Salt-tolerant hydrocarbon-removing marinobacter capable of producing biosurfactant and application of salt-tolerant hydrocarbon-removing marinobacter

Abstract

The invention discloses salt-tolerant hydrocarbon-removing marinobacter capable of producing a biosurfactant and application of the salt-tolerant hydrocarbon-removing marinobacter, and relates to the technical field of microorganisms. The marinobacter hydrocarbonaceous classicus DH5 is preserved in the China Center for Type Culture Collection on June 3, 2024, and the preservation number of the marinobacter hydrocarbonaceous classicus DH5 is CCTCC (China Center for Type Culture Collection) NO: M20241128. Crude oil and petroleum hydrocarbon can be degraded, dispersed and emulsified under the hypersalinity condition, a surfactant can be generated, the fluidity of the crude oil in a stratum is effectively improved, and the recovery rate of the crude oil is increased.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to a salt-tolerant hydrocarbon-degrading Marinobacter that produces biosurfactants and its application. Background Art

[0002] Microbial Enhanced Oil Recovery (MEOR) utilizes the principle that certain microorganisms can degrade petroleum, increase the emulsification degree of petroleum, and reduce the viscosity of petroleum. By injecting microbial liquid into the formation, the physical and chemical properties of reservoir rocks and crude oil are changed through the microorganisms and their own metabolic activities and metabolites, so as to achieve the effect of displacing and stripping petroleum from depleted and high-water-cut oil reservoirs. It is a method with strong adaptability, low cost, easy construction and environmental friendliness, and can be used for the exploitation of unconventional petroleum resources, which is of great significance for the exploitation of remaining oil in reservoirs close to the economic limit.

[0003] In related technologies, usually after fermenting and culturing microorganisms with substrates such as oils and fats, starches, etc., they are then injected into the formation as oil displacement agents. However, this operation method has problems such as complex construction operation, short validity period, and the nutrient components in the culture medium may affect the composition of the indigenous microbial community in the formation, thereby further affecting the microbial oil displacement effect. Therefore, it is very important to isolate microorganisms that can metabolize and produce biosurfactants using crude oil as the sole carbon source. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. For this purpose, the present invention provides a strain of hydrocarbon-degrading Marinobacter ( Marinobacter hydrocarbonoclasticus ) DH5.

[0005] The present invention also provides a microbial agent.

[0006] The present invention also provides a biosurfactant.

[0007] The present invention also provides a preparation method of the above biosurfactant.

[0008] The present invention also provides the application of the above hydrocarbon-degrading Marinobacter DH5, microbial agent or biosurfactant.

[0009] A strain of hydrocarbon-degrading Marinobacter ( Marinobacter hydrocarbonoclasticus ) DH5 according to the first aspect embodiment of the present invention, the hydrocarbon-degrading Marinobacter DH5 was deposited at the China Center for Type Culture Collection on June 3, 2024, and the deposit number is CCTCC NO: M20241128.

[0010] The hydrocarbon-degrading Marinobacter DH5 according to the embodiment of the present invention has at least the following beneficial effects: The hydrocarbon-degrading Marinobacter DH5 of the embodiment can degrade, disperse and emulsify crude oil, petroleum hydrocarbons and liquid paraffin under high salinity conditions, and can also produce biosurfactants, effectively improving the fluidity of crude oil in the formation and enhancing the crude oil recovery rate.

[0011] According to some embodiments of the present invention, the nucleotide sequence of the 16S rRNA of the hydrocarbon-degrading Marinobacter DH5 is as shown in SEQ ID NO: 1.

[0012] A bacterial agent according to an embodiment of the second aspect of the present invention, the bacterial agent contains the hydrocarbon-degrading Marinobacter DH5 described in the embodiment of the first aspect of the present invention. Since the bacterial agent adopts all the technical solutions of the hydrocarbon-degrading Marinobacter DH5 of the above embodiment, it has at least all the beneficial effects brought by the technical solutions of the above embodiment.

[0013] According to some embodiments of the present invention, the bacterial agent further contains other bacteria or fungi.

[0014] According to some embodiments of the present invention, the active ingredient of the bacterial agent is the hydrocarbon-degrading Marinobacter DH5.

[0015] According to some embodiments of the present invention, the bacterial agent further includes at least one of a carrier, a surfactant, a stabilizer and a pH regulator.

