Hydrophobically modified bacillus licheniformis multilayer biological membrane, osmotic regulator and application

Abstract

The invention discloses a hydrophobic modified bacillus licheniformis multilayer biological membrane, an osmotic regulator and application, and belongs to the field of petroleum development. Bacillus licheniformis is used as a production strain to obtain a biological membrane, hydrophobic groups are grafted to the surface of the biological membrane, the surface characteristics are changed, and the biological membrane shows super-hydrophobicity, so that the flow resistance of a water phase in a pore throat is increased, and the effect of controlling water without blocking is achieved; the hydrophobic modified bacillus licheniformis multi-layer biological membrane has a nanoscale size, so that the hydrophobic modified bacillus licheniformis multi-layer biological membrane can permeate into nano-micro pores of a low-permeability reservoir to form a compact and stable hydrophobic biological membrane; and in larger pores, a high-permeability channel can be blocked through the polymerization characteristic, so that the fluid flow is adjusted, and the oil displacement effect is improved. Performance determination results show that when the hydrophobic modified bacillus licheniformis multilayer biological membrane prepared by the invention is applied to a low-permeability reservoir as an osmotic regulator, the osmotic effect can be remarkably improved, and a novel osmotic regulator is provided for future oilfield development.

Description

Technical Field

[0001] The invention relates to the field of petroleum development, and in particular to a hydrophobically modified Bacillus licheniformis multilayer biofilm, an osmotic regulator and applications thereof. Background Art

[0002] As one of the important technologies to increase oil production after primary oil production, water injection development is widely used by oil fields at home and abroad. Due to the limited natural energy of the reservoir, this method can not only make up for the underground deficit caused by primary oil production, maintain or increase the oil layer pressure, and achieve high and stable production of the oil field, but also play the role of displacing reservoir crude oil and obtain a higher recovery rate. With the rapid growth of oil demand at home and abroad, the objects of oil and gas exploration and development are becoming increasingly complex, the grade of reserves is getting worse and worse, and more and more low-permeability oil fields are being developed. In recent years, among the proven reserves in my country, the proportion of low-permeability oil reservoirs accounts for more than 2 / 3 of the national reserves, with huge development potential, and will become the main resource for increasing reserves and production. According to the "SY / T 6285-2011 Standard for Oil and Gas Reservoir Evaluation Methods", the permeability of low-permeability oil fields is generally 10-50mD, the reservoir has developed natural fractures, fine lithology, poor physical properties, small pore throat radius, complex pore structure, and strong heterogeneity. After long-term water injection, the surface of the rock pores in the near-well zone of the water injection well has been scoured for a long time, and the wettability of the pore surface has changed from the initial oil-philicity to hydrophilicity, forming a hydration layer. The hydration layer reduces the effective diameter of the core pores, hindering the seepage of oil and gas. The aggregation and retention of water droplets in the seepage channel will cause a significant increase in water saturation, directly affecting the effective flow and output of oil and gas fluids. At the same time, the injected water is easy to escape in the dominant channel, shielding the remaining oil between the wells, resulting in high water content in the oil well, that is, the relative permeability of water increases and the relative permeability of oil decreases, which not only reduces the effective sweep of the reservoir matrix, but also may lead to uneven distribution of reservoir fluids and reduce the overall recovery rate. And due to the wetting characteristics of the reservoir, a large amount of injected water is easily retained in the matrix, and some problems of reservoir damage in the water injection process are gradually exposed, such as water lock damage, hydration expansion of sensitive minerals, particle migration blocking nanopore throat channels, incompatibility of injected water and reservoir fluids, etc., which will lead to a decrease in reservoir permeability.

[0003] At present, most oil fields have entered the medium-high water content period or even the ultra-high water content period. Chemical methods have become a common method to solve the problem of water affinity in low permeability reservoirs and high water content in oil wells. One method is to set up a barrier in the reservoir to completely block the flow of unwanted fluids, that is, profile control and water plugging technology. Due to the simple construction and high economic benefits of this method, it is widely used in medium and high permeability reservoirs. The common point of existing plugging agents is to block or reduce the crossflow channel to achieve fluid flow diversion. However, due to the complexity of low permeability reservoirs and high reservoir injection pressure, profile control will further increase the injection pressure, resulting in the failure of water injection into the reservoir. At the same time, due to the low fluid volume in the low permeability reservoir, water plugging will further reduce the fluid production of the oil well, resulting in no oil production. Therefore, whether it is the "complete blocking" of gel / cement or the "blocking but not dead" of particles, it will reduce the permeability of the reservoir and affect the seepage capacity. Another method is to add a regulator to the system to control the relative permeability. The chemical agent is adsorbed and deposited on the pores or surface of the rock to form a hydrophobic chemical film to prevent or reduce the filtrate from entering the formation, improve the wettability of the rock surface, and make the surface highly oil-wet. Under the action of capillary resistance, it is difficult for water to enter the pore throat and flow. Under the condition of keeping the overall permeability unchanged, the relative permeability of oil is increased and the relative permeability of water is reduced, so as to achieve water control in unconventional oil reservoirs such as low permeability, dense, and shale. At present, permeability regulators are mainly polymers and nanomaterials. However, since the injection of chemical permeability regulators will inevitably cause damage to the reservoir, the urgent need for replacement technology has made biorecovery technology widely concerned because of its biodegradability, no re-pollution, non-toxic or low-toxicity, generally non-sensitizing, better environmental compatibility, better selectivity and specificity under extreme temperatures, pH values, and salt concentrations, and structural diversity. It can be applied to special fields and is considered to be one of the most economical and effective technologies for stabilizing oil and controlling water and improving recovery in the later stage of oil field development in the future. Summary of the invention

