Denitrified Jones strain LU-8 as well as culture medium and application thereof

Abstract

The invention belongs to the technical field of oil displacement of microbial oil reservoirs, and particularly relates to a Jones denitrificans LU-8 strain as well as a culture medium and application of the Jones denitrificans LU-8 strain. The invention provides a microorganism. The nucleotide sequence of 16SrRNA of the microorganism is as shown in SEQ ID NO: 1. The invention also provides a Jones denitrifying bacterium LU-8 strain which is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.27833. The invention also provides the Jones denitrifying bacterium LU-8 strain. The microorganism and the strain provided by the invention occupy advantages when being co-cultured with a strain in injected water, and have high bacterium concentration and proportion under a facultative anaerobic condition; the oil displacement agent has good emulsifying performance, can significantly improve the oil extraction efficiency, and has wide prospects in the field of microbial oil reservoir oil displacement.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial reservoir oil recovery, and in particular relates to a denitrifying Jonesia (Jonesia denitrificans) LU-8 strain and a culture medium and application thereof. Background Art

[0002] Oil production generally goes through three stages: primary oil production is to rely on natural energy to produce oil, such as rock expansion, edge water drive, gravity, natural gas expansion, etc.; secondary oil production is to increase the pressure of the oil layer by injecting water and gas to produce oil; tertiary oil production is to change the viscosity of crude oil or the interfacial tension with the underlying medium by physical and chemical methods to displace the discontinuous or difficult-to-extract crude oil in the oil layer. Microbial Enhanced Oil Recovery (MEOR) technology is extremely important in tertiary oil production. It is an important successor technology after thermal drive, chemical drive and polymer drive.

[0003] Microbial oil recovery technology is a technology to enhance oil recovery by strengthening water flooding. Its main content is to use microorganisms in the reservoir or specially selected natural flora to produce specific metabolism, thereby improving oil recovery. In this method, microorganisms contribute to the improvement of crude oil recovery in two main ways. On the one hand, microorganisms grow in rock layers, produce gases, biosurfactants, biopolymers, organic solvents, acids and other non-toxic biochemical substances, which can solubilize crude oil, reduce surface viscosity or interfacial tension, improve crude oil fluidity and thus recover and capture oil; on the other hand, they can form biofilms through bacterial enrichment and metabolize macromolecular biopolymers to selectively block high permeability channels, thereby improving the sweep efficiency of the extraction process.

[0004] Compared with chemical agents, microbial oil recovery has unique advantages. First, microorganisms are mobile and have local enrichment effects. They can move autonomously to find nutrients, and proliferate in large quantities under suitable environmental conditions and rich nutrients to form biofilms and metabolites. Secondly, microorganisms can use cheap industrial and agricultural by-products as nutrients for large-scale fermentation. Under conditions of insufficient nutrition in the oil reservoir, microorganisms will die in large numbers and will not be retained in large quantities in the formation. Their metabolites can be biodegraded and are harmless to humans and animals. Its simple process, low cost, and green environmental protection make it have broad application prospects.

[0005] Chinese patent application 201710003922.1 discloses a strain of Bacillus subtilis AnPL-1 that can produce lipopeptide surfactants under anaerobic conditions. The deposit number of the strain is CGMCC NO.13461. The strain AnPL-1 disclosed in the present invention is a facultative anaerobic bacterium. The temperature range for efficient production of lipopeptides under anaerobic conditions is 30°C-40°C, the pH range is 6.0-7.5, and the tolerance range of NaCl is 0-5%. The emulsification coefficient of the anaerobic fermentation liquid of the strain for crude oil is greater than 56%, the diameter of the oil drainage ring is greater than 15mm, and the strain AnPL-1 produces lipopeptide surfactants in situ in physical simulation cores, which increases the crude oil recovery rate by 9.9%.

[0006] Chinese patent application 201911303973.1 discloses a high temperature resistant microbial oil recovery bead XJ-50 and its application in heavy oil reservoir exploitation and a heavy oil reservoir exploitation method. The high temperature resistant microbial oil recovery bead XJ-50, Latin name Bacillus kokeshiiformis, deposit number CGMCC NO: 17891. The high temperature resistant microbial oil recovery bead XJ-50 provided can emulsify heavy oil at 50°C and can be applied to the field of microbial oil recovery technology.