[0016] According to some embodiments of the present invention, the dosage form of the bacterial agent includes but is not limited to liquid agent, emulsion, suspension, powder, granule, wettable powder or water dispersible granule.

[0017] A biosurfactant according to an embodiment of the third aspect of the present invention is prepared by fermentation using the hydrocarbon-degrading Marinobacter DH5 described in the embodiment of the first aspect of the present invention or the bacterial agent described in the embodiment of the second aspect of the present invention.

[0018] A preparation method of a biosurfactant according to an embodiment of the fourth aspect of the present invention includes the following steps: Inoculate the hydrocarbon-degrading Marinobacter DH5 described in the embodiment of the first aspect of the present invention or the bacterial agent described in the embodiment of the second aspect of the present invention into a fermentation medium, and cultivate to obtain a fermentation broth. The fermentation broth contains a biosurfactant.

[0019] According to some embodiments of the present invention, the temperature of the cultivation is 20°C to 50°C. For example: it can be 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, or 50°C.

[0020] According to some embodiments of the present invention, the culturing time is 2 to 7 days. For example, it can be 2, 3, 4, 5, 6, or 7 days.

[0021] According to some embodiments of the present invention, the fermentation medium includes, but is not limited to, LB medium. Those skilled in the art can select a medium well-known in the art that can be used for culturing Marinobacter hydrocarbonoclasticus as the fermentation medium.

[0022] According to some embodiments of the present invention, the pH of the fermentation medium is 5 - 9.5. For example, it can be 5, 6.5, 7, 7.5, 8, 8.5, 9, or 9.5.

[0023] According to some embodiments of the present invention, the fermentation medium includes a carbon source, a nitrogen source, and inorganic salts.

[0024] According to some embodiments of the present invention, the carbon source includes at least one of sodium acetate, soybean oil, glucose, sucrose, crude oil, and liquid paraffin.

[0025] According to some embodiments of the present invention, the nitrogen source includes at least one of peptone, urea, sodium nitrate, diammonium hydrogen phosphate, ammonium chloride, and peanut meal.

[0026] According to some embodiments of the present invention, the inorganic salts include at least one of Na2MoO4, MgSO4, KH2PO4, NaCl, and FeSO4 According to some embodiments of the present invention, the fermentation medium includes the following components: Sodium acetate 1 g / L - 50 g / L, soybean oil 1 g / L - 40 g / L, NaNO3 1 g / L - 10 g / L, Na2MoO4 0.02 g / L - 0.08 g / L, MgSO4 0.05 g / L - 0.2 g / L, KH2PO4 0.05 g / L - 1 g / L, (NH4)2HPO4 0.05 g / L - 1 g / L, NaCl 1 g / L - 50 g / L, pH 7.0 - 7.2.

[0027] According to some embodiments of the present invention, the preparation method further includes: removing the bacteria in the fermentation broth, collecting the liquid phase, adjusting the pH of the liquid phase to 2.0 - 8.0, and then separating to obtain a solid phase. For example, the pH can be adjusted to 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8, 5, 5.2, 5.4, 5.6, 5.8, 6, 6.2, 6.4, 6.6, 6.8, 7, 7.2, 7.4, 7.6, 7.8, or 8.

[0028] According to some embodiments of the present invention, the preparation method further includes: preparing a mixed solution of the solid phase and an organic solvent, filtering, and removing the solvent.

[0029] According to some embodiments of the present invention, the organic solvent includes at least one of methanol, ethanol, dichloromethane, and ethyl acetate.

[0030] Use of Marinobacter hydrocarbonoclasticus DH5 described in the first aspect embodiment of the present invention according to the fifth aspect embodiment of the present invention or the microbial agent described in the second aspect embodiment of the present invention in the preparation of a biosurfactant.

[0031] Use of Marinobacter hydrocarbonoclasticus DH5 described in the first aspect embodiment of the present invention according to the sixth aspect embodiment of the present invention or the microbial agent described in the second aspect embodiment of the present invention in A1) or A2): A1) Degrading petroleum or petroleum hydrocarbons; A2) Preparing a product for degrading petroleum or petroleum hydrocarbons.

