Bacillus and screening method and application thereof

Abstract

The invention provides bacillus as well as a screening method and application thereof, and belongs to the technical field of microbial strain fermentation. The bacillus is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.33396, the preservation date is January 13, 2025, the preservation address is Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, and the postcode is 100101; the strain is classified and named as bacillus sp., and the strain number is DQ-4. The bacillus DQ-4 provided by the invention can be used for synthesizing rhamnolipid through fermentation, and the rhamnolipid can be used as an oil displacement biosurfactant, so that the rhamnolipid can be used for oil displacement of offshore oilfields. The bacillus DQ-4 disclosed by the invention overcomes the defects of bacillus aeruginosa, improves the oil extraction rate of an offshore oil field, and can reduce the offshore oil displacement cost.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial strain fermentation, and specifically relates to a bacillus and a screening method and application thereof. Background Art

[0002] Rhamnolipids, as a typical biosurfactant, has good biodegradability and environmental friendliness, and is therefore widely used in environmental remediation, oil pollution treatment, agricultural prevention and control, and oilfield development. In the process of offshore oilfield exploitation, rhamnolipids, as a natural biosurfactant, can effectively reduce the water-oil interfacial tension, promote oil-water separation, and enhance oil recovery (Enhanced Oil Recovery, EOR). It also has low toxicity and excellent biodegradability, and is an ideal green chemical.

[0003] Traditional rhamnolipid production methods, such as chemical synthesis or plant extraction, are costly, inefficient, and may cause environmental pollution, limiting their large-scale application. In the current technological development process, microbial fermentation has become the mainstream method for rhamnolipid production, among which Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) has attracted attention for its efficient synthesis of rhamnolipids, but it also has some significant disadvantages. Pseudomonas aeruginosa has strong pathogenicity and is an opportunistic pathogen that may cause pneumonia, urinary tract infections and other diseases in immunocompromised individuals. It also has strong drug resistance and can be used through efflux pumps, β-lactamase secretion and biofilm formation, which poses a challenge to drug control during production. In addition, Pseudomonas aeruginosa is highly adaptable and can survive in extreme environmental conditions. Although this feature is advantageous in some applications, it may also compete with other microorganisms during fermentation, leading to contamination and reduced yield. Therefore, screening for more stable and biosafe microorganisms has become the key to producing rhamnolipids. Summary of the invention

[0004] The purpose of the present invention is to provide a Bacillus Bacillus sp. , and provides a screening method and specific application of the Bacillus to make up for the shortcomings of the existing technology.

[0005] Bacillus Bacillus sp.As a common fermentation microorganism, it has low pathogenicity and is usually a non-pathogenic strain. It can reproduce under mild conditions and has strong tolerance to harsh environments such as high salt and low temperature. Bacillus has weak biofilm formation ability, low drug resistance, and the production process is more stable and easy to control. Therefore, the development of a process for synthesizing rhamnolipids by efficient fermentation based on Bacillus can overcome the various shortcomings of Pseudomonas aeruginosa in industrial applications, such as pathogenicity, strong drug resistance, and high pollution risk. At the same time, it can improve the production efficiency of rhamnolipids and provide a green and sustainable additive for the unique environmental conditions of offshore oil fields, which has broad application prospects.

[0006] In order to achieve the above object, the specific technical scheme adopted by the present invention is as follows: A Bacillus, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC), with the deposit number: CGMCC No.33396, the deposit date: January 13, 2025, and the deposit address: Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, Postal Code: 100101; Classification and name: Bacillus Bacillus sp. , strain number: DQ-4.

[0007] The Bacillus DQ-4 was screened from the activated sludge of Daqing Oilfield. The 16S rRNA of the strain is shown in SEQ ID NO.1. By comparison, it was found that the gene sequence similarity between DQ-4 and Bacillus was 100%.

[0008] The basic characteristics of the Bacillus DQ-4 are as follows: Gram staining is positive, spore staining is positive, starch hydrolysis test is negative, gelatinization test is positive, catalase test is positive, indole test is negative, VP test is negative, methyl red test is negative, hydrogen sulfide production test is negative, and glucose oxidation / fermentation test is oxidative non-fermentative.