[0004] The purpose of the present invention is to provide a hydrophobically modified Bacillus licheniformis multilayer biofilm, an osmotic regulator and its application to solve the problems existing in the above-mentioned prior art. The biofilm is obtained by using Bacillus licheniformis as a production strain, and then excessive hydrophobic groups are grafted onto the biofilm to make it super hydrophobic. The application of the biofilm in low-permeability oil reservoirs can significantly improve the reservoir permeability.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides a hydrophobically modified Bacillus licheniformis multilayer biofilm, which is obtained by grafting hydrophobic groups on a biofilm secreted by Bacillus licheniformis, wherein the hydrophobic groups include long-chain alkyl groups with more than 7 carbon atoms.

[0007] The present invention also provides a method for preparing a hydrophobically modified Bacillus licheniformis multilayer biofilm, comprising the step of grafting hydrophobic groups on the biofilm secreted by Bacillus licheniformis, wherein the hydrophobic groups include long-chain alkyl groups with more than 7 carbon atoms.

[0008] Optionally, the long chain alkyl group is an octyl group (C8H 17 -), dodecyl group (C 12 H 25 -) and hexadecyl groups (C 16 H 33 -) one or more.

[0009] Optionally, the preparation method comprises the following steps:

[0010] dissolving the multilayer biofilm secreted by Bacillus licheniformis with water and adjusting the pH value to be alkaline to obtain a biofilm aqueous solution;

[0011] Dissolving a long-chain alkyl compound containing a hydrophobic group, N,N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide in an organic solvent, and then dripping the mixture into the biofilm aqueous solution, stirring the mixture in a water bath to carry out a grafting reaction;

[0012] After adjusting the pH value of the reaction solution to neutral to terminate the reaction, ethanol is added to the reaction solution, the solution is allowed to stand, centrifuged, and the precipitate is collected to obtain a hydrophobically modified Bacillus licheniformis multilayer biofilm.

[0013] Optionally, the biofilm secreted by Bacillus licheniformis is obtained by the following steps:

[0014] After inoculating the fermentation medium with Bacillus licheniformis, the culture medium is shaken and cultured, the culture medium is separated, and the supernatant is collected;

[0015] The supernatant is mixed with ethanol, stirred and separated to obtain a precipitate, which is then dialyzed and dried to obtain the multilayer biofilm secreted by the Bacillus licheniformis.

[0016] Optionally, the ratio of the biofilm secreted by the Bacillus licheniformis to water is (1-4) g: (50-150) mL; and / or the alkaline pH value is 7.0-8.0.

[0017] Optionally, the ratio of the long-chain alkyl compound containing a hydrophobic group, N,N'-dicyclohexylcarbodiimide, N-hydroxysuccinimide and the organic solvent is 0.1g: (0.2-0.8)g: (0.1-0.5)g: (40-50)mL; and / or the conditions of the grafting reaction are: stirring speed of 400-500rpm, water bath temperature of 40-45°C, reaction time of 4-6h.

[0018] The present invention also provides an application of the hydrophobically modified Bacillus licheniformis multilayer biofilm in preparing a permeability regulator for low-permeability oil reservoirs.

[0019] The invention also provides a permeability regulator for low-permeability oil reservoirs, which contains the hydrophobic modified Bacillus licheniformis multilayer biofilm.

[0020] The present invention also provides the use of the hydrophobically modified Bacillus licheniformis multilayer biofilm or the permeability regulator in any of the following:

[0021] (1) Application in improving oil recovery effect in low permeability reservoirs;

[0022] (2) Application in improving the water control and oil production increase effect of low permeability core;

[0023] (3) Application in improving the compressive strength of low-permeability cores;

[0024] (4) Application in improving the permeability of low permeability reservoirs.

[0025] The present invention also provides a method for improving the permeability of a low-permeability oil reservoir, comprising the step of applying the hydrophobically modified Bacillus licheniformis multilayer biofilm or the permeability regulator to the low-permeability oil reservoir.