[0032] According to some embodiments of the present invention, the degradation can be carried out in an NaCl environment of 0 (w / v)% - 18 (w / v)%. For example: the NaCl concentration is 0 (w / v)%, 1 (w / v)%, 2 (w / v)%, 3 (w / v)%, 4 (w / v)%, 5 (w / v)%, 6 (w / v)%, 7 (w / v)%, 8 (w / v)%, 9 (w / v)%, 10 (w / v)%, 11 (w / v)%, 12 (w / v)%, 13 (w / v)%, 14 (w / v)%, 15 (w / v)%, 16 (w / v)%, 17 (w / v)% or 18 (w / v)%.

[0033] According to some embodiments of the present invention, the petroleum hydrocarbons include at least one of alkanes and polycyclic aromatic hydrocarbons.

[0034] According to some embodiments of the present invention, the alkane includes C 16 -C 32 . The alkane is a straight-chain saturated alkane.

[0035] According to some embodiments of the present invention, the alkane includes C 16 , C 17 , C 18 , C 19 , C 20 , C 21 , C 22 , C 23 , C 24 , C 25 , C 26 , C 27 , C 28 , C 29 , C30 and C 31 and C 32 at least one of them.

[0036] According to some embodiments of the present invention, the alkane includes C 16 and C 20 and C 24 and C 28 and C 32 at least one of them.

[0037] According to some embodiments of the present invention, the polycyclic aromatic hydrocarbon includes at least one of naphthalene, anthracene, phenanthrene, and pyrene.

[0038] According to some embodiments of the present invention, the petroleum hydrocarbon is liquid paraffin.

[0039] According to some embodiments of the present invention, the product is selected from reagents and reagent kits.

[0040] Application of Marinobacter hydrocarbonoclasticus DH5 described in the first aspect embodiment of the present invention, the bacterial agent described in the second aspect embodiment of the present invention, or the biosurfactant described in the third aspect embodiment of the present invention in B1) or B2) according to the seventh aspect embodiment of the present invention: B1) Emulsifying petroleum, petroleum hydrocarbon or liquid paraffin; B2) Preparing a product for emulsifying petroleum, petroleum hydrocarbon or liquid paraffin.

[0041] According to some embodiments of the present invention, the emulsification can be carried out in an environment of 0 (w / v)% - 35 (w / v)% Na + environment.

[0042] According to some embodiments of the present invention, the emulsification can be carried out in an environment of 0 (w / v)% - 35 (w / v)% Ca 2+ environment.

[0043] According to some embodiments of the present invention, the emulsification can be carried out in an NaCl environment of 0 (w / v)% - 35 (w / v)%. For example: the concentration of NaCl is 0 (w / v)%, 1 (w / v)%, 2 (w / v)%, 3 (w / v)%, 4 (w / v)%, 5 (w / v)%, 6 (w / v)%, 7 (w / v)%, 8 (w / v)%, 9 (w / v)%, 10 (w / v)%, 11 (w / v)%, 12 (w / v)%, 13 (w / v)%, 14 (w / v)%, 15 (w / v)%, 16 (w / v)%, 17 (w / v)%, 18 (w / v)%, 19 (w / v)%, 20 (w / v)%, 21 (w / v)%, 22 (w / v)%, 23 (w / v)%, 24 (w / v)%, 25 (w / v)%, 26 (w / v)%, 27 (w / v)%, 28 (w / v)%, 29 (w / v)%, 30 (w / v)%, 31 (w / v)%, 32 (w / v)%, 33 (w / v)%, 34 (w / v)% or 35 (w / v)%.

[0044] According to some embodiments of the present invention, the emulsification can be carried out in a CaCl2 environment of 0 (w / v)% - 35 (w / v)%. For example: the concentration of CaCl2 is 0 (w / v)%, 1 (w / v)%, 2 (w / v)%, 3 (w / v)%, 4 (w / v)%, 5 (w / v)%, 6 (w / v)%, 7 (w / v)%, 8 (w / v)%, 9 (w / v)%, 10 (w / v)%, 11 (w / v)%, 12 (w / v)%, 13 (w / v)%, 14 (w / v)%, 15 (w / v)%, 16 (w / v)%, 17 (w / v)%, 18 (w / v)%, 19 (w / v)%, 20 (w / v)%, 21 (w / v)%, 22 (w / v)%, 23 (w / v)%, 24 (w / v)%, 25 (w / v)%, 26 (w / v)%, 27 (w / v)%, 28 (w / v)%, 29 (w / v)%, 30 (w / v)%, 31 (w / v)%, 32 (w / v)%, 33 (w / v)%, 34 (w / v)% or 35 (w / v)%.