[0009] The screening method of Bacillus DQ-4 comprises the following steps: (1) Initial screening of bacterial strains: Take the Daqing Oilfield sludge and add it to the inorganic salt culture medium, and cultivate it at a constant temperature for 30 o C, 150 rpm oscillation, so that the bacteria in the sludge are fully released; after the oscillation is completed, it is left to stand, and the sludge is re-precipitated to obtain the supernatant as the bacterial suspension, and the bacterial suspension is added to the LB medium and cultured at a constant temperature for 30 o C, 150 rpm shaking culture to obtain fermentation broth; (2) The fermentation broth was diluted gradiently and spread on LB plates and placed at 30 o After culturing at constant temperature for 48 h, different colonies were picked and placed in LB medium for 30 o C, 150 rpm shaking culture for 3 days; (3) Rescreening of strains: The strains obtained from the initial screening were repeatedly streaked to separate the single colonies, which were then inoculated into the fermentation medium for 30 min. o C, 150 rpm shaking culture for 3 days; after the culture, the fermentation liquid was centrifuged at 8000 rpm for 10 min, and the supernatant was collected; the supernatant was tested for oil ring, surface tension and emulsification index, and the bacteria with larger oil ring, lowest surface tension and better emulsification index were selected, purified by streaking and preserved, which was Bacillus DQ-4.

[0010] The application of the Bacillus DQ-4 in the preparation of rhamnolipid.

[0011] The application of the Bacillus DQ-4 in offshore oil recovery; the Bacillus DQ-4 is inoculated in a fermentation medium, and after the bacteria grow to a stable period, a large amount of secondary metabolites (DQ-Rha) are produced, that is, an oil recovery biosurfactant is obtained, which is used for offshore oil recovery, especially offshore oil field oil recovery.

[0012] A bacterial agent, comprising the Bacillus DQ-4; the bacterial agent is the Bacillus DQ-4 inoculated in LB medium, 10-55 o C, obtained by culturing at 150 rpm for 24 h.

[0013] The method for preparing rhamnolipid by Bacillus DQ-4 is as follows: the Bacillus DQ-4 is inoculated in a fermentation medium for fermentation, and separated and purified to obtain rhamnolipid; the fermentation medium comprises a substrate, which is glucose, sucrose, n-hexadecane, glycerol, olive oil, sodium citrate, and liquid paraffin, specifically comprising: 20 g / L glucose, 2 g / L ammonium chloride, 5 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 2 g / L dipotassium hydrogen phosphate, 1.2 g / L beef extract, 4 g / L yeast powder, and 8 g / L peptone.

[0014] Furthermore, the inoculation amount is 5%, and the fermentation temperature is 10~55 o C, the fermentation speed is 150 rpm; the culture time is 24~168; the initial pH value of the culture and fermentation is 4~10.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The Bacillus DQ-4 provided by the present invention can synthesize rhamnolipids by fermentation, and rhamnolipids can be used as oil recovery biosurfactants, thereby being used for oil recovery in offshore oil fields. The Bacillus DQ-4 of the present invention overcomes the shortcomings of Bacillus aeruginosa, improves the oil recovery rate of offshore oil fields, and can reduce the cost of offshore oil recovery. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a physical diagram of the oil-draining ring experiment during strain screening of the present invention.

[0017] Figure 2 This is the growth curve of the strain DQ-4 screened in the present invention in the enriched medium.

[0018] Figure 3 This is the phylogenetic tree of strain DQ-4 screened in the present invention.

[0019] Figure 4 Scanning electron microscope image of strain DQ-4 screened for the present invention.

[0020] Figure 5 This is the metabolic growth of the strain after condition optimization.

[0021] Figure 6 This is the thin layer chromatography analysis diagram of DQ-Rha.

[0022] Figure 7 This is the Fourier transform infrared chromatogram (FTIR) analysis of DQ-Rha.

