Streptococcus sp. Strain and application thereof

Abstract

The invention belongs to the technical field of oil reservoir microorganisms, and particularly relates to a zoococcus strain and application thereof. The bacterial strain is a Planococcus sp. Bacterial strain WL-9, and is a new strain of the Planococcus sp. The strain is separated from an oil reservoir source, can reduce interfacial tension between crude oil and water, plays a role in emulsifying the crude oil, is good in oil reservoir adaptability, and can convert the crude oil into methane in an extreme environment of the oil reservoir and in the presence of CO2. The invention provides a new high-quality strain resource for microbial oil recovery, and has great significance for improving the oil recovery rate, reducing carbon emission by CCUS microbial carbon conversion and treating and repairing petroleum pollution.

Description

Technical Field

[0001] This invention belongs to the field of oil reservoir microbiology technology, specifically relating to a zoococcal strain and its application. Background Technology

[0002] Oil reservoir microorganisms convert crude oil and CO2 into methane, which is one of the main natural phenomena of methane production. After tertiary oil recovery, 30-40% of the residual oil in the reservoir remains unrecoverable. Oil reservoir microorganisms have enormous potential for methane conversion; globally, 22 sedimentary basins have been reported to exhibit clear characteristics of microbial degradation of crude oil for methane regeneration, and 12 basins may exhibit similar characteristics. These include the Junggar Basin, Ordos Basin, Songliao Basin, and Liaohe Basin in China; the Olla, Nebo-Hemphill, and Cushing oil fields in the United States; the Yabase oil field in Japan; the Mildred Lake Settling Basin tailings in Canada; the Eocene reservoir oil field in Australia; and the Pannonian Basin in Hungary. Based on research by Russian scientists, it is estimated that over geological periods, the crude oil in existing reservoirs has cumulatively produced approximately 655 trillion cubic feet of methane through biodegradation, equivalent to 4.3 times the amount of methane gas from conventional extraction plus dissolved gases from crude oil (Milkov AV, 2011). Therefore, microbial degradation of crude oil to regenerate methane has enormous potential and economic benefits globally.

[0003] CO2 capture, utilization, and storage (CCUS) refers to the industrial process of capturing, separating, and utilizing CO2 from industrial emission sources while simultaneously reducing CO2 emissions. Global assessments of carbon reduction technologies consistently indicate that CCS / CCUS, with their massive carbon sequestration capacity, are a crucial safety net for achieving carbon neutrality and serve as the primary technology for large-scale carbon utilization and emission reduction. Injecting waste CO2 into oil reservoirs allows reservoir microorganisms to utilize the CO2 and residual oil to produce methane, reducing carbon emissions and further enhancing oil recovery through crude oil conversion, generating usable clean energy—methane; achieving a win-win situation in terms of both economic and social benefits.

[0004] However, the exogenous microbial strains that have been developed so far have adaptability problems when facing the extreme environment of oil reservoirs, such as temperature, pH and salinity, and have low carbon conversion efficiency, which limits their application in microbial oil recovery and carbon emission reduction. There is an urgent need to develop new high-efficiency endogenous microbial strains that are suitable for various types of oilfields. Summary of the Invention

[0005] Based on the shortcomings of existing technologies that use exogenous microbial strains, this invention provides a novel Zoococcus strain, Planococcus sp. WL-9, isolated from the reservoir itself. It is isolated from a highly efficient methanogenic system of crude oil produced fluid, has good reservoir adaptability, and can be applied to improve crude oil recovery.

[0006] The technical solution of the present invention is as follows:

[0007] Firstly, a *Planococcus sp.* strain WL-9 is provided, which was patented by the China General Microbiological Culture Collection Center (CGMCC) on March 11, 2024, with accession number CGMCCNo.30008 and classification name: *Planococcus sp.*. Preferably, it has a 16S rRNA sequence as shown in SEQ ID NO: 1.

[0008] Secondly, a culture of the aforementioned Plantococcus sp. strain WL-9 or an extract thereof is provided.

[0009] Thirdly, a method for preparing a culture of the aforementioned Plantococcus sp. strain WL-9 is provided, comprising: inoculating Plantococcus sp. strain WL-9 into a culture medium and culturing it at 28–32°C for 70–80 h.

[0010] Fourthly, a microbial agent is provided, which contains the aforementioned Plantococcus sp. strain WL-9 or a culture or an extract of the culture.

[0011] Fifthly, the application of the aforementioned Plantococcus sp. strain WL-9 or its culture, extracts of the culture, or bacterial agents in microbial enhanced oil recovery or improved oil recovery is provided.

