A Bacillus bacterium, its screening method and application

Abstract

The present invention provides a Bacillus bacterium, its screening method and application, belonging to the technical field of microbial strain fermentation. A Bacillus bacterium, which is preserved in the China General Microbiological Culture Collection Center (CGMCC), with the preservation number: CGMCC No. 33396, the preservation date: January 13, 2025, and the preservation address: Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, Zip Code: 100101; classified and named as Bacillus Bacillus sp. , and the strain number is: DQ-4. The Bacillus bacterium DQ-4 provided by the present invention can synthesize rhamnolipid through fermentation, and rhamnolipid can be used as a biosurfactant for oil displacement, thus being used for oil displacement in offshore oilfields. The Bacillus bacterium DQ-4 of the present invention overcomes the disadvantages of Bacillus aeruginosa, improves the oil production rate of offshore oilfields, and can reduce the cost of offshore oil displacement.

Description

Technical Field

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

[0002] Rhamnolipids, as a typical biosurfactant, have good biodegradability and environmental friendliness, and thus are widely used in fields such as environmental remediation, petroleum pollution treatment, agricultural control, and oilfield development. During the exploitation of marine oilfields, rhamnolipids, as a natural biosurfactant, can effectively reduce the water-oil interfacial tension, promote oil-water separation, and improve the enhanced oil recovery (EOR). At the same time, they have low toxicity and excellent biodegradation performance, and are an ideal green chemical.

[0003] Traditional production methods of rhamnolipids, such as chemical synthesis or plant extraction, are costly, inefficient, and may cause environmental pollution, which limits their large-scale application. In the current technological development process, the microbial fermentation method has become the mainstream method for rhamnolipid production. Among them, Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) has received attention for its high-efficiency synthesis of rhamnolipids, but there are also some significant disadvantages. Pseudomonas aeruginosa has strong pathogenicity and is an opportunistic pathogen that may cause diseases such as pneumonia and urinary tract infections in immunocompromised individuals. It also has strong drug resistance and can resist antibiotics through mechanisms such as efflux pumps, β-lactamase secretion, and biofilm formation, which poses challenges for drug control during the production process. In addition, Pseudomonas aeruginosa has very strong adaptability and can survive in extreme environmental conditions. Although this characteristic has advantages in some applications, it may also compete with other microorganisms during the fermentation process, resulting in contamination and yield reduction. Therefore, screening more stable and biosafe microorganisms has become the key to rhamnolipid production. Summary of the Invention

[0004] The object of the present invention is to propose a Bacillus Bacillus sp. , and provide a screening method and specific application of the Bacillus to make up for the deficiencies of the prior art.

[0005] Bacillus Bacillus sp.As a common fermenting microorganism, it has low pathogenicity, usually being a non-pathogenic strain. It can reproduce under mild conditions and has strong tolerance, adapting to harsh environments such as high salt and low temperature. The ability of Bacillus to form biofilms is weak, its drug resistance is low, and the production process is more stable and easy to control. Therefore, developing a process for synthesizing rhamnolipids by an efficient fermentation method based on Bacillus can overcome various disadvantages of Pseudomonas aeruginosa in industrial applications, such as high pathogenicity, strong drug resistance, high pollution risk, etc. At the same time, it can improve the production efficiency of rhamnolipids and provide a green and sustainable auxiliary agent for the unique environmental conditions of offshore oilfields, with broad application prospects.

[0006] To achieve the above object, the specific technical solutions adopted by the present invention are as follows:

[0007] A Bacillus strain, which is preserved in the China General Microbiological Culture Collection Center (CGMCC), with the preservation number: CGMCC No. 33396, the preservation date: January 13, 2025, and the preservation address: Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, Postcode: 100101; classified and named as Bacillus Bacillus sp. , and the strain number is: DQ-4.

[0008] The Bacillus DQ-4 is screened from the activated sludge of Daqing Oilfield. The 16S rRNA of this strain is as shown in SEQ ID NO.1. Through comparison, it is found that the gene sequence similarity between DQ-4 and Bacillus is 100%.

