A cholesterol-degrading bacterium, HY-2, and its application in environmental remediation.

Abstract

This invention discloses a strain of *Gordonella cholerae* capable of degrading n-tetradecane. Gordonia cholesterolivorous HY-2 and its application in environmental remediation. The strain is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20252335. The strain has rod-shaped, Gram-positive cells, no flagella, no spores, and possesses a capsule structure. Single-factor degradation experiments showed that the strain can grow using n-tetradecane as the sole carbon source and exhibits degradation ability under initial n-tetradecane concentrations of 0.1%–5% (v / v); it shows a high degradation rate at an initial n-tetradecane concentration of 0.5% (v / v) and an inoculum size of 2% (v / v); and it maintains good degradation activity within a pH range of 6–10. This invention can be used to prepare microbial agents for degrading n-tetradecane and is suitable for the remediation of water or soil contaminated with n-tetradecane.

Description

Technical Field

[0001] This invention relates to the fields of environmental bioengineering, microbiology and pollution ecological remediation technology, and more specifically, to a cholesterol Gordon's bacterium HY-2 that degrades n-tetradecane and its application in environmental remediation. Background Technology

[0002] n-Tetradecane ( n Tetradecane (n-tetradecane) is a typical medium- to long-chain straight-chain alkane, widely found in industrial solvents, chemical raw materials, lubricating oils, and fossil fuels. During industrial production, chemical product storage and transportation, and oily wastewater discharge, n-tetradecane may leak, be discharged, or migrate into aquatic and soil environments. Due to its low hydrophilicity and relatively stable chemical properties, n-tetradecane does not readily volatilize or degrade in the natural environment, easily accumulating in soil pores or oily wastewater systems. This can negatively impact soil structure, pore aeration, and the aquatic ecosystem, placing pressure on the ecological restoration of polluted areas.

[0003] Traditional physical and chemical remediation technologies for treating alkane pollution typically include thermal desorption, chemical oxidation, solvent elution, and adsorption separation. While these technologies can reduce pollutant concentrations under certain conditions, they often suffer from high engineering costs, significant disturbance during the remediation process, substantial impact on the native microbial ecosystem, and potential secondary pollution in practical applications. In contrast, microbial remediation technology utilizes the metabolic activity of functional microorganisms to transform or mineralize alkane pollutants such as n-tetradecane into low-toxicity products, cell biomass, carbon dioxide, and water. It offers advantages such as better environmental compatibility, milder operating conditions, and suitability for in-situ remediation, thus showing promising application prospects in the treatment of alkane pollutants.

[0004] However, in actual site remediation, existing degrading strains targeting hydrophobic alkanes such as n-tetradecane still face certain limitations. First, the bioavailability and mass transfer efficiency of pollutants are low. n-Tetradecane has low hydrophilicity and easily exists in the oil phase or adsorbed form in soil particles, suspended solids in water, or at the oil-water interface, making it difficult for conventional microorganisms to fully contact it, thus limiting substrate uptake and degradation efficiency. Second, the environmental adaptability of functional strains still needs improvement. Actual polluted environments are often accompanied by fluctuations in organic pollutant concentrations, changes in nutrient conditions, and various stress factors. Some degrading bacteria lack sufficient colonization and sustained degradation capabilities in complex polluted substrates, making it difficult to maintain stable remediation effects over the long term. Third, changes in environmental pH conditions affect degradation efficiency. Many industrial waste sites or chemical wastewater spill areas may be alkaline. Some conventional degrading bacteria are quite sensitive to pH changes; their growth activity and degradation capacity may significantly decrease after deviating from the suitable range.

[0005] Therefore, screening functional strains from polluted environments that can utilize n-tetradecane and maintain degradation activity within a certain concentration range and under neutral to slightly alkaline conditions has certain practical significance and application value for enriching alkane-degrading microbial resources and expanding the bioremediation technology pathways for n-tetradecane-polluted water and soil. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing bioremediation technologies, such as low utilization rate of hydrophobic substrates like n-tetradecane, weak environmental resistance, and poor alkali tolerance by degrading strains, and to provide a *Gordonella cholerae* strain that degrades n-tetradecane. Gordonia cholesterolivorans HY-2 and its application in environmental remediation.

[0007] This strain can grow using n-tetradecane as the sole carbon source and exhibits n-tetradecane degradation capabilities. Its oil expulsion zone assay was positive, suggesting that the strain may produce extracellular active substances that facilitate the emulsification or dispersion of hydrophobic substrates. The strain possesses a capsule structure and maintains its degradation activity within a certain range of n-tetradecane concentrations and under neutral to slightly alkaline conditions.