[0007] At present, compared with chemical oil recovery technology, microbial oil recovery technology has a smaller improvement in recovery rate and requires continuous activation and supplementation of oil recovery functional bacteria; the cost of microbial fermentation is still high, and the injection amount of microorganisms and nutrients is low; the number, activity, and action cycle of the dominant functional bacteria in the entire metabolic chain are low, which are key issues that restrict the large-scale promotion of this technology. Forming a low-cost continuous injection strategy and reducing the comprehensive cost of microbial oil recovery technology have become key technical issues that need to be urgently solved in production. Therefore, the strategy of screening dominant oil recovery functional bacteria on the ground and optimizing the fermentation medium to expand the concentration of bacteria injected into the underground reservoir environment and increase the production of metabolites can theoretically promote the increase in oil production. Summary of the invention

[0008] In view of this, the object of the present invention is to provide a strain of Jonesia denitrificans LU-8 that can be exogenously injected into oil reservoirs and efficiently drive oil. The strain can take advantage when co-cultured with strains in oil reservoir injection water, has the highest bacterial concentration and proportion under facultative anaerobic conditions, and grows faster, which can significantly improve crude oil recovery. The present invention also provides a culture medium for fermenting the strain, as well as a microbial agent, an emulsifier and an activator for microbial oil reservoir oil recovery.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] In the first aspect of the present invention, a microorganism is provided, and the gene sequence of the 16S rRNA of the microorganism is as shown in SEQ ID NO: 1:

[0011] 1gagtttgatc ctggctcagg acgaacgctg gcggcgtgct taacacatgc aagtcgaacg

[0012] 61atgatgaccg gagcttgctt tggttggatt agtggcgaac gggtgagtaa cacgtgagta

[0013] 121acctgccccc ttcttcggga taaccattgg aaacggtggc taataccggg tatgacatca

[0014] 181gatcgcatgg tttggtgtgg aaaggttttt tctggtgggg gatgggctcg cggcctatca

[0015] 241gcttgttggt ggggtgatgg cctaccaagg ctttgacggg tagccggcct gagagggcga

[0016] 301ccggccacac tgggactgag acacggccca gactcctacg ggaggcagca gtggggaata

[0017] 361ttgcacaatg ggggaaaccc tgatgcagcg acgccgcgtg ggggatgaag gccttcgggt

[0018] 421tgtaaacccc tttcagtaag gaacaaggcc atgattgttg ttgtggtgag ggtacttgca

[0019] 481gaagaacggc cggctaacta cgtgccagca gccgcggtaa tacgtagggc gcaacggttg

[0020] 541tccggaatta ttgggcgtaa agagctcgta ggcggtttgt cgcgtctgtc gtgaaatccc

[0021] 601gatgcttaac gtcgggcttg cggtgggtac gggcaggctg gagtgcggta ggggtgactg

[0022] 661gaattcctgg tgtagcggtg gaatgcgcag atatcaggag gaacaccgat ggcgaaggca

[0023] 721ggtcactggg ccgttactga cgctgaggag cgaaagcatg gggagcgaac aggattagat

[0024] 781accctggtag tccatgccgt aaacgttggg cactaggtgt ggggattgtt ccacgattcc

[0025] 841tgtaccgtac gtaacgcatt aagtgccccg cctggggagt acggccgcaa ggctaaaact

[0026] 901caaaggaatt gacgggggcc cgcacaagcg gcggagcatg cggattaatt cgatgcaacg

[0027] 961cgaagaacct taccaaggct tgacatacgc tagatcgctc cagagatggg gttttccttt

[0028] 1021ggactggtgt acaggtggtg catggttgtc gtcagctcgt gtcgtgagat gttgggttaa

[0029] 1081gtcccgcaac gagcgcaacc cttgttctat gttgccagca cgtaatggtg gggactcatg

[0030] 1141ggagactgcc ggggtcaact cggaggaagg tggggacgac gtcaaatcat catgcccctt

[0031] 1201atgtcttggg cttcacgcat gctacaatgg ccggtacaat gggttgcgat actgtgaggt

[0032] 1261ggagcgaatc cctaaaagcc ggtctcagtt cggattgggg tctgcaactc gaccccatga

[0033] 1321agtcggagtc gctagtaatc gcagatcagc aacgctgcgg tgaatacgtt cccgggcctt

[0034] 1381gtacacaccg cccgtcaagt cacgaaagtt ggtaacaccc gaagccggcg gcctaacccc

[0035] 1441ttttggggag ggcgttctcg aaggtgggac tggcgattgg gactaagtcg taacaaggta

[0036] 1501gccgtaccgg aagg.