[0045] According to some embodiments of the present invention, the petroleum hydrocarbon includes at least one of alkanes and polycyclic aromatic hydrocarbons.

[0046] According to some embodiments of the present invention, the alkane includes C 16 -C 32 . The alkane is a straight-chain saturated alkane.

[0047] According to some embodiments of the present invention, the alkane includes C 16 , C 17 , C18 and C 19 and C 20 and C 21 and C 22 and C 23 and C 24 and C 25 and C 26 and C 27 and C 28 and C 29 and C 30 and C 31 and C 32 at least one of

[0048] According to some embodiments of the present invention, the alkane includes C 16 and C 20 and C 24 and C 28 and C 32 at least one of

[0049] According to some embodiments of the present invention, the polycyclic aromatic hydrocarbon includes at least one of naphthalene, anthracene, phenanthrene, and pyrene.

[0050] According to some embodiments of the present invention, the petroleum hydrocarbon is liquid paraffin.

[0051] According to some embodiments of the present invention, the product is selected from reagents and reagent kits.

[0052] Application of Marinobacter hydrocarbonoclasticus DH5 described in the first aspect embodiment of the present invention, the bacterial agent described in the second aspect embodiment of the present invention, or the biosurfactant described in the third aspect embodiment of the present invention in oil recovery according to the seventh and eighth aspect embodiments of the present invention.

[0053] Other features and advantages of the present invention will be described in the subsequent specification, and will be partially apparent from the specification, or understood by implementing the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 shows the emulsification and degradation effects of strain DH5 on crude oil; Figure 2 shows the growth of Marinobacter hydrocarbonoclasticus DH5 in a crude oil inorganic salt medium with different salinities; Figure 3 shows the emulsification effect of the biosurfactant produced by Marinobacter hydrocarbonoclasticus DH5 on liquid paraffin; where 1, 2, 3, 4, 5, 6, 7, 8 respectively refer to the biosurfactant crude product containing 1000, 900, 800, 700, 600, 500, 400, 300 mg / L in the biosurfactant solution used; Figure 4Effect of different concentrations of NaCl on the emulsifying activity of biosurfactant produced by Marinobacter hydrocarbonoclasticus DH5; Figure 5 Effect of different concentrations of CaCl2 on the emulsifying activity of biosurfactant produced by Marinobacter hydrocarbonoclasticus DH5. Specific implementation mode

[0055] The following will clearly and completely describe the concept and technical effects of the present invention in combination with embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts shall fall within the scope of protection of the present invention.

[0056] For those not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0057] When a numerical range is disclosed herein, the above range is considered continuous and includes the minimum and maximum values of the range, as well as each value between such minimum and maximum values. Further, when the range refers to an integer, it includes each integer between the minimum and maximum values of the range. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0058] "And / or" is used to indicate that either or both of the described situations may occur. For example, A and / or B includes (A and B) and (A or B).

[0059] Unless otherwise specified, the formula of the crude oil inorganic salt medium is as follows: Na2MoO4 0.08 g / L, MgSO4 0.2 g / L, KH2PO4 1 g / L, (NH4)2HPO4 1 g / L, crude oil 0.5 (w / v)%, pH 7.0; The formula of the LB medium is as follows: Tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7.0; The formula of the high salinity inorganic salt medium is as follows: Na2MoO4 0.08 g / L, MgSO4 0.2 g / L, KH2PO4 1 g / L, (NH4)2HPO4 1 g / L, NaCl 80 g / L, pH 7.0; C 16 、C20 , C 24 , C 28 , C 32 are all straight-chain saturated alkanes.

[0060] Example 1 (Isolation, Identification, and Preservation of Hydrocarbon-Removing Marinobacter) 1. Transfer 10 mL of the water sample collected from the oilfield into a triangular flask containing 100 mL of crude oil inorganic salt medium, and incubate it with shaking at 35 °C and 150 rpm for 1 week. Through preliminary screening, it was found that there are microorganisms in some samples that can emulsify crude oil.

