Microbial agent for microbial oil recovery and preparation method thereof

Abstract

The invention relates to the technical field of oil recovery microbial agents, in particular to a microbial agent for microbial oil recovery and a preparation method, and the microbial agent comprises the following components: a compound bacteria solution consisting of a pseudomonas aeruginosa microbial agent, a bacillus licheniformis microbial agent and a methane bacteria microbial agent, and a carrier; the carrier comprises modified nano silicon dioxide; a first crosslinking agent; the surfactant is polyvinyl alcohol; sodium carboxymethyl cellulose; a fiveleaf gynostemma herb compound; the preparation method comprises the following steps: S1, preparing a compound bacteria solution; S2, preparing a gel solution; S3, preparing a carrier; and S4, preparing a microbial agent. Pseudomonas aeruginosa, bacillus licheniformis and methane bacteria are selected as microorganisms, the oil-water interfacial tension is reduced, the formation pressure is increased, and the oil displacement effect is improved; the components in the carrier are used for promoting propagation and protecting strains, so that the shelf life of the microbial agent is prolonged, and the microbial oil extraction efficiency is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial oil recovery, in particular to a microbial agent for microbial oil recovery and a preparation method thereof. BACKGROUND

[0002] Common microbial species in microbial oil recovery include Bacillus subtilis, Bacillus licheniformis and Pseudomonas aeruginosa. Microbial enhanced oil recovery is mainly due to the action of bacteria and metabolic products, which mainly include biosurfactants, biopolymers, biological enzymes, biological acids, solvents and biological gases.

[0003] The oil displacement mechanism of microorganisms mainly includes: 1. producing biosurfactants to reduce oil-water interfacial tension: the biosurfactants produced by microbial metabolism can reduce the oil-water interfacial tension, promote the emulsification of crude oil and separate from the rock surface, and improve the oil washing efficiency; 2. metabolizing to generate gas (such as CO2 and CH4) to increase the pressure of the oil layer: microorganisms produce gas under anaerobic conditions, increase the formation pressure and promote the flow of crude oil, and the gas dissolved in the crude oil can reduce its viscosity; 3. secreting organic acids to dissolve rock pores to improve permeability: organic acids (such as formic acid and propionic acid) produced by microorganisms can dissolve carbonate minerals, expand pore throats and improve reservoir permeability; 4. biodegradation of crude oil to reduce viscosity: microorganisms can degrade long-chain hydrocarbons and heavy components in crude oil through enzymatic action, reduce viscosity and freezing point, and enhance the flowability of crude oil; 5. microbial cells and metabolic products block high permeability layers: the bacterial cells and biological polymers produced by them can selectively block high permeability channels, adjust the swept volume of injected water and improve oil displacement efficiency; 6. producing organic solvents to improve the physical properties of crude oil: organic solvents such as alcohols and ketones produced by microbial metabolism can change the composition of crude oil and the wettability of rock surface, reduce adsorption resistance and improve the flowability of crude oil in the pore.

[0004] The microbial agent for oil recovery needs to be added regularly and quantitatively during actual oil recovery, so the agent is required to have a long shelf life, and the current oil recovery agent has a short shelf life. In addition, due to high temperature and high salinity in the underground, the agent is difficult to survive in the oil recovery formation. Therefore, in order to solve the above problems, the present application designs a microbial agent for microbial oil recovery and a preparation method thereof. SUMMARY

[0005] In order to solve the above problems, the present application provides a microbial agent for microbial oil recovery and a preparation method thereof.

[0006] A microbial agent for microbial oil recovery comprises the following components:

[0007] A complex bacterial liquid composed of Pseudomonas aeruginosa bacterial agent, Bacillus licheniformis bacterial agent, and methane bacterial agent in a volume ratio of 1:1:0.8-1, and a carrier in a liquid-solid ratio of 1-2 mL:1 g to the complex bacterial liquid, wherein the concentration of the Pseudomonas aeruginosa bacterial agent, the Bacillus licheniformis bacterial agent, and the methane bacterial agent is 10 8～10 cfu / ml respectively.

[0008] The carrier comprises the following components in percentage by mass:

[0009] Modified nanosilica: 55-60%;

[0010] First crosslinking agent: 0.5-1%;

[0011] Surfactant: 1-3%;

[0012] Polyvinyl alcohol: 3-5%;

[0013] Sodium carboxymethyl cellulose: 1-2%;

[0014] Gynostemma pentaphyllum complex: the rest.

[0015] Further, the preparation method of the Gynostemma pentaphyllum complex is as follows:

[0016] The Gynostemma pentaphyllum is crushed to 40 mesh to obtain a powder, water is added to the powder to obtain a water content of 15-25%, and 5.5-6 wt% of a complex enzyme is added to the powder, wherein the complex enzyme comprises cellulase and pectinase in a mass ratio of 3:1, and the Gynostemma pentaphyllum is enzymolyzed at pH=4.5 and 50-55℃ for 1.8-2 h, and then enzyme inactivation and primary centrifugation are performed to obtain supernatant and precipitate, the precipitate is redissolved with an equal amount of deionized water and mixed with the supernatant again to obtain a mixed liquid, 95% ethanol is added to the mixed liquid until the mass concentration of the ethanol is 30-35%, and then secondary centrifugation is performed at 4-6℃ for 1-1.5 h, and the supernatant is subjected to supercritical extraction to obtain an extraction liquid, and the precipitate is redissolved with an equal amount of 0.1-0.2 mol / L NaHCO3 solution to obtain a redissolution liquid.

[0017] The molecularly imprinted polymer specifically recognizing gypenoside is synthesized by using gypenoside XLIX as a template molecule, acrylamide as a functional monomer, and divinylbenzene as a second cross-linking agent at a molar ratio of 1:5:18-19, and then the molecularly imprinted polymer after elution of the template molecule is obtained and filled into a molecularly imprinted column; the extract liquid is passed through the molecularly imprinted column, and then eluted and impurity-removed by using an ethanol solution with a mass fraction of 35-40%, and then desorbed by using a methanol-acetic acid mixed solution with a volume ratio of 7-9:1 to obtain a desorption liquid, the desorption liquid is mixed with a re-dissolution liquid, and concentrated at 30-35 DEG C under reduced pressure to a solid content of 17-18% to obtain a concentrated liquid, and then the concentrated liquid is subjected to spray drying, the inlet air temperature of the spray drying is 80-85 DEG C, and the outlet air temperature is 50-55 DEG C, to obtain the gynostemma extract;

[0018] The agarose and sodium alginate are mixed at a mass ratio of 1:2-5 and heated to 85-90 DEG C, then cooled to 40-45 DEG C to obtain the sodium alginate-coated agarose, and then the gynostemma extract is mixed with the sodium alginate-coated agarose at a mass ratio of 1:1-2, and then ultrasonic oscillation is performed for 25-30 min to obtain the gynostemma compound.