[0037] In some embodiments, the microorganisms include, but are not limited to, bacteria, actinomycetes, fungi, viruses, protozoa, algae, and the like.

[0038] In some specific embodiments, the microorganism can be a bacterium.

[0039] In some more specific embodiments, the microorganism is Jonesia denitrificans.

[0040] The second aspect of the present invention provides a denitrifying Jonesella LU-8 strain, which is deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC No.27833.

[0041] The LU-8 strain of the present invention is isolated from crude oil in an oil reservoir, and has a 16S rRNA gene sequence similar to that of Jonesia denitrificans of 99%, and can be identified as the genus Jonesia denitrificans. The morphological characteristics of the strain are as follows: the colonies on nutrient agar are small, convex, and smooth, and are arranged in a branched Y and rod shape. As the culture time increases, the colonies turn yellow, and products are secreted around them.

[0042] The third aspect of the present invention provides a culture medium for fermenting the microorganism described in the first aspect or the strain described in the second aspect, which comprises a carbon source, a nitrogen source and trace elements.

[0043] In some embodiments, the carbon source is molasses.

[0044] In some embodiments, the nitrogen source is ammonium sulfate and yeast powder.

[0045] In some embodiments, the trace element is magnesium sulfate.

[0046] In some embodiments, the mass ratio of the carbon source, the nitrogen source and the trace elements is approximately (1-5): 1: 0.05.

[0047] In some specific embodiments, the mass ratio of ammonium sulfate to yeast powder is about 1:1.

[0048] In some specific embodiments, the concentration of molasses can be about 1.0 g / L, 1.5 g / L, 2.0 g / L, 2.5 g / L, 3.0 g / L, 3.5 g / L, 4.0 g / L, 4.5 g / L, or 5.0 g / L.

[0049] In some specific embodiments, the concentration of ammonium sulfate is 0.5 g / L;

[0050] The concentration of the yeast powder is 0.5 g / L;

[0051] The concentration of the magnesium sulfate is 0.05 g / L.

[0052] The fourth aspect of the present invention provides a microbial agent, which comprises the microorganism described in the first aspect and / or the strain described in the second aspect, and a nutrient.

[0053] In some embodiments, the nutrient may be a powder and / or a liquid formulation.

[0054] In some specific embodiments, the powder may be the culture medium described in the third aspect.

[0055] In some specific embodiments, the liquid preparation may be a liquid prepared from the culture medium described in the third aspect.

[0056] A fifth aspect of the present invention provides a method for producing an emulsifier, the method comprising one or more of the following steps:

[0057] (1) uniformly mixing the microbial agent described in the fourth aspect and then amplifying and culturing the microorganism described in the first aspect and / or the strain described in the second aspect;

[0058] (2) Inoculating the microorganism described in the first aspect or the strain described in the second aspect into the culture medium described in the third aspect for amplification culture.

[0059] In some embodiments, the step (2) further comprises the following steps:

[0060] 1) culturing the microorganism and / or strain in the culture medium to obtain a seed solution;

[0061] 2) inoculating the seed solution into the culture medium for expansion culture.

[0062] In some embodiments, the seed solution has an inoculation density of 2%-10%. In some specific embodiments, the seed solution has an inoculation density of about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%. In some more specific embodiments, the seed solution has an inoculation density of 5%.

[0063] In some embodiments, the expansion culture comprises the following conditions:

[0064] (i) the temperature may be 24-37°C;

[0065] (ii) The rotation speed can be 200 rpm;

[0066] (iii) The time can be 24-72 hours.

[0067] In some embodiments, the temperature of the expansion culture can be about 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. In some specific embodiments, the temperature of the expansion culture is 30° C.

[0068] In some embodiments, the time of the amplification culture can be about 24-48 hours. In some embodiments, the time of the amplification culture can be 24h, 25h, 26h, 27h, 28h, 29h, 30h, 31h, 32h, 33h, 34h, 35h, 36h, 37h, 38h, 39h, 40h, 41h, 42h, 43h, 44h, 45h, 46h, 47h, 48h.

