NL2028866A

NL2028866A - Brevibacillus borstelensis, preparation and use thereof, and preparation method of biosurfactant

Info

Publication number: NL2028866A
Application number: NL2028866A
Authority: NL
Inventors: Dong Hao; Feng Yangyang; Sun Shanshan; Yao Puyong; Zheng Anying; Hu Linqi; Yu Gaoming; Zhang Zhi; Li Yang; Yi Shaojin; Li Fei; She Yuehui; Zhang Fan; Zhang Wenda
Original assignee: Univ Yangtze
Priority date: 2020-08-13
Filing date: 2021-07-28
Publication date: 2022-04-04
Also published as: CN112195115A; CN112195115B; NL2028866B1

Abstract

The present disclosure relates to the technical field of use of a bioengineering technology in microbial oil recovery and discloses brevibacillus borslelensis, a preparation and use thereof, and a preparation method of a surfactant. The brevibacillus borslelensis is deposited in China General Microbiological Culture Collection Center and has an accession number of CGMCC No. 9981. The brevibacillus borslelensis and the preparation thereof may effectively increase an oil recovery rate. The lipopeptide biosurfactant prepared by the preparation method of a 10 surfactant has a good physical property, effectively reduces surface tension and has a good emulsifying property on petroleum, hydrocarbons and lipids.

Description

BREVIBACILLUS BORSTELENSIS, PREPARATION AND USE THEREOF, AND PREPARATION METHOD OF BIOSURFACTANT TECHNICAL FIELD

[01] The present disclosure relates to the technical field of use of a bioengineering technology in microbial oil recovery and specifically relates to a brevibacillus borstelensis, a preparation and use thereof, and a preparation method of a biosurfactant.

BACKGROUND ART

[02] When an oil field is in a middle and late period of development, it becomes more difficult to increase oil production. People are continuously searching for economical, effective and environmentally-friendly new technologies to improve oil recovery rate. Microbial oil recovery receives more concerns due to the advantages of a wide application range, a simple process, a low cost and environmental friendliness. The microbial oil recovery generally refers to injecting appropriate bacterial strains, nutrient agents or fermentation broth into an oil reservoir to enable the strains to regenerate and metabolize in the oil reservoir. In this way, oil or active substances are generated to reduce oil-water interfacial tension to improve oil recovery rate.

[03] Inthe past several decades, there have been many patents reporting that a variety of species of microorganisms use different carbon sources to metabolize and produce surfactants and their applications in the petroleum industry. There are many microorganisms capable of producing biosurfactants and the most studied microorganisms are mainly pseudomonas, bacillus, acinetobacter, rhodococcus, candida, etc. Biosurfactants generally falls into two categories: bioemulsifiers with a relatively large molecular weight and micromolecular biosurfactants. The bioemulsifiers with a relatively large molecular weight are generally amphipathic polysaccharides and proteins, and the most studied bioemulsifiers are emulsan and alasan generated by acinetobacter. The micromolecular biosurfactants primarily include glycolipids mainly generated by pseudomonas, rhodococcus, candida and burkholderia and lipopeptides mainly generated by bacillus. The oil reservoir contains hydrocarbon-degrading and surfactant-producing microorganisms. It is generally considered that a single microorganism isolated from the oil reservoir can better adapt to an extreme environment. These microorganisms are a hotspot for microbial oil recovery. Screening and isolating biosurfactant-producing microorganisms from an oil reservoir, or adjusting the ratio of biosurfactant-producing microorganisms in an oil layer by injecting and culturing bacteria and nutrients is one of the main research contents in microbial oil recovery.

[04] In a process of microbial oil recovery, different oil reservoirs also have certain requirements on strains. It is reported that currently there are many biosurfactant- producing microorganisms, so are those isolated from the oil reservoir. However, there are rare reports in literatures and patents on biosurfactant-producing brevibacillus borstelensis in the oil reservoir. In the prior art, there are some patents related to brevibacillus horstelensis. Uses of the brevibacillus borstelensis in brewing, pharmaceutical and food industry, animal husbandry, organic pollutant degradation, kitchen waste treatment and oil exploitation are respectively reported. Yet, there is no report on production of biosurfactants by brevibacillus borstelensis metabolism.

[05] Based on the technological blank existing in the prior art, the present disclosure provides brevibacillus borstelensis, a preparation and use thereof, and a preparation method of a biosurfactant.

