Halomonas sp. DH1 as well as mixed preparation and application thereof

Abstract

The invention discloses a halophilic hydrocarbon degrading bacterium Halomonas sp. DH1 as well as a mixed preparation and application thereof, and relates to the technical field of microorganisms. The invention specifically discloses a strain of Halomonas sp. DH1, which is preserved in the China Center for Type Culture Collection on June 3, 2024, and the preservation number is CCTCC (China Center for Type Culture Collection) NO: M20241125. The strain can degrade, disperse, emulsify and solubilize crude oil and degrade various petroleum hydrocarbons under the condition of hypersalinity, can generate a surfactant, can effectively improve the fluidity of the crude oil in a stratum, and improves the recovery rate of the crude oil.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to halophilic hydrocarbon-degrading bacteria Halomonas sp. DH1 and its mixed preparation and 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 highly water-cut 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 grease and starch, they are then injected into the formation as oil-displacing agents. However, this operation method has problems such as complex construction operation, short effective period, and the nutrient components in the culture medium may affect the composition of the endogenous microbial community in the formation, thereby further affecting the microbial oil-displacement effect. Therefore, it is very important to isolate microorganisms that can metabolize 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 Halomonas ( Halomonas sp. ) DH1.

[0005] The present invention provides a strain of Dietzia ( Dietzia lutea ) DH3.

[0006] The present invention also provides a microbial agent.

[0007] The present invention also provides a biosurfactant.

[0008] The present invention also provides a preparation method of the above biosurfactant.

[0009] The present invention also provides the application of the above Halomonas DH1, Dietzia DH3, microbial agent, composition or biosurfactant.

[0010] A strain of Halomonas ( Halomonas sp. ) DH1 according to the first aspect embodiment of the present invention, the Halomonas DH1 was deposited at the China Center for Type Culture Collection on June 3, 2024, and the deposit number is CCTCC NO: M20241125. The address of the deposit unit is Wuhan University, Wuhan, China

[0011] Halomonas sp. DH1 according to an embodiment of the present invention has at least the following beneficial effects: The Halomonas sp. DH1 of the embodiment can degrade, disperse, solubilize and emulsify crude oil under high salinity conditions, as well as degrade petroleum hydrocarbons, and can also produce biosurfactants, which can effectively improve the fluidity of crude oil in the formation and increase the oil recovery rate.

[0012] According to some embodiments of the present invention, the nucleotide sequence of the 16S rRNA of the Halomonas sp. DH1 is as shown in SEQ ID NO: 1.

[0013] A Dietzia sp. ( Dietzia lutea ) DH3 according to an embodiment of the second aspect of the present invention was deposited at the China Center for Type Culture Collection on June 3, 2024, with the deposit number CCTCC NO: M20241127. The address of the depository is Wuhan University, Wuhan, China

[0014] According to some embodiments of the present invention, the nucleotide sequence of the 16S rRNA of the Dietzia sp. DH3 is as shown in SEQ ID NO: 2.

[0015] Dietzia sp. DH3 according to an embodiment of the present invention has at least the following beneficial effects: The Dietzia sp. DH3 of the embodiment is adapted to grow in a high salinity environment and can degrade crude oil under high salinity conditions.

[0016] A bacterial agent according to an embodiment of the third aspect of the present invention, the bacterial agent contains the Halomonas sp. DH1 described in the first aspect embodiment of the present invention and / or the Dietzia sp. DH3 described in the second aspect embodiment of the present invention.

[0017] The bacterial agent according to an embodiment of the present invention has at least the following beneficial effects: Since the bacterial agent adopts all the technical solutions of the Halomonas sp. DH1 and / or Dietzia sp. DH3 of the above embodiments, it has at least all the beneficial effects brought by the technical solutions of the above embodiments. Moreover, Halomonas sp. DH1 and Dietzia sp. DH3 can synergistically promote each other's growth in a high salinity environment, improve the tolerance to NaCl and the degradation effect on petroleum hydrocarbons and crude oil.

[0018] According to some embodiments of the present invention, the bacterial agent includes Halomonas sp. DH1 and Dietzia sp. DH3.

[0019] According to some embodiments of the present invention, the bacterial agent is obtained by mixing equal volumes of the Halomonas sp. DH1 bacterial solution and the Dietzia sp. DH3 bacterial solution, and the OD of the Halomonas sp. DH1 bacterial solution and the Dietzia sp. DH3 bacterial solution 600The ratio is 1:(0.9 - 1.1). For example, it can be 1:1.