[0012] Sixthly, the application of the aforementioned Plantococcus sp. strain WL-9 or its culture or extract or agent in emulsified crude oil is provided.

[0013] Seventhly, the application of the aforementioned Plantococcus sp. strain WL-9 or its culture or extracts or agents in the degradation of petroleum hydrocarbons to produce methane is provided.

[0014] Eighthly, the application of the aforementioned Plantococcus sp. strain WL-9 or its culture or extracts or agents in the microbial degradation of petroleum hydrocarbon pollutants is provided.

[0015] Ninth aspect, a method for enhancing oil recovery is provided, comprising: injecting the aforementioned Plantococcus sp. strain WL-9 or its culture or extract or agent into an oil reservoir.

[0016] The beneficial effects of this invention are as follows:

[0017] The *Planococcus* sp. strain WL-9 provided by this invention is a novel species of the genus *Planococcus*. This strain, isolated from the reservoir source, can reduce the interfacial tension between crude oil and water, thus emulsifying the crude oil. It also exhibits good reservoir adaptability, capable of converting crude oil into methane even in extreme reservoir environments and in the presence of CO2. This invention provides a new and high-quality microbial strain resource for microbial enhanced oil recovery (MEOR), and is of great significance for improving crude oil recovery, reducing carbon emissions through microbial carbon conversion (CCUS), and remediating oil pollution. Attached Figure Description

[0018] Figure 1 This is a morphological diagram of strain WL-9 of the present invention;

[0019] Figure 2 This is the ribosomal protein fingerprint of strain WL-9 of the present invention;

[0020] Figure 3 The phylogenetic tree of strain WL-9 of this invention based on the 16S rRNA gene sequence;

[0021] Figure 4 Comparison of interfacial tension data between each group of sample solutions and crude oil. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Example 1: Isolation and Screening of Strain WL-9

[0024] The strain WL-9 of this invention was isolated from the crude oil produced fluid of Shengli Oilfield. The specific isolation procedures were as follows:

[0025] Solid culture medium for bacterial isolation (hereinafter referred to as "solid culture medium"): LB agar medium (components: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 15 g / L agar) prepared using the aqueous phase of crude oil produced fluid, pH 7.0-7.2.

[0026] Isolation and screening method: 1 mL of liquid was taken from the methanogenic system and diluted 10-fold, 100-fold, and 1000-fold, respectively. 100 μL of each diluted solution was then spread onto solid culture medium and incubated at 30°C for 3 days. Single colonies were picked and cultured on fresh solid culture medium using the three-step streak method for 3 days at 30°C. This process was repeated. After isolation and purification, several pure bacterial strains were obtained for subsequent research. These included the novel *Planococcus* sp. WL-9, a strain of the *Planococcus* species described in this invention.

[0027] Strain preservation information: Preservation number: CGMCC No.30008, Preservation institution: China General Microbiological Culture Collection Center (CGMCC), Preservation address: No.3, No.1 Beichen West Road, Chaoyang District, Beijing, Preservation date: March 11, 2024.

[0028] Example 2: Detection of physiological and biochemical characteristics of strain WL-9

[0029] After 3 days of growth under culture conditions, the physiological and biochemical functions of strain WL-9 were assessed using the GEN III detection system manufactured by Biolog Corporation, USA. Other physiological characteristics of the strain were determined according to Bergey's Manual of Systematic Bacteriology.

[0030] according to Figure 1 As shown, after growth on solid culture medium, the strain forms pale yellow colonies with a moist surface. Identification results show that strain WL-9 is a Gram-positive bacterium with spherical cells. It is catalase-positive and oxidase-negative. BIOLOG GENIII assays for strain WL-9 are shown in Table 1, experiments using a single carbon source and a single nitrogen source are shown in Table 2, and antibiotic resistance results are shown in Table 3. Strain WL-9 tolerates growth in acidic environments above pH 6 and 1-8% NaCl.

[0031] Table 1. Chemical sensitivity test for Biolog GEN III detection

[0032]

[0033] Table 2. Growth experiments of Biolog GEN III on single carbon or nitrogen sources.

[0034]

[0035] Table 3 Results of antibiotic resistance testing

[0036]

[0037]

[0038] Note: Antibiotic sensitivity: 's' indicates sensitive, 'r' indicates resistant, and 'm' indicates moderate or weak resistance.