[0023] Figure 8 is the hydrogen NMR spectrum of DQ-Rha ( 1 H-NMR) analysis.

[0024] Fig. 9 Predicted H NMR spectrum for the DQ-Rha predicted structure ( 1 H-NMR) analysis.

[0025] Fig.10 This is the MALDI-TOF MS analysis of DQ-Rha. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described in detail below in conjunction with the drawings in the embodiments of the present invention. It is worth noting that the described embodiments are only part of the embodiments of the present invention, not all of the embodiments.

[0027] Therefore, based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work shall fall within the scope of protection of the present invention.

[0028] The culture medium used in the embodiments of the present invention is as follows: Inorganic salt culture medium: 3.48 g potassium dihydrogen phosphate, 1.5 g sodium hydrogen phosphate dodecahydrate, 3.96 g ammonium sulfate, 0.7 g magnesium sulfate, 1000 mL deionized water, at 121o C sterilize for 30 min.

[0029] LB liquid medium: 10 g tryptone, 5 g yeast extract powder, 10 g sodium chloride, 1000 mL deionized water, at 121 o C sterilize for 30 min.

[0030] LB solid medium: 10 g tryptone, 5 g yeast extract powder, 10 g sodium chloride, 20 g agar, 1000 mL deionized water, at 121 o C sterilize for 30 min.

[0031] Fermentation medium: 20 g glucose, 2 g ammonium chloride, 5 g sodium acetate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, 2 g dipotassium hydrogen phosphate, 1.2 g beef extract, 4 g yeast powder, 8 g peptone, 1000 mL deionized water, at 105 o C sterilize for 30 min.

[0032] Embodiment 1: The screening method of Bacillus DQ-4 is as follows: (1) Initial screening of bacterial strains In November 2022, Daqing Oilfield sludge was collected in Daqing City, Heilongjiang Province. 5 g of Daqing Oilfield sludge was added to a conical flask containing 100 mL of inorganic salt culture medium. The conical flask was placed in a constant temperature shaker for 30 min. o C, shake at 150 rpm for 3 days to fully release the bacteria in the sludge; let it stand for 20 minutes after the shaking, wait for the sludge to re-precipitate, and obtain the supernatant as the bacterial suspension. Take 5 mL of the bacterial suspension and add it to the conical flask of LB medium. Place the conical flask on a constant temperature shaker for 30 o C, shake culture at 150 rpm for 3 days.

[0033] The fermentation liquid was diluted and spread on LB plate at 30 o After culturing in a constant temperature incubator at 40 °C for 48 h, different colonies were selected to obtain 33 strains of bacteria, which were then placed in LB medium for 30 min. o C, 150 rpm shaking culture for 3 days.

[0034] (2) Rescreening of bacterial strains The strains obtained from the initial screening were separated into single colonies by repeated streaking and inoculated into fermentation medium for 30 min. o C, 150 rpm shaking culture for 3 days. After the culture is completed, the fermentation broth is centrifuged at 8000 rpm for 10 min, and the supernatant is collected. The above supernatant is tested for oil ring, surface tension and emulsification index, and the supernatant with a larger oil ring (such as Figure 1As shown in the figure), the bacteria DQ-4 with the lowest surface tension and the best emulsification index were purified and stored by streaking.

[0035] Embodiment 2: Determination of enrichment growth curve of strain DQ-4: The strain DQ-4 obtained in Example 1 was seeded in LB medium and placed in a constant temperature shaker for 30 o C 150 rpm shaking culture. The growth curve of microorganisms is divided into adjustment period, logarithmic period, stable period and decay period. The bacterial concentration of DQ-4 grown at different time periods was measured (OD 600 ),like Figure 2 As shown in the figure, DQ-4 enters the logarithmic phase within 2 hours after inoculation, and the number of microorganisms increases greatly at a constant geometric progression. The bacteria during this period have good adaptability. Inoculating bacteria in the logarithmic phase into the culture medium is conducive to the growth of bacteria and can shorten the culture time of bacteria. After 24 hours, the bacteria begin to enter the stable phase, during which the bacteria produce a large number of secondary metabolites. Bacteria DQ-4 has a longer stable period in this culture medium, which is conducive to the production of secondary metabolites such as surfactants. Therefore, the seed solution cultured for 24 hours was selected for subsequent experiments.