[0061] In the aseptic operation bench, take 100 μL of the fermentation broth of the experimental group with obvious crude oil emulsification and spread it on the LB agar plate medium, and incubate it at 35 °C for 48 h. Pick different-shaped single colonies on the LB agar plate medium, streak and purify them on the LB agar plate medium, and incubate at 35 °C for 48 h. Pick the single colonies after purification culture, perform enrichment culture, and then inoculate them into the crude oil inorganic salt medium, and incubate them with shaking at 35 °C and 150 rpm for 72 h, and select the strains corresponding to the experimental group with obvious crude oil emulsification.

[0062] As Figure 1 shown (left: crude oil inorganic salt medium; right: fermentation broth after culturing Hydrocarbon-Removing Marinobacter for 72 h). Strain DH5 has good dispersion, emulsification, and degradation effects on crude oil.

[0063] 2. According to the conventional method for identifying bacterial strains, extract the genomic DNA of strain DH5, design primer pairs for PCR amplification of 16S rRNA, detect the PCR amplification products by agarose gel electrophoresis, and send them to Nanjing Personal Biotechnology Co., Ltd. for DNA sequencing. And submit the sequencing results of the PCR amplification products to NCBI and use BLAST for retrieval and homology comparison.

[0064] The nucleotide sequence of the 16S rRNA of strain DH5 is shown in SEQ ID NO: 1. After comparison, the similarity between strain DH5 and Marinobacter hydrocarbonoclasticus is 100% (the representative strain corresponding to it in Genbank is MW965527.1 Marinobacter hydrocarbonoclasticus ), and it is determined that strain DH5 is Marinobacter hydrocarbonoclasticus .

[0065]

[0066] Halobacillus hydrocarbonoclasticus ( Marinobacter hydrocarbonoclasticus ) DH5 was deposited at the China Center for Type Culture Collection (CGMCC) (Address: Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province) on June 3, 2024, with the deposit number: CCTCC NO: M20241128.

[0067] Halobacillus hydrocarbonoclasticus DH5 can be preserved by the following methods: (1) Short-term preservation: Streak Halobacillus hydrocarbonoclasticus DH5 on a slant LB medium, culture at 35 °C for 48 h, and then preserve at 4 °C.

[0068] (2) Long-term preservation: (2.1) Glycerol cryopreservation method: Scrape several loops of freshly cultured Halobacillus hydrocarbonoclasticus DH5 from the slant LB medium, transfer it into a 1.5 mL sterilized glycerol tube (prepared by mixing glycerol and sterile water at a volume ratio of 3:7) at 30 (w / v)%, and preserve at -70 °C.

[0069] (2.2) Skim milk cryopreservation method: Scrape several loops of freshly cultured Halobacillus hydrocarbonoclasticus DH5 from the slant LB medium, transfer it into a glycerol tube containing sterilized skim milk, and preserve at -70 °C.

[0070] Preparation Example 1 This example provides a method for preparing a crude biosurfactant produced by Halobacillus hydrocarbonoclasticus DH5, and the steps are as follows: Inoculate 100 μL of Halobacillus hydrocarbonoclasticus DH5 preserved by the glycerol cryopreservation method into 50 mL of LB medium, shake and culture at 35 °C and 150 rpm for 12 h to activate the strain. Take 2 mL of the activated bacterial liquid, centrifuge to collect the bacterial cells, wash and resuspend with sterile water, and then inoculate it into 100 mL of crude oil inorganic salt medium. After culturing at 37 °C for 6 days, centrifuge at 12000 rpm for 3 min to remove the bacterial cells, and collect the supernatant. Adjust the pH of the supernatant to 2.0 with 6 mol / L hydrochloric acid, centrifuge at 12000 rpm for 10 min, collect the precipitate, dissolve the precipitate with methanol, filter through a 0.22 μm microporous filter membrane, and then rotary evaporate to dryness to obtain the crude biosurfactant.

[0071] Test Example 1 1. After adding 2 (w / v)%, 4 (w / v)%, 6 (w / v)%, 8 (w / v)%, 10 (w / v)%, 12 (w / v)%, 14 (w / v)%, 16 (w / v)%, 18 (w / v)%, 20 (w / v)% NaCl to the crude oil inorganic salt medium respectively, the medium was sterilized at 121 °C for 20 min to obtain crude oil inorganic salt media with different salinities.