[0026] The present invention discloses the following technical effects:

[0027] The present invention grafts a large number of multilayer biofilms with hydrophobic groups of "pinning" structures with different roughness secreted by the biological main raw material Bacillus licheniformis, so that the biofilm has super hydrophobicity. Its characteristics enable it to effectively block hypertonic channels in large pores. At the same time, the nanoscale biofilm can enter the nanoscale pores of low permeability reservoirs, effectively adjust the flow of reservoir fluids, and significantly improve the oil recovery efficiency. At the same time, the Bacillus licheniformis multilayer biofilm has excellent properties such as high temperature resistance, salt resistance, and anaerobic resistance, adapts to a variety of complex oil reservoir environments, has long-term stability, and ensures that it can continue to play a role under harsh conditions. This technology can be used for large-scale production using mature fermentation equipment, with controllable production costs, and is non-toxic and harmless, meeting the dual requirements of modern petroleum industry for environmental protection and economy. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1The SEM images of the hydrophobically modified Bacillus licheniformis multilayer biofilms prepared in Examples 2 to 4; (a) to (c) correspond to the hydrophobically modified Bacillus licheniformis multilayer biofilms prepared in Examples 2 to 4, respectively;

[0030] Figure 2 This is a SEM image of a hydrophobic modified Bacillus licheniformis multilayer biofilm covering the rock surface. DETAILED DESCRIPTION

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] A variety of oilfield chemical plugging agents have been disclosed in the prior art. Traditional oilfield chemical plugging agents have gradually developed from the initial cement slurry, resin and particle plugging to water-soluble polymers and their gel gel plugging, which have achieved significant recovery and economic benefits. However, with the conventional oilfield approaching the development limit and the vigorous development of unconventional resources, especially unconventional oil reservoirs such as low permeability, the development efficiency of traditional plugging agents is limited. Multiple plugging and adjustment allow the profile control agent to enter the high and low permeability layers, resulting in a reduction in the profile control radius and a bypass phenomenon after water injection. Not only does it not expand the oil displacement area, but it also reduces the oil increase effect. After the injection of the chemical profile control agent, the physical properties of the reservoir are prone to change, such as changing the wettability and interfacial tension, which reduces the adsorption and service life of the subsequent profile control agent. Permeability regulators are special in oilfield water plugging agents. Their key components are mainly high molecular weight water-soluble polymers or their weakly cross-linked gel systems, which can form hydrophobic chemical films adsorbed on the rock pore walls. When hydrophobic groups are added to the polymer chain, the properties of ionic and non-ionic water-soluble polymer solutions (such as rheology and viscosity) will change. When hydrophobic groups are added to the polymer chain, the properties of ionic and non-ionic water-soluble polymer solutions (such as rheology and viscosity) will change. When water molecules pass through the hydrophobic groups, the hydrophobic groups tend to attract and repel each other, resulting in a higher viscosity of the hydrophobically modified polymer solution. The hydrophobic association promotes more polymer adsorption on the hydrophobically modified polymer, which reduces the polymer's permeability to the water phase. Therefore, the permeability regulator is different from conventional water plugging materials and is a new type of selective plugging material. However, no matter which of the above plugging materials, there are certain defects, such as:

[0037] (1) The pore throat size of unconventional oil reservoirs such as low-permeability reservoirs is at the nanometer level, which makes it difficult for conventional polymer or gel permeability regulators to enter, and there is a problem of poor adaptability to pores of different sizes;

[0038] (2) Polymer permeability regulator materials have poor salt resistance and are prone to "salting out" under high-salinity conditions; silicate materials will have an adverse effect on the rheology and filtration properties of drilling fluids;

[0039] (3) The reservoir conditions are complex. Due to the adsorption of polymers, the rock permeability is easily reduced, and the concentration and velocity of the polymer front flow are reduced. Therefore, most materials involved in permeability regulators have low regulation efficiency in high-permeability reservoirs and heterogeneous reservoirs. There may also be problems such as incompatibility between polymers and fracturing fluid additives.