SUMMARY

[06] The present disclosure provides hrevibacillus borstelensis, a preparation and use thereof, and a preparation method of a biosurfactant.

[07] The present disclosure adopts the following technical solutions.

[08] A brevibacillus borstelensis is named YZ-2 and has an accession number of CGMCC No. 9981.

[09] In one aspect, the present disclosure provides a brevibacillus borstelensis preparation, the brevibacillus borstelensis preparation contains brevibacillus borstelensis having an accession number of CGMCC No. 9981 and the brevibacillus borstelensis preparation may be solid or liquid.

[10] In another aspect, the present disclosure provides a preparation method of a lipopeptide biosurfactant and the method includes fermenting and cultivating brevibacillus borstelensis or a brevibacillus borstelensis preparation in a nutrient medium to generate a lipopeptide biosurfactant.

[11] Furthermore, the nutrient medium may be an LB medium.

[12] Furthermore, the nutrient medium may include 10-50 g/L of sucrose, 10-40 g/L of dried corn steep liquor powder, 3-20 g/L of peptone, 0.1-2 g/L of MgSO, 2-18 g/L of KCI, 5-9 g/L of MnSO,4, 5-10 g/L of CuSOy4, 5-12 g/L of ZnSO: and 1-7 g/L of KH>POy4 and the lipopeptide biosurfactant may be generated at a pH of 5-9.5 and a fermentation temperature of 20-60°C.

[13] Furthermore, the method may include a step of removing bacteria after fermentation.

[14] Furthermore, fermenting and cultivating the brevibacillus borstelensis in the nutrient medium may include:

[15] step 1.1: streaking a strain deposited in a slant medium on a plating medium for activating by using an inoculating loop, cultivating the strain at a constant temperature of 30°C for 20 h, picking three loops of the strain from the plating medium, and inoculating the strain in a first-stage seed shake flask for culture at 30°C and 180 r/min for 18 h;

[16] step 1.2: inoculating 2% of volume of first-stage seeds in the first-stage seed shake flask into a second-stage seed shake flask for a second-stage seed culture for 18 h under the same condition as step 1.1; and

[17] step 1.3: inoculating 2% of volume of second-stage seeds in the second-stage seed shake flask into a fermentation shake flask until a sporation rate reaching 100% under the same condition as step 1.1.

[18] In still another aspect, the present disclosure provides use of a lipopeptide biosurfactant in oil displacement.

[19] In yet another aspect, the present disclosure further provides a preparation method of a lipopeptide biosurfactant and brevibacillius borstelensis metabolizes to generate the lipopeptide biosurfactant.

[20] In still yet another aspect, the present disclosure provides use of brevibacillus borstelensis in improving oil recovery rate.

[21] In still yet another aspect, the present disclosure provides a brevibacillus borstelensis preparation in improving oil recovery rate.

[22] Compared with the prior art, the embodiments may have the following advantages:

[23] 1. The lipopeptide biosurfactant prepared by the preparation method of the present disclosure has a good physical property, effectively reduced surface tension and good emulsifying property on petroleum, hydrocarbons and lipids.

[24] 2. The brevibacillus borstelensis preparation may effectively improve oil recovery rate.

[25] Description of deposit information

[26] Deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China;

[27] Date of deposit: November 18, 2014;

[28] Strain name: Brevibacillus borstelensis,

[29] Latin name: Brevibacillus borstelensis,

[30] Deposit institute: China General Microbiological Culture Collection Center;

[31] Abbreviation of deposit institute: CGMCC;

[32] Accession number: CGMCC No. 9981.

BRIEF DESCRIPTION OF DRAWINGS

[33] FIG. 1is a schematic diagram of an oil spreading circle of fermentation broth of the strain YZ-2 in example 1 of the present disclosure;

[34] FIG. 21s a phylogenetic tree of 16S rRNA-based surfactant-producing strain YZ-2 in example 1 of the present disclosure;

[35] FIG. 3 is an infrared spectrogram of crude metabolites of the strain YZ-2 in example 2 of the present disclosure;

[36] FIG. 4-1 is a total ion chromatogram (TIC) of a local 1 of metabolites of a strain YZ-2 in example 2 of the present disclosure;

