CN111763240B - Sodium lauroyl sarcosinate-diglucoside peptide compound and composite oil displacement agent - Google Patents
Sodium lauroyl sarcosinate-diglucoside peptide compound and composite oil displacement agent Download PDFInfo
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- CN111763240B CN111763240B CN201910261710.2A CN201910261710A CN111763240B CN 111763240 B CN111763240 B CN 111763240B CN 201910261710 A CN201910261710 A CN 201910261710A CN 111763240 B CN111763240 B CN 111763240B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0802—Tripeptides with the first amino acid being neutral
- C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
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- C09K8/00—Compositions for drilling of boreholes or wells; Compositions for treating boreholes or wells, e.g. for completion or for remedial operations
- C09K8/58—Compositions for enhanced recovery methods for obtaining hydrocarbons, i.e. for improving the mobility of the oil, e.g. displacing fluids
- C09K8/584—Compositions for enhanced recovery methods for obtaining hydrocarbons, i.e. for improving the mobility of the oil, e.g. displacing fluids characterised by the use of specific surfactants
Abstract
The invention provides a sodium lauroyl sarcosinate-diglucoside peptide compound and a composite oil displacement agent. The chemical structural formula of the sodium lauroyl sarcosinate-diglucoside peptide compound is as follows:
the invention provides a sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent containing a sodium lauroyl sarcosinate-diglucoside peptide compound obtained by reacting sodium lauroyl sarcosinate with diglucoside peptide. The compound oil displacement agent can be well compounded with oil extraction functional bacteria to form a microorganism-chemical compound oil displacement agent. The microbial-chemical compound oil displacement agent has the effects of degrading and reducing viscosity of crude oil by microbial flooding and the effect of emulsifying and reducing viscosity of chemical flooding, enlarges swept volume and dispersion efficiency of the chemical oil displacement agent in an oil layer by a biological carrying mode, effectively improves synergistic reaction efficiency of microbes and chemical agents, and realizes 1+1>2, in the process.
Description
Technical Field
The invention belongs to the technical field of tertiary oil recovery, relates to preparation and application of a microorganism-chemical compound oil displacement agent, and particularly relates to a sodium lauroyl sarcosinate-diglucoside peptide compound and a compound oil displacement agent.
Background
Along with the rapid development of national economy, the demand of petroleum is continuously increased, the contradiction between petroleum consumption and supply is more prominent, and in order to solve the contradiction, besides increasing exploration strength and continuously searching petroleum resources, the method is also one of effective ways for improving the crude oil recovery ratio of developed oil fields.
The Microbial enhanced oil recovery-MEOR (Microbial enhanced oil recovery-MEOR) is a technology for improving the recovery of crude oil by utilizing the natural biochemical action (biodegradation and metabolites) of living cells of microorganisms. The microorganism and/or nutrient solution with oil extraction function is injected into the oil reservoir, so that oil extraction functional bacteria grow and reproduce in the oil reservoir, the physical (surface and interface properties) and chemical properties (crude oil components) of crude oil are changed through the biochemical action of the microorganism, the molecular chain length and the shear viscosity of the crude oil are reduced, the fluidity of the crude oil is improved, and the recovery ratio of the crude oil is improved.
Although the microbial oil recovery technology can well solve the problem of crude oil fluidity of heavy oil reservoirs such as Xinjiang oil fields, the chemical oil displacement agent is still required to be matched for oil displacement in order to achieve higher crude oil recovery. Although the simple mixing of the two components has the advantages of simple operation and low operation cost, the diffusion speeds of the two components after entering an oil layer are greatly different, the separation is easy to occur, and the synergistic oil displacement effect cannot be achieved. How to realize the good combination of microbial oil displacement and chemical oil displacement and improve the recovery ratio of crude oil become important problems which people pay attention to widely and need to solve urgently.
The chemical oil-displacing agent is modified on the surface of the microorganism with the oil extraction function, and enters an oil layer through the synergistic loading of the microorganism, so that the problem of separation of the chemical oil-displacing agent and the microorganism is solved, and the important way of realizing the synergistic oil displacement of the chemical oil-displacing agent and the microorganism is realized.