[0008] Another objective of this invention is to investigate the degradation patterns of the HY-2 strain under different initial substrate concentrations, different inoculum amounts, and different initial environmental pH values, so as to provide parameter references for its subsequent preparation of bacterial agents and environmental remediation applications.

[0009] To achieve the above objectives, the specific technical solution adopted by the present invention is as follows: This invention provides a strain of *Gordonia cholesterolivorans* HY-2 with the ability to degrade n-tetradecane. *Gordonia cholesterolivorans* HY-2 is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, 430072, China, accession number: CCTCC NO: M 20252335, deposit date: October 24, 2025.

[0010] Furthermore, the present invention described Gordonia cholesterolivorans The morphological and physiological-biochemical characteristics of strain HY-2 are as follows: Cell morphology characteristics: Under a light microscope, the cells of this HY-2 strain exhibited a rod-shaped morphology; after standard Gram staining, the result was positive (G). + Further observation confirmed that this strain lacks flagella and the ability to produce spores, but it has a distinct capsule structure around its cells.

[0011] Physiological and biochemical metabolic characteristics: Biochemical reaction tests showed that the strain was positive for VP test, starch hydrolysis test, and catalase test; and its oil expulsion ring test was positive, suggesting that the strain has a strong oil film diffusion ability and may produce extracellular active substances that are beneficial to the emulsification of hydrophobic substrates. Gelatin liquefaction test and methyl red (MR) test were negative.

[0012] The present invention also provides a microbial inoculant containing the above-mentioned *Gordonella cholerae* HY-2 as an active ingredient.

[0013] The present invention also provides the application of the above-mentioned Cholesterol Gordon's bacterium HY-2 and microbial agents in the degradation of n-tetradecane.

[0014] Furthermore, the application is for in-situ and ex-situ ecological restoration of alkaline soils or water bodies contaminated with n-tetradecane.

[0015] The present invention also provides a method for degrading n-tetradecane, wherein the bacterial suspension of the above-mentioned Cholesterol Gordon's HY-2 is inoculated into an environment containing n-tetradecane.

[0016] Furthermore, the initial volume concentration of the n-tetradecane is 0.1~5% (v / v), and the bacterial suspension... OD 600 The concentration ranges from 0.95 to 1.05, and the inoculation rate is 0.1% to 5% (v / v).

[0017] Furthermore, the environment is a body of water and / or soil, and the pH of the environment is 5-10.

[0018] The beneficial effects of this invention are as follows: 1. The HY-2 strain provided by this invention can grow using n-tetradecane as the sole carbon source and exhibits high degradation capacity under low concentrations of n-tetradecane, making it a candidate functional strain for the remediation of n-tetradecane-contaminated environments.

[0019] 2. The HY-2 strain showed a positive oil film diffusion test, indicating that it possesses a certain oil film diffusion capability, suggesting that this strain may produce extracellular active substances that facilitate the emulsification or dispersion of hydrophobic n-tetradecane. This characteristic helps increase the contact opportunities between the bacterial cells and the hydrophobic substrate, thereby alleviating to some extent the problem of limited mass transfer of n-tetradecane in aqueous systems.

[0020] 3. The HY-2 strain exhibited high n-tetradecane degradation activity under neutral to slightly alkaline conditions, with the pH 9 treatment group showing the highest degradation rate under the conditions of this experiment. This result indicates that the HY-2 strain has a certain degree of adaptability to slightly alkaline environments and can provide a candidate strain for the bioremediation of slightly alkaline n-tetradecane-contaminated water or soil.

[0021] 4. Under the conditions of this experiment, the degradation rate of n-tetradecane was relatively high when the inoculum amount was 2% (v / v), which can be used as a reference for subsequent optimization of the bacterial agent dosage. This result indicates that within a certain inoculum amount range, further increasing the amount of bacterial suspension added has limited effect on improving the degradation efficiency. Therefore, 2% (v / v) can be used as a reference value for subsequent optimization of application parameters. Attached Figure Description

[0022] Figure 1 This is a SEM image of strain HY-2 of the present invention.

[0023] Figure 2 This is a phylogenetic tree diagram of the strain HY-2 of this invention.

[0024] Figure 3 The trend graph shows the effect of different initial n-tetradecane concentrations as a single-factor variable on the degradation rate of strain HY-2.