[0009] The basic characteristics of the Bacillus DQ-4: 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, V-P test is negative, methyl red test is negative, hydrogen sulfide production test is negative, and glucose oxidation / fermentation test is oxidative non-fermentative.

[0010] The screening method of the Bacillus DQ-4 includes the following steps:

[0011] (1) Primary screening of strains: Take the oil sludge of Daqing Oilfield and add it to the inorganic salt medium, incubate at a constant temperature, 30 o °C, shake at 150 rpm to fully release the bacteria in the oil sludge; after the shaking ends, let it stand still, wait for the oil sludge to redeposit, and the supernatant obtained is the bacterial suspension. Take the bacterial suspension and add it to the LB medium, incubate at a constant temperature, 30 o °C, shake and culture at 150 rpm to obtain the fermentation broth;

[0012] (2) Take the fermentation broth, dilute it in gradients and spread it on the LB plate, and place it at 30 oAfter culturing at a constant temperature of o 48 h, different colonies were picked and transferred into LB medium respectively, and cultured at 30

[0013] °C with shaking at 150 rpm for 3 days; o (3)Re-screening of strains: Each strain obtained from the primary screening was purified into single colonies by repeated streaking, and then inoculated into the fermentation medium respectively. Cultured at 30

[0014] °C with shaking at 150 rpm for 3 days; After the culture was completed, the fermentation broth was centrifuged at 8000 rpm for 10 min, and the supernatant was collected; The supernatant was tested for oil displacement circle, surface tension and emulsification index. Bacteria with a larger oil displacement circle, the lowest surface tension and a better emulsification index were selected, streaked and purified for preservation, which was Bacillus DQ-4.

[0015] Application of the Bacillus DQ-4 in the preparation of rhamnolipid.

[0016] Application of the Bacillus DQ-4 in offshore oil displacement; The Bacillus DQ-4 was inoculated into the fermentation medium. When the bacteria grew to the stationary phase, a large amount of secondary metabolites (DQ-Rha) began to be produced, and then the oil-displacing biosurfactant was obtained, which was used for offshore oil displacement, especially for oil displacement in offshore oilfields.

[0016] A bacterial agent, which includes the Bacillus DQ-4; The bacterial agent was obtained by inoculating the Bacillus DQ-4 into LB medium and culturing at 10-55 o °C with shaking at 150 rpm for 24 h.

[0017] The method for preparing rhamnolipid by the Bacillus DQ-4 is as follows: The Bacillus DQ-4 was inoculated into the fermentation medium for fermentation, and then separated and purified to obtain rhamnolipid; The fermentation medium includes substrates, and the substrates are glucose, sucrose, n-hexadecane, glycerol, olive oil, sodium citrate, liquid paraffin. Specifically, it includes: glucose 20 g / L, ammonium chloride 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, dipotassium hydrogen phosphate 2 g / L, beef extract 1.2 g / L, yeast powder 4 g / L, peptone 8 g / L.

[0018] Furthermore, the inoculation amount is 5%, the fermentation temperature is 10-55 o °C, the fermentation rotation speed is 150 rpm; The culture time is 24-168 h; The initial pH value of the culture fermentation is 4-10.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] The Bacillus DQ-4 provided by the present invention can synthesize rhamnolipids through fermentation. Rhamnolipids can be used as oil-displacing biosurfactants and thus are used for oil displacement in offshore oilfields. The Bacillus DQ-4 of the present invention overcomes the disadvantages of Bacillus aeruginosa, improves the oil recovery rate of offshore oilfields, and can reduce the cost of offshore oil displacement. Description of the Drawings

[0021] Figure 1 It is a physical diagram of the oil-displacement circle experiment during the strain screening of the present invention.

[0022] Figure 2 It is the growth curve of the strain DQ-4 screened by the present invention in the enrichment medium.

[0023] Figure 3 It is the phylogenetic tree of the strain DQ-4 screened by the present invention.

[0024] Figure 4 It is the scanning electron microscope image of the strain DQ-4 screened by the present invention.