[0040] The prior art solution also discloses a hydrophobic modified nanomaterial, which is an emerging technology as a permeability regulator for pressure reduction and injection enhancement. The nanomaterial has a small particle size and can enter the nanoscale pore throat size of low-permeability, dense, shale reservoirs. By injecting a hydrophobic nano-silica dispersion into the formation, reservoir pressure reduction and injection enhancement can be achieved. The modified nano-drag reduction and injection enhancement permeability regulator uses nanoparticles as the core and introduces wettability-changing functional groups as modifiers to form modified nano-polysilicon. The "in-situ surface modification" + "chemical deaggregation" technology is used to obtain small-particle, modified nano-polysilicon that can be dispersed in water. The surface of the modified nano-silica particles has unsaturated bonds and strong adsorption capacity. They enter the hydration layer on the surface of the rock pores in the formation and undergo competitive adsorption, replacing the original hydration film to form a strong new nano-particle adsorption layer. Specifically, the hydrophobic nanoparticles change the flow characteristics of the pores, break through the repulsive force of the water film on the pore surface, and the nano-lattice structure of the nanoparticles enhances the lubrication effect of the rock surface, increases the overall flow rate, and increases the water flow, which has the effect of reducing pressure and increasing injection. The formation of the nano-layer of the lattice structure also effectively prevents the contact between water and clay minerals on the rock surface, reduces the probability of clay hydration expansion and the shedding of surface clay particles, reduces the chance of particle migration blocking the formation pores, reduces the flow resistance, and achieves the effect of reducing pressure and increasing injection. Based on the liquid phase in-situ surface modification technology, when the nanoparticles are initially generated, the organic compounds are combined with their surface atoms by chemical bonds to obtain surface modified nanomaterials. By using functional modifiers, the nanomaterials are given lipophilic and hydrophobic groups, water-dispersible groups and reactive functional groups, etc. The modifiers and nanomaterials can be combined through weak chemical bonds such as covalent bonds, hydrogen bonds or dangling bonds and adsorption, etc., to derive a variety of nanomaterials with special functions, thereby realizing the controllable surface structure of nanoparticles and developing functional nanomaterials suitable for oil field development. However, this emerging hydrophobic modified nanomaterial also has certain defects, such as:

[0041] (1) Nanomaterials can effectively plug unconventional formations such as nano-micrometer pore throats, but nanoparticles have high surface energy and a serious tendency to agglomerate, which greatly limits their compatibility with dispersants. Stable and well-dispersed modified nanomaterial permeability regulators are affected by the size and shape of the nanomaterials themselves, modification, nanomaterial concentration, pH, and temperature.

[0042] (2) The amount of nanomaterials required to play the role of highly efficient modified nano-drag reduction and injection permeability regulator in mine applications is large and costly, and they can rarely be directly used in oil field development.

[0043] Based on the defects of the plugging agents used in oil fields in the prior art, the technical solution of the present invention is proposed, which will be described in the following in the form of specific embodiments.

[0044] the term:

[0045] CHCl3: chloroform;

[0046] DCC: N,N'-dicyclohexylcarbodiimide;

[0047] NHS: N-hydroxysuccinimide.

[0048] Example 1 Bacillus licheniformis multilayer biofilm extraction

[0049] 1. Culture of Bacillus licheniformis

[0050] 1.1 Culture medium

[0051] LB medium: 3 g tryptone, 0.5 g yeast extract, 1 g sodium chloride, 5 h urea, 4 g glucose, and 1000 mL distilled water, pH 7.0 ± 0.5;

[0052] Seed culture medium: 1 g tryptone, 13 g sucrose, 1 g sodium chloride, 15 g corn starch, 16 g corn syrup powder, 20 g soybean cake powder, 0.5 g beef extract and 1000 mL distilled water, pH 7.0 ± 0.5;

[0053] Fermentation medium: 20 g glucose, 5 g corn starch, 20 g soybean cake powder, 5 g urea, 6 g dipotassium hydrogen phosphate, 3 g potassium dihydrogen phosphate, 0.5 g yeast extract, 1 g magnesium sulfate, 1 g calcium carbonate, make up to 1000 mL with distilled water, pH 7.0±0.5.

[0054] 1.2 Activation and cultivation of bacterial strains

[0055] Take out the tube of Bacillus licheniformis (common commercial strain) stored in the ultra-low temperature refrigerator, inoculate it into LB culture medium with an inoculation loop under sterile conditions, maintain at 37°C, and culture for 24 hours to activate the strain; make the activated strain into a suspension with sterilized physiological saline under sterile conditions, and bathe it in 90°C water for 8 minutes; take a loop of activated and sterilized strain, inoculate it into the seed culture medium with an inoculation loop under sterile conditions, maintain at 37°C, 180rpm, and culture for 15 to 24 hours (20 hours in this experiment).

[0056] 1.3 Fermentation culture

[0057] The cultured bacteria were inoculated into the fermentation medium at a rate of 1%, maintained at 37°C, 260 rpm, and shaken for 18 to 24 h (20 h in this experiment). The turbidity of the culture solution was observed or the optical density (OD 600 ) to monitor the growth of the bacteria until the bacteria in the culture medium reach the logarithmic growth phase. The containers of the seed culture medium and the fermentation medium are both triangular flasks.

[0058] Based on phenotypic characteristics and 16S rDNA gene sequence analysis, it was identified as Bacillus licheniformis, a Gram-positive bacillus.