[0019] Description: The cellulase effectively destroys the cellulose structure in the cell wall of gynostemma, and promotes the release of saponins and polysaccharides; the supercritical extraction selectively extracts non-polar saponins, and avoids the residue of organic solvents; the gynostemma extract containing various saponins similar to gypenoside XLIX can be obtained by using gypenoside XLIX as a template; the gynostemma extract obtained in the above steps contains saponins and polysaccharides, the triterpene nucleus of saponins is a hydrophobic group, and the sugar chain is a hydrophilic group, and has good amphiphilicity, and can directly reduce the oil-water interfacial tension, and the saponin structure of gynostemma is special, the polycyclic triterpenoid structure is more resistant to high temperature and high salinity environment in oil reservoir than the steroidal saponin or pentacyclic triterpenoid saponin, the polycyclic triterpenoid structure contains a polar sugar chain and a hydrophobic nucleus, and can be embedded into the cell membrane of pseudomonas aeruginosa, the polar sugar chain forms a hydrophilic layer on the surface of the cell membrane, hinders the direct contact of salt ions in the formation with membrane proteins, and the sugar chain of saponins can be partially degraded into glucose by the β-glycosidase of pseudomonas aeruginosa, as a carbon source to promote the breeding activity; the acidic polysaccharides are slowly hydrolyzed into monosaccharides at high temperature, and provide sustained energy for pseudomonas aeruginosa; saponins reduce the oil-water interfacial tension, so that small oil droplets are more easily contacted with lipase and protease secreted by bacillus licheniformis for degradation; the carbon source provided by polysaccharides promotes the synthesis of these enzymes, the strong hydrogen bond formed by the hydroxyl group of polysaccharides and water molecules increases the proportion of bound water, forms a low ionic strength micro area, and the low ionic micro area can reduce the Na + / Cl-attack to ether bond, maintain the fluidity of the methane bacterial cell membrane; agarose can be slowly degraded into glucose by glycosidase secreted by microorganisms, which can be used as a carbon source to promote the reproduction of various bacteria, and sodium alginate is an anionic polysaccharide (containing carboxyl), which can be combined with saponins and polysaccharides in gynostemma pentaphyllum extract through hydrogen bonds, and its coating layer can form a more dense network through ionic crosslinking in high salinity reservoirs, slowly releasing the internal extract.

[0020] Further, the parameters of the supercritical extraction include: the pressure is 200-300 bar, the flow rate of carbon dioxide is 6-8 g / min, the extraction temperature is 45-50℃, the carrying agent is a mixed solution of ethanol and water mixed in a volume ratio of 2-3:1, the flow rate of the carrying agent is 0.2-0.4 mL / min, and the extraction time is 70-90 min.

[0021] Description: The above parameters can ensure high solubility and low degradation; ethanol breaks down the polarity barrier; the extraction time balances efficiency and energy consumption, preventing the increase of impurity ratio.

[0022] Further, the preparation method of the modified nanosilica is as follows:

[0023] Pulse laser irradiation is performed on nanosilica particles with a particle size of 60-80 nm to obtain pretreated nanosilica;

[0024] Polyurethane and hydroxyethyl acrylate are mixed in a molar ratio of 1:0.9-1, and anhydrous tetrahydrofuran is added thereto, under nitrogen protection and condensation reflux conditions, the temperature is raised to 80-85℃, and dibutyltin dilaurate is added to catalyze the reaction for 1.6-2h to obtain a copolymer, wherein the mass ratio of the total mass of polyurethane and hydroxyethyl acrylate to the mass of anhydrous tetrahydrofuran is 1:4-5, and the addition amount of dibutyltin dilaurate is 0.04-0.05wt% of the copolymer;

[0025] The copolymer accounts for 3-4wt% of the pretreated nanosilica, and the pretreated nanosilica is subjected to supercritical CO2 reaction at a temperature of 42-45℃ and a pressure of 16-18MPa for 3.5-4h to obtain modified nanosilica.

[0026] Description: The hydroxyl groups on the surface of the silicon dioxide are removed by the pulse laser pretreatment, which enhances the grafting rate of the copolymer. The surface of the modified nano-silicon dioxide is grafted with the polyurethane-hydroxyethyl acrylate copolymer containing hydrophilic and oleophilic groups. The interfacial tension between the crude oil and water can be significantly reduced, and the emulsification and stripping of the crude oil can be promoted. The supercritical CO2 reaction enables the copolymer to be uniformly anchored on the surface of the silicon dioxide. The modified nano-silicon dioxide is used as a carrier skeleton to adsorb the composite bacteria solution (Pseudomonas aeruginosa, Bacillus licheniformis, and Methanobacterium) and the gynostemma pentaphyllum compound, thereby improving the stability of the system. The amphiphilic property enables the system to gather at the oil-water interface, which helps the bacteria to be uniformly distributed in the oil reservoir environment. The porous structure can protect the bacteria from being damaged by the high temperature, high pressure, and complex ion environment in the oil reservoir.

[0027] Further, the wavelength of the pulse laser irradiation is 1064 nm, the power is 18-24 mW, and the spot diameter is 3-5 mm.

[0028] Description: The above parameters can increase the specific surface area of the modified nano-silicon dioxide, thereby loading more microorganisms.

[0029] Further, the first crosslinking agent is glutaraldehyde.

[0030] Description: Glutaraldehyde binds the components such as nano-silicon dioxide, polyvinyl alcohol, and sodium carboxymethyl cellulose, thereby enhancing the mechanical strength of the carrier, preventing the carrier from disintegrating rapidly under the water flow scouring or high pressure in the oil reservoir, and controlling the release rate of the components (such as gynostemma pentaphyllum extract and bacteria) to achieve a slow-release effect.

[0031] Further, the surfactant includes a mixture of mannose erythritol lipid and sophorolipid at a mass ratio of 1:1-1.2.

[0032] Description: Mannose erythritol lipid is a glycolipid biosurfactant with good surface activity and special biological activity. Sophorolipid exhibits good surface activity characteristics such as emulsification, wetting, dispersion, and low foamability.

[0033] A preparation method of a bacteria agent for microbial oil recovery according to any one of the preceding embodiments, comprising the following steps:

[0034] S1, preparing a composite bacteria solution:

[0035] Pseudomonas aeruginosa, Bacillus licheniformis, and Methanobacterium are respectively subjected to liquid culture to obtain Pseudomonas aeruginosa agent, Bacillus licheniformis agent, and Methanobacterium agent, which are uniformly mixed at a volume ratio to obtain a composite bacteria solution;

[0036] S2, preparing a gel solution:

[0037] According to the ratio, polyvinyl alcohol and sodium carboxymethyl cellulose are mixed, and then dissolved in water at 90-100 DEG C with a solid-liquid ratio of 1g:20-25ml, stirred uniformly to obtain a mixed solution, and then cooled to 55-65 DEG C, and then 30-40wt% of the gynostemma pentaphyllum compound is added and uniformly mixed to obtain a gel solution;

[0038] S3, preparing a carrier:

[0039] S3-1, mixing the remaining gynostemma pentaphyllum compound with modified nano-silica, and then adding a second crosslinking agent and crosslinking for 4-5h to obtain a crosslinked material;

[0040] S3-2, pouring the crosslinked material obtained in S3-1 and the gel solution obtained in S2 into a mold, first cooling the mold at-15 to-10 DEG C to form, and then performing 3 cycles of freezing and thawing, wherein the freezing and thawing method is: first freezing at-25 to-20 DEG C for 10-12h, and then thawing at 25-30 DEG C for 5-10h to obtain a carrier;

[0041] S4, preparing a microbial agent:

[0042] Mixing the carrier and the compound microbial solution at a ratio of 30-35 DEG C, 180-200rpm for 4-5h, and then drying to obtain a microbial agent.