[0025] Figure 5 It is the metabolic growth of the strain after condition optimization.

[0026] Figure 6 It is the thin-layer chromatography analysis chart of DQ-Rha.

[0027] Figure 7 It is the Fourier transform infrared chromatography (FTIR) analysis chart of DQ-Rha.

[0028] Figure 8 It is the nuclear magnetic resonance hydrogen spectrum ( 1 1H-NMR) analysis of DQ-Rha.

[0029] Figure 9 It is the predicted nuclear magnetic resonance hydrogen spectrum ( 1 1H-NMR) analysis drawn for the predicted structure of DQ-Rha.

[0030] Figure 10 It is the time-of-flight mass spectrometry (MALDI-TOF MS) analysis chart of DQ-Rha. Detailed Embodiments

[0031] 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 described in detail below with reference to the accompanying drawings in the embodiments of the present invention. It should be noted that the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments.

[0032] Therefore, based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0033] The culture media used in the embodiments of the present invention are as follows:

[0034] Inorganic salt medium: 3.48 g of potassium dihydrogen phosphate, 1.5 g of disodium hydrogen phosphate dodecahydrate, 3.96 g of ammonium sulfate, 0.7 g of magnesium sulfate, 1000 mL of deionized water, sterilized at 121 o °C for 30 min.

[0035] LB liquid medium: 10 g of tryptone, 5 g of yeast extract powder, 10 g of sodium chloride, 1000 mL of deionized water, sterilized at 121 o °C for 30 min.

[0036] LB solid medium: 10 g of tryptone, 5 g of yeast extract powder, 10 g of sodium chloride, 20 g of agar, 1000 mL of deionized water, sterilized at 121 o °C for 30 min.

[0037] Fermentation medium: 20 g of glucose, 2 g of ammonium chloride, 5 g of sodium acetate, 0.2 g of magnesium sulfate, 0.05 g of manganese sulfate, 2 g of dipotassium hydrogen phosphate, 1.2 g of beef extract, 4 g of yeast powder, 8 g of peptone, 1000 mL of deionized water, sterilized at 105 o °C for 30 min.

[0038] Example 1:

[0039] The screening method of Bacillus DQ-4 is as follows:

[0040] (1) Preliminary screening of strains

[0041] In November 2022, oil sludge from Daqing Oilfield was collected in Daqing City, Heilongjiang Province. 5 g of the oil sludge from Daqing Oilfield was added to a conical flask containing 100 mL of inorganic salt medium. The conical flask was placed in a constant temperature shaker and shaken at 30 o °C and 150 rpm for 3 days to fully release the bacteria in the oil sludge; after shaking, it was left to stand for 20 min. After the oil sludge redeposited, the supernatant was obtained as a bacterial suspension. 5 mL of the bacterial suspension was added to a conical flask containing LB medium. The conical flask was placed in a constant temperature shaker and shaken at 30 o °C and 150 rpm for 3 days.

[0042] The above fermentation broth was serially diluted and spread on LB plates, and placed in a constant temperature incubator at 30 o °C for 48 h. Then, different colonies were picked, and 33 strains of bacteria were obtained. They were respectively picked into LB medium and cultured at 30 oC, shake culture at 150 rpm for 3 days.

[0043] (2)Rescreening of strains

[0044] For each strain obtained from the primary screening, single colonies were isolated by repeated streaking, and then each was inoculated into the fermentation medium. Incubate at 30 o °C, shake culture at 150 rpm for 3 days. After the culture, centrifuge the fermentation broth at 8000 rpm for 10 min, and collect the supernatant. Test the oil displacement circle, surface tension and emulsification index of the above supernatant, and select the bacterium DQ-4 with a larger oil displacement circle (as Figure 1 shown), the surface tension reduced to the lowest, and a better emulsification index. Purify it by streaking and preserve it.