[0020] According to some embodiments of the present invention, the bacterial agent further contains other bacteria or fungi.

[0021] According to some embodiments of the present invention, the active ingredient of the bacterial agent is Halomonas sp. DH1 and / or Dietzia sp. DH3.

[0022] 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.

[0023] According to some embodiments of the present invention, the dosage form of the bacterial agent includes but is not limited to liquid, emulsion, suspension, powder, granule, wettable powder, or water dispersible granule.

[0024] A biosurfactant according to the embodiments of the fourth aspect of the present invention is prepared by fermentation using Halomonas sp. DH1 described in the embodiments of the first aspect of the present invention or the bacterial agent including Halomonas sp. DH1 described in the embodiments of the third aspect of the present invention.

[0025] A method for preparing a biosurfactant according to the embodiments of the fifth aspect of the present invention includes the following steps: Inoculate Halomonas sp. DH1 described in the embodiments of the first aspect of the present invention or the bacterial agent including Halomonas sp. DH1 described in the embodiments of the third aspect of the present invention into a fermentation medium, and culture to obtain a fermentation broth. The fermentation broth contains the biosurfactant.

[0026] According to some embodiments of the present invention, the temperature of the culture is 20°C to 60°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, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, or 60°C.

[0027] According to some embodiments of the present invention, the time of the culture is 2 to 7 days. For example, it can be 2, 3, 4, 5, 6, or 7 days.

[0028] 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 known in the art that can be used for culturing Halomonas sp. as the fermentation medium.

[0029] According to some embodiments of the present invention, the pH of the fermentation medium is 4-10. For example, it can be 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, 8, 8.2, 8.4, 8.6, 8.8, 9, 9.2, 9.4, 9.6, 9.8 or 10.

[0030] According to some embodiments of the present invention, the fermentation medium comprises a carbon source, a nitrogen source and inorganic salts.

[0031] According to some embodiments of the present invention, the carbon source comprises at least one of sodium acetate, soybean oil, glucose, sucrose, crude oil, liquid paraffin.

[0032] According to some embodiments of the present invention, the nitrogen source comprises at least one of peptone, urea, sodium nitrate, diammonium hydrogen phosphate, ammonium chloride, peanut meal.

[0033] According to some embodiments of the present invention, the inorganic salts comprise at least one of Na2MoO4, MgSO4, KH2PO4, NaCl, FeSO4.

[0034] According to some embodiments of the present invention, the fermentation medium comprises crude oil.

[0035] According to some embodiments of the present invention, the fermentation medium comprises the following components: Na2MoO4 0.02 g / L - 0.15 g / L, MgSO4 0.05 g / L - 0.4 g / L, KH2PO4 0.5 g / L - 2 g / L, (NH4)2HPO4 0.5 g / L - 2 g / L, crude oil 100 mg / L - 800 mg / L, pH 6.8 - 7.2.

[0036] According to some embodiments of the present invention, the fermentation medium comprises the following components: Na2MoO4 0.08 g / L, MgSO4 0.2 g / L, KH2PO4 1 g / L, (NH4)2HPO4 1 g / L, crude oil 500 mg / L, pH 7.0.

[0037] According to some embodiments of the present invention, the preparation method further comprises: removing the thalli in the fermentation broth, collecting the liquid phase, preparing a mixture of the liquid phase and an organic solvent, and separating to obtain a solid phase, thus obtaining the biosurfactant.

[0038] According to some embodiments of the present invention, the preparation method further includes: preparing a mixed solution of the solid phase and water, and removing the solvent. Those skilled in the art can select methods well-known in the art to remove the solvent, including but not limited to freeze-drying, drying, or rotary evaporation.

[0039] According to some embodiments of the present invention, the solvent includes at least one of ethanol, ethyl acetate, and methanol.

[0040] According to some embodiments of the present invention, the volume ratio of the liquid phase to the organic solvent is 1:(2 - 5). For example, it can be 1:2, 1:2.2, 1:2.4, 1:2.6, 1:2.8, 1:3, 1:3.2, 1:3.4, 1:3.6, 1:3.8, 1:4, 1:4.2, 1:4.4, 1:4.6, 1:4.8, or 1:5.