[0043] Further, in S4, the drying is vacuum freeze-drying at-35 to-25 DEG C, 5-10Pa for 12-24h.

[0044] Description: Vacuum freeze-drying can avoid ice crystal damage to cells and improve the survival rate of living bacteria.

[0045] Compared with the existing microbial oil recovery microbial agent, the present application has the following advantages:

[0046] (1) The microorganism of the present application selects Pseudomonas aeruginosa, which produces high-yield rhamnolipid and other biological surfactants, promotes the emulsification and stripping of crude oil, degrades long-chain alkanes, and reduces the viscosity of crude oil; Bacillus licheniformis secretes lipase and protease, efficiently degrades asphaltene and colloid, increases the content of saturated hydrocarbons in crude oil, and significantly enhances the flowability; and the malonic acid metabolized by Bacillus licheniformis and the surfactants produced by Pseudomonas aeruginosa synergistically reduce the oil-water interfacial tension, enhance the emulsification ability of crude oil, promote the stripping of crude oil from rock pores, and provide metabolic substrates such as malonic acid for methanogens to accelerate the gas production process, and the methanogens metabolize CO2 and CH4 to increase the formation pressure and drive residual oil to migrate to production wells.

[0047] (2) The carrier of the present application contains gynostemma complex, the gynostemma complex contains gynostemma extract, the gynostemma extract contains saponins and polysaccharides, the polycyclic triterpenoid structure of saponins contains a polar sugar chain and a hydrophobic core, which can be embedded in the cell membrane of Pseudomonas aeruginosa, and the polar sugar chain forms a hydrophilic layer on the surface of the cell membrane, which hinders the direct contact of salt ions in the layer with membrane proteins, and the sugar chain of saponins can be partially degraded into glucose by the β-glycosidase of Pseudomonas aeruginosa, which promotes the reproductive activity as a carbon source; the acidic polysaccharide is slowly hydrolyzed into monosaccharide at high temperature, providing sustained energy for Pseudomonas aeruginosa; saponins reduce the oil-water interfacial tension, making it easier for small oil droplets to be contacted and degraded by lipase and protease secreted by Bacillus licheniformis; the carbon source provided by polysaccharides promotes the synthesis of these enzymes, and the strong hydrogen bond formed between the hydroxyl group of polysaccharides and water molecules increases the proportion of bound water, forming a low ionic strength microzone, which can reduce the attack of Na + / Cl- on ether bonds, maintaining the fluidity of the cell membrane of methanobrevibacter; agarose can be slowly degraded into glucose by glycosidase secreted by microorganisms, which can be used as a carbon source to promote the reproductive activity of each strain, and sodium alginate is an anionic polysaccharide that can be combined with saponins and polysaccharides in gynostemma extract through hydrogen bonds, and its coating layer will form a more dense network due to ionic crosslinking in high salinity reservoirs, slowly releasing the internal extract.

[0048] (3) In the carrier of the present application, modified nanosilica forms an amphiphilic structure by surface grafting of polyurethane / hydroxyethyl acrylate copolymer to stabilize Pickering emulsion, reduce crude oil adsorption strength, and has a high specific surface area for loading bacteria and metabolites, delaying the inactivation of biosurfactants, and the surface copolymer of modified nanosilica adsorbs saponin molecules through hydrogen bonds, slowly releases after injection into the oil reservoir, prolongs the action time, and saponins and modified nanosilica work together on heavy crude oil to reduce viscosity by destroying the aggregation structure of asphaltene, the carrier uses polyvinyl alcohol-carboxymethyl cellulose sodium to form a temperature-sensitive hydrogel network that slowly swells at the temperature of the oil reservoir to control the release rate of the bacteria, and the film-forming property of polyvinyl alcohol can wrap the bacteria to reduce the damage of harmful ions (such as high concentration of salt) in the oil reservoir to the bacteria, and carboxymethyl cellulose sodium can be metabolized by Bacillus licheniformis and other bacteria as a carbon source precursor to supplement nutrients; its hydrophilicity can improve the wettability of the rock surface of the oil reservoir (from oleophilic to hydrophilic) to promote the detachment of crude oil from the rock; part of the gynostemma complex is premixed in the gel network to be released preferentially to the oil-water interface to emulsify crude oil quickly; modified nanosilica is combined with gynostemma complex through a crosslinking agent to improve the mechanical strength and salt tolerance of the carrier, and the carrier forms a stable porous network structure after repeated freezing and thawing, and then combines with microorganisms to protect them from inactivation in high temperature and high pressure environments, so the above carrier components can protect the target microorganisms in the bacterial agent and prolong the shelf life of the bacterial agent. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 is a comparison chart of the results of the invention exploration 1;

[0050] Figure 2 is a comparison chart of the results of the invention exploration 2;

[0051] Figure 3 is a comparison chart of the results of the invention exploration 3;

[0052] Figure 4 is a comparison chart of the results of the invention exploration 4. DETAILED DESCRIPTION

[0053] In order to further illustrate the manner in which the application is to be performed and the results achieved by its use, the following examples are given.

[0054] Example 1: A microbial agent for microbial enhanced oil recovery, comprising the following components:

[0055] A composite microbial solution composed of Pseudomonas aeruginosa microbial agent, Bacillus licheniformis microbial agent, and methane bacteria microbial agent in a volume ratio of 1:1:0.9, and a carrier in a liquid-solid ratio of 1.5 mL:1 g with the composite microbial solution, the concentration of the Pseudomonas aeruginosa microbial agent, the Bacillus licheniformis microbial agent, and the methane bacteria microbial agent is 10 9 cfu / ml;

[0056] The carrier comprises the following components in mass percentage:

[0057] Modified nano-silicon dioxide: 58%;

[0058] First crosslinking agent: 0.8%;

[0059] Surfactant: 2%;

[0060] Polyvinyl alcohol: 4%;

[0061] Sodium carboxymethyl cellulose: 1.5%;

[0062] Gynostemma pentaphyllum complex: the balance;

[0063] The preparation method of the Gynostemma pentaphyllum complex is:

[0064] The gynostemma pentaphyllum is crushed to 40 mesh to obtain a powder, water is added to the powder to obtain a water content of 20%, and 5.8wt% of the powder is added to the powder. The composite enzyme includes cellulase and pectinase with a mass ratio of 3:1, and the gynostemma pentaphyllum is enzymolyzed at 52℃ and pH=4.5 for 1.9h. After the enzymolysis, the material liquid is placed in a 80℃ water bath for 15min for enzyme inactivation, and once centrifugation is performed. The temperature of the once centrifugation is 4℃, the speed is 8000rpm, and the time is 15min. The supernatant and the precipitate are obtained. The precipitate is redissolved with an equal amount of deionized water and mixed with the supernatant again to obtain a mixed liquid. A 95% mass concentration of ethanol is added to the mixed liquid until the mass concentration of ethanol is 32%. After standing at 5℃ for 1.2h, secondary centrifugation is performed. The temperature of the secondary centrifugation is 4℃, the speed is 9000rpm, and the time is 20min. The supernatant is taken for supercritical extraction. The parameters of the supercritical extraction include: the pressure is 250bar, the flow rate of carbon dioxide is 7g / min, the extraction temperature is 48℃, the carrying agent is a mixed solution of ethanol and water with a volume ratio of 2.5:1, the flow rate of the carrying agent is 0.3mL / min, and the extraction time is 80min. The extraction liquid is obtained, and the precipitate is redissolved with an equal amount of 0.15mol / L NaHCO3 solution to obtain a redissolution liquid.

[0065] A molecularly imprinted polymer specifically recognizing gypenoside XLIX is synthesized by using gypenoside XLIX as a template molecule, acrylamide as a functional monomer, and divinylbenzene as a second cross-linking agent, with a molar ratio of 1:5:18.5. The template molecule is eluted from the molecularly imprinted polymer, which is then filled into a molecularly imprinted column. The template molecule is eluted by Soxhlet extraction using a methanol-acetic acid mixture with a volume ratio of 9:1 as a solvent at 60℃ for 14h. After extraction, the molecularly imprinted polymer is washed with distilled water until the washing liquid is neutral, and then vacuum dried at 40℃ for 24h.

[0066] The extraction liquid is passed through the molecularly imprinted column, and then eluted with an ethanol solution with a mass fraction of 38% to remove impurities. A methanol-formic acid mixture with a volume ratio of 8:1 is used to desorb the molecularly imprinted polymer, obtaining a desorption liquid. The desorption liquid and the redissolution liquid are mixed and concentrated under reduced pressure at 33℃ to a solid content of 17.5%, obtaining a concentrated liquid. The concentrated liquid is then spray dried, with an inlet air temperature of 83℃ and an outlet air temperature of 53℃, obtaining a gynostemma pentaphyllum extract.

[0067] Agarose and sodium alginate are mixed at a mass ratio of 1:3.5 by heating to 88℃, and then cooled to 43℃ to obtain sodium alginate-coated agarose. The gynostemma pentaphyllum extract is mixed with the sodium alginate-coated agarose at a mass ratio of 1:1.5, and then ultrasonic oscillation is performed for 28min at a frequency of 25kHz and a power of 200W, obtaining a gynostemma pentaphyllum complex.

[0068] The preparation method of the modified nanometer silicon dioxide is as follows:

[0069] The nanometer silicon dioxide particles with a particle size of 65-75 nm are subjected to pulse laser irradiation, the wavelength of the pulse laser irradiation is 1064 nm, the power is 20 mW, and the spot diameter is 4 mm, to obtain pretreated nanometer silicon dioxide;

[0070] The polyurethane and hydroxyethyl acrylate are mixed at a molar ratio of 1:0.95, and anhydrous tetrahydrofuran is added thereto, and under the conditions of nitrogen protection and condensation reflux, the temperature is raised to 82℃ and dibutyltin dilaurate is added to catalyze the reaction for 1.8 h, to obtain a copolymer, wherein the mass ratio of the total mass of the polyurethane and the hydroxyethyl acrylate to the mass of the anhydrous tetrahydrofuran is 1:4.5, and the addition amount of the dibutyltin dilaurate is 0.045wt% of the copolymer; wherein the nitrogen flow is 15 L / min, and the condensation reflux uses water cooling at a flow rate of 2 L / min and a temperature of 72℃;

[0071] The copolymer accounts for 3.5wt% of the pretreated nanometer silicon dioxide, and the pretreated nanometer silicon dioxide is subjected to supercritical CO2 reaction at a temperature of 43℃ and a pressure of 17 MPa for 3.8 h, to obtain modified nanometer silicon dioxide;

[0072] The first crosslinking agent is glutaraldehyde, and the surfactant includes a mixture of mannose erythritol lipid and sophorolipid at a mass ratio of 1:1.1.

[0073] Example 2: A preparation method for preparing a microbial agent for microbial enhanced oil recovery using the components of Example 1, comprising the following steps:

[0074] S1, preparing a composite bacterial solution:

[0075] The Pseudomonas aeruginosa, Bacillus licheniformis and methane bacteria are respectively subjected to liquid culture to obtain Pseudomonas aeruginosa agent, Bacillus licheniformis agent and methane bacteria agent, and the three are mixed uniformly according to the volume ratio to obtain a composite bacterial solution;

[0076] The method for liquid culture of Pseudomonas aeruginosa is as follows: the preserved Pseudomonas aeruginosa slant strain is taken and a single colony is picked up with an inoculation loop, inoculated into LB solid culture medium, and incubated at 37℃ for 24 h to obtain activated strain; the LB liquid culture medium is sterilized at 121℃ for 20 min, cooled to room temperature, and used as needed, 1-2 single colonies are picked up from the activated LB solid culture medium, inoculated into the above-mentioned LB liquid culture medium, and the inoculation amount is 3% of the volume of the culture medium, and the culture is carried out at 37℃ under shaking at 200 rpm (in an aerobic environment) for 20 h to obtain Pseudomonas aeruginosa agent;

[0077] Method for liquid culture of Bacillus licheniformis: Take the slant culture of Bacillus licheniformis, pick a single colony and inoculate it into beef extract peptone solid medium, and incubate at 30℃ for 24h to obtain activated strain. Prepare an optimized liquid medium (beef extract 5g / L, peptone 15g / L, glucose 2g / L, sodium chloride 3g / L, magnesium sulfate 0.5g / L, pH 7.2), sterilize at 121℃ for 20min, and cool for later use. Take the activated liquid seed culture (pre-cultured in shake flasks for 12h in advance), inoculate it into the above medium at a volume ratio of 5%, and culture at 30℃ and 150rpm for 24h with shaking. The resulting Bacillus licheniformis inoculum is obtained.