[0036] Embodiment 3: Strain identification: The above strains were inoculated into LB medium at 37 o C constant temperature shaker shaking culture for 24 hours, collect the bacteria, use the genomic DNA extraction kit to extract the bacterial genomic DNA of the DQ-4 strain; use the 16S rDNA universal primer to perform PCR amplification reaction on 27F / 1492R, and use the 16S rDNA universal primer to perform PCR amplification reaction on 27F / 1492R. The total volume of the PCR reaction system is 25 , PCR amplification conditions: 96 o C 5 min; 96 o C 30 s, 56 o C 30 s, 72 o C 1 min, 35 cycles; 72 o C for 5 min; the size of the PCR product was identified by 1% agarose gel electrophoresis, and the principle that magnetic beads can adsorb or release charged substances was used to adsorb DNA in a high-salt, low-pH solution, and release DNA in a low-salt, high-pH solution to obtain purified PCR amplified fragments, which were sequenced to obtain sequence information: the obtained sequence information was compared with NCBI-BLAST, where 16SrDNA is shown in SEQ ID NO.1 ( Figure 3 ), strain DQ-4 was identified as Bacillus.

[0037] The strain DQ-4 was activated 1-5 times, with an inoculation volume of 5%, an initial pH value of 4-10, and a temperature of 10-55 o C, and the culture time is 24~168h.

[0038] Embodiment 4: Purification and preservation: The strain DQ-4 obtained in Example 1 was inoculated into LB medium and placed in a constant temperature shaker at 30 o C, shake culture at 150 rpm for 24 h, streak the bacterial solution on the LB plate, and place it in a constant temperature incubator for 30 o C for 48 h, and a single colony was picked, which was the deposited strain Bacillus DQ-4 of the present application. Bacillus The cell morphology of strain DQ-4 is as follows: on the LB plate, the colonies are round, regular, viscous, dark yellow in color, and Figure 4 As shown, it is in the shape of a short rod, about 2 .

[0039] Embodiment 5: (1) Extraction of metabolites The strain DQ-4 obtained in Example 1 was inoculated into LB medium and placed in a constant temperature shaker at 30 o C, 150 rpm shaking culture for 24 h, as the seed solution, inoculated into the fermentation medium at 5% v / v, 15 o C, 150 rpm for 72 h. After the culture was completed, the fermentation broth was centrifuged at 12000 rpm for 10 min to remove most of the bacteria, and the filtrate was adjusted to pH 2 with 6 mol / L hydrochloric acid and then heated to 4 o C for 24 h. Equal volumes of ethyl acetate were added for extraction several times, and the organic phases were combined and then o C, and remove the organic solvent by rotary evaporation to obtain a crude product of the surfactant (DQ-Rha). The weight of the crude product is accurately weighed.

[0040] (2) Purification of metabolites The crude product DQ-Rha was dissolved in anhydrous methanol by ultrasonication at a speed of 12000 rpm and a temperature of 4 o C in a refrigerated centrifuge for 10 min, and the supernatant was heated at 46 o C to obtain a pure product, and accurately weigh the weight of the pure DQ-Rha.

[0041] Embodiment 6: Optimization of bacterial strain conditions: The strain DQ-4 was inoculated into LB medium and placed in a constant temperature shaker at 30 oC, 150 rpm shaking culture for 24 h, as seed solution. Change the carbon source, initial pH value, temperature and culture time of strain DQ-4 during the culture process, and analyze the growth and metabolism of strain DQ-4, such as OD 600 , surface tension, emulsification index and DQ-Rha yield.