[0072] 2. The hydrocarbon-degrading bacterium Marinobacter DH5 was inoculated into a 50 mL Erlenmeyer flask containing 30 mL of LB medium and cultured at a constant temperature of 35 °C for 12 h for activation. The activated bacterial solution was centrifuged at 20000 rpm for 2 min, the cells were collected, washed with sterile water and resuspended, and adjusted to an OD 600 of about 1.000 to obtain a bacterial suspension.

[0073] 3. 1 mL of the bacterial suspension was respectively inoculated into 100 mL of crude oil inorganic salt media with different salinities, placed in a constant temperature shaker, and cultured at 35 °C and 180 rpm for 96 h. 1 mL of the culture solution was taken, centrifuged to collect the cells, washed 3 times with sterile water, and then resuspended with 1 mL of sterile water, and its OD 600 value was measured to investigate the effect of salinity on the growth of Marinobacter DH5.

[0074] The results are as Figure 2 shown.

[0075] Marinobacter DH5 grew well in the medium with a salinity of 2 (w / v)% - 16 (w / v)% using crude oil as the sole carbon source. Marinobacter DH5 had a certain preference for high salinity. Compared with the low salinity environment, increasing the salinity of the medium was helpful for the growth of Marinobacter DH5.

[0076] Test Example 2 1. After adding naphthalene, anthracene, phenanthrene, pyrene, C 16 , C 20 , C 24 , C 28 , C 32 , crude oil or liquid paraffin to the high salinity inorganic salt medium respectively, the medium was sterilized at 121 °C for 20 min to obtain high salinity inorganic salt media containing different carbon sources.

[0077] 2. After activating Marinobacter DH5 by streaking on a plate, three single colonies were picked and inoculated into a 50 mL Erlenmeyer flask containing 30 mL of LB medium, and cultured at a constant temperature of 35 °C and 180 rpm for 8 h. An appropriate amount of the bacterial solution was centrifuged to collect the cells, washed with sterile water and resuspended, and adjusted to an OD 600 of about 1.000 to obtain a bacterial suspension.

[0078] 3. Inoculate 1 mL of bacterial suspension into 100 mL of high-mineralization inorganic salt medium containing different carbon sources to be tested, and culture at 35°C and 150 rpm for 4 days. Take 1 mL of culture medium and centrifuge at 10,000 rpm for 2 min, collect the bacteria, wash them with sterile water 3 times, resuspend them with 1 mL of sterile water, and measure their OD 600 The culture solution was extracted three times with CH2Cl2, the organic phase was collected and dried with nitrogen, and the degradation rate (A) was calculated by weighing method.

[0079] A=m0 / m1×100%; Wherein: m0 is the mass of the carbon source to be tested collected after extraction with organic solvent and drying with nitrogen, and m1 is the mass of the carbon source to be tested added initially.

[0080] The results are shown in Table 1.

[0081] Table 1

[0082] The hydrocarbon-removing Bacillus DH5 can grow with saturated hydrocarbons of different carbon chain lengths, various polycyclic aromatic hydrocarbons, crude oil, and liquid paraffin as the sole carbon source, and can degrade a variety of petroleum hydrocarbons, crude oil, and liquid paraffin.

[0083] Test Example 3 The crude biosurfactant obtained in Preparation Example 1 was dissolved in water to obtain biosurfactant solutions with crude biosurfactant concentrations of 300, 400, 500, 600, 700, 800, 900 and 1000 mg / L, respectively; 3 mL of the biosurfactant solution was mixed with liquid paraffin in a volume ratio of 1:1, and the mixture was fully oscillated and dispersed on a vortex oscillator for 5 min, then allowed to stand for 24 h, and photographed to evaluate the emulsification properties of biosurfactants of different concentrations on liquid paraffin.

[0084] The results are as follows Figure 3 shown.

[0085] The biosurfactant produced by Marinobacterium dehydrogenase DH5 can effectively emulsify liquid paraffin.