[0041] Use of the Halomonas sp. DH1 described in the first aspect embodiment of the present invention according to the sixth aspect embodiment of the present invention or the bacterial agent including the Halomonas sp. DH1 described in the third aspect embodiment of the present invention in the preparation of biosurfactant.

[0042] Use of the Halomonas sp. DH1 described in the first aspect embodiment of the present invention according to the seventh aspect embodiment of the present invention, the Dietzia sp. DH3 described in the second aspect embodiment of the present invention, or the bacterial agent described in the third 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.

[0043] According to some embodiments of the present invention, the degradation can be carried out in an NaCl environment of 0 (w / v)% - 20 (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)%, 18 (w / v)%, 19 (w / v)%, or 20 (w / v)%.

[0044] According to some embodiments of the present invention, the petroleum hydrocarbons include at least one of alkanes and polycyclic aromatic hydrocarbons.

[0045] According to some embodiments of the present invention, the alkane includes C 16 -C 32 . The alkane is a straight-chain saturated alkane.

[0046] 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 , C 30 , C 31 , C 32 or at least one of them.

[0047] According to some embodiments of the present invention, the alkane includes C 16 , C 20 , C 24 , C 28 , C 32 or at least one of them.

[0048] According to some embodiments of the present invention, the polycyclic aromatic hydrocarbon includes at least one of naphthalene, anthracene, phenanthrene, and pyrene.

[0049] According to some embodiments of the present invention, the petroleum hydrocarbon is liquid paraffin.

[0050] According to some embodiments of the present invention, the product is selected from reagents and reagent kits.

[0051] Use of the Halomonas DH1 described in the first aspect embodiment of the present invention, the microbial agent described in the second aspect embodiment of the present invention, or the biosurfactant described in the third aspect embodiment of the present invention according to the seventh aspect embodiment of the present invention in any one of B1) to B3): B1) Solubilizing or emulsifying a hydrophobic matrix; B2) Preparing a product for solubilizing or emulsifying a hydrophobic matrix; B3) Oil recovery.

[0052] According to some embodiments of the present invention, the hydrophobic matrix includes at least one of kerosene, peanut oil, olive oil, liquid paraffin, crude oil, n-heptane, and toluene.

[0053] According to some embodiments of the present invention, the product is selected from reagents and reagent kits.

[0054] Other features and advantages of the present invention will be described in the subsequent specification, and, in part, will be obvious from the specification, or will be understood by practicing the present invention. Brief Description of the Drawings

[0055] Figure 1 is the emulsification effect of strain DH1 on crude oil; Figure 2 is the total ion current chromatogram of saturated hydrocarbons before and after the degradation of crude oil by the mixture of Halomonas sp. DH1 and Dietzia sp. DH3 at a salinity of 10 (w / v)%; A: before degradation, B: after degradation; Figure 3 is the total ion current chromatogram of aromatic hydrocarbons before and after the degradation of crude oil by the mixture of Halomonas sp. DH1 and Dietzia sp. DH3 at a salinity of 10 (w / v)%; A: before degradation, B: after degradation; Figure 4 is the emulsification effect of the biosurfactant produced by Halomonas sp. DH1 on different substrates; among them, 1: kerosene, 2: peanut oil, 3: olive oil, 4: liquid paraffin, 5: crude oil, 6: n-heptane, 7: toluene. Detailed implementation manners

[0056] The following will clearly and completely describe the concept and technical effects of the present invention in combination with the embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only 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.

[0057] For those not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0058] 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 integers, 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.

[0059] "And / or" is used to indicate that either or both of the stated situations may occur. For example, A and / or B includes (A and B) and (A or B).

[0060] 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, NaCl 50 g / L, Xinjiang Oilfield crude oil 2 (w / v)%, pH 7.0; The formulation of LB medium is as follows: Tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7.0; The formulation of fermentation 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 500 mg / L, pH 7.0; C 16 、C 20 、C 24 、C 28 、C 32 are all straight-chain saturated alkanes.