However, the addition of chemical oil displacement agents and the reaction process thereof often destroy the physiological functions of microorganisms, reduce the oil displacement effect of the microorganisms, and even cause the microorganisms to die in a large scale, so that the microbial oil displacement cannot be realized. In addition, the addition of the chemical oil displacement agent can also cause the reduction of the movement capacity and the permeability of microorganisms, and the substantial oil displacement effect on low-permeability oil reservoirs is difficult to generate. Therefore, the selection of chemical agents, the addition method, the control of the addition amount and the control of reaction conditions become the key points of the microbial-chemical complex oil displacement.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a sodium lauroyl sarcosinate-diglucoside peptide compound.
The invention also aims to provide the sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent which can be well compounded with oil extraction functional bacteria to form a microorganism-chemical composite oil displacement agent.
The invention also aims to provide a microbial-chemical composite oil displacement agent suitable for oil displacement of oil fields, in particular to oil displacement of high-viscosity oil fields.
In order to solve the technical problems, the invention is realized by adopting the following technical scheme.
The invention provides a sodium lauroyl sarcosinate-diglucoside peptide compound. The structural formula of the sodium lauroyl sarcosinate-diglucoside peptide compound is as follows:
the sodium lauroyl sarcosinate-diglucoside peptide compound with the structure can be obtained by reacting sodium lauroyl sarcosinate with diglucoside peptide, and the chemical reaction formula is as follows:
the invention provides a sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent, which contains a sodium lauroyl sarcosinate-diglucoside peptide compound obtained by reacting sodium lauroyl sarcosinate with diglucoside peptide; preferably, the sodium lauroyl sarcosinate-diglucoside peptide compound is the sodium lauroyl sarcosinate-diglucoside peptide compound with the structure.
The invention provides a preparation method of the sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent, which comprises the following steps:
adding sodium lauroyl sarcosinate into a diglucoside peptide solution to react to obtain the sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent, wherein the molar ratio of the diglucoside peptide to the sodium lauroyl sarcosinate is 1: 1-1.8.
In the above preparation method, preferably, the reaction is carried out at 37 to 40 ℃ for 6 to 12 hours.
In the above preparation method, preferably, the mass concentration of the diglucoside peptide solution is 40% -60%; in the preparation method, the mass concentration of the diglucoside peptide solution does not affect the reaction, but the final yield of the reaction is influenced to a certain extent.
In the above preparation method, preferably, the diglucoside peptide solution is formed by dissolving diglucoside peptide in water, wherein the diglucoside peptide is prepared by the following steps:
1) culturing the bacterial strain inoculated into the culture medium in an air bath shaker until the bacteria grow to the late logarithmic phase, centrifuging, and collecting the bacteria;
2) washing the collected thallus with dilute sulfuric acid to remove impurities, and suspending the thallus to prepare a suspension;
3) subpackaging the suspension, centrifuging and collecting lower layer bacterial bodies;
4) eluting the collected lower layer bacterial body, performing centrifugal separation, taking the upper layer capsular solution, performing chromatographic separation and purification, and obtaining the diglucoside peptide.
In the above method for preparing the diglucoside peptide, preferably, the centrifugation in step 1) is performed at 6000-10000 rpm for 10-20 min; further preferably, the centrifugation is at 8000 rpm for 15 min.
In the above-mentioned method for producing a diglucoside peptide, preferably, the medium used in step 1) is 200 mL.
In the above-mentioned method for producing a diglucoside peptide, preferably, the strain inoculated into the culture medium in step 1) is cultured in a 500mL flask.
In the above-mentioned method for producing a diglucoside peptide, preferably, the strain in step 1) is any strain containing a diglucoside peptide in a metabolite.
In the above-mentioned method for preparing a diglucoside peptide, preferably, the culturing in step 1) is carried out at 63-68 ℃ and 180r/min (160-); further preferably, the culturing is carried out at 65 ℃ and 170 r/min.
In the above-mentioned method for producing a diglucoside peptide, preferably, the suspension in step 2) has an absorbance of 1 as measured at a wavelength of 600nm using a 1cm cuvette.
In the above method for producing a diglucoside peptide, preferably, the pH of the dilute sulfuric acid in step 2) is 2 to 3; further preferably, the pH of the dilute sulfuric acid is 2.
In the above method for preparing the diglucoside peptide, preferably, the centrifugation in step 3) is performed at 6000-; further preferably, the centrifugation is at 8000 revolutions per minute for 10 min.
In the above-mentioned method for producing a diglucoside peptide, the split charging in step 3) is preferably 20mL per portion.