[0069] In some specific embodiments, the expansion culture time is 48 hours.

[0070] The sixth aspect of the present invention provides an emulsifier produced by the method according to the fifth aspect, wherein the EI of the emulsifier is 24 Greater than 80%.

[0071] In some embodiments, the EI of the emulsifier is 24It can be about 80%-200%.

[0072] In some embodiments, the emulsifier can be culture broth, culture broth extract, whole bacteria, whole bacteria extract, fermentation broth and / or fermentation broth extract.

[0073] In some specific embodiments, the emulsifier is fermentation broth and / or fermentation broth extract.

[0074] The seventh aspect of the present invention provides use of the microorganism described in the first aspect, the strain described in the second aspect, the microbial agent described in the fourth aspect and / or the emulsifier described in the sixth aspect in reservoir flooding.

[0075] The eighth aspect of the present invention provides the use of the microorganism described in the first aspect, the strain described in the second aspect, the microbial agent described in the fourth aspect and / or the emulsifier described in the sixth aspect in improving crude oil recovery.

[0076] In some embodiments, the microorganisms, strains, microbial agents and / or emulsifiers are activated by an activator to emulsify crude oil into oil droplets.

[0077] The present invention has at least the following beneficial effects:

[0078] (1) The denitrifying Jonesella LU-8 strain provided by the present invention has an advantage when co-cultured with the strain in the injected water, has a high bacterial concentration and proportion under facultative anaerobic conditions, and the pure culture bacterial concentration can reach 10 9 , accounting for more than 50%.

[0079] (2) High emulsification efficiency: the crude oil in the crude oil culture medium was completely emulsified within 4 days, and the oil droplets with a particle size of ≤15 μm accounted for more than 95%, showing good emulsification properties;

[0080] (3) Stable performance, emulsification index EI 24 The result is greater than 80%, and the emulsification performance is stable;

[0081] (4) Significantly improve oil recovery efficiency. Short core physical simulation experiments found that the oil recovery efficiency was increased by 11%. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1 This is a photo of LU-8 strain LB plate colonies.

[0083] Figure 2 This is a Gram staining photo of the LU-8 strain LB plate colony.

[0084] Figure 3 This is the 16S rRNA evolutionary tree of the LU-8 strain.

[0085] Figure 4This is a graph showing the percentage of co-culture of LU-8 strain and injected water.

[0086] Figure 5 This is a statistical chart of bacterial concentration of self-cultivated bacteria in the injected water.

[0087] Figure 6 LU-8 strain EI 24 Result diagram, where A is the blank control group, B is a schematic diagram of the liquid level height of the pure culture group after 48 hours of culture, and C is a schematic diagram of the liquid level height of the experimental group (emulsifier added) after 48 hours of culture.

[0088] Figure 7 This is the result of emulsification of crude oil in the shake flask.

[0089] Figure 8 This is the result of observing emulsified crude oil under a microscope.

[0090] Fig. 9 This is a statistical chart of crude oil emulsification particle size analyzed by microscopic image analysis software.

[0091] Fig.10 The figure shows the change in the proportion of LU-8 strain at different inoculation sizes.

[0092] Fig.11 This is the growth curve of LU-8 strain at different inoculation sizes.

[0093] Fig.12 This is the growth curve of LU-8 strain under facultative conditions at different inoculation sizes.

[0094] Fig.13 This is the growth curve of LU-8 strain at different temperatures.

[0095] Fig.14 The percentage changes of LU-8 strain at different temperatures. DETAILED DESCRIPTION

[0096] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0097] Unless the context clearly dictates otherwise, the expressions "a", "an" and "an" as used herein include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0098] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0099] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0100] The fermentation medium of the present invention comprises: 1-5 g / L molasses, 0.5 g / L ammonium sulfate, 0.05 g / L magnesium sulfate and 0.5 g / L yeast powder.

[0101] The formula of the solid LB plate of the invention is: 0.15% of corn steep liquor dry powder, 0.6% of sodium nitrate, 0.25% of ammonium chloride and 1.5% of agar powder.