[0078] Method for liquid culture of methanogenic bacteria: Pure cultures of methanogenic bacteria were inoculated onto a solid anaerobic medium (containing 5 g / L yeast extract, 3 g / L peptone, 2 g / L sodium bicarbonate, 0.5 g / L sodium sulfide·9H2O, 15 g / L agar, pH 7.0) using anaerobic techniques. The medium was anaerobically cultured at 35°C for 5 days to obtain activated colonies. A modified Hungate medium was then prepared (basal solution: 0.5 g / L KH2PO4, 1 g / L NH4Cl, 1 g / L NaCl, 0.1 g / L MgCl2·6H2O, 0.1 g / L CaCl2·6H2O, 0.5 g / L CaCl2·6H2O, 0.1 g / L CaCl2·6H2O, 0.5 g / L NH4Cl, 0.5 g / L NaCl, 0.5 g / L MgCl2·6H2O, 0.5 g / L CaCl2·6H2O ...

[0079] The culture medium was sterilized with 0.1 g / L H₂O, 3 g / L yeast extract, and 2 g / L peptone. 1 mol / L sodium sulfide solution (final concentration 0.05%) and 1 mol / L ascorbic acid solution (final concentration 0.02%) were added to remove dissolved oxygen. Methanol (final concentration 0.5%) was added as a carbon source. The base solution was dispensed and purged with high-purity nitrogen (99.99%) for 15 min to remove oxygen. The solution was then sealed and sterilized at 121°C for 20 min. After cooling to room temperature, the reducing agent and carbon source were added aseptically. In an anaerobic glove box, activated methanogenic bacteria culture was inoculated into the above liquid culture medium at a volume ratio of 10%. The culture was then statically incubated at 35°C (strictly anaerobic environment) for 7 days to obtain the methanogenic bacteria inoculum.

[0080] S2. Preparation of gel solution:

[0081] According to the above ratio, polyvinyl alcohol and sodium carboxymethyl cellulose are mixed and dissolved in water at 95°C at a solid-liquid ratio of 1g:23ml. The mixture is stirred evenly to obtain a solution. Then, the solution is cooled to 60°C and 35wt% of the Gynostemma pentaphyllum complex is added and mixed evenly to obtain a gel solution.

[0082] S3. Preparation of the carrier:

[0083] S3-1. Mix the remaining Gynostemma pentaphyllum complex with modified nano-silica, then add a second crosslinking agent and crosslink for 4.5 h to obtain a crosslinked product;

[0084] S3-2, pour the crosslinking material obtained in S3-1 and the gel liquid obtained in S2 into a mold, first cool the mold at -12℃ to form, and then freeze-thaw for 3 cycles, the freeze-thaw method is: first freeze at -22℃ for 11h, then thaw at 28℃ for 8h, to obtain a carrier;

[0085] S4, prepare a bacterial agent:

[0086] Mix the carrier and the complex bacterial liquid at a ratio of 32℃, 190rpm for 4.5h, and then dry to obtain a bacterial agent, the drying is vacuum freeze-drying at -30℃, 8Pa for 18h.

[0087] Example 3: The difference between this example and Example 1 is that the complex bacterial liquid is composed of Pseudomonas aeruginosa bacterial agent, Bacillus licheniformis bacterial agent, and Methanobacterium bacterial agent with a volume ratio of 1:1:0.8, and the liquid-solid ratio of the carrier to the complex bacterial liquid is 1mL:1g, the concentration of the Pseudomonas aeruginosa bacterial agent, Bacillus licheniformis bacterial agent, and Methanobacterium bacterial agent is 10 8 cfu / ml.

[0088] Example 4: The difference between this example and Example 1 is that the complex bacterial liquid is composed of Pseudomonas aeruginosa bacterial agent, Bacillus licheniformis bacterial agent, and Methanobacterium bacterial agent with a volume ratio of 1:1:1, and the liquid-solid ratio of the carrier to the complex bacterial liquid is 2mL:1g, the concentration of the Pseudomonas aeruginosa bacterial agent, Bacillus licheniformis bacterial agent, and Methanobacterium bacterial agent is 10 10 cfu / ml.

[0089] Example 5: The difference between this example and Example 1 is that the carrier includes the following components by mass percentage: modified nanometer silicon dioxide: 55%; first crosslinking agent: 0.5%; surfactant: 1%; polyvinyl alcohol: 3%; sodium carboxymethyl cellulose: 1%; gynostemma pentaphyllum complex: the balance; the surfactant includes a mixture of mannose erythritol lipid and sophorolipid with a mass ratio of 1:1.

[0090] Example 6: The difference between this example and Example 1 is that the carrier includes the following components by mass percentage: modified nanometer silicon dioxide: 60%; first crosslinking agent: 1%; surfactant: 3%; polyvinyl alcohol: 5%; sodium carboxymethyl cellulose: 2%; gynostemma pentaphyllum complex: the balance; the surfactant includes a mixture of mannose erythritol lipid and sophorolipid with a mass ratio of 1:1.2.

[0091] Example 7: The difference between this example and Example 1 is that the gynostemma is crushed to 40 mesh to obtain a powder, water is added to the powder to a moisture content of 15%, and then a composite enzyme is added, which includes cellulase and pectinase in a mass ratio of 3:1, accounting for 5.5% of the weight of the powder, and the gynostemma is enzymatically hydrolyzed at pH = 4.5 and 50°C for 1.8 h.

[0092] Example 8: The difference between this example and Example 1 is that the gynostemma is crushed to 40 mesh to obtain a powder, water is added to the powder to a moisture content of 25%, and then a composite enzyme is added, which includes cellulase and pectinase in a mass ratio of 3:1, accounting for 6% of the weight of the powder, and the gynostemma is enzymatically hydrolyzed at pH = 4.5 and 55°C for 2 h.

[0093] Example 9: The difference between this example and Example 1 is that ethanol with a mass concentration of 95% is added to the mixed solution until the mass concentration of ethanol is 30%, and then the solution is centrifuged again after standing at 4°C for 1 h, and the precipitate is resuspended with an equal volume of 0.1 mol / L NaHCO3 solution to obtain a resuspension solution.

[0094] Example 10: The difference between this example and Example 1 is that ethanol with a mass concentration of 95% is added to the mixed solution until the mass concentration of ethanol is 35%, and then the solution is centrifuged again after standing at 6°C for 1.5 h, and the precipitate is resuspended with an equal volume of 0.2 mol / L NaHCO3 solution to obtain a resuspension solution.

[0095] Example 11: The difference between this example and Example 1 is that the parameters of supercritical extraction include a pressure of 200 bar, a flow rate of carbon dioxide of 6 g / min, an extraction temperature of 45°C, a carrying agent that is a mixed solution of ethanol and water in a volume ratio of 2:1, a flow rate of the carrying agent of 0.2 mL / min, and an extraction time of 70 min.

[0096] Example 12: The difference between this example and Example 1 is that the parameters of supercritical extraction include a pressure of 300 bar, a flow rate of carbon dioxide of 8 g / min, an extraction temperature of 50°C, a carrying agent that is a mixed solution of ethanol and water in a volume ratio of 3:1, a flow rate of the carrying agent of 0.4 mL / min, and an extraction time of 90 min.