[0025] Figure 4 Trend graph showing the effect of different initial inoculum amounts as a single-factor variable on the degradation efficiency of HY-2 strain.

[0026] Figure 5 Trend graph showing the effect of different initial environmental pH values ​​as a single-factor variable on the n-tetradecane degradation performance of strain HY-2. Detailed Implementation

[0027] 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.

[0028] The culture medium formulation used in the specific embodiments of the present invention is as follows: LB medium (g / L): NaCl 10.0, tryptone 10.0, yeast extract 5.0, pH 7.0, autoclaved at 121 ℃ for 20 min.

[0029] Inorganic salt culture medium (g / L): K2HPO4 1.0, KH2PO4 1.0, NH4NO3 1.0, MgSO4·7H2O 0.3, CaCl2 0.03, FeSO4 0.005, ZnSO4 0.002, MnSO4 0.0002, autoclaved at 121 ℃ for 20 min.

[0030] Example 1: Isolation, purification, microbiological morphology, physiological and biochemical characteristics, and molecular identification of strain HY-2 1. Enrichment, isolation, and purification of bacterial strains: Activated sludge was collected from wastewater at an oil refinery in Jiangsu Province, China. After settling for 24 hours, 5 mL of the supernatant was inoculated into 100 mL of inorganic salt medium with n-tetradecane as the sole carbon source. After culturing at 30 °C and 160 rpm for 7 days, 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. This process was repeated 3-5 times to progressively screen for dominant microorganisms capable of utilizing n-tetradecane for growth. After enrichment, an appropriate amount of bacterial suspension was serially diluted 10-fold with sterile inorganic salt medium, with the dilution range set at 10-10. -1 10 -2 10 -3 10 -4 10 -5 10 -6 and 10 -7 100 μL of bacterial suspensions at different dilutions were plated onto the surface of an inorganic salt solid selective medium with n-tetradecane as the sole carbon source and incubated at 30 °C. Plates with moderate colony numbers and significant morphological differences were selected for repeated streaking isolation and purification until uniform single colonies were obtained, which were then considered the primary culture medium. Gordonia cholesterolivorans HY-2 strain.

[0031] 2. Morphological and microscopic observation: Fresh single colonies isolated and purified were selected, smeared, dried, fixed, Gram-stained, and observed under a light microscope. Results confirmed that HY-2 cells under the microscope exhibited typical rod-shaped structures; the cells were stained purple, clearly indicating Gram-positive (G...). + Further observation confirmed that this strain lacked flagella and did not produce spores, but it possessed a distinct capsule structure. The electron microscopy results of the strain are as follows: Figure 1 As shown.

[0032] 3. Determination of key physiological and biochemical indicators: The metabolic capacity of the purified HY-2 strain was analyzed using conventional bacterial physiological and biochemical identification methods. The tests included gelatin liquefaction test, methyl red test, VP test, starch hydrolysis test, catalase test, and oil expulsion ring test.

[0033] The identification results showed that the strain was negative for gelatin liquefaction and methyl red (MR) tests. Notably, it was positive for VP, starch hydrolysis, and catalase tests. Furthermore, after inoculating HY-2 bacterial suspension on a plate covered with a hydrophobic oil film, a clear transparent oil-extraction zone appeared around the colony, indicating a positive oil-extraction zone test. The specific physiological and biochemical characteristics of this strain are shown in Table 1.

[0034] Table 1 Summary of physiological and biochemical characteristics of strain HY-2 4. Molecular identification and preservation of strain HY-2: HY-2 strain was identified through 16S rRNA sequence analysis and identification. Gordonia cholesterolivorans The specific steps are as follows: Microbial DNA of strain HY-2 was obtained using PrepMan. TM Extraction was performed using Ultra Sample Preparation Reagent (Thermo Fisher Scientific, USA; Cat. No. 4318930), following the product instructions. The purified DNA was amplified by PCR using universal bacterial primers F27 (SEQ ID NO.1) and 1492R (SEQ ID NO.2). The primer sequences are as follows: F27 (SEQ ID NO.1): 5'-AGAGTTTGATCCTGGCTCAG-3' 1492R (SEQ ID NO.2): 5'-GGTTACCTTGTTACGACTT-3' The PCR reaction system consisted of (25 μL): 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.

[0035] 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 sent to Zhejiang Tianke High-Tech Development Co., Ltd. for Sanger sequencing.