[0102] Core specifications: length 30 cm, diameter 3.8 cm, pore volume 110 mL (saturated oil volume 72 mL);

[0103] The core was prepared by the following method:

[0104] The core is an artificial cemented core made of quartz sand and a binder. Natural sands of different particle sizes are fully mixed and stirred evenly. Preheat the epoxy resin in a constant temperature water bath 2 hours in advance, weigh anhydrous ethanol, epoxy resin, dibutyl phthalate, and ethylenediamine in turn, add them to a 5000mL glass beaker, and stir them evenly to make a binder. Slowly add the prepared binder to the natural sand, stir it evenly, and fill it evenly in the mold in 3-6 times. Place the core mold under the press, start the press, slowly increase the pressure to 15MPa, press for 15 minutes, and then slowly release the pressure. Place the pressed artificial core at room temperature, and drill a core with a diameter of 3.80cm and a length of 30cm according to the experimental requirements.

[0105] Example 1 Screening of Denitrifying Jonesella LU-8 Strain

[0106] Take 100mL of reservoir formation water, 2% crude oil, 0.15% corn steep liquor powder, 0.6% sodium nitrate and 0.25% ammonium chloride in a sterile Erlenmeyer flask and culture at 37°C, 200rpm for 7 days. Dilute the culture solution in a gradient manner and apply it to a solid LB plate. Culture at 37°C for 48h, pick out single colonies with different morphologies, and inoculate them into the above-mentioned sterilized screening medium. A total of 8 strains were screened and preserved, numbered LU-1 to LU-8, for the next step of verification. Crystal violet stained micrographs of the strains and colony morphology ( Figure 1 ). The colonies of LU-1, LU-2, LU-3, and LU-6 have regular and complete edges, and are smooth, opaque, convex, and white; the colonies of LU-4 are beige and translucent with a halo; the colonies of LU-5 are smaller and bright brown; the colonies of LU-7 are grayish yellow and flat; the colonies of LU-8 are small, convex, and smooth on nutrient agar, and are arranged in a branched Y and rod shape. As the culture time increases, the colonies turn yellow, and products are secreted around them ( Figure 2 ).

[0107] Example 2 Identification of Denitrifying Jonesella LU-8 Strain

[0108] Pick a single colony and 100 μL sterile water, boil in boiling water for 10 min, then freeze at -20℃ for 30 min, take out, and after thawing, centrifuge at 12000rpm for 2 min, take 1 μL supernatant as template for PCR amplification, PCR amplification system (25 μL) is: 1 μL of primers 27F / 1492R, 12.5 μL of 2× PrimeStar premix, 1 μL of template, 9.5 μL of ddH2O. The reaction procedure is: 98℃ 15s, 55℃ 15s, 72℃ 20s, 72℃ 10min, 30 cycles. The sequence of primer 27F is shown in SEQ ID NO: 2: AGAGTTTGATCCTGGCTCAG; the sequence of primer 1492R is shown in SEQ ID NO: 3: TACGGCTACCTTGTTACGACTT.

[0109] The centrifuge tube containing the PCR product was stored in a refrigerator at -20°C and then sequenced using a gene sequencer. After obtaining the 16S rRNA sequence of the strain, BLAST was performed in the NCBI database to query and download the 16S rRNA sequences of microbial species with high similarity to the sequenced strain sequence for homology analysis and comparison. The phylogenetic tree of the strain was constructed using MEGA 6.0 software. The analysis results are shown in the figure. Figure 3 As shown, the LU-8 strain has a similarity of 99% with Jonesia denitrificans and can be identified as a denitrifying Jonesia genus.

[0110] Example 3 Evaluation of LU-8 strain growth performance

[0111] Pick a single colony on the LB plate, inoculate it in LB liquid medium with the same ingredients, and culture it at 30℃ for 24h as seed liquid. The experimental group is inoculated with 10% in the fermentation medium for producing emulsifiers prepared from reservoir injection water (no sterilization is required), and the shake flask is cultured for 48h under facultative conditions or aerobic conditions, 30℃, and 200rpm. The control group does not add target bacteria, and only the bacteria in the injection water are cultured under facultative conditions and aerobic conditions at 37℃ and 200r / min. Samples are taken from the experimental group and the control group every 12h for real-time fluorescence quantitative PCR, and the total bacteria 16S and LU-8 bacteria are absolutely quantified, and the growth curve and proportion curve of LU-8 bacteria under facultative conditions and aerobic conditions are drawn.