[37] FIG. 4-2 is a total ion chromatogram (TIC) of a local 2 of a metabolite of the strain YZ-2 in example 2 of the present disclosure;

[38] FIG. 5-1 is a schematic diagram of mass spectrometry of a local 1 of metabolites of the strain YZ-2 in example 2 of the present disclosure;

[39] FIG. 5-2 is a schematic diagram of mass spectrometry of a local 2 of metabolites of the strain YZ-2 in example 2 of the present disclosure;

[40] FIG. 6 is a flowchart of the oil-displacing simulation test in example 4 of the present disclosure; and

[41] FIG. 7 is a schematic diagram of an effect of the YZ-2 surfactant on improving oil recovery rate in example 4 of the present disclosure.

DETAILED DESCRIPTION OF THE EMBODIMENTS 5 [42] To better explain technical effects of brevibacillus borstelensis (YZ-2) and features and use thereof, the present disclosure will be further explained accompanying with specific examples. The following examples are not intended to limit the present disclosure, but merely to illustrate the present disclosure. The experiment methods used in the following examples are conventional, unless otherwise specified; the experimental methods in the examples without specified conditions are generally conducted under conventional conditions; and the used materials, agents, etc. in the examples are generally commercially available, unless otherwise specified.

[43] The present disclosure provides a brevibacillus horstelensis strain isolated from an oil reservoir environment. Research data showed that the strain had a good oil recovery characteristic. A physical oil-displacing simulation experiment showed that the brevibacillus borstelensis biological preparation may greatly improve the oil recovery rate in the rock core. The present disclosure provides a preparation method of a lipopeptide biosurfactant by using fermentation of brevibacillus horstelensis, a technology for extracting metabolites of the brevibacilius borstelensis, and a brevibacillus horstelensis preparation and use thereof in improving oil recovery rate.

[44] Example 1 Separation, identification and deposit of a surfactant-producing strain

[45] 1. Separation of a surfactant-producing strain

[46] According to a conventional strain screening method, 10 mL of a water sample collected from an oil field was inoculated to a medium containing 100 mL of sterilized crude oil (2% crude oil, v:v). Constant temperature shaking culture was conducted at 35°C and 150 rpm for 72 h. An experimental group with a high degree of emulsification and dispersion of crude oil was selected. 100 pL of fermentation broth was spread on an LB agar plate medium and culture was conducted at 35°C for 48 h.

Single colonies of different morphologies were picked and streaked and purified on an LB agar plate medium and culture was conducted at 35°C for 48 h. Single colonies were picked and inoculated into an original inorganic salt medium with crude oil as a carbon source. Culture was conducted at 35°C and 150 rpm for 72 h. A degree of emulsification and dispersion of crude oil was observed and a surface tension of the fermentation broth was measured, and a strain with the largest reduction in surface tension was selected.

[47] In this example, it was found that there were microorganisms in the sample that could emulsify and disperse crude oil and reduce the surface tension of a culture solution through a preliminary screening. After the above-mentioned culture and domestication, a biosurfactant-producing strain was isolated and named YZ-2. As shown in FIG. 1, the diameter of an oil-displacing circle of the YZ-2 was 7.21 cm and the surface tension of the fermentation broth was reduced to 30.103 mN/m. As shown in Table 1, YZ-2 metabolites have an excellent surface activity and could be used in microbial enhanced oil recovery rate.

[48] Table 1 Determination of surfactant-producing microorganism

[49] Strain | Optimum Surface Tension Diameter of Oil-Displacing ie Jo

[50] 2. Identification and deposit of strain [SI] According to the "Manual for Systematic Identification of Common Bacteria", the strain YZ-2 isolated in the present disclosure was identified to the genus mainly through individual morphological characteristics, colony characteristics, staining reaction, physiological and biochemical reaction, etc. of the strain and by a morphological observation, a gram Dyeing, a hydrogen peroxide contact enzyme reaction, an oxidase experiment, a glucose oxidation fermentation experiment and a methyl red experiment. The identification result was shown in Table 2.