[0045] Example 2:

[0046] Determination of the enrichment growth curve of strain DQ-4: Inoculate the strain DQ-4 screened in Example 1 into LB medium, and place it in a constant temperature shaker. Incubate at 30 o °C, shake culture at 150 rpm. The growth curve of microorganisms is divided into the adjustment period, logarithmic period, stationary period and decline period. Measure the cell concentration (OD 600 ) of DQ-4 growing at different time periods. As Figure 2 shown, DQ-4 enters the logarithmic period within 2 h after inoculation, and the number of microorganisms increases greatly in a constant geometric progression. The bacteria during this period have good adaptability. Inoculating the bacteria in the logarithmic period into the medium is beneficial to the growth of bacteria and can shorten the culture time of bacteria. After 24 h, the bacteria begin to enter the stationary period. During this interval, the bacteria produce a large amount of secondary metabolites. Bacterium DQ-4 has a relatively long stationary period in this medium, which is beneficial to the production of substances such as surfactants in secondary metabolites. Therefore, the seed solution cultured for 24 h is selected for subsequent experiments.

[0047] Example 3:

[0048] Strain identification: Inoculate the above strain into LB medium, incubate in a constant temperature shaker at 37 o °C for 24 hours, collect the bacterial cells, and extract the bacterial genomic DNA of strain DQ-4 using a genomic DNA extraction kit; perform a PCR amplification reaction using the 16S rDNA universal primer pair 27F / 1492R. The total volume of the PCR reaction system is 25 , and the PCR amplification conditions are: 96 o °C for 5 min; 96 o °C for 30 s, 56 o °C for 30 s, 72 oC 1 min, 35 cycles; 72 o C 5 min; The size of the PCR product was identified by 1% agarose gel electrophoresis. Based on the principle that magnetic beads can adsorb or release charged substances, DNA was adsorbed in a high-salt and low-pH solution and released in a low-salt and high-pH solution to obtain purified PCR amplification fragments. The sequence information was obtained by sequencing. The obtained sequence information was compared and analyzed with NCBI-BLAST. Among them, 16S rDNA is shown as SEQ ID NO.1 ( Figure 3 ), and the strain DQ-4 was identified as Bacillus.

[0049] The activation times of the strain DQ-4 were between 1 and 5 times, the inoculation amount was 5%, the initial pH value was between 4 and 10, and the temperature was between 10 and 55 o °C, and the culture time was 24 - 168 h.

[0050] Example 4:

[0051] Purification and preservation: The strain DQ-4 screened in Example 1 above was inoculated into LB medium and placed in a constant temperature shaker at 30 o °C, and cultured with shaking at 150 rpm for 24 h. The bacterial liquid was taken and streaked on an LB plate, and placed in a constant temperature incubator at 30 o °C for 48 h. Single colonies were picked, which were the preserved strain Bacillus DQ-4 of this application Bacillus , and the cell morphology of the strain DQ-4 was: on the LB plate, the colonies were round, regular, relatively viscous on the surface, and dark yellow. As shown in Figure 4 , it was short rod-shaped, about 2 .

[0052] Example 5:

[0053] (1) Extraction of metabolites

[0054] The strain DQ-4 screened in Example 1 was inoculated into LB medium and placed in a constant temperature shaker at 30 o °C, and cultured with shaking at 150 rpm for 24 h as the seed liquid, and inoculated into the fermentation medium at 5% v / v for culture, and cultured at 15 o °C and 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. The filtrate was adjusted to pH 2 with 6 mol / L hydrochloric acid and refrigerated at 4 o °C for 24 h. Equal volume of ethyl acetate was added for extraction multiple times, and the organic phases were combined and rotary evaporated at 46 o °C to remove the organic solvent, and the crude product of the surfactant (DQ-Rha) was obtained, and the weight of the crude product was accurately weighed.

[0055] (2)Purification of metabolites

[0056] The above-mentioned crude product DQ-Rha was ultrasonically dissolved in anhydrous methanol, centrifuged at 12,000 rpm and 4 o °C for 10 min in a refrigerated centrifuge, and the supernatant was rotary evaporated at 46 o °C to obtain the pure product, and the weight of the pure product DQ-Rha was accurately weighed.