[0112] The 16S qPCR quantitative system (20 μL) was as follows: primer 100 μM 8F / 338R 0.08 μL each, 2×RealStar GreenFast Mixture 10 μL, template 1 μL, ddH2O 8.84 μL. The reaction procedure was as follows: 95°C pre-denaturation for 3 min; 95°C 30 s, 55°C 30 s, 72°C 30 s, 81°C 10 s, 40 cycles.

[0113] The LU-8 quantitative system (20 μL) is: primer 100 μM JonesiaF / JonesiaR 0.1 μL each, 2×RealStarGreen Fast Mixture 10 μL, template 1 μL, ddH2O 8.8 μL. The reaction procedure is: 95°C pre-denaturation for 3 min; 95°C 30 s, 56°C 30 s, 72°C 30 s, 81°C 10 s, 40 cycles. The sequence of primer 8F is shown in SEQ ID NO: 4: AGAGTTTGATTGGCTC, and the sequence of primer 338R is shown in SEQ ID NO: 5: GCTGCCTCCCGTAGGAGT. The sequence of primer JonesiaF is shown in SEQ ID NO: 6: TCCTACGGGAGGCAGCAGT, and the sequence of primer JonesiaR is shown in SEQ ID NO: 7: CATCGGGATTTCACGACAGA.

[0114] The results are as follows Figure 4 and Figure 5 As shown in the figure, in the control group, the bacterial flora grew rapidly under aerobic conditions and reached a plateau within 24 hours, with the highest bacterial concentration reaching 10 9 / mL; the concentration under facultative conditions at 24h was significantly lower than that under aerobic conditions, indicating that facultative conditions can effectively inhibit the growth of bacteria in the injected water to a certain extent. Figure 5As shown, the experimental group accounted for the highest 30.08% at 24h and still had an advantage at 48h.

[0115] Example 4 Evaluation of emulsification performance of LU-8 strain

[0116] A single colony on the LB plate was picked up, inoculated into an LB liquid culture medium with the same composition, and cultured at 30°C for 24 hours as a seed solution. The seed solution was inoculated into a fermentation medium for producing an emulsifier prepared from reservoir injection water, and cultured at 30°C and 200 rpm for 48 hours to obtain an emulsifier.

[0117] The obtained emulsifier was mixed with diesel in a stoppered test tube at a volume ratio of 1:1, vortexed for 3 minutes, and allowed to stand for 48 hours. The results showed that the EI of the mixed group was 24 More than 80% ( Figure 6 ), and the emulsification effect can last for more than 1 month. 24 The calculation formula is: EI 24 =(height of emulsified layer in experimental group / height of organic layer in blank control group)×100%.

[0118] In the reservoir injection water, the medium was prepared according to the formula of 2g / 100mL of reservoir crude oil, 0.48g / 100mL of dipotassium hydrogen phosphate trihydrate, 0.15g / 100mL of potassium dihydrogen phosphate, 0.1g / 100mL of ammonium sulfate, 0.05g / 100mL of sodium citrate, 0.02g / 100mL of magnesium sulfate heptahydrate, 0.5g / 100mL of glucose, 0.01g / 100mL of yeast powder, and 0.01mL / 100mL of trace elements, pH 7.0. After culturing at 37°C and 200rpm in a shaking table for 4 days, the shake flask crude oil showed a good emulsification effect. The results are as follows: Figure 7 A small amount of culture solution was dropped onto a glass slide and observed under an optical microscope. The collected image was binarized and analyzed using microscopic image analysis software to obtain the size and proportion of the emulsified particles. Figure 8 and Fig. 9 As shown, oil droplets with a particle size of ≤15 μm account for more than 95%.

[0119] Example 5 Evaluation of Environmental Adaptability of LU-8

[0120] The LU-8 strain was co-cultured with bacteria injected into the reservoir water, and the environmental adaptability of LU-8 was evaluated from three aspects: oxygen conditions, inoculation conditions and temperature conditions.

[0121] The results of Example 3 show that compared with aerobic conditions, facultative conditions can better inhibit the growth of bacteria in injection water. The LU-8 strain was co-cultured with reservoir injection water for 48 hours. At 24 hours, the proportion of the LU-8 strain reached a maximum of 30.08%, and it still had an advantage at 48 hours. The seed liquid was inoculated at 2%, 5% and 10% inoculation respectively. The proportion of the LU-8 strain was the highest at 10% inoculation, reaching more than 40%, but the bacterial concentration was 10 8 -10 9 / mL, which is lower than that under 2% and 5% inoculation conditions (such as Fig.10 and 11 ), but under facultative conditions, the 5% inoculum concentration was higher (as shown in Fig.12 LU-8 strain was cultured at 24℃, 30℃ and 37℃ respectively. At 30℃, the proportion of LU-8 strain was the highest, reaching more than 50%, and the bacterial concentration was close to 10 9 (like Fig.13 and 14 shown).