[0059] 2. Extraction of multilayer biofilm of Bacillus licheniformis

[0060] (1) Bacterial cell removal: After the fermentation culture is completed, the culture medium contains a large amount of biofilm and bacterial cells. The bacterial cells are separated from the culture medium using a centrifuge (8000 rpm, 20 min) and the supernatant is collected.

[0061] (2) Precipitation and collection: Slowly add the supernatant to ice ethanol at a volume ratio of 1:2 (1 part supernatant, 2 parts ethanol), stirring gently during the addition process to allow the biofilm to precipitate from the solution. Leave the mixture at 4°C overnight to promote the precipitation of the biofilm. Centrifuge again using a centrifuge (8000rpm, 20min~30min), and the precipitate is the crude biofilm. Pour off the supernatant and carefully collect the precipitate.

[0062] (3) Purification and preservation: Resuspend the precipitated biofilm in a small amount of distilled water and gently stir until completely dissolved. Place the dissolved biofilm solution in a dialysis bag and dialyze with distilled water for 24 to 26 hours, changing the dialysis water several times during the process. Dialysis removes low molecular weight impurities, salts, and small molecular compounds. After dialysis, place the biofilm solution in a freeze-drying bottle and use a freeze dryer to completely remove the water in the biofilm solution to obtain a powdered product. The obtained product powder is divided into sterile bottles, sealed and stored in a dry room or a refrigerator at 4°C for long-term storage.

[0063] Example 2 Preparation of hydrophobically modified Bacillus licheniformis multilayer biofilm

[0064] Modified Bacillus licheniformis biofilm hydrophobic group grafting: Take 1g of biofilm dry powder, dissolve it in 50mL of deionized water, stir evenly, and use a magnetic stirrer to heat at 50℃ until it is completely dissolved to form a biofilm aqueous solution. Adjust the pH value of the biofilm aqueous solution to pH 7.5 using 0.1M sodium carbonate solution. The slightly alkaline environment helps to deprotonate the carboxyl or amino groups in the biofilm and increase the reaction activity. Dissolve 0.1g of dodecyl chloride (dodecyl chloride was used in this experiment, CHCl3) in 50mL of CHCl3. 12 H 25Cl), stir evenly. Add 0.2g DCC and 0.1NHS to the CHCl3 solution to promote carboxyl activation and stir until completely dissolved. The mixed solution of dodecyl chloride, DCC and NHS is slowly added dropwise to the biofilm aqueous solution through a burette, the stirring speed is controlled at 400rpm, the temperature is maintained at 40°C, and the temperature is maintained by heating in a water bath. The dropping process is completed in about 30min. Continue to stir the reaction mixture and keep it at 40°C for 4h. Dodecyl chloride undergoes amidation reaction with the carboxyl or amino group in the biofilm to achieve covalent grafting.

[0065] Add 100 mL of phosphate buffer (PBS, pH 7.4) to terminate the reaction and restore the pH value of the solution to neutral to prevent overreaction. The biofilm aqueous solution was precipitated with 100 mL of ice ethanol and allowed to stand for 4 hours to allow the grafted biofilm to precipitate from the solution. The reaction mixture was centrifuged at 8000 rpm for 20 minutes and the precipitate was collected. Repeat the washing with ice ethanol for 3 to 5 times to remove the unreacted dodecylating agent and by-products. The washed biofilm precipitate was placed in a freeze dryer for drying until a dry powdery modified product was obtained, which was a hydrophobically modified Bacillus licheniformis multilayer biofilm.

[0066] Example 3 Preparation of hydrophobically modified Bacillus licheniformis multilayer biofilm

[0067] Modified Bacillus licheniformis multilayer biofilm hydrophobic group grafting: Take 2.5g biofilm dry powder, dissolve it in 80mL deionized water, stir evenly, and use a magnetic stirrer to heat at 55℃ until it is completely dissolved to form a biofilm aqueous solution. Adjust the pH value of the biofilm aqueous solution to pH 7.8 using 0.2M sodium carbonate solution. The slightly alkaline environment helps to deprotonate the carboxyl or amino groups in the biofilm and increase the reaction activity. Dissolve 0.1g octyl chloride (1-octyl chloride was used in this experiment, C8H 17 Cl), stir evenly. Add 0.5g DCC and 0.25g NHS to the CHCl3 solution to promote carboxyl activation and stir until completely dissolved. The mixed solution of octyl chloride, DCC and NHS is slowly added dropwise to the biofilm aqueous solution through a burette, the stirring speed is controlled at 400rpm, the temperature is maintained at 45°C, and the temperature is maintained by heating in a water bath. The addition process is completed in about 30 minutes. Continue to stir the reaction mixture and keep it at 45°C for 4 hours. Octyl chloride will undergo amidation reaction with the carboxyl or amino group in the biofilm to achieve covalent grafting.

