 days. Compared to existing strains, this patented strain, with a lower inoculum size in an environment with an initial n-hexadecane concentration of 0.5% (v / v), achieved a degradation rate of over 90%, demonstrating a significant breakthrough advantage and providing a more industrially viable solution for the efficient and low-cost remediation of n-hexadecane contamination.

[0092] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make changes or modifications to the above-described technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

[0093] The amino acid and nucleotide sequences involved in this invention are as follows:

[0094] SEQ ID NO.1;

[0095] Name: 16S rDNA sequence

[0096] Sequence type: DNA (genomic DNA)

[0097] Source: Gordonia cholesterolivorans YDY-4

[0098] AAGTCGAACGGAAAGGCCCAGCTTGCTGGGTACTCGAGTGGCGAACGGGTGAGTAACACGTGGGTGATCTGCCCTGGACTCTGGGATAAGCCTGGGAAACTGGGTCTAATACCGGATAGGACCACTGATTGCATGGTTGGTGGTGGAAAGCTTTTGCGGTTCAGGATGGGCCCGCGGCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCGCTAGGGACGAAGCGTAAGTGACGGTACCTGGAGAAGAAGCACCGGCCAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTGTCCGGAATTACTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCGTCTGTGAAATTCTGCAACTCAATTGCAGGCGTGCAGGCGATACGGGCAGACTTGAGTACTACAGGGGAGACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGTAGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGTACTAGGTGTGGGGCTCATTTCACGAGTTCCGTGCCGTAGCTAACGCATTAAGTACCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCC

[0099] SEQ ID NO: 2

[0100] Name: Primer F27

[0101] Sequence Type: DNA (other DNA)

[0102] Source: synthetic construct

[0103] AGAGTTTGATCCTGGCTCAG

[0104] SEQ ID NO: 3

[0105] Name: Primer 1492R

[0106] Sequence type: DNA (other DNA)

[0107] Source: synthetic construct

[0108] GGTTACCTTGTTACGACTT.

Claims

1. A strain of cholesterol-eating Gordon's bacterium YDY-4, characterized in that: The cholesterol-eating Gordon's bacterium YDY-4 is named *Gordonia cholesterolivorans* YDY-4 and is deposited at the China Center for Type Culture Collection (CCTCC) on June 6, 2025, with accession number CCTCC M20251290.

2. The cholesterol-eating Gordon's bacterium YDY-4 strain according to claim 1, characterized in that: The nucleotide sequence of the 16S rDNA of the cholesterol-eating Gordon's YDY-4 is shown in SEQ ID NO:

1.

3. A bacterial solution, characterized in that: The active ingredient in the bacterial solution includes *Gordonella oryzae* YDY-4 as described in claim 1 or 2.

4. A method for preparing bacterial suspension as described in claim 3, characterized in that: The cholesterol-eating Gordon's bacterium YDY-4 was cultured in LB medium to obtain a bacterial culture.

5. The method for preparing bacterial culture according to claim 4, characterized in that: The LB medium comprises deionized water and the following components at the following concentrations: NaCl 10.0 g / L, tryptone 10.0 g / L, yeast extract 5.0 g / L, and pH 7.

0.

6. The application of *Gordonella oryzae* YDY-4 as described in claim 1 or 2, the bacterial suspension as described in claim 3, or the bacterial suspension obtained by the preparation method as described in claim 4 or 5, characterized in that: Application in the degradation of n-hexadecane.

7. A method for degrading n-hexadecane, characterized in that: The cholesterol-eating Gordon's bacterium YDY-4 as described in claim 1 or 2, the bacterial solution as described in claim 3, or the bacterial solution obtained by the preparation method as described in claim 4 or 5 is inoculated into an environment containing n-hexadecane to be degraded, and the n-hexadecane is degraded under conditions of pH = 8.0 to 10.0 and temperature of 28 to 32°C.

8. The method for degrading n-hexadecane according to claim 7, characterized in that: The cholesterol-eating Gordon's bacterium YDY-4 or its bacterial culture was prepared into OD. 600 The bacterial suspension has a concentration of 0.8–1.5, and the inoculation is performed using the bacterial suspension at a volume inoculation rate of 1%–5%.

9. The method for degrading n-hexadecane according to claim 7, characterized in that: In the environment containing the desired degradation of n-hexadecane, the concentration of n-hexadecane is 0.1% to 1.0% (v / v).

10. A method for degrading n-hexadecane according to claim 7, characterized in that: The environment described is wastewater and / or contaminated soil.

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