[52] Table 2 Identification of physiological-biochemical characteristics of strain YZ-2

[53] Characteristics | YZ-2 Characteristics | YZ-2 Strain a see [OW | [weed ei oer ov [wen Nitrate + D-xylose hes | Fe Starch + Growth Optimum: 30°C; lowest: 15°C, ee ee

[54] In addition, according to a conventional bacterial identification method, a genomic DNA of the strain YZ-2 was separately extracted. Primers were designed for a PCR amplification, amplified products were detected by an agarose gel electrophoresis, and an ideal PCR product was selected and sent to Sangon Biotech for DNA sequencing. A sequencing result of the PCR amplified product of YZ-2 was submitted to NCBI and subjected to search and a homology detection by using BLAST. Clustalx1.83, Mega and other software were used to construct a phylogenetic tree. As shown in FIG. 2, the YZ-2 strain was determined to be brevibacillus borstelensis (having a similarity of 99% to a brevibacillus borstelensis strain 1CK49) IO by a 16S rDNA sequence analysis (the corresponding representative strain in Genbank was shown in Table 3 below). [S5] Table 3 Alignment result of 16S rRNA sequence of YZ-2 with sequence in Genbank database

[56] Strain | Base Species/genus with most similar sequence in Homology lo

[57] The YZ-2 strain of the present disclosure could be preserved by the following methods:

[58] (1) Short-term preservation: the above-mentioned strain was streaked on a slant medium, cultured at 35°C for 48 h, and preserved at 4°C.

[59] (2) Long-term preservation: a glycerin cryopreservation method: a few rings of the strain were scraped from the fresh slant medium and the strain was transferred to a glycerol tube containing 1.5 mL of 30% sterilized glycerin and cryopreserved at -80°C. Or a skim milk cryopreservation method: 2 rings of the strain were scraped from the fresh slant medium again and the strain was transferred to a glycerol tube containing sterilized skim milk and cryopreserved at -80°C.

[60] The screened brevibacillus borstelensis YZ-2 in the example was deposited in the China General Microbiological Culture Collection Center at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China on November 18, 2014 and had an access number of CGMCC No. 9981.

[61] Example 2 Extraction and analysis of YZ-2 metabolites

[62] 1. Fermentation of YZ-2

[63] The brevibacillus borstelensis YZ-2 provided in the example was cultured through a fermentation medium. The fermentation medium included 10-50 g/L of sucrose, 10-40 g/L of dried corn steep liquor powder, 3-20 g/L of peptone, 1-5 g/L of MgSO,4, 2-18 g/L of KCl, 5-9 g/L of MnSO,, 5-10 g/L of CuSOy4, 5-12 g/L of ZnSO4 and 1-7 g/L of KH;PO,, had a pH of 6-8 and was sterilized at 115°C for 30 min.

[64] A strain deposited in a slant medium was streaked on a plating medium for activating, the strain was cultivated at a constant temperature of 30°C for 20 h. Three loops of the strain (each inoculating loop contains more than 3 single colonies with obvious characteristics) from the plating medium was inoculated into a first-stage seed shake flask (a 250-mL triangular flask with a liquid-loading volume of 50 mL) for culture at 30°C and 180 r/min for 18 h. 2% of volume of first-stage seeds in the first- stage seed shake flask was inoculated into a second-stage seed shake flask (a 250-mL conical flask with a liquid-loading volume of 100 mL) for culture for 18 h under the same condition. 2% of volume of second-stage seeds in the second-stage seed shake flask were inoculated into a fermentation shake flask (a 500-mL conical flask with a liquid-loading volume of 150 mL) until a sporation rate reaching 100% under the same condition.

[65] 2. Extraction and identification of lipopeptide generated by YZ-2

[66] 10 L of fermentation broth was centrifuged at room temperature and 8,000 r/min for 10 min. A precipitate was removed. pH was adjusted to 2.0 with 6 mol/mL of hydrochloric acid. An obtained acidified solution was stored at 4°C for 24 h. The acidified solution was centrifuged at room temperature and 8,000 r/min for 10 min. The supernatant was removed. The precipitate was collected and extracted for 3 times or more with an appropriate amount of methanol. The methanol extract was centrifuged under the same condition. The supernatant was taken and subjected to a concentration by a rotary evaporation (at 45°C). The concentrate was dissolved with an appropriate amount of distilled water and pH was adjusted to be 7.0. A suction filtration was conducted. The supernatant was taken and pH was adjusted to 2.0 again. The supernatant was put still at 4°C overnight and centrifuged. The precipitate was taken, collected and extracted for 3 times with a same volume of the fermentation broth. The extract was centrifuged and the supernatant was taken and subjected to vacuum concentration by using a rotary evaporator. The concentrate was placed in a constant temperature electric oven at 60°C until it reached a constant weight and the IO weight was measured. The obtained product was yellow-brown solid powder. The obtained yellow-brown solid powder and potassium bromide were ground in a mortar, and tablets were prepared. The tablets were subjected to an infrared spectroscopy analysis as shown in FIG. 3. The result showed that there were a typical cyclic lactam bonds in lipopeptides and nitrogen and hydrogen bonds of amino acids, it was inferred that the strain YZ-2 metabolized to generate a lipopeptide biosurfactant.