[0057] Example 6:

[0058] Optimization of strain conditions: The strain DQ-4 was inoculated into LB medium and placed in a constant temperature shaker at 30 o °C and shaken at 150 rpm for 24 h as the seed solution. The carbon source, initial pH value, temperature, culture time and other condition factors of the strain DQ-4 were changed during the culture process, and the growth and metabolism of the strain DQ-4 were analyzed, such as OD 600 , surface tension, emulsification index and the yield of DQ-Rha.

[0059] The growth and metabolism results of the strain DQ-4 under different conditions are as Figure 5 shown. The results show that: after condition optimization, the best carbon source is glucose, the best culture temperature is 15 o °C, the best initial pH value is 7, and the best culture time is 72 h. Finally, the surface tension after optimization of the conditions is 34.7 mN / m, OD 600 is 1.724, the emulsification index is 72.3%, and the yield of DQ-Rha is 14.263 g / L.

[0060] Example 7:

[0061] Qualitative analysis of a metabolite of Bacillus:

[0062] (1)Thin layer chromatography (TLC) analysis

[0063] A small amount of the pure product DQ-Rha obtained in Example 5 above was dissolved in methanol. A horizontal line was drawn with a pencil 0.5 cm from the bottom edge of the silica gel plate. The sample was dipped with a capillary tube and spotted on the drawn line. Each time the sample was spotted after the previously spotted sample had evaporated to dryness, and the diameter of the spotted sample was controlled within 2 mm. After the sample was completely dried, the thin layer plate was placed in a developing tank for development. The ratio of the developing agent was V 氯仿 :V 甲醇 :V 水 =65:25:4. The lid was sealed with petroleum jelly, and it was appropriate that the developing agent did not cover the spotted area. The development was stopped until the developing agent rose to 1 cm from the upper edge of the silica gel plate.

[0064] Colorimetric test was carried out with phenol-sulfuric acid color reagent. Phenol-sulfuric acid color reagent: 3.0 g of phenol and 5 mL of sulfuric acid solution were dissolved in 95 mL of ethanol. After spraying the color reagent, it needed to be placed in an oven at 110 o °C for 10 min. Carbohydrates showed brown spots, while lipopeptides did not show color. As Figure 6 shown, after spraying the phenol-sulfuric acid color reagent, brown spots appeared, and compared with the purchased rhamnolipid finished product, it showed that the Rf value was consistent with the color development degree. Therefore, through preliminary determination, the metabolite DQ-Rha was a rhamnolipid biosurfactant.

[0065] (2) Fourier transform infrared spectroscopy (FTIR) analysis

[0066] The pure product DQ-Rha obtained in Example 5 was measured by a Fourier transform infrared spectrometer using the KBr tablet method. According to the infrared absorption spectrum, the purified DQ-Rha was identified and analyzed. As Figure 7 shown, it could be seen that the characteristic absorption peaks of carbohydrates were at 3151, 3045, 2815, 1407 cm -1 ⁻¹, the stretching vibration of -CH and the bending motion of -CH, and the characteristic peak at 1727 cm -1 ⁻¹ indicated the presence of a carbonyl group (-C=O in -COOH), and 1143 cm -1 ⁻¹ was the symmetric stretching vibration of C-O-C, indicating the presence of glycosidic bonds and cyclic lactone structures in the molecule, further determining that DQ-Rha was a rhamnolipid biosurfactant.

[0067] (3) Nuclear magnetic resonance hydrogen spectroscopy ( 1 ¹H-NMR) analysis

[0068] The pure product DQ-Rha obtained in Example 2 above was measured and analyzed using a 500 MHz Bruker Avance IIIHD NMR spectrometer. It was operated at 14.1 Tesla (500 MHz, 1 ¹H), equipped with a 5 mm inverse probe (QXI), and deuterated chloroform was used as the solvent for 1 ¹H analysis.