[0097] Example 13: The difference between this example and Example 1 is that gynostemma saponin XLIX is used as a template molecule, acrylamide is used as a functional monomer, and divinylbenzene is used as a second cross-linking agent, and a molecularly imprinted polymer that specifically recognizes gynostemma saponin is synthesized in a molar ratio of 1:5:18, and then elution and impurity removal are performed using an ethanol solution with a mass fraction of 35%, and then desorption is performed using a methanol-formic acid mixed solution in a volume ratio of 7:1.

[0098] Example 14: The difference between this example and Example 1 is that the molecularly imprinted polymer specifically recognizing gypenoside is synthesized with gypenoside XLIX as the template molecule, acrylamide as the functional monomer, divinylbenzene as the second cross-linking agent, and in a molar ratio of 1:5:19, and the impurities are removed by elution with an ethanol solution having a mass fraction of 40%, and then desorbed with a methanol-formic acid mixed solution having a volume ratio of 9:1.

[0099] Example 15: The difference between this example and Example 1 is that the desorption solution and the redissolution solution are mixed and concentrated under reduced pressure at 30°C to a solid content of 17% to obtain a concentrated solution.

[0100] Example 16: The difference between this example and Example 1 is that the desorption solution and the redissolution solution are mixed and concentrated under reduced pressure at 35°C to a solid content of 18% to obtain a concentrated solution.

[0101] Example 17: The difference between this example and Example 1 is that the inlet air temperature of the spray drying is 80°C and the outlet air temperature is 55°C.

[0102] Example 18: The difference between this example and Example 1 is that the inlet air temperature of the spray drying is 85°C and the outlet air temperature is 50°C.

[0103] Example 19: The difference between this example and Example 1 is that agarose and sodium alginate are heated to 85°C and mixed in a mass ratio of 1:2, and after mixing is completed, the mixture is cooled to 40°C to obtain agarose coated with sodium alginate, and then the gypenoside extract and the agarose coated with sodium alginate are mixed in a mass ratio of 1:1, and then ultrasonic oscillation is performed for 25 min to obtain a gypenoside compound.

[0104] Example 20: The difference between this example and Example 1 is that agarose and sodium alginate are heated to 90°C and mixed in a mass ratio of 1:5, and after mixing is completed, the mixture is cooled to 45°C to obtain agarose coated with sodium alginate, and then the gypenoside extract and the agarose coated with sodium alginate are mixed in a mass ratio of 1:2, and then ultrasonic oscillation is performed for 30 min to obtain a gypenoside compound.

[0105] Example 21: The difference between this example and Example 1 is that nanometer-sized silicon dioxide particles having a particle size of 75-80 nm are subjected to pulsed laser irradiation, the wavelength of the pulsed laser irradiation is 1064 nm, the power is 18 mW, and the spot diameter is 3 mm to obtain pretreated nanometer-sized silicon dioxide.

[0106] Example 22: The difference between this example and Example 1 is that nanometer-sized silicon dioxide particles having a particle size of 60-65 nm are subjected to pulsed laser irradiation, the wavelength of the pulsed laser irradiation is 1064 nm, the power is 24 mW, and the spot diameter is 5 mm to obtain pretreated nanometer-sized silicon dioxide.

[0107] Example 23: The difference between this example and Example 1 is that the polyurethane and hydroxyethyl acrylate are mixed in a molar ratio of 1:0.9, and anhydrous tetrahydrofuran is added thereto, and under the conditions of nitrogen protection and condensation reflux, the temperature is raised to 80°C and dibutyl tin dilaurate is added to catalyze the reaction for 1.6 h, to obtain a copolymer, wherein the mass ratio of the total mass of polyurethane and hydroxyethyl acrylate to the mass of anhydrous tetrahydrofuran is 1:4, and the addition amount of dibutyl tin dilaurate is 0.04wt% of the copolymer.

[0108] Example 24: The difference between this example and Example 1 is that the polyurethane and hydroxyethyl acrylate are mixed in a molar ratio of 1:1, and anhydrous tetrahydrofuran is added thereto, and under the conditions of nitrogen protection and condensation reflux, the temperature is raised to 85°C and dibutyl tin dilaurate is added to catalyze the reaction for 2 h, to obtain a copolymer, wherein the mass ratio of the total mass of polyurethane and hydroxyethyl acrylate to the mass of anhydrous tetrahydrofuran is 1:5, and the addition amount of dibutyl tin dilaurate is 0.05wt% of the copolymer.

[0109] Example 25: The difference between this example and Example 1 is that 3wt% of the copolymer based on the pretreated nano-silica is reacted with the pretreated nano-silica in supercritical CO2 at a temperature of 42°C and a pressure of 16MPa for 3.5h to obtain modified nano-silica.

[0110] Example 26: The difference between this example and Example 1 is that 4wt% of the copolymer based on the pretreated nano-silica is reacted with the pretreated nano-silica in supercritical CO2 at a temperature of 45°C and a pressure of 18MPa for 4h to obtain modified nano-silica.

[0111] Example 27: The difference between this example and Example 2 is that polyvinyl alcohol and sodium carboxymethyl cellulose are mixed, and dissolved in water at 90°C at a solid-liquid ratio of 1g:25ml, and stirred uniformly to obtain a mixed solution, and then cooled to 55°C and 30wt% of the gynostemma pentaphyllum compound is added and mixed uniformly to obtain a gel solution.

[0112] Example 28: The difference between this example and Example 2 is that polyvinyl alcohol and sodium carboxymethyl cellulose are mixed, and dissolved in water at 100°C at a solid-liquid ratio of 1g:20ml, and stirred uniformly to obtain a mixed solution, and then cooled to 65°C and 40wt% of the gynostemma pentaphyllum compound is added and mixed uniformly to obtain a gel solution.

[0113] Example 29: The difference between this example and Example 2 is that S3-1, the remaining gynostemma pentaphyllum compound is mixed with the modified nano-silica, and then a second crosslinking agent is added and crosslinked for 4h to obtain a crosslinked material.

[0114] Example 30: The difference between this example and Example 2 is that S3-1, after mixing the remaining gynostemma pentaphyllum compound with modified nano-silica, a second crosslinking agent is added and crosslinked for 5 h to obtain a crosslinked product.

[0115] Example 31: The difference between this example and Example 2 is that S3-2, the crosslinked product obtained in S3-1 and the gel liquid obtained in S2 are both poured into a mold, the mold is first cooled and shaped at -15℃, and then 3 cycles of freezing and thawing are performed, the freezing and thawing method being: first frozen at -25℃ for 12 h, and then thawed at 30℃ for 10 h, to obtain a carrier.