[0061] Unless otherwise specified, in the embodiments of the present invention, the degradation rate (A) of the strain to different carbon sources (naphthalene, anthracene, phenanthrene, pyrene, C 16 、C 20 、C 24 、C 28 、C 32 、crude oil or liquid paraffin) is tested by the weighing method, and the specific steps are as follows: Take the culture solution after inoculating different test strains, centrifuge, extract the supernatant three times with CH2Cl2, collect the organic phase and dry it with nitrogen to obtain the extraction product, and weigh to obtain the mass of the extraction product (m0); ; wherein, m1 is the mass of the corresponding carbon source initially added in the corresponding volume of the culture solution.

[0062] Example 1 (Isolation, identification and preservation of Halomonas) 1. Transfer 10 mL of the water sample collected from the oilfield into a triangular flask of crude oil inorganic salt medium (inoculate multiple samples), and shake and culture at 35 °C and 150 rpm for 1 week. Through preliminary screening, it is found that: there are microorganisms in some samples that can significantly emulsify the crude oil.

[0063] In a sterile operating bench, 100 μL of the fermentation broth of the experimental group with obvious crude oil emulsification was taken and spread on an LB agar plate medium, and cultured at 35 °C for 48 h. Single colonies with different morphologies on the LB agar plate medium were picked, streaked and purified on the LB agar plate medium, and cultured at 35 °C for 48 h. After enrichment culture of the picked purified single colonies, they were inoculated into a crude oil inorganic salt medium and cultured with shaking at 35 °C and 150 rpm for 72 h, and the strains corresponding to the experimental group with obvious crude oil emulsification were selected.

[0064] The emulsification and dispersion effect of strain DH1 on crude oil is as Figure 1 shown (left: the fermentation broth after culturing Halomonas in a crude oil inorganic salt medium for 4 days; right: the crude oil inorganic salt medium).

[0065] Through culture and domestication, strains DH1 and DH3 with good dispersion and emulsification effects on crude oil were isolated, and they can produce biosurfactants using crude oil as the sole carbon source.

[0066] 2. According to the conventional strain identification method, the genomic DNA of strains DH1 and DH3 was extracted, primers were designed for PCR amplification of 16S rRNA, the PCR amplification products were detected by agarose gel electrophoresis, and sent to Nanjing Personal Biotechnology Co., Ltd. for DNA sequencing. And the sequencing results of the PCR amplification products were submitted to NCBI and retrieved and compared for homology using BLAST.

[0067] The nucleotide sequence of 16S rRNA of strain DH1 is shown in SEQ ID NO: 1. After comparison, the homology of strain DH1 with Halomonas sp. is 99.92% (the representative strain / species of the most similar sequence in Genbank is MG561378.1 Halomonas sp. B35), and strain DH1 was determined to be Halomonas sp. . Strain DH1 was named Halomonas ( Halomonas sp. ) DH1 and was deposited in the China Center for Type Culture Collection (CCTCC) on June 3, 2024 (address: Wuhan University, Bayi Road, Wuchang District, Hubei Province), deposit number: CCTCC NO: M20241125.

[0068]

[0069] The nucleotide sequence of the 16S rRNA of strain DH3 is shown in SEQ ID NO: 2. After comparison, strain DH3 was named Dietzia ( Dietzia lutea ) DH3 and was deposited at the China Center for Type Culture Collection (CCTCC) (Address: Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province) on June 3, 2024, with the deposit number: CCTCC NO: M20241127.

[0070]

[0071] Halomonas sp. DH1 and Dietzia sp. DH3 can be preserved by the following methods: (1) Short-term preservation: Streak Halomonas sp. DH1 or Dietzia sp. DH3 on a slant LB medium, culture at 35 °C for 48 h, and then store at 4 °C.

[0072] (2) Long-term preservation: (2.1) Glycerol cryopreservation method: Scrape several loops of freshly cultured Halomonas sp. DH1 or Dietzia sp. DH3 from the slant LB medium, transfer them into a 1.5 mL sterilized glycerol tube (prepared by mixing glycerol and sterile water at a volume ratio of 3:7) at 30 (v / v)%, and store at -70 °C.

[0073] (2.2) Skim milk cryopreservation method: Scrape several loops of freshly cultured Halomonas sp. DH1 or Dietzia sp. DH3 from the slant LB medium, transfer them into a glycerol tube containing sterilized skim milk, and store at -70 °C.