[0122] In summary, the functional bacteria LU-8 is a facultative anaerobic bacteria that grows well under aerobic and facultative anaerobic conditions. After culturing at 30°C with 5% inoculated water for 24 hours, the target functional bacteria accounted for nearly 60% and the bacterial concentration was 10 9 More than pcs / mL.

[0123] Example 6 Application of LU-8 strain in oil recovery

[0124] The obtained emulsifier was used to perform physical simulation of oil recovery, including the following steps: injecting the fermentation liquid into the core at an injection rate of 1 mL / min, standing at 37° C. for 5 days, and performing oil recovery after 5 days. The results showed that the oil recovery efficiency was increased by 11%.

[0125] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A microorganism, characterized in that The nucleotide sequence of the 16S rRNA of the microorganism is shown in SEQ ID NO:

1.

2. The microorganism according to claim 1, characterized in that The microorganism is denitrifying Jonesella.

3. A denitrifying Jonesia LU-8 strain, characterized in that It is deposited in the China General Microbiological Culture Collection Center with the deposit number CGMCC No.27833.

4. The strain according to claim 3, characterized in that The gene sequence of 16S rRNA of the strain is shown as SEQ ID NO:

1.

5. A culture medium for fermenting the microorganism according to any one of claims 1-2 or the strain according to any one of claims 3-4, characterized in that: Contains carbon source, nitrogen source and trace elements; Preferably, the carbon source is molasses; And / or, the nitrogen source is ammonium sulfate and yeast powder; And / or, the trace element is magnesium sulfate; Preferably, the mass ratio of the carbon source, nitrogen source and trace elements is (1-5): 1: 0.05; Preferably, the mass ratio of ammonium sulfate to yeast powder is 1:

1.

6. The culture medium according to claim 5, characterized in that The concentration of the molasses is 1-5 g / L; The concentration of ammonium sulfate is 0.5 g / L; The concentration of the yeast powder is 0.5 g / L; The concentration of the magnesium sulfate is 0.05 g / L.

7. A microbial agent, characterized in that: Comprising the microorganism according to any one of claims 1-2 and / or the strain according to any one of claims 3-4, and a nutrient; Preferably, the nutrient is a powder and / or a liquid preparation.

8. The microbial agent according to claim 7, characterized in that: The nutrient agent is the culture medium according to claim 5 or 6.

9. A method for producing an emulsifier, characterized in that The method comprises at least one of the following steps: (1) after uniformly mixing the microbial agent according to any one of claims 7 to 8, amplifying and culturing the microorganism according to any one of claims 1 to 2 or the strain according to any one of claims 3 to 4; (2) inoculating the microorganism according to any one of claims 1 to 2 or the strain according to any one of claims 3 to 4 into the culture medium according to any one of claims 5 to 6 for amplification and cultivation; Preferably, the step (2) further comprises the following steps: 1) culturing the microorganism and / or strain in the culture medium to obtain a seed solution; 2) inoculating the seed solution into the culture medium for expansion culture.

10. The method according to claim 9, characterized in that The expansion culture includes one or more of the following conditions: (i) Temperature is 24-37°C; (ii) the rotation speed is 200 rpm; (iii) The duration is 24-72 hours.

11. An emulsifier produced according to any one of claims 9 to 10, characterized in that: The EI of the emulsifier 24 80%-200%; Preferably, the emulsifier is culture broth, culture broth extract, whole bacteria, whole bacteria extract, fermentation broth and / or fermentation broth extract.

12. Use of the microorganism according to any one of claims 1-2, the strain according to any one of claims 3-4, the microbial agent according to any one of claims 7-8, and / or the emulsifier according to claim 11 in oil reservoir flooding.

13. Use of the microorganism according to any one of claims 1-2, the strain according to any one of claims 3-4, the microbial agent according to any one of claims 7-8, and / or the emulsifier according to claim 11 in improving crude oil recovery.

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