[67] 3. Mass spectrometry of YZ-2 metabolites

[68] Chromatographic conditions: mobile phase: 0.1% acetic acid aqueous solution + acetonitrile; flow rate: 0.2 ml/min; column temperature: 35°C; injection volume: 5 uL; and chromatographic column: WatersAcquity UPLC C18 1.7 um 2.1x100 mm.

[69] Conditions of mass spectrometry: ion source: ESI (+); capillary voltage: 3.0 KV; cone hole voltage: 40 V; de-solvent temperature: 300°C; ion source: 150°C; atomizing gas: 650 L/h; and acquisition mode: Msl Scan.

[70] A liquid chromatographic separation was shown in FIG. 4-1 and FIG. 4-2. An analysis of the metabolites of the strain YZ-2 showed that there were 8 time periods for producing peaks (respectively 1.101, 6.173, 6.814, 7.249, 7.602, 7.936 and 8.354 min). As the analysis shown in FIG. 5-2, there were 8 fragment peaks with a molecular weight of 965.96-1078.48 and main peaks with a molecular weight of 993.95 and

994.99, indicating that there were surfactin and iturin with multiple homologues including short-chain lipopeptides. In addition, there were four small peaks of

1463.99-1702.25, indicating a fengycin lipopeptide existed.

[71] The analysis of the metabolites of the strain YZ-2 indicated that there were 8 time periods for producing peaks (respectively 1.101, 6.173, 6.814, 7.249, 7.602, 7.936 and 8.354 min). As an analysis shown in FIG. 5-1, there were 9 fragment peaks with a molecular weight of 979.92-1052.22 and main peaks with a molecular weight of

1008.06, 1022.06, 1036.15 and 1037.14, indicating that there were surfactin and iturin with multiple homologues.

[72] Example 3 Optimization of fermentation system of lipopeptide generated by YZ-2

[73] Seed bacteria: seed bacteria was inoculated on an enrichment medium which included basic compositions of 5 g/L of yeasts, 10 g/L. of NaCl and 10 g/L of peptone. The bacteria were cultivated to a concentration of 108-10 cells/mL and used as seed liquid for experiment study of the present example.

[74] 1. Optimization of carbon source

[75] Preparation of medium: 1,000 mL of distilled water was measured, 10-40 g/L of dried corn steep liquor powder, 1-5 g/L. of MgSO,4, 2-18 g/L of KCI, 5-9 g/L of MnSO,4, 5-10 g/L of CuSO,4, 5-12 g/L of ZnSO: and 1-7 g/L of KH2PO4 and pH was adjusted to 6-8. The medium was subpackaged into 100 mL each in eight 250-mL conical flasks and 2% of the following eight carbon sources were added respectively: sucrose (No.1), glucose (No.2), dried corn steep liquor powder (No.3), starch (No.4), olives oil (No.5), lactose (No.6), glycerin (No.7) and liquid paraffin (No.8). After sterilization, 5% of the seed liquid was inoculated, shaking culture was conducted below 45°C and at 150 rpm for 72 h. Fermentation broth was diluted to different gradients and a surface tension of the fermentation broth (unit: mN/m) was measured. The result was shown in Table 4:

[76] Table 4 Influence of different carbon sources on lipopeptide production by fermentation of strain YZ-2

[77] Carbon Not Diluted for 5 | Diluted for Diluted for Diluted for

EE EE EC KE Ez es ew Ee EE

[78] Measured surface tension data was observed. The surface tension was 38.2 mN/m and the dry weight of the bacteria was 1.39 g/L after the medium containing bacteria added with the No.1 carbon source was diluted 20 times and the surface tension was 37.1 mN/m and a dry weight of the bacteria was 0.89 g/L after the medium containing bacteria added with the No.3 carbon source was diluted 20 times. Considering the dry weight of the bacteria and reduction of the surface tension of the fermentation broth, the sucrose was selected as the best carbon source for fermentation. The addition amount of the sucrose was optimized. The product was collected by an acid precipitation method, dried and weighed. At the same time, the bacteria were collected by ultracentrifugation and dried to a dry weight. It was found that when the addition amount of the sucrose was 1-4 g/L, the yield of a surfactant reached the highest value, such that 1%-4% of the sucrose was selected as the best carbon source for lipopeptide production by the strain YZ-2.