[0036] The 16S rDNA sequence of strain HY-2 is shown in SEQ ID NO.3: Homology comparison of the 16S rDNA sequence of strain HY-2 with gene sequences in GenBank revealed that it belongs to... Gordonia cholesterolivorans ,and Gordonia cholesterolivorans strain Chol-3 showed the highest homology, reaching 99.86%. Figure 2 This is a phylogenetic tree diagram of strain HY-2.

[0037] Based on sequencing results and physiological and biochemical test results, strain HY-2 was determined to belong to... Gordonia cholesterolivorans Therefore, strain HY-2 was named *Gordonella cholesterolis*. Gordonia cholesterolivorans HY-2, deposited at the China Center for Type Culture Collection (CCTCC), with accession number CCTCCNO: M 20252335, deposited on October 24, 2025, address: Wuhan University, Wuhan, China, postal code: 430072.

[0038] Example 2: Effect of initial n-tetradecane concentration on the degradation performance of HY-2 strain Standard inorganic salt liquid culture medium was prepared, and different volume percentages of n-tetradecane were added as the sole carbon source to achieve initial concentrations of 0.1%, 0.5%, 1%, 2%, 3%, and 5%, respectively. The HY-2 seed culture was centrifuged to collect the bacterial cells, which were then washed twice with sterile inorganic salt culture medium and resuspended. The concentration of the bacterial suspension was adjusted to OD0.05. 600 = 1.0 ± 0.05. Subsequently, bacterial suspension was added at an inoculum rate of 1% (v / v), i.e., 1 mL of bacterial suspension was added to each bottle. Each treatment group was incubated at 30 ℃ and 160 rpm with constant temperature shaking for 15 days. After incubation, n-tetradecane in the culture system was extracted with the organic solvent n-hexane, and the residual amount of n-tetradecane was determined by gas chromatography. The degradation rate was calculated based on the initial concentration. The n-tetradecane degradation rate was calculated using the following formula: Degradation rate (%) = (C0 - C) t ) / C0 × 100% Where C0 is the initial concentration of n-tetradecane, C t This represents the residual concentration of n-tetradecane after the culture period.

[0039] Experimental results show (e.g.) Figure 3As shown in the figure: Strain HY-2 exhibits strong degradation ability of n-tetradecane at low concentrations (0.1%), with a degradation rate as high as 97.42%-97.69%. The degradation rate decreases with increasing substrate concentration. At a concentration of 0.5%, the degradation rate remains between 65.98% and 71.60%; even under a high concentration of 5%, the degradation rate remains around 8%. These results indicate that strain HY-2 has a high degradation ability under low concentrations of n-tetradecane.

[0040] Example 3: Effect of inoculum ratio on the n-tetradecane degradation efficiency of strain HY-2 In an inorganic salt liquid medium containing a uniform initial concentration of n-tetradecane, the effect of different inoculum sizes on the degradation of n-tetradecane was investigated using strain HY-2 as the inoculum source. The HY-2 seed culture was centrifuged to collect the bacterial cells, washed twice with sterile inorganic salt medium, and resuspended. The concentration of the bacterial suspension was adjusted to... OD 600 = 1.0 ± 0.05. The initial addition of n-tetradecane to each treatment group was 0.5 mL, i.e., the initial pollutant concentration was 0.5% (v / v). Subsequently, the inoculum was added to the culture system at inoculum rates of 0.1%, 0.5%, 1%, 1.5%, 2%, 3%, and 5% (v / v), respectively. Each treatment group was cultured at 30 ℃ and 160 rpm for 15 days with constant temperature shaking. After the culture was completed, n-tetradecane in the culture system was extracted with the organic solvent n-hexane, and the residual amount of n-tetradecane was determined by gas chromatography. The degradation rate was calculated based on the initial concentration. The n-tetradecane degradation rate was calculated using the following formula: Degradation rate (%) = (C0 - C) t ) / C0 × 100% Where C0 is the initial concentration of n-tetradecane, C t This represents the residual concentration of n-tetradecane after the culture period.

[0041] Experimental results show (e.g.) Figure 4 As shown in the figure: With the increase of the initial inoculum amount, the degradation rate of n-tetradecane in the system showed a clear trend of first increasing and then slightly decreasing. At a low inoculum amount (0.1%), the degradation rate was less than 1%; when the inoculum amount increased to 1.5% (v / v), the degradation rate significantly increased to about 82%; when the inoculum amount reached 2% (v / v), the degradation rate reached 90%–92%, which is a relatively high level under the experimental conditions. Subsequently, when the inoculum amount continued to increase to 3% and 5%, the degradation rate did not increase further, but instead slightly decreased to the 83%–84% range. Therefore, under the experimental conditions, a 2% (v / v) inoculum amount can be regarded as a more suitable initial inoculum ratio for the degradation of n-tetradecane by the HY-2 strain.