[0074] Preparation Example 1 This example provides a method for preparing a crude biosurfactant produced by Halomonas sp. DH1, and the steps are as follows: Take 2 mL of the activated Halomonas sp. DH1 bacterial solution, inoculate it into the fermentation medium at an inoculation amount of 5 (v / v)%, culture at 35 °C for 6 days, centrifuge at 10000 rpm for 3 min to remove the bacteria, and collect the supernatant. Mix the supernatant with absolute ethanol at a volume ratio of 1:4, let it stand overnight at 4 °C, centrifuge at 10000 rpm, collect the precipitate, dissolve the precipitate with an appropriate amount of water, and freeze-dry to obtain the crude DH1 surfactant.

[0075] Test Example 1 1. Add 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)%, and 20 (w / v)% NaCl to the crude oil medium (Na2MoO4 0.08 g / L, MgSO4 0.2 g / L, KH2PO4 1 g / L, (NH4)2HPO4 1 g / L, Xinjiang Oilfield crude oil 0.5 (w / v)%, pH 7.0) respectively, sterilize the medium at 121 °C for 20 min to obtain crude oil media with different salinities.

[0076] 2. Use an inoculation loop to activate the Halomonas sp. DH1 and Dietzia sp. DH3 preserved on the slant by streaking on a plate, and incubate at a constant temperature of 35 °C for 20 h. Pick three loops (each loop of the bacterial strain contains more than 3 distinct single colonies) of the bacterial strain and inoculate it into a 50 mL Erlenmeyer flask containing 30 mL of LB medium, and culture at 35 °C and 180 rpm for 8 h. Take 10 mL of the bacterial liquid, centrifuge it, collect the cells, wash them with sterile water and resuspend them, and adjust the OD 600 to approximately 1.000. Thus, a Halomonas sp. DH1 bacterial suspension and a Dietzia sp. DH3 bacterial suspension are obtained.

[0077] 3. Respectively inoculate "1 mL of Halomonas sp. DH1 bacterial suspension", "1 mL of Dietzia sp. DH3 bacterial suspension" or "mixed bacteria (0.5 mL of Halomonas sp. DH1 bacterial suspension + 0.5 mL of Dietzia sp. DH3 bacterial suspension)" into 100 mL of crude oil media with different salinities, place them in a constant temperature shaker, and culture at 35 °C and 150 rpm for 7 days. Take 1 mL of the culture solution, centrifuge to collect the cells, wash them 3 times with sterile water, resuspend them with 1 mL of sterile water, and measure its OD 600 value to investigate the effect of salinity on the growth of Halomonas sp. DH1 and Dietzia sp. DH3. And calculate the degradation rate of crude oil by the weighing method.

[0078] 4. Take the residual oil sample collected from the 10 (w / v)% salinity experimental group treated with the inoculated mixed bacteria and separate the four components of crude oil. The separation method of the four components of crude oil refers to the standard NB / SH / T 0509-2010, and perform GC-MS analysis on saturated hydrocarbons and aromatic hydrocarbons. Use the crude oil medium before inoculating the bacterial strain for the same treatment as the blank control (before degradation). The GC-MS analysis is completed by the Geochemistry Laboratory of Yangtze University.

[0079] The growth conditions of different strains and the degradation conditions of crude oil in crude oil media with different salinities are shown in Table 1 and Table 2 respectively. Through GC-MS analysis of the saturated hydrocarbon components of the 10 (w / v)% salinity crude oil medium before and after degradation by Halomonas sp. DH1 and Dietzia sp. DH3, the total ion chromatogram obtained is as Figure 2 shown; through GC-MS analysis of the aromatic hydrocarbon components of the 10 (w / v)% salinity crude oil medium before and after degradation by Halomonas sp. DH1 and Dietzia sp. DH3, the total ion chromatogram obtained is as Figure 3 shown. The statistical results of the change data of the four components of crude oil are shown in Table 3.

[0080] Table 1

[0081] Note: "-" indicates that the growth of Dietzia sp. DH3 almost stops in the high salinity environment of 16%, 18%, and 20%.

[0082] Table 2

[0083] Table 3

[0084] As shown in Table 1 and Table 2, Halomonas sp. DH1 and Dietzia sp. DH3 can both adapt to the high salinity environment and tend to grow, reproduce and degrade crude oil in the environment with higher salinity. Halomonas sp. DH1 grows well in the salinity range of 0-20 (w / v)%, and its optimal growth salinity range is 2 (w / v)% - 14 (w / v)%. Dietzia sp. DH3 grows well in the salinity range of 0-14 (w / v)%, and its optimal growth salinity range is 2 (w / v)% - 8 (w / v)%. Halomonas sp. DH1 and Dietzia sp. DH3 can synergistically promote growth at high salinity and increase the degradation rate of crude oil; in the salinity environment of 2 (w / v)% - 12 (w / v)%, the degradation rate of crude oil by the composite bacteria reaches over 50%.