[79] 2. Optimization of nitrogen source

[80] Preparation of medium: 1,000 mL of distilled water was measured and 10-40 g/L of sucrose, 1-5 g/L of MgSO, 2-18 g/L of KCI, 5-9 g/L of MnSOy, 5-10 g/L of CuSO,, 5-12 g/L of ZnSO, and 1-7 g/L of KH:PO:. After the materials were evenly stirred, an obtained mixture was subpackaged into 100 mL each in 6 250-mL conical flasks. According to the following table, 0.5% of 6 different nitrogen sources were prepared and added respectively to the medium: ammonium chloride (No.1), ammonium nitrate (No.2), peptone (No.3), dried corn steep liquor powder (No.4), sodium glutamate (No.5) and sodium nitrate (No.6). After sterilization, 5% of the seed liquid was inoculated, shaking culture was conducted below 45°C and at 150 rpm for 72 h. Fermentation broth was diluted to different gradients and the surface tension of the fermentation broth was measured. The result was shown in Table 5:

[81] Table 5 Influence of different nitrogen sources on lipopeptide production by fermentation of strain YZ-2

[82] Nitrogen Fermentation Diluted for 5 Diluted for 10 | Diluted for 20 pw NE Tee co fwe [|

[83] The study result indicated that when inorganic nitrogen sources NaNOs, ammonium chloride (No.1) and ammonium nitrate (No.2) were added, the surface tensions of the fermentation broth were not significantly reduced after three days of culture, while when organic nitrogen sources were used for fermentation, the surface tensions of the fermentation broth were all reduced to about 30 mN/m.

[84] Example 4 Oil-displacing simulation test

[85] 1. Preparation of oil-displacing bacteria solution

[86] 150 ml of a fermentation medium for surfactant-producing bacteria and 3 L of an inorganic salt medium were prepared. The prepared medium was subjected to a high-pressure steam sterilization at 121°C for 20 min. The YZ-2 strain was activated and transferred to the fermentation medium, shaking culture was conducted at 40°C and 150 r/min. After 24 h, fermentation broth was added to 3 L of an oil-displacing medium at an addition amount of 10%, shaking culture was conducted at 40°C and 150 r/min. Two days later, the cultivated strain was taken out and put into a refrigerator at 4°C for later use.

[87] 2. Physical oil-displacing simulation test of cores

[88] As shown in FIG. 6, an oil-displacing process was connected (in FIG. 6, 1- distilled water container, 2-advection pump, 3-six-way valve, 4-intermediate container, 5-pressure gauge, 6-core holder, 7-core, 8-confining pressure pump and 9-measuring cylinder) and consisted of a micro-value speed pump (a gas cylinder for constant- pressure displacement of a pressure source), a high-pressure container, a core model and an oil-water separation metering pipe. The above four parts were connected with pipelines and valves. A pressure value was displayed by the pressure gauge arranged as required. The parts that need to maintain an experimental temperature were put into aincubator.

[89] Two artificial cores, D21 and D22, with a gas permeability of about 300 md were selected, 2 pore volume (PV) of the YZ-2 bacteria liquid was injected, a well was closed for 2 days, and oil-displacing effects were measured according to the following steps.

[90] (1) Core saturated water

[91] Dry weights of the artificial cores with the gas permeability of 300 md were measured. The weighed artificial cores were put into a jar and evacuation was conducted with a vacuum pump for three hours. Simulated formation water was added to enable the cores to be completely soaked in the water. The vacuum pump was turned on until a vacuum gauge read zero. The soaked cores were taken out and wet weights of the soaked cores were measured to obtain the core pore volume.