[0068] 125 mL of phosphate buffer (PBS, pH 7.4) was added to terminate the reaction, and the pH value of the solution was restored to neutral to prevent overreaction. The biofilm aqueous solution was precipitated with 200 mL of ice ethanol and allowed to stand for 5 hours to allow the grafted biofilm to precipitate from the solution. The reaction mixture was centrifuged at 8000 rpm for 25 min and the precipitate was collected. The washing was repeated 4 times with ice ethanol to remove unreacted octylating agent and by-products. The washed biofilm precipitate was placed in a freeze dryer and dried until a dry powdered modified biofilm product was obtained, which was a hydrophobic modified Bacillus licheniformis multilayer biofilm.

[0069] Example 4 Preparation of hydrophobically modified Bacillus licheniformis multilayer biofilm

[0070] Modified Bacillus licheniformis biofilm hydrophobic group grafting: Take 4g of biofilm dry powder, dissolve it in 100mL of deionized water, stir evenly, and use a magnetic stirrer to heat at 60℃ until it is completely dissolved to form a biofilm aqueous solution. Adjust the pH value of the biofilm aqueous solution to pH 8.0 using 0.3M sodium carbonate solution. The slightly alkaline environment helps to deprotonate the carboxyl or amino groups in the biofilm and increase the reaction activity. Dissolve 0.1g of hexadecyl chloride (hexadecyl chloride was used in this experiment, CHCl3) in 50mL of CHCl3. 16 H 33 Cl), stir evenly. Add 0.8g DCC and 0.5g NHS to the CHCl3 solution to promote carboxyl activation and stir until completely dissolved. The mixed solution of hexadecyl chloride, DCC and NHS is slowly added dropwise to the biofilm aqueous solution through a burette, the stirring speed is controlled at 400rpm, the temperature is maintained at 45°C, and the temperature is maintained by heating in a water bath. The addition process is completed in about 30 minutes. Continue to stir the reaction mixture and keep it at 45°C for 6 hours. Hexadecyl chloride will undergo amidation reaction with the carboxyl or amino group in the biofilm to achieve covalent grafting.

[0071] Add 150 mL of phosphate buffer (PBS, pH 7.4) to terminate the reaction and restore the pH value of the solution to neutral to prevent overreaction. The biofilm aqueous solution was precipitated with 300 mL of ice ethanol and allowed to stand for 6 hours to allow the grafted biofilm to precipitate from the solution. The reaction mixture was centrifuged at 8000 rpm for 30 minutes and the precipitate was collected. Repeat the washing with ice ethanol for 3 to 5 times to remove the unreacted hexadecylating agent and by-products. The washed biofilm precipitate was placed in a freeze dryer for drying until a dry powdered modified biofilm product was obtained, which was a hydrophobically modified Bacillus licheniformis multilayer biofilm.

[0072] During the modification process of the above-mentioned Examples 2 to 4, it should be noted that: be careful when ethanol precipitation is performed to avoid stirring too fast to cause co-precipitation of proteins or other macromolecular impurities. Keep sufficient time during dialysis to ensure the complete removal of small molecule impurities. The dripping speed is appropriate to prevent excessive reaction or excessive local concentration, which leads to uneven grafting. The use of organic solvents must be carried out in a fume hood. Controlling the temperature and pH value is the key to the success of the grafting reaction.

[0073] The hydrophobically modified Bacillus licheniformis multilayer biofilm prepared in Examples 2 to 4 above was used as a permeability regulator to perform performance verification, as follows:

[0074] 1. Super hydrophobic effect

[0075] (1) Methods

[0076] The grafted multilayer biofilm is dried and prepared into powder or film, and the sample is treated with gold spraying to make its surface conductive. Scanning is performed using SEM, and the surface morphology is observed at an appropriate magnification. Representative strongly hydrophilic dry reservoir cores are selected and immersed in a solution with hydrophobic modified Bacillus licheniformis multilayer biofilm as a permeability regulator for 1 to 3 days. The contact angle of the treated core is measured using a contact angle meter. The contact angle changes of the core before and after treatment are compared.

[0077] (2) Results

[0078] Through scanning electron microscopy, the morphology of Examples 2 to 4 was successfully grafted with hydrophobic modification groups. Due to the presence of hydrophobic groups, the surface of the grafted multilayer biofilm showed a rougher, irregular, and obviously protruding morphology and clustering phenomenon, with a layered distribution, such as Figure 1 As shown in Table 1, the hydrophobic ability of the hydrophobically modified Bacillus licheniformis multilayer biofilm as a permeability regulator is shown in Table 1. The formed super-hydrophobic biofilm permeability regulator changes the wettability, and the water flow forms an outward spherical surface. When the pore diameter changes, a liquid resistance phenomenon (Jiamin effect) is generated to increase the water flow resistance. After Examples 2 to 3 are soaked in the permeability regulator for 24 hours, the contact angle is always greater than 120°, showing good super-hydrophobicity, among which Example 2 has the best super-hydrophobic performance.