[0116] Example 32: The difference between this example and Example 2 is that S3-2, the crosslinked product obtained in S3-1 and the gel liquid obtained in S2 are both poured into a mold, the mold is first cooled and shaped at -10℃, and then 3 cycles of freezing and thawing are performed, the freezing and thawing method being: first frozen at -20℃ for 10 h, and then thawed at 25℃ for 5 h, to obtain a carrier.

[0117] Example 33: The difference between this example and Example 2 is that the carrier and the composite bacterial liquid are mixed at a ratio at 30℃ and 180 rpm for 4 h, and then dried to obtain a bacterial agent, the drying being vacuum freeze drying at -35℃ and 5 Pa for 12 h.

[0118] Example 34: The difference between this example and Example 2 is that the carrier and the composite bacterial liquid are mixed at a ratio at 35℃ and 200 rpm for 5 h, and then dried to obtain a bacterial agent, the drying being vacuum freeze drying at -25℃ and 10 Pa for 24 h.

[0119] Experimental Example: The description of this experimental example is based on the description in Example 2, and is intended to illustrate the actual application effect of the present application.

[0120] 20 mL and 100 mL of dehydrated crude oil obtained from each embodiment of the bacterial agent of the present application are mixed uniformly, water is used as a blank group, and the mixture is cultured at 35℃ for 72 h, the temperature of a rotational viscometer is set to 40℃, the viscosity of each group of thick oil is determined, the viscosity reduction rate % is calculated, and the average value of three results is taken.

[0121] The bacterial agent obtained from each embodiment of the bacterial agent of the present application is prepared into a 1 wt% aqueous solution, surface tension testing is performed, the interfacial tension reduction rate % is calculated, and the average value of three results is taken.

[0122] A porous medium containing crude oil with a porosity of 20% and a water permeability of 110 mD is selected, the bacterial agent of the present application is injected into the oil-containing porous medium for displacement, the total injection amount is 1.5 times the pore volume, the recovery rate % is calculated, and the average value of three results is taken.

[0123] Investigation 1: Investigate the effects of the components of the microbial agent on the viscosity reduction rate and interfacial tension reduction rate of microbial enhanced oil recovery.

[0124] The difference between Comparative Example 1 and Example 1 is that the carrier does not contain the Gynostemma pentaphyllum complex;

[0125] from Figure 1 The results show that, since the control example 1 lacks the Gynostemma pentaphyllum complex, the supply of nutrients to the bacteria and the effect on promoting bacterial reproduction are greatly reduced. As a result, the viscosity reduction rate and interfacial tension reduction rate of the control example 1 are significantly reduced compared with the example 1.

[0126] Comparing Examples 1 and 3-6, it can be seen that too low an overall bacterial concentration and too low a proportion of Gynostemma pentaphyllum complex in the carrier will reduce the viscosity reduction rate and interfacial tension reduction rate of microbial oil recovery. In Example 4, the bacterial concentration is higher and in Example 5, the proportion of Gynostemma pentaphyllum is larger, which is better than Example 1, but the improvement is slight. Therefore, from an economic point of view, the parameters of Example 1 are relatively better.

[0127] Investigation 2: Investigate the effect of the preparation method of Gynostemma pentaphyllum complex on the viscosity reduction rate and interfacial tension reduction rate of microorganisms.

[0128] The difference between Comparative Example 2 and Example 1 is that Gynostemma pentaphyllum saponin XLIX was directly used as one of the raw materials of the Gynostemma pentaphyllum complex;

[0129] from Figure 2 The results show that, compared with the multiple Gynostemma pentaphyllum saponins of the same type as Gynostemma pentaphyllum saponins obtained by molecular imprinting, the number of saponins in Control Example 2, which directly uses Gynostemma pentaphyllum saponin XLIX as a single component, is significantly reduced. As a result, the provision of nutrients to the bacteria and the effect on promoting bacterial reproduction are greatly weakened. Thus, the viscosity reduction rate and interfacial tension reduction rate of Control Example 2 are significantly weaker than those of Example 1.

[0130] Comparing Examples 1 and 7-20, it can be seen that excessively small or large parameters in enzymatic hydrolysis, alcohol precipitation, supercritical extraction, molecular imprinting, concentration, spray drying, and agarose and sodium alginate coating will reduce the viscosity reduction rate and interfacial tension reduction rate of microbial oil recovery. Therefore, from a comprehensive perspective, the parameters in Example 1 are relatively better.

[0131] Investigation 3: Investigate the effects of the preparation method of modified nano-silica on the viscosity reduction rate and interfacial tension reduction rate of microorganisms.

[0132] The difference between Comparative Example 3 and Example 1 is that pulsed laser irradiation was not performed;

[0133] Depend on Figure 3The results show that, in Comparative Example 3, the nano-silica material was not pretreated with pulsed laser irradiation, which resulted in a decrease in the surface porosity of the nano-silica material, a decrease in its binding with other carriers, and a decrease in its adsorption capacity for saponin molecules. As a result, the viscosity reduction rate and interfacial tension reduction rate of Comparative Example 3 were significantly weaker than those of Example 1.

[0134] Comparing Examples 1 and Examples 21 to 26, it can be seen that excessively small or large pulsed laser irradiation parameters, excessively small or large polyurethane content in the copolymer, and excessively small or large supercritical CO2 reaction parameters will all reduce the viscosity reduction rate and interfacial tension reduction rate of microbial oil recovery. Therefore, from a comprehensive perspective, the parameter effect of Example 1 is relatively better.

[0135] Investigation 4: Investigate the effect of the preparation method of the inoculant on the recovery rate of microbial enhanced oil recovery.

[0136] The difference between Comparative Example 4 and Example 1 is that the Gynostemma pentaphyllum complex is not added in advance in step S2, and the Gynostemma pentaphyllum complex is completely mixed in S3-1;

[0137] Depend on Figure 4 The results show that, compared with the control example 2, the Gynostemma pentaphyllum complex was mixed in one go in control example 4, which may have led to a decrease in bacterial activity due to local nutrient deficiency. As a result, the viscosity reduction rate and interfacial tension reduction rate of control example 4 were significantly weaker than those of example 1.

[0138] Comparing Examples 2 and 27 to 34, it can be seen that if the preparation parameters of the gel solution are too small or too large, the cross-linking time is too short or too long, the mold forming parameters are too small or too large, and the mixing and drying parameters are too small or too large, the viscosity reduction rate and interfacial tension reduction rate of microbial oil recovery will be reduced. Therefore, from a comprehensive perspective, the parameters of Example 2 are relatively better.

Claims

1. A microbial agent for microbial enhanced oil recovery, characterized in that, The carrier comprises the following components by mass percentage: A complex bacterial liquid composed of Pseudomonas aeruginosa bacterial agent, Bacillus licheniformis bacterial agent, and methane bacterial agent in a volume ratio of 1:1:0.8-1, and a carrier in a liquid-solid ratio of 1-2 mL:1 g to the complex bacterial liquid, wherein the concentrations of the Pseudomonas aeruginosa bacterial agent, the Bacillus licheniformis bacterial agent, and the methane bacterial agent are all 10 8～10 cfu / ml 8～10 The carrier comprises the following components by mass percentage: Modified nano-silica: 55-60%; First cross-linking agent: 0.5-1%; Surfactant: 1-3%; Polyvinyl alcohol: 3-5%; Sodium carboxymethyl cellulose: 1-2%; Gynostemma pentaphyllum complex: the balance.