[0042] The growth and metabolism results of strain DQ-4 under different conditions are shown in Figure 5 The results showed that after optimization, the best carbon source was glucose and the best culture temperature was 15 o C, the optimal initial pH value was 7, and the optimal culture time was 72h. The surface tension after the optimized conditions was finally 34.7mN / m, OD 600 is 1.724, the emulsification index is 72.3%, and the DQ-Rha yield is 14.263 g / L.

[0043] Embodiment 7: Qualitative analysis of a Bacillus metabolite: (1) Thin layer chromatography (TLC) analysis A small amount of pure DQ-Rha obtained in Example 5 was dissolved in methanol. A horizontal line was drawn on the silica gel plate at a distance of 0.5 cm from the bottom edge with a pencil. The capillary was dipped into the sample and dotted on the dotted line. Each time the sample was dotted, the diameter of the sample was controlled within 2 mm after the last sample was evaporated. After the sample was completely dried, the thin layer plate was placed in a development tank for development. The ratio of the developing agent was V 氯仿 :V 甲醇 :V 水 =65:25:4, the lid is sealed with vaseline, and the developing agent should not cover the sample spot, until the developing agent rises to 1 cm from the upper edge of the silica gel plate and stops.

[0044] Use phenol-sulfuric acid colorimetric test. Phenol-sulfuric acid colorimetric test: 3.0 g phenol and 5 mL sulfuric acid solution are dissolved in 95 mL ethanol. After spraying the colorimetric test, place the mixture in an oven at 110 °C. o C color development takes 10 min, sugars show brown spots, lipopeptides do not show color. Figure 6 As shown, phenol-sulfuric acid color developer showed brown spots after spraying, and compared with the purchased rhamnolipid finished product, it was shown that the Rf value was consistent with the color development degree. Therefore, it was preliminarily determined that the metabolite DQ-Rha was a rhamnolipid biosurfactant.

[0045] (2) Fourier transform infrared spectroscopy (FTIR) analysis The pure DQ-Rha obtained in Example 5 was determined by a Fourier transform infrared spectrometer using a KBr pellet method. The purified DQ-Rha was identified and analyzed based on the infrared absorption spectrum. Figure 7As shown, we can see 3151, 3045, 2815, 1407 cm -1 The characteristic absorption peaks of sugars are at 1727 cm -1 The characteristic peak at 1143 cm indicates the presence of a carbonyl group (-C=O in -COOH). -1 The results show that the symmetric stretching vibration of COC indicates the existence of glycosidic bonds and cyclic lactone structures in the molecule, which further indicates that DQ-Rha is a rhamnolipid biosurfactant.

[0046] (3) Nuclear magnetic resonance hydrogen spectroscopy ( 1 H-NMR) analysis The pure DQ-Rha obtained in Example 2 was measured and analyzed using a 500 MHz Bruker Avance IIIHD NMR spectrometer. 1 H) with a 5 mm reverse probe (QXI) and deuterated chloroform as solvent. 1 H analysis.

[0047] The molecular structure of rhamnolipids was confirmed by nuclear magnetic resonance spectroscopy. Figure 8 As shown, the hydrogen spectrum shows 3.2 ppm to At 5.5 ppm, multiple peaks appear, each showing the structural characteristics of the sugar ring, representing the sugar ring part of rhamnose. This observation confirms that rhamnose is a monosaccharide ring. 0.8 ppm to The peaks of methyl (CH3) and methane (CH2) protons in the fatty acid chain are observed in the 2.5 ppm range, reflecting the type and length of the fatty acid chain. 0.8ppm) comes from the methyl group (CH3) at the end of the fatty acid chain, with a relatively high intensity. At the same time, the structure predicted by DQ-Rha was drawn in Chemdraw software to predict the chemical shift of hydrogen elements in DQ-Rha and compared with the experimental nuclear magnetic hydrogen spectrum. Fig. 9 It can be seen that the peak position of hydrogen in the experimental results is basically consistent with the software prediction. It is determined again that DQ-Rha is a monosaccharide monoester type biosurfactant.