[0079] Table 1 Hydrophobicity of Bacillus licheniformis multilayer biofilms with different hydrophobic modifications

[0080]

[0081] 2. Biofilm analysis

[0082] (1) Methods

[0083] A standard microscope slide (75×25 mm) was immersed in a 250 mL conical flask containing 50 mL LB medium, the flask was sterilized and inoculated with the hydrophobically modified Bacillus licheniformis multilayer biofilm prepared in Examples 2 to 4. After static culture at 37°C for 72 hours, the slide was removed from the flask and washed with distilled water. The biofilm formed on the slide was stained with LIVE / DEAD Baclight. Images of the biofilm formed on the slide were collected by confocal microscopy. The image analysis software package COMSTAT was used to quantify the biofilm structure.

[0084] (2) Results

[0085] COMSTAT analysis of the biofilm structure showed that the biofilms formed in Examples 2 to 4 had a thickness of 27.72 to 36.23 μm, a roughness factor of 0.06 to 0.08, and a biomass of 8.91 to 10.58 μm 3 / μm 2 The hydrophobically modified Bacillus licheniformis multilayer biofilm formed a dense, uniform and fused biofilm. Among them, the biofilm formed in Example 2 had the largest thickness, a moderate roughness coefficient, and a large number of organisms, as shown in Table 2.

[0086] Table 2 Biofilm structure of multilayer biofilms of Bacillus licheniformis with different hydrophobic modifications

[0087]

[0088] 3. Ability to inhibit hydration expansion

[0089] (1) Methods

[0090] After the cores were immersed in pure water and 0.6% hydrophobically modified Bacillus licheniformis multilayer biofilm (Examples 2 to 4) for 72 hours, the cores were placed in a triaxial compression test device to simulate the real conditions in the formation. By controlling different lateral pressures, the compressive resistance of the cores under multi-axial stress was analyzed, and the inhibition effect of core hydration expansion was evaluated.

[0091] (2) Results

[0092] The 0.6% hydrophobically modified Bacillus licheniformis multilayer biofilm can have a good protective effect on the original state of hair and flakes of shale illite and illite-montmorillonite mixed layers and their primary pore structure, e.g. Figure 2 The hydration expansion inhibition effects of Examples 2 to 4 are shown in Table 3. The results show that the compressive strength of the original core is 164Mpa. After being soaked in Examples 2 to 4, the compressive strength of the core is improved to varying degrees.

[0093] Table 3 Compressive strength of cores after immersion in multilayer biofilms of different hydrophobically modified Bacillus licheniformis

[0094]

[0095] 4. Permeability adjustment and water control and oil increase capabilities

[0096] (1) Methods

[0097] The oil displacement experiment of the hydrophobically modified Bacillus licheniformis multilayer biofilm prepared in Examples 2 to 4 was carried out in a low permeability (porosity of about 10 to 20%, permeability of about 20 mD) core. Compared with the injection of formation water or simulated liquid into the core, the experimental oil was kerosene, and the temperature was about 25°C, and the injection pressure and core recovery rate were recorded.

[0098] (2) Results

[0099] The oil-water permeability experiment found that the hydrophobic modified Bacillus licheniformis multilayer biofilm prepared in Examples 2 to 4 has a good water control and oil increase effect in low permeability cores, and can effectively reduce the relative permeability of the water phase and increase the relative permeability of the oil phase, as shown in Table 4, where n w and n o are the phase permeability shape factors of the water phase and the oil phase, respectively. The higher the shape factor, the worse the fluidity of the fluid; K rw * and K ro * are the permeability endpoint values ​​of the water phase and the oil phase respectively. The lower the endpoint value, the worse the fluidity of the fluid. Due to the formation of a superhydrophobic biofilm, the kinetic energy and flow energy of the crude oil are greatly improved, and the flow resistance of the water phase will increase to a certain extent.

[0100] Table 4 Changes in phase permeability parameters of multilayer biofilms of Bacillus licheniformis with different hydrophobic modifications

[0101] Example <![CDATA[n w ]]> <![CDATA[n o ]]> <![CDATA[K rw ]]> <![CDATA[K ro ]]> Example 2 2.8 1.8 0.889 0.209 Example 3 2.5 2.5 0.834 0.260 Example 4 2.7 2 0.872 0.252

[0102] The recovery rate in the low permeability core is shown in Table 5. The recovery rate of the first water drive is only 10.13% to 12.61%. After the permeability regulator is injected, the recovery rate is improved, and the improvement rate is more than 20%. Example 2 has the highest recovery rate of 37.13%, and the recovery rate is increased by 24.55%. This is because the formed biofilm is adsorbed on the surface of the reservoir rock pores and repairs the surface roughness, so that the flow resistance during water injection is reduced and the injection pressure is reduced. At the same time, the hydrophobic effect causes the residual oil droplets in the unswept or low-swept residual oil zone to aggregate and form oil segment plugs, thereby enhancing the flow capacity of the oil phase.