[0085] Collect the four components. The recovery rate of the blank control sample is 99.04%, and the recovery rate of the sample after degradation is 94.05%. As shown in Table 3, Figure 2 、 Figure 3As shown. After treatment with the composite bacteria, the saturated hydrocarbon content in the crude oil decreased from 77.10% to 66.73%, and the aromatic hydrocarbon content decreased from 10.87% to 6.88%. Combining with the overall degradation rate of the crude oil, the composite bacteria had a significant degradation effect on the saturated hydrocarbon and aromatic hydrocarbon components. From the GC-MS analysis results, after the combined degradation of Halomonas sp. DH1 and Dietzia sp. DH3, Pr / Ph decreased by 10.73%, indicating that the crude oil was degraded. The two parameters of Pr / nC17 and Ph / nC18 increased after the action, and the value of ∑nC21- / ∑nC22+ decreased from the original 0.976 to 0.772. Before and after degradation, the oil samples contained various aromatic hydrocarbon compounds such as naphthalene, phenanthrene, triaromatic steranes, fluorene, dibenzofuran, biphenyl, dibenzothiophene, and chrysene. Naphthalene, phenanthrene, and triaromatic steranes in the samples were the main components of aromatic hydrocarbons, and their contents accounted for more than 85% of the total aromatic hydrocarbons. Compared with the oil sample before degradation, after the degradation effect, the content change of naphthalene was more obvious, decreasing from 46.74% to 37.03%; among the tricyclic aromatic compounds, except for the content of dibenzofuran decreasing by 4.7%, the contents of dibenzothiophene and fluorene increased; the content of biphenyl compounds decreased by 33.9%, and the contents of aromatic compounds such as phenanthrene increased. This shows that the composite bacteria have a good degradation effect on naphthalene. Considering the relatively high overall degradation rate of the composite bacteria on petroleum hydrocarbons, although the relative contents of dibenzothiophene and fluorene increased slightly, the overall degradation effect on aromatic hydrocarbon compounds was still obvious, especially for aromatic hydrocarbon compounds such as naphthalene, biphenyl, and dibenzofuran. Halomonas sp. DH1 not only had a significant degradation effect on the saturated hydrocarbon components, but also had an obvious degradation effect on various aromatic hydrocarbon compounds such as naphthalene, phenanthrene, triaromatic steranes, fluorene, dibenzofuran, biphenyl, dibenzothiophene, and chrysene in the crude oil.

[0086] Test Example 2 1. On the basis of the inorganic salt medium (Na2MoO4 0.08 g / L, MgSO4 0.2 g / L, KH2PO4 1 g / L, (NH4)2HPO4 1 g / L, NaCl 100 g / L, pH 7.0), 300 mg / L of naphthalene, anthracene, phenanthrene, pyrene, C 16 , C 20 , C 24 , C 28 , C 32 were added respectively, and then the medium was sterilized at 121 °C for 20 min to obtain an inorganic salt medium containing different carbon sources to be tested.

[0087] 2. Inoculate Halomonas sp. DH1 and Dietzia sp. DH3 preserved on the slant with an inoculation loop for activation by streaking on a plate, and incubate at 35 °C for 20 h. Pick three loops of bacteria (each loop of bacteria contains more than 3 distinct single colonies) from the plate and transfer them into a 50 mL Erlenmeyer flask containing 30 mL of LB medium, and culture at 35 °C and 180 rpm for 8 h. Take 10 mL of the bacterial solution, centrifuge, collect the cells, wash with sterile water and resuspend, and adjust to OD 600 to approximately 1.000. Thus, a Halomonas sp. DH1 bacterial suspension and a Dietzia sp. DH3 bacterial suspension are obtained.

[0088] 3. Transfer 1 mL of the Halomonas sp. DH1 bacterial suspension into 100 mL of an inorganic salt medium containing different carbon sources to be tested, and incubate with constant shaking at 35 °C and 150 rpm for 7 days. Take 1 mL of the culture solution, centrifuge at 10000 rpm for 2 min, collect the cells, wash 3 times with sterile water, resuspend with 1 mL of sterile water, and measure its OD 600 value. Calculate the degradation rate by the weighing method.