[0048] (4) MALDI-TOF MS analysis The pure DQ-Rha obtained in Example 5 was determined and analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry on a Brukerautboflex speed analyzer with a laser intensity of 3200-5500. An equal volume of matrix solution was mixed, wherein the matrix solution was 70% acetonitrile aqueous solution containing 0.1% trifluoroacetic acid (TFA). -Cyano-4-hydroxycinnamic acid (CHCA), masses in the range of 200-4000 were measured.

[0049] According to the time-of-flight mass spectrum, the purified DQ-Rha was identified and structurally analyzed. Fig.10 As shown in the figure, the mass-to-charge ratio (m / z) 651 is the molecular cluster ion formed by the combination of rhamnolipid molecules and sodium ions, representing the whole molecule of monorhamnolipid, which is a molecular cluster ion formed by the combination of a rhamnose molecule (molecular weight of 146 Da) and a longer fatty acid chain (such as C10 or C12, etc.). The mass-to-charge ratio (m / z) 254 may be a fragment ion of a fatty acid chain of rhamnolipid that is cleaved, that is, DQ-Rha is a monorhamnolipid biosurfactant.

[0050] Embodiment 8: Acute oral toxicity test of a Bacillus metabolite: The acute oral toxicity test is based on GB / T 21804-2008 fixed-dose test method for acute oral toxicity of chemicals. Before the test, the animals were adapted in the barrier environment animal room for 6 days. The animals were fasted for 4 hours before exposure. After the test began, the animals were exposed to the poison once by oral gavage. One animal was first taken for pre-test exposure (the exposure dose was 5040.6 mg / kg). If the experimental animal survived within 96 hours after exposure, the formal test dose was determined to be 5000 mg / kg. Then another 4 animals were taken for exposure (the exposure dose was 5070.3 mg / kg), and the test was terminated (i.e., 14 days of observation were conducted, and no more animals were taken for exposure). The animals were fasted for 1 hour after each exposure. The exposure process and the poisoning and death of the animals during the observation period were observed and recorded, and the animals were weighed once a week. The observation period was 14 days. After the observation period, the surviving animals were killed and grossly dissected.

[0051] The test results showed that no poisoning symptoms or poisoning deaths were observed in the experimental animals in the 5040.6 mg / kg dose group within 14 days after exposure; no poisoning symptoms or poisoning deaths were observed in the experimental animals in the 5070.3 mg / kg dose group within 14 days after exposure. The weight of all surviving animals was normal (see Table 1). At the end of the experimental observation, no abnormal changes were found in the gross anatomical examination of the test animals.

[0052] Table 1 Results of acute oral toxicity test

[0053] Under the conditions of this experiment, no poisoning symptoms or deaths were observed in the experimental animals within 14 days after exposure. The acute oral LD50 of the tested samples to ICR mice 50 >5000 mg / kg. The results show that the Bacillus metabolites are non-toxic and harmless, providing a green and sustainable additive for the unique environmental conditions of offshore oil fields, and have broad application prospects.

[0054] Embodiment 9: Oil washing performance test of a Bacillus metabolite: Crude oil was mixed with quartz sand of different mesh sizes in a mass ratio of 1:5 and heated at 65 o C for 48 h, and then put into the oil washing bottle. Aged oil sand (dry weight about 15.0 g) was placed in an oil washing bottle and 30 mL of 1% DQ-Rha solution was added. o C, shake for 2 h, take out the washing oil bottle, add the corresponding concentration of DQ-Rha solution to the top mark of the washing oil bottle, shake well and place it at 65 o C incubator for 2 h, and read the volume of washed oil. At the same time, a control group was set up, and the mass of aged oil sand and the volume of washed oil were recorded in the control group (distilled water). Compared with the control group, the oil washing efficiency of the experimental group increased by 54.6%. The results show that DQ-Rha, a Bacillus metabolite, has oil washing performance at low doses and has application prospects in improving oil recovery.