[0103] Table 5 Changes in recovery rate after infiltration of multilayer biofilms with different hydrophobic modifications of Bacillus licheniformis

[0104]

[0105] It can be seen from the experimental schemes and experimental data of the above embodiments that the present invention uses Bacillus licheniformis as a production strain of biofilm to produce a multilayer biofilm with specific properties. The multilayer biofilm has high viscosity and high molecular weight, can form a stable continuous gel-like film on the rock surface, and has good compatibility with the reservoir. By excessively grafting hydrophobic groups onto the multilayer biofilm, its surface properties are changed to make it exhibit superhydrophobicity, thereby increasing the flow resistance of the water phase in the pore throats and achieving the effect of "controlling water without blocking". The nanoscale size of the hydrophobically modified Bacillus licheniformis multilayer biofilm enables it to penetrate into the nano-micro pores of the low permeability reservoir to form a dense and stable hydrophobic biofilm; in larger pores, the high permeability channels can be blocked by polymerization characteristics, thereby regulating the flow of fluids and improving the oil recovery effect.

[0106] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A hydrophobically modified Bacillus licheniformis multilayer biofilm, characterized in that: The hydrophobic group is obtained by grafting a hydrophobic group onto the biofilm secreted by Bacillus licheniformis, wherein the hydrophobic group includes a long-chain alkyl group with more than 7 carbon atoms.

2. A method for preparing a hydrophobically modified Bacillus licheniformis multilayer biofilm, characterized in that: The method comprises the steps of grafting a hydrophobic group on the biofilm secreted by Bacillus licheniformis, wherein the hydrophobic group comprises a long-chain alkyl group with more than 7 carbon atoms.

3. The preparation method according to claim 2, characterized in that: The preparation method comprises the following steps: dissolving the biofilm secreted by Bacillus licheniformis with water and adjusting the pH value to be alkaline to obtain a biofilm aqueous solution; Dissolving a long-chain alkyl compound containing a hydrophobic group, N,N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide in an organic solvent, and then dripping the mixture into the biofilm aqueous solution, stirring the mixture in a water bath to carry out a grafting reaction; After adjusting the pH value of the reaction solution to neutral to terminate the reaction, ethanol is added to the reaction solution, the solution is allowed to stand, centrifuged, and the precipitate is collected to obtain a hydrophobically modified Bacillus licheniformis multilayer biofilm.

4. The preparation method according to claim 3, characterized in that: The biofilm secreted by Bacillus licheniformis is obtained by the following steps: After inoculating the fermentation medium with Bacillus licheniformis, the culture medium is shaken and cultured, the culture medium is separated, and the supernatant is collected; The supernatant is mixed with ethanol, stirred and separated to obtain a precipitate, which is then dialyzed and dried to obtain the biofilm secreted by the Bacillus licheniformis.

5. The preparation method according to claim 3, characterized in that: The ratio of the biofilm secreted by the Bacillus licheniformis to water is (1-4) g: (50-150) mL; and / or the alkaline pH value is 7.0-8.

0.

6. The preparation method according to claim 3, characterized in that: The ratio of the long-chain alkyl compound containing a hydrophobic group, N,N'-dicyclohexylcarbodiimide, N-hydroxysuccinimide and the organic solvent is 0.1g: (0.2-0.8)g: (0.1-0.5)g: (40-50)mL; and / or the conditions of the grafting reaction are: a stirring speed of 400-500rpm, a water bath temperature of 40-45°C, and a reaction time of 4-6h.

7. Use of the hydrophobically modified Bacillus licheniformis multilayer biofilm according to claim 1 in preparing a permeability regulator for low-permeability oil reservoirs.

8. A permeability regulator for low permeability oil reservoirs, characterized in that: A multilayer biofilm containing the hydrophobically modified Bacillus licheniformis according to claim 1.

9. Use of the hydrophobically modified Bacillus licheniformis multilayer biofilm according to claim 1 or the permeability regulator according to claim 8 in any of the following: (1) Application in improving oil recovery effect in low permeability reservoirs; (2) Application in improving the water control and oil production increase effect of low permeability core; (3) Application in improving the compressive strength of low-permeability cores; (4) Application in improving the permeability of low permeability reservoirs.

10. A method for improving the permeability of a low-permeability oil reservoir, characterized in that: The method comprises the step of applying the hydrophobically modified Bacillus licheniformis multilayer biofilm according to claim 1 or the permeability regulator according to claim 8 to a low permeability oil reservoir.

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