[0042] Example 4: Effects of environmental pH stress on the n-tetradecane degradation performance of strain HY-2 Multiple groups of inorganic salt liquid culture systems containing equal amounts of n-tetradecane were initially pHed to 5, 6, 7, 8, 9, and 10 to investigate the effect of different initial pH on the ability of strain HY-2 to degrade n-tetradecane. 0.5 mL of n-tetradecane was added to each group to achieve an initial concentration of 0.5% (v / v). The HY-2 seed culture was centrifuged to collect the bacterial cells, washed twice with sterile inorganic salt medium, and resuspended. The concentration of the bacterial suspension was adjusted to... OD 600 = 1.0 ± 0.05. Subsequently, 2.0 mL of HY-2 bacterial suspension was added to each treatment group, representing an inoculum size of 2% (v / v). Each group was cultured at 30 ℃ and 160 rpm with constant temperature shaking for 15 days. After culture, n-tetradecane in the culture system was extracted with the organic solvent n-hexane, and the residual amount of n-tetradecane was determined by gas chromatography. The degradation rate was calculated based on the initial concentration. The n-tetradecane degradation rate was calculated using the following formula: Degradation rate (%) = (C0 - C) t ) / C0 × 100% Where C0 is the initial concentration of n-tetradecane, C t This represents the residual concentration of n-tetradecane after the culture period.

[0043] Experimental results show (e.g.) Figure 5 As shown in the figure, strain HY-2 exhibited differentiated degradation responses to different initial pH conditions. Under slightly acidic conditions (pH=5), the degradation rate of n-tetradecane was 18.61%, indicating that the slightly acidic environment had a certain inhibitory effect on the degradation activity of this strain. When the initial pH was adjusted to 6 and 7, the degradation rate recovered to 77.48% and 85.66%, respectively. Under slightly alkaline conditions, strain HY-2 showed high degradation activity, with degradation rates of 94.88%, 97.17%, and 96.56% in the pH=8, 9, and 10 treatment groups, respectively. Among them, the pH=9 treatment group had the highest degradation rate. The above results indicate that strain HY-2 has good n-tetradecane degradation capacity under neutral to slightly alkaline conditions and can be used as a candidate functional strain for bioremediation of slightly alkaline n-tetradecane-contaminated environments.

[0044] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the present invention. Any equivalent substitutions, modifications, or alterations made by those skilled in the art based on the technical content disclosed in the present invention, as long as they do not depart from the technical solution and spirit of the present invention, should fall within the protection scope of the present invention.

Claims

1. A strain of *Gordonella hy-2* capable of degrading n-tetradecane, characterized in that, The cholesterol Gordon's bacterium HY-2 was named Gordonia cholesterolivorans HY-2 was deposited at the China Center for Type Culture Collection on October 24, 2025, with accession number CCTCC NO: M 20252335.

2. The *Gordonella hy-2* strain according to claim 1, characterized in that, The 16S rDNA sequence of strain HY-2 is shown in SEQ ID NO.

3.

3. The *Gordonella hy-2* strain according to claim 1, characterized in that, The strain has rod-shaped cells that are Gram-positive, with a capsule-like outer layer, and no flagella or spores.

4. A microbial inoculant, characterized in that, The microbial agent uses Cholesterol Gordon's HY-2 as the active ingredient as described in claim 1.

5. The use of Cholesterol Gordon's HY-2 as described in any one of claims 1 to 3 or the microbial agent as described in claim 4 in the degradation of n-tetradecane.

6. The application according to claim 5, characterized in that, The application is for in-situ or ex-situ bioremediation of alkaline soils or water bodies contaminated with n-tetradecane.

7. A method for degrading n-tetradecane, characterized in that, The bacterial suspension prepared from the cholesterol Gordon's bacterium HY-2 as described in claim 1 was inoculated into an environment containing n-tetradecane at an initial volume concentration of 0.1%-5%.

8. The method according to claim 7, characterized in that, The bacterial suspension OD 600 The concentration ranges from 0.95 to 1.05, and the inoculation rate is 0.1% to 5% (v / v).

9. The method according to claim 7, characterized in that, The environment is a body of water and / or soil, with a pH of 5-10.

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