[0055] Embodiment 10: Core physical simulation oil recovery experiment: oil recovery experiment of the strain of the present invention on a simulated marine oil field environment.

[0056] The core physical simulation oil recovery experiment simulates the "micro-reservoir" environment of marine oil fields through artificial cores, and simulates the potential of DQ-Rha to improve crude oil recovery in marine reservoirs. The oil recovery potential evaluation experiment was carried out using artificial sandfill cores, and 3 cores were selected, numbered #1, #2, and #3. Core #1 was used to evaluate the oil recovery potential of DQ-Rha, the metabolite of strain DQ-4; core #2 was used to evaluate the oil recovery potential of the purchased finished product rhamnolipid (Rha); core #3 was used as a blank control (injection of reservoir formation water instead of fermentation liquid). The basic parameters of the cores are shown in Table 2, and the recovery data of the three cores are shown in Table 3.

[0057] Table 2 Basic parameters of the core model in the physical simulation experiment

[0058] Table 3 Recovery factor data in physical simulation experiment

[0059] As shown in Table 3, the results of the oil recovery experiments of cores #1 and #2 show that the DQ-Rha biological agent can increase the crude oil recovery by 10.77% on the basis of a single water drive, and the purchased finished rhamnolipid (Rha) can increase the crude oil recovery by 11.35% on the basis of a single water drive, both showing good oil recovery potential; core #3, which serves as a blank control, was directly subjected to a secondary water drive after the primary water drive, and at this time, the crude oil recovery was only increased by 1.1%; the results show the feasibility of using rhamnolipid for oil recovery in a marine reservoir environment.

[0060] The above experiments show that the strain DQ-4 provided by the present invention is a rhamnolipid-producing Bacillus, which can improve the recovery rate of offshore oil fields.

[0061] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A Bacillus, characterized in that The strain is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration (CGMCC) with the deposit number: CGMCC No.33396, the deposit date: January 13, 2025, and the deposit address: Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, Postal Code: 100101; the classification name is Bacillus Bacillus sp. , strain number: DQ-4.

2. The bacillus according to claim 1, characterized in that The Bacillus DQ-4 was screened from activated sludge of Daqing Oilfield, and the 16S rRNA of the strain is shown in SEQ ID NO.

1.

3. The bacillus according to claim 1, characterized in that The characteristics of the Bacillus DQ-4 are as follows: Gram staining is positive, spore staining is positive, starch hydrolysis test is negative, gelatinization test is positive, catalase test is positive, indole test is negative, VP test is negative, methyl red test is negative, hydrogen sulfide production test is negative, and glucose oxidation / fermentation test is oxidative non-fermentative.

4. The screening method for Bacillus DQ-4 according to claim 1, characterized in that: The following steps are involved: (1) Initial screening of bacterial strains: Take the Daqing Oilfield sludge and add it to the inorganic salt culture medium, and incubate it at a constant temperature to fully release the bacteria in the sludge; after the oscillation is completed, let it stand and wait for the sludge to re-precipitate, and obtain the supernatant as the bacterial suspension. Take the bacterial suspension and add it to the LB culture medium, and incubate it at a constant temperature to obtain the fermentation liquid; (2) The fermentation broth was diluted gradiently and spread on LB plates and placed at 30 o After constant temperature culture at C, different bacterial colonies were picked and placed into LB medium respectively, and cultured at constant temperature with shaking; (3) Rescreening of strains: The strains obtained from the initial screening were separated into single colonies by repeated streaking, and the colonies were inoculated into fermentation medium and cultured at a constant temperature with shaking. After the culture was completed, the fermentation broth was centrifuged and the supernatant was collected. The supernatant was tested for oil ring, surface tension and emulsification index, and Bacillus DQ-4 was screened.

5. Use of the Bacillus DQ-4 according to claim 1 in the preparation of rhamnolipid.

6. Use of the Bacillus DQ-4 described in claim 1 in offshore oil recovery.

7. A bacterial agent, characterized in that: The bacterial agent comprises the Bacillus DQ-4 described in claim 1.

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