2. The microbial agent for microbial enhanced oil recovery according to claim 1, wherein The preparation method of the gynostemma pentaphyllum complex is as follows: The gynostemma pentaphyllum is crushed to 40 mesh to obtain a powder, water is added to the powder to obtain a water content of 15-25%, and 5.5-6 wt% of a complex enzyme is added to the powder, the complex enzyme comprising cellulase and pectinase in a mass ratio of 3:1, and the gynostemma pentaphyllum is enzymolyzed at pH=4.5 and 50-55°C for 1.8-2 h, and then enzyme inactivation and primary centrifugation are performed to obtain supernatant and precipitate, the precipitate is redissolved with an equal amount of deionized water and mixed with the supernatant again to obtain a mixed solution, 95% ethanol is added to the mixed solution until the mass concentration of ethanol is 30-35%, and the mixture is placed at 4-6°C for 1-1.5 h and then subjected to secondary centrifugation, the supernatant is subjected to supercritical extraction to obtain an extract, and the precipitate is redissolved with an equal amount of 0.1-0.2 mol / L NaHCO3 solution to obtain a redissolved solution; Gynostemma pentaphyllum saponin XLIX is used as a template molecule, acrylamide is used as a functional monomer, and divinylbenzene is used as a second cross-linking agent to synthesize a molecularly imprinted polymer that specifically recognizes gynostemma pentaphyllum saponin at a molar ratio of 1:5:18-19, and then the molecularly imprinted polymer after elution of the template molecule is obtained and filled into a molecularly imprinted column; the extract is passed through the molecularly imprinted column, and then eluted and impurity-removed with an ethanol solution with a mass fraction of 35-40%, and then desorbed with a methanol-formic acid mixed solution with a volume ratio of 7-9:1 to obtain a desorption solution, the desorption solution and the redissolved solution are mixed and concentrated under reduced pressure at 30-35°C to a solid content of 17-18% to obtain a concentrated solution, and then the concentrated solution is subjected to spray drying, the inlet air temperature of the spray drying is 80-85°C, and the outlet air temperature is 50-55°C, to obtain a gynostemma pentaphyllum extract; Agarose and sodium alginate are mixed at a mass ratio of 1:2-5 and heated to 85-90°C, and then cooled to 40-45°C to obtain agarose coated with sodium alginate, and then the gynostemma pentaphyllum extract and the agarose coated with sodium alginate are mixed at a mass ratio of 1:1-2, and then ultrasonic oscillation is performed for 25-30 min to obtain a gynostemma pentaphyllum complex.

3. The microbial agent for microbial enhanced oil recovery of claim 2, wherein, The parameters of the supercritical extraction include: a pressure of 200-300 bar, a flow rate of carbon dioxide of 6-8 g / min, an extraction temperature of 45-50°C, a carrying agent being a mixed solution of ethanol and water mixed at a volume ratio of 2-3:1, a flow rate of the carrying agent being 0.2-0.4 mL / min, and an extraction time of 70-90 min.

4. The microbial agent for microbial enhanced oil recovery of claim 1, wherein, The preparation method of the modified nano-silica is as follows: Nano-silica particles with a particle size of 60-80 nm are subjected to pulsed laser irradiation to obtain pretreated nano-silica; The polyurethane and hydroxyethyl acrylate are mixed in a molar ratio of 1:0.9-1, and anhydrous tetrahydrofuran is added thereto, and under the conditions of nitrogen protection and condensation reflux, the temperature is raised to 80-85 DEG C and dibutyl tin dilaurate is added to catalyze the reaction for 1.6-2 h, to obtain a copolymer, wherein the mass ratio of the total mass of the polyurethane and hydroxyethyl acrylate to the mass of anhydrous tetrahydrofuran is 1:4-5, and the addition amount of dibutyl tin dilaurate is 0.04-0.05 wt% of the copolymer; The copolymer accounting for 3-4 wt% of the pretreated nano-silica is reacted with the pretreated nano-silica in supercritical CO2 at a temperature of 42-45 DEG C and a pressure of 16-18 MPa for 3.5-4 h to obtain modified nano-silica.

5. A microbial agent for microbial enhanced oil recovery as claimed in claim 4, wherein, The wavelength of the pulsed laser irradiation is 1064 nm, the power is 18-24 mW, and the spot diameter is 3-5 mm.

6. The microbial agent for microbial enhanced oil recovery of claim 1, wherein, The first crosslinking agent is glutaraldehyde.

7. The microbial agent for microbial enhanced oil recovery of claim 1, wherein, The surfactant comprises a mixture of mannose erythritol lipid and sophorolipid in a mass ratio of 1:1-1.

2.

8. A method for preparing a microbial agent for microbial enhanced oil recovery as described in any one of claims 1 to 7, characterized in that, The method comprises the following steps: S1, preparing a composite bacteria solution: Pseudomonas aeruginosa, Bacillus licheniformis and methane bacteria are respectively subjected to liquid culture to obtain Pseudomonas aeruginosa inoculum, Bacillus licheniformis inoculum and methane bacteria inoculum, and the three are mixed uniformly in a volume ratio to obtain a composite bacteria solution; S2, preparing a gel solution: According to the ratio, polyvinyl alcohol and sodium carboxymethyl cellulose are mixed, and then dissolved in water at 90-100 DEG C in a solid-liquid ratio of 1 g:20-25 ml, stirred uniformly to obtain a mixed solution, and then cooled to 55-65 DEG C, and then 30-40 wt% of the gynostemma pentaphyllum compound is added and mixed uniformly to obtain a gel solution; S3, preparing a carrier: S3-1, the remaining gynostemma pentaphyllum compound is mixed with the modified nano-silica, and then a second crosslinking agent is added and crosslinked for 4-5 h to obtain a crosslinked product; S3-2, the crosslinked product obtained in S3-1 and the gel solution obtained in S2 are both poured into a mold, the mold is first cooled and formed at -15 to -10 DEG C, and then subjected to 3 cycles of freezing and thawing, wherein the freezing and thawing method is: first frozen at -25 to -20 DEG C for 10-12 h, and then thawed at 25-30 DEG C for 5-10 h, to obtain a carrier; S4, preparing an inoculum: The carrier and the composite bacteria solution are mixed at 30-35 DEG C and 180-200 rpm for 4-5 h, and then dried to obtain an inoculum.

9. The method for preparing a microbial agent for microbial enhanced oil recovery as described in claim 8, characterized in that, In S4, the drying is vacuum freeze-drying at -35 to -25 DEG C and 5-10 Pa for 12-24 h.

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