[0039] Example 3: Detection of cytochemical characteristics of strain WL-9

[0040] The cell wall fatty acid composition of strain WL-9 was analyzed by gas chromatography (HP6890) (Sasser M. 1990, Identification of bacteria by gas chromatography of cellular fatty acids. USFCC News Lett, 20:1-6.). In strain WL-9 of this invention, the highest content of cell wall fatty acids was isopentdecanoic acid (C15:Oiso), with a content of 34.79%; followed by ω7c-hexadecanoic acid monounsaturated alcohol (C15:Oiso). 16:1 ω7c alcohol), with a content of 12.35%; followed by iso-tetradecanoic saturated fatty acids (C 14:0 The content of isocyanate is 11.78%. The content of other fatty acids is all below 10%, as shown in Table 4.

[0041] Table 4. Cellular fatty acid composition of strain WL-9

[0042]

[0043]

[0044] Example 4: Determination of the phylogenetic position of strain WL-9

[0045] Genomic DNA was extracted from strain WL-9 of the present invention and amplified, cloned and sequenced its 16S rRNA gene sequence. The sequencing results are shown in SEQ ID NO: 1.

[0046] SEQ ID NO:1:GCGGAAACCACGGGAGCTTGCTCCTGTTGGTTTTAG CGGCGGACGGGTGAGTAACACGTGGGCAACCTGCCCTGCAGATCGGGATAACTCCGGGAAACCGGTGCTAATACCGAATAGTTTGGCGCCTCTCCTGAGGCGCTACGGAAAGACGGTTTCGGCTGTCACTGCAGGATGGGCCCGCGGCGCATTAGCTAGTTGGGGGGGTAACGGCCTCCCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGCAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAACTCTGTTGTAAGGGAAGAAACCGTGCCAGTTAACTACTGGCACCTTGACGGTACCTTACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTCCTTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGGGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCGCTGCCCGGTGCAGAGATGCGCCTTTCCCTTCGGGGACAGCGGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAAAGGGCAGCCAACCCGCGAGGGGGAGCCAATCCCAGAAAACCGTTCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGCCGGAATCGCTAGTAATCGTGGATCAGCATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCAC

[0047] The obtained sequences were compared online in the international authoritative bacterial classification and analysis database (http: / / www.ezbiocloud.net / ) (Kim OS, Cho YJ, Lee K, et al. 2012, Introducing EzTaxon-e: aprokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol, 62:716-721.). The results showed that the strain WL-9 of this invention had the highest similarity to species of the genus *Planococcus* (98.21%). Among them, the species with the highest pairwise sequence similarity were *Planococcus sp. (in:firmicutes)strain MJ058 (98.21%), *Planococcus sp. (in:firmicutes)strain MJ058 (98.21%), and *Planomicrobium sp. MDT1-79 (98.14%). Therefore, it can be seen that the 16S rRNA gene sequence of strain WL-9 of this invention has a maximum similarity to known bacterial species below the 98.7% threshold for distinguishing different bacterial species. To further clarify the phylogenetic position of the strain, a phylogenetic tree was constructed using the 16S rRNA gene of a representative type strain of the genus Zoococcus. For example... Figure 3 As shown, the invented strain WL-9 forms a separate clade with a certain phylogenetic gap, clustering with other strains in the genus *Planococcus*, fully demonstrating that it is a new species of the genus *Planococcus*. Its taxonomic position is: Kingdom Bacteria; Phylum Bacillota; Class Bacilli; Order Bacillales; Family Planococcaceae; Genus *Planococcus*.

[0048] Example 5: Detection of characteristic peaks of cellular ribosome proteins

[0049] 1. Detection method: The EXS3000 fully automated bacterial mass spectrometry identification system of Zhongyuan Huiji Company was used to collect the ribosomal protein fingerprint of the bacterial strain.

[0050] (1) Preparation of reagents

[0051] Preparation of matrix solution: Weigh 15 mg of α-cyano-4-hydroxycinnamic acid, add 500 μl of acetonitrile, 475 μl of deionized water and 25 μl of trifluoroacetic acid in sequence, vortex for 3 min, store at 4℃, and the shelf life is 1-2 weeks.

[0052] Preparation of 70% formic acid solution: Mix 7ml formic acid and 3ml deionized water thoroughly and store at 4℃ away from light.

[0053] (2) Extraction method

[0054] Add 300 μl of sterile water to a 1.5 ml centrifuge tube. Using a sterile inoculation loop, take an appropriate amount of fresh bacterial sample and suspend it fully in water. Add 900 μl of anhydrous ethanol, vortex to mix, centrifuge at 12000 rpm for 2 min, and discard the supernatant.

[0055] Centrifuge twice at 12,000 rpm for 1 minute, remove the residual supernatant with a pipette, and dry at room temperature for about 3 minutes.

[0056] Add 50 μl of 70% formic acid solution, mix thoroughly by pipetting, and let stand for 1-2 min. Then add 50 μl of acetonitrile, mix thoroughly, centrifuge at 12000 rpm for 3 min, and retain the supernatant.