[0069] The molecular structure of rhamnolipid was confirmed by nuclear magnetic resonance spectroscopy. As Figure 8 shown, the hydrogen spectrum showed that multiple peaks would appear from 3.2 ppm to 5.5 ppm, respectively showing the structural characteristics of the sugar ring, representing the sugar ring part of rhamnose, and this observation confirmed that rhamnose was a single sugar ring. In addition, at 0.8 ppm to Peaks of the protons in the fatty acid chain, namely the methyl (CH3) and methylene (CH2) protons, can be observed within the range of 2.5 ppm, reflecting the type and length of the fatty acid chain. The methyl peak ( 0.8 ppm) comes from the methyl (CH3) at the end of the fatty acid chain and has a relatively high intensity. Meanwhile, the predicted structure of DQ-Rha was drawn in Chemdraw software to predict the chemical shifts of hydrogen elements in DQ-Rha, and it was compared with the 1H NMR spectrum obtained from the experiment. By Figure 9 It can be seen that the peak positions of hydrogen elements in the experimental results are basically consistent with those predicted by the software. It was determined again that this DQ-Rha is a monosaccharide monoester biosurfactant.

[0070] (4) Analysis by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS)

[0071] The matrix-assisted laser desorption / ionization time-of-flight mass spectrometry was used to determine and analyze the pure DQ-Rha obtained in Example 5 above on a Brukerautboflex speed analyzer with a laser intensity of 3200 - 5500. It was mixed with an equal volume of matrix solution, and the matrix solution was α-cyano-4-hydroxycinnamic acid (CHCA) containing 0.1% trifluoroacetic acid (TFA) in 70% aqueous acetonitrile solution, and the mass within the range of 200 - 4000 was measured. The purified DQ-Rha was identified and structurally analyzed based on the time-of-flight mass spectrum. As

[0072] shown, the mass-to-charge ratio (m / z) 651 is the molecular cluster ion after the rhamnolipid molecule binds to a sodium ion, representing the overall molecule of monorhamnolipid, which is the molecular cluster ion formed by the binding of a rhamnose molecule (molecular weight of 146 Da) and a relatively long fatty acid chain (such as C10 or C12, etc.). The fragment ion at the mass-to-charge ratio (m / z) 254 may be a fragment ion cleaved from a fatty acid chain part of the rhamnolipid, that is, DQ-Rha is a monorhamnolipid biosurfactant. Figure 10 shown, the mass-to-charge ratio (m / z) 651 is the molecular cluster ion after the rhamnolipid molecule binds to a sodium ion, representing the overall molecule of monorhamnolipid, which is the molecular cluster ion formed by the binding of a rhamnose molecule (molecular weight of 146 Da) and a relatively long fatty acid chain (such as C10 or C12, etc.). The fragment ion at the mass-to-charge ratio (m / z) 254 may be a fragment ion cleaved from a fatty acid chain part of the rhamnolipid, that is, DQ-Rha is a monorhamnolipid biosurfactant.

[0073] Example 8:

[0074] Acute oral toxicity test of a Bacillus metabolite:

[0075] The acute oral toxicity test was conducted in accordance with GB / T 21804-2008 Chemicals - Fixed-dose procedure for acute oral toxicity testing. Before the test, the animals were acclimated in the barrier environment animal house for 6 days. The animals were fasted for 4 hours before dosing. After the test started, the animals were dosed once by oral gavage. First, one animal was taken for a pre-test dose (the dosing dose was 5040.6 mg / kg). The experimental animal survived within 96 hours after dosing; that is, the formal test dose was determined to be 5000 mg / kg. Then, another 4 animals were taken for dosing (the dosing dose was 5070.3 mg / kg), and the test was terminated (i.e., a 14-day observation was carried out, and no other animals were taken for dosing). The animals were fasted for 1 hour after each dosing. The poisoning and death conditions of the animals during the dosing process and the observation period were observed and recorded. The animals were weighed once a week, and the observation period was 14 days. After the observation period ended, the surviving animals were sacrificed and subjected to gross dissection.

[0076] 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 dosing; no poisoning symptoms or poisoning deaths were observed in the experimental animals in the 5070.3 mg / kg dose group within 14 days after dosing. The body weights of all surviving animals were normal (see Table 1). After the experimental observation ended, gross dissection examination of the test animals showed no abnormal changes.