[0089] The results are shown in Table 4.

[0090] Table 4

[0091] Halomonas sp. DH1 can grow using saturated hydrocarbons with different carbon chain lengths and various polycyclic aromatic hydrocarbons as the sole carbon source, and degrade various petroleum hydrocarbons, especially naphthalene, anthracene, C 16 -C 28 with good degradation effects.

[0092] Test Example 3 Dissolve 0.1 g of the crude DH1 surfactant prepared in Preparation Example 1 in 20 mL of water to obtain a crude DH1 surfactant solution. Mix 3 mL of the crude DH1 surfactant solution with kerosene, peanut oil, olive oil, liquid paraffin, crude oil, n-heptane, and toluene at a volume ratio of 1:1, shake and disperse thoroughly on a vortex oscillator for 2 min, then let stand for 2 h, observe the changes, take pictures, and evaluate the emulsifying performance of the biosurfactant at different concentrations on liquid paraffin.

[0093] The results are as Figure 3 shown.

[0094] After the crude solution of surfactant DH1 was mixed with oil phases such as kerosene respectively, the total volume of the liquid did not change significantly, the volume of the water layer decreased, and the water layer became turbid. Halomonas sp. DH1 can produce biosurfactant using crude oil as the sole carbon source. The biosurfactant it produced has a good emulsifying and dispersing effect on petroleum hydrocarbons, indicating that it can solubilize petroleum hydrocarbons in the environment and improve the fluidity of crude oil, and can be used for microbial enhanced oil recovery.

[0095] The embodiments of the present invention have been described in detail above in conjunction with the embodiments, 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 spirit of the present invention.

Claims

1. A Halomonas sp. Halomonas sp. ), DH1, characterized in that The Halomonas sp. DH1 was deposited at the China Center for Type Culture Collection on June 3, 2024, with the deposit number CCTCC NO: M20241125.

2. A Dietzia bacterium ( Dietzia lutea ), characterized in that The Dietzia sp. DH3 was deposited at the China Center for Type Culture Collection on June 3, 2024, with the deposit number CCTCC NO: M20241127.

3. A microbial agent, characterized in that, The microbial agent contains the Halomonas sp. DH1 described in claim 1 and / or the Dietzia sp. DH3 described in claim 2.

4. A biosurfactant, characterized in that, It is prepared by fermentation using the Halomonas sp. DH1 described in claim 1 or the microbial agent containing the Halomonas sp. DH1 in claim 3.

5. The preparation method of the biosurfactant according to claim 4, characterized in that, It includes the following steps: Inoculate the Halomonas sp. DH1 described in claim 1 or the microbial agent containing the Halomonas sp. DH1 in claim 3 into a fermentation medium and culture to obtain a fermentation broth.

6. Use of the Halomonas sp. DH1 described in claim 1 or the microbial agent containing the Halomonas sp. DH1 in claim 3 in the preparation of a biosurfactant.

7. Use of the Halomonas sp. DH1 described in claim 1, the Dietzia sp. DH3 described in claim 2, or the microbial agent described in claim 3 in A1) or A2): A1) Degrading petroleum or petroleum hydrocarbons; A2) Preparing a product for degrading petroleum or petroleum hydrocarbons.

8. The application according to claim 7, characterized in that, The petroleum hydrocarbons include at least one of alkanes and polycyclic aromatic hydrocarbons; Preferably, the alkane includes C 16 -C 32 ; Preferably, the polycyclic aromatic hydrocarbons include at least one of naphthalene, anthracene, phenanthrene, and pyrene.

9. Use of the Halomonas sp. DH1 described in claim 1, the microbial agent containing the Halomonas sp. DH1 in claim 3, or the biosurfactant described in claim 4 in any one of B1) to B3): B1) Solubilizing or emulsifying a hydrophobic matrix; B2) Preparing a product for solubilizing or emulsifying a hydrophobic matrix; B3) Oil recovery.

10. The application according to claim 9, wherein The hydrophobic matrix includes at least one of kerosene, peanut oil, olive oil, liquid paraffin, crude oil, n-heptane, and toluene.

Images