[0086] Test Example 4 Dissolve NaCl in water to prepare NaCl solutions with NaCl concentrations of 5 (w / v)%, 10 (w / v)%, 15 (w / v)%, 20 (w / v)%, 25 (w / v)%, 30 (w / v)%, and 35 (w / v)%; dissolve CaCl₂ in water to prepare CaCl₂ solutions with CaCl₂ concentrations of 5 (w / v)%, 10 (w / v)%, 15 (w / v)%, 20 (w / v)%, 25 (w / v)%, 30 (w / v)%, and 35 (w / v)%. Dissolve the crude biosurfactant prepared in Preparation Example 1 with NaCl solutions or CaCl₂ solutions of different salt concentrations to prepare biosurfactant solutions with a crude biosurfactant concentration of 100 mg / L. Mix 3 mL of the biosurfactant solution with liquid paraffin at a volume ratio of 1:1, fully oscillate and disperse it on a vortex oscillator for 5 min, then let it stand for 1, 2, 7, 10, 20, and 30 days, read the height of the emulsion layer, and calculate the E 24 index.

[0087] E 24 index = height of the emulsion layer / total height of the liquid × 100%.

[0088] The results are as Figure 4 , Figure 5 shown.

[0089] Sodium salts will affect the emulsifying activity of biosurfactants. When the Na + concentration is below 30%, different Na + concentrations have little effect on the emulsifying performance of biosurfactants in the initial stage; when the Na + concentration reaches above 30%, the emulsifying performance of biosurfactants for liquid paraffin slightly decreases, and the E 24 index drops from 58.3% to 54.2%. In different experimental groups, the height of the emulsion layer slightly decreases within the first 1 - 2 days, but as time extends, the height of the emulsion layer basically remains unchanged. This indicates that the emulsion has good stability.

[0090] Calcium salts will affect the emulsifying activity of biosurfactants. In the initial stage, the emulsifying performances of biosurfactant solutions containing different concentrations of Ca 2+ differ greatly. The emulsifying effect of the biosurfactant added with Ca 2+ will stabilize within five days.

[0091] In summary, Marinobacter hydrocarbonoclasticus DH-5 can grow and degrade petroleum hydrocarbons in a high salinity environment. At the same time, it can utilize petroleum hydrocarbons as the sole carbon source to metabolize and produce biosurfactants, and the biosurfactants have excellent salt tolerance and good emulsifying activity in high concentrations of NaCl and CaCl₂ environments.

[0092] The embodiments of the present invention have been described in detail in combination with the embodiments above, but the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those of ordinary skill in the art, various changes can be made without departing from the gist of the present invention.

Claims

1. A strain of Marinobacter hydrocarbonoclasticus ( Marinobacter hydrocarbonoclasticus ), DH5, characterized in that The hydrocarbon-degrading Marinobacter DH5 was deposited at the China Center for Type Culture Collection on June 3, 2024, with the deposit number CCTCC NO: M20241128.

2. A bacterial agent, characterized in that, The bacterial agent contains the hydrocarbon-degrading Marinobacter DH5 described in claim 1.

3. A biosurfactant, characterized in that, It is prepared by fermentation using the hydrocarbon-degrading Marinobacter DH5 described in claim 1 or the bacterial agent described in claim 2.

4. The preparation method of the biosurfactant according to claim 3, characterized in that, It includes the following steps: Inoculate the hydrocarbon-degrading Marinobacter DH5 described in claim 1 or the bacterial agent described in claim 2 into a fermentation medium and culture to obtain a fermentation broth.

5. Use of the hydrocarbon-degrading Marinobacter DH5 described in claim 1 or the bacterial agent described in claim 2 in the preparation of a biosurfactant.

6. Use of the hydrocarbon-degrading Marinobacter DH5 described in claim 1 or the bacterial agent described in claim 2 in A1) or A2): A1) Degrade petroleum or petroleum hydrocarbons; A2) Prepare a product for degrading petroleum or petroleum hydrocarbons.

7. Use of the hydrocarbon-degrading Marinobacter DH5 described in claim 1, the bacterial agent described in claim 2, or the biosurfactant described in claim 3 in B1) or B2): B1) Emulsify petroleum or petroleum hydrocarbons; B2) Prepare a product for emulsifying petroleum or petroleum hydrocarbons.

8. The application according to claim 6 or 7, characterized in that, The petroleum hydrocarbons include at least one of alkanes and polycyclic aromatic hydrocarbons.

9. The application according to claim 8, wherein The alkane includes C 16 -C 32 ; And / or, the polycyclic aromatic hydrocarbons include at least one of naphthalene, anthracene, phenanthrene, and pyrene.

10. Use of the hydrocarbon-degrading Marinobacter DH5 described in claim 1, the bacterial agent described in claim 2, or the biosurfactant described in claim 3 in petroleum recovery.

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