[92] (2) Saturated oil

[93] The cores were put into the core holder and put in a 40°C incubator. Dehydrated crude oil was added to a steel container, pipelines were connected, and the constant-flux pump was started to press the crude oil into the cores. When 3-4 mL of the crude oil flowed out from an outlet end of the core holder, an oil saturation displacement ended. A crude oil saturation was obtained according to a volume of displaced water.

[94] (3) Water displacement

[95] The simulated formation water was put into the steel container, pipelines for oil displacement were connected, the advection pump was started for oil displacement, crude oil was collected with a 5-mL test tube at the outlet end of the core holder, and count was conducted once about every other 10 min until the water content reached 95%.

[96] (4) Microbial displacement

[97] The oil-displacing bacteria liquid was put into a steel container, pipelines for oil displacement were connected, the amount of displaced crude oil was recorded, the advection pump was turned off after the amount reached a specified PV number, and the well was closed at 40°C for 2 days as planned.

[98] (5) Follow-up water displacement

[99] After the specified well-closing time, follow-up water displacement was conducted, the amount of the displaced crude oil was recorded, and the value of an improved oil recovery rate was calculated. The cores used for the biosurfactant- producing strain YZ-2 were D21 and D22. Determination results of physical properties of the two homogeneous artificial cores were shown in Table 6. After subjected to the oil saturation displacement, the two cores were subjected to the water replacement to the water content of 95% and the biosurfactant-producing bacteria solution was injected. The well was closed for 2 days. The follow-up water displacement was conducted. The recovery rate was calculated and the results were shown in Table 6.

[100] Table 6 Determination results of physical properties of homogeneous cores

[101] Diameter Length Pore Porosity/% | Permeability/10 | Ne TT 50 25 70.4 8.8 25.46 161.32 wT 90 25 69.3 8.64 25.40 158.33 a

[102] According to the determination results of physical properties of the D21 and D22 in Table 6, physical parameters of the two cores were almost the same. The gas permeability was about 300 md, the core pore volume was about 14%, and the oil saturation was about 83%. According to experiment data of the oil displacement by the YZ-2 in FIG. 7, for the two cores of D21 and D22, after 120 mg/L of a surfactant solution generated by the YZ-2 was used for displacement, the oil recovery rate was improved by 5.6% and 11.69% and the oil-displacing effect was obvious.

[103] The present disclosure is not limited by the aforementioned examples. The aforementioned examples and the description only illustrate the principle of the present disclosure. Various changes and modifications may be made to the present disclosure without departing from the spirit and scope of the present disclosure. Such changes and modifications all fall within the claimed scope of the present disclosure. The protection scope of the present disclosure is defined by the appended claims.

Claims

_15- Conclusions

1. Brevibacillus brushensis, with deposit number CGMCC No. 9981.

The Brevibacillus brushensis preparation, wherein the brevibacillus brushensis preparation contains brevibacillus brushensis having deposit number CGMCC No. 9981 according to claim 1, and the brevibacillus brushensis preparation is solid or liquid.

A method of preparing a lipopeptide biosurfactant, the method comprising fermenting and culturing the brevibacillus brushensis according to claim 1 or the brevibacillus horstelensis preparation according to claim 2 in a nutrient medium to generate a lipopeptide biosurfactant.

The preparation method according to claim 3, wherein the nutrient medium is an LB medium.

The preparation method according to claim 3, wherein the nutrient medium is 10 - 50 g/L sucrose, 10 - 40 g/L dried corn steep liquor powder, 3 - 20 g/L peptone, 0.1 - 2 g/L MgSO 4 , 2-18 g/L L KCl, 5 — 9 g/L MnSO4, 5 — 10 g/L CuSO,4, 5 — 12 g/L ZnSO, and 1 — 7 g/L KH;PO; and the lipopeptide biosurfactant is generated at a pH of 5-9.5 and a fermentation temperature of 20-60 °C.

The preparation method of claim 3, wherein the method further comprises a step of removing bacteria after fermentation.

Use of the lipopeptide biosurfactant according to any one of claims 3 to 6 in oil displacement.

A method of preparation of a lipopeptide biosurfactant comprising using the brevibacillus brushensis of claim 1 to metabolize and generate the lipopeptide biosurfactant.

Use of the brevibacillus brushensis according to claim 1 for the

- 16 - improving the oil recovery rate.

Use of the brevibacillus brushensis preparation according to claim 2 for improving the oil recovery rate.