[0077] Table 1 Results of acute oral toxicity test

[0078]

[0079] Under the conditions of this test, no poisoning symptoms or poisoning deaths were observed in the experimental animals within 14 days after dosing. The acute oral LD 50 > 5000 mg / kg of the test sample for ICR mice. The results showed that the metabolite of this Bacillus was non-toxic and harmless, providing a green and sustainable additive for the unique environmental conditions of offshore oilfields and having broad application prospects.

[0080] Example 9:

[0081] Testing the oil-washing performance of a metabolite of Bacillus:

[0082] Crude oil was mixed with quartz sand of different mesh ratios and mixed at a mass ratio of 1:5. It was aged for 48 h in an environment of 65 o °C and then filled into an oil-washing bottle. At the same time, the density of the crude oil ( ) was measured. The aged oil sand (dry weight about 15.0 g) was filled into the oil-washing bottle, and 30 mL of a 1% DQ-Rha solution was added. Under the conditions of 90 rpm and 65 o °C, after shaking for 2 h, the oil-washing bottle was taken out, and the corresponding concentration of DQ-Rha solution was added to the top scale of the oil-washing bottle. After shaking well, it was placed back at 65o Continue the cultivation in an incubator at C for 2 h, and read the volume of the washed-out oil. At the same time, set up a control group. In the control group (distilled water), record the mass of the aged oil sand and the volume of the washed-out oil. Compared with the control group, the oil washing efficiency of the experimental group increased by 54.6%. The results show that a metabolite DQ-Rha of a Bacillus strain has oil washing performance at a low dose and has application prospects in improving oil recovery.

[0083] Example 10:

[0084] Core physical simulation oil displacement experiment: An oil displacement experiment of the strain of the present invention on a simulated marine oilfield environment.

[0085] The core physical simulation oil displacement experiment simulates the "micro-oil reservoir" environment of a marine oilfield through an artificial core, and simulates and studies the potential of DQ-Rha to improve crude oil recovery in a marine oil reservoir. An oil displacement potential evaluation experiment was carried out using an artificial sand-packed core. Three cores were selected and numbered #1, #2, and #3. Core #1 was used to evaluate the oil displacement potential of the metabolite DQ-Rha of strain DQ-4; Core #2 was used to evaluate the oil displacement potential of the purchased commercial rhamnolipid (Rha); Core #3 was used as a blank control (injecting reservoir formation water instead of fermentation broth). The basic parameters of the cores are shown in Table 2, and the oil recovery data of the three cores are shown in Table 3.

[0086] Table 2 Basic parameters of the core model in the physical simulation experiment

[0087]

[0088] Table 3 Oil recovery data in the physical simulation experiment

[0089]

[0090] As shown in Table 3, the results of the oil displacement experiments on Cores #1 and #2 show that the DQ-Rha biological agent can increase the crude oil recovery by 10.77% on the basis of primary water flooding, and the purchased commercial rhamnolipid (Rha) can increase the crude oil recovery by 11.35% on the basis of primary water flooding, both showing good oil displacement potential; Core #3 as a blank control was directly subjected to secondary water flooding after primary water flooding, and at this time, the crude oil recovery only increased by 1.1%; the results show the feasibility of using rhamnolipid for oil displacement in a marine oil reservoir environment.

[0091] The above experiments illustrate that the strain DQ-4 provided by the present invention is a Bacillus strain producing rhamnolipid and can improve oil recovery for marine oilfields.

[0092] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A Bacillus ( Bacillus sp. ), characterized in that This strain is deposited in the China General Microbiological Culture Collection Center (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, Postcode: 100101; it is classified and named as Bacillus Bacillus sp. , and the strain number is: DQ-4.

2. Use of the Bacillus DQ-4 as claimed in claim 1 in the preparation of rhamnolipid.

3. Use of the Bacillus DQ-4 as claimed in claim 1 in offshore oil displacement.

4. A bacterial agent, characterized in that, The bacterial agent comprises the Bacillus DQ-4 as claimed in claim 1.

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