[0057] (3) Mass spectrometry analysis

[0058] Sample application: Take 1 μl of the supernatant extracted above and drop it into the target site. After it is allowed to air dry naturally at room temperature, add 1 μl of matrix solution to cover the sample and allow it to air dry naturally for detection.

[0059] Mass spectrometry data acquisition: Spectroscopy data were acquired using an EXS3000 matrix-assisted laser desorption / ionization time-of-flight mass spectrometer (MA LDI-TOF-MS).

[0060] 2. Detection Results: The characteristic peaks of the bacterial ribosomal proteins are shown in Table 5, and the ribosomal protein fingerprint is shown in the figure below. Figure 2 As shown.

[0061] Table 5. Characteristic peaks of ribosomal proteins in strain WL-9

[0062] Mass-to-charge ratio (m / z) Relative peak area (%) 6392.1 100 5153.45 93 9570.79 75 4785.55 73 3195.6 59 6415.97 41 6289.56 35 2576.55 25

[0063] Example 6: Experimental Study on the Emulsifying Effect of Strain WL-9 on Crude Oil

[0064] 1. Experimental Groups: Three strains of bacteria, Pesudomonas sp., Arthrobacter sp., and Planococcus sp. WL-9, were isolated from the methanogenic system of Shengli Oilfield crude oil produced fluid and enriched. Each strain was plated separately on LB agar medium and incubated statically at 30°C for 72 h. Then, each strain was inoculated into 3 mL of LB liquid medium and incubated at 30°C and 150 rpm (with sufficient oxygen contact) for 72 h to obtain the culture medium for each strain, which served as the experimental groups. LB liquid medium without inoculation served as the control group.

[0065] 2. Experimental Methods: Sample solutions from each experimental group and control group were added to crude oil from Daqing Oilfield. At 0 min, 10 min, 20 min, 30 min, 40 min, and 50 min after addition, the changes in interfacial tension of oil droplets in each sample solution were measured using a spin drop interfacial tension meter (model: CNGTX-601, CNG ENTERPRISESE LIMITED, MD, USA). Experimental conditions: spin speed 5000 rpm, temperature 45℃, density difference 0.15 g / cm³. 3 (Refer to the reservoir temperature and crude oil density of Daqing Oilfield.)

[0066] 3. Experimental results: such as Figure 4 As shown in the figure, among the three strains, Planococcus sp. WL-9 exhibits a more significant ability to reduce interfacial tension, indicating its remarkable effect in emulsifying crude oil. It can be applied to emulsifying non-flowable residual oil under water-drive conditions, increasing crude oil fluidity, and altering rock surface wettability, making it easier for the oil film to peel off from the rock surface and become the mobile phase, thereby improving oil recovery.

[0067] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A Planococcus sp. strain WL-9, characterized in that, It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 30008.

2. The Planococcus sp. strain WL-9 according to claim 1, characterized in that, It has a 16S rRNA sequence as shown in SEQ ID NO:

1.

3. A culture of Planococcus sp. strain WL-9 as described in claim 1 or 2, or an extract thereof.

4. The method for preparing the culture of Planococcus sp. strain WL-9 according to claim 3, characterized in that, The preparation method includes: inoculating Planococcus sp. strain WL-9 into a culture medium and culturing it at 28–32°C for 70–80 h.

5. A microbial agent, characterized in that, The bacterial agent contains the Plantococcus sp. strain WL-9 as described in claim 1 or 2, or the culture as described in claim 3, or an extract of the culture thereof.

6. The application of the Plantococcus sp. strain WL-9 of claim 1 or 2, or the culture of claim 3 or an extract thereof, or the bacterial agent of claim 4, in microbial enhanced oil recovery or improved oil recovery.

7. The use of the Planococcus sp. strain WL-9 of claim 1 or 2, or the culture of claim 3 or an extract thereof, or the bacterial agent of claim 4, in emulsified crude oil.

8. The use of the Plantococcus sp. strain WL-9 of claim 1 or 2, or the culture of claim 3 or an extract thereof, or the bacterial agent of claim 4, in the degradation of petroleum hydrocarbons to produce methanogens.

9. The application of the Planococcus sp. strain WL-9 of claim 1 or 2, or the culture of claim 3 or an extract thereof, or the microbial agent of claim 4, in the microbial degradation of petroleum hydrocarbon pollutants.

10. A method for enhancing oil recovery, characterized in that, The method includes injecting the Plantococcus sp. strain WL-9 of claim 1 or 2, or the culture of claim 3 or an extract thereof, or the bacterial agent of claim 4 into an oil reservoir.

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