In the above method for preparing the diglucoside peptide, preferably, the elution in step 4) is performed by eluting with clear water at 38-40 ℃ for 10-12 h; further preferably, the elution is carried out at 40 ℃ for 12h with clear water.
In the above method for preparing the diglucoside peptide, when the chromatographic separation is performed in the step 4), the retention time of the upper layer capsular solution can be determined according to the retention time of a diglucoside peptide standard sample. In chromatographic analysis, the time from the start of sample injection to the time when the concentration of the component is at its maximum after the column, i.e. the time from the start of sample injection to the time when the peak of a chromatographic peak of a component appears, is referred to as the retention time of the component.
The lauroyl sarcosine sodium-diglucoside peptide compound oil displacement agent obtained by the preparation method can be purified to obtain the lauroyl sarcosine sodium-diglucoside peptide compound.
The invention also provides a microbial-chemical composite oil-displacing agent, which is prepared by compounding the sodium lauroyl sarcosine-diglucoside peptide composite oil-displacing agent and oil-producing functional bacteria.
In the microbial-chemical composite oil-displacing agent, the sodium lauroyl sarcosinate-diglucoside peptide composite oil-displacing agent is preferably prepared by the preparation method of the sodium lauroyl sarcosinate-diglucoside peptide composite oil-displacing agent.
In the above-mentioned microbial-chemical complex oil-displacing agent, the sodium lauroyl sarcosinate-diglucoside peptide compound in the sodium lauroyl sarcosinate-diglucoside peptide complex oil-displacing agent is preferably modified on the surface of the oil-producing functional bacteria.
In the above-mentioned microbial-chemical complex oil-displacing agent, preferably, the microbial-chemical complex oil-displacing agent is prepared by the following preparation method:
1) adding 200mL of culture medium inoculated with the strain into a 500mL triangular flask, placing the triangular flask in an air bath shaker for culture at 65 ℃ and 170r/min, when the bacteria grow to the late logarithmic phase, centrifuging at 8000r/min for 15min to collect cells, washing with dilute sulfuric acid (pH value is 2) to remove impurities, suspending the bacteria, preparing suspension (1cm cuvette) with the light absorption value of 1 at the wavelength of 600nm, subpackaging 20mL of each part, centrifuging at 8000 rpm for 10min, and collecting the bacteria at the lower layer;
2) eluting the bacterial body with clear water at 40 deg.C for 12h, centrifuging, collecting the upper layer of capsular solution, performing chromatographic separation, and purifying to obtain diglucoside peptide (in the chromatographic separation process, the retention time of the upper layer of capsular solution can be determined according to the retention time of diglucoside peptide standard sample);
3) placing a diglucoside peptide solution with the mass concentration of 40-60% in a water bath at 39 ℃, adding sodium lauroyl sarcosinate with the mass of 3 times that of the diglucoside peptide, and reacting for 12 hours to obtain the sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent;
4) adding the sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent obtained in the step 3) into a bacterial culture medium, transferring into an oil extraction functional bacterial agent to form a mixture, wherein the volume ratio of the sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent to the oil extraction functional bacterial agent is 10:1-5:1, culturing and proliferating for 24-36h until the mixture in the culture medium has an absorbance value of 1 measured by a 1cm cuvette under the wavelength of 600nm, and obtaining the microorganism-chemical composite oil displacement agent.
The invention also provides a preparation method of the microbial-chemical composite oil displacement agent, which comprises the following steps:
adding the sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent into a bacterial culture medium, transferring oil extraction functional bacteria to form a mixture, and culturing and proliferating to obtain the microorganism-chemical composite oil displacement agent.
In the above method for preparing a microbial-chemical composite oil-displacing agent, preferably, the culture proliferation is a culture proliferation in which the mixture in the culture medium has an absorbance of 1 measured at a wavelength of 600nm with a 1cm cuvette.
In the above method for preparing the microbial-chemical composite oil-displacing agent, the culture and proliferation time is preferably 24-36 h.
In the preparation method of the microorganism-chemical compound oil-displacing agent, preferably, the transferring of the oil-producing functional bacteria is performed by transferring an oil-producing functional bacteria microbial inoculum, wherein the volume ratio of the sodium lauroyl sarcosinate-diglucoside peptide compound oil-displacing agent to the oil-producing functional bacteria microbial inoculum is 10:1-5: 1; more preferably, the volume ratio is 10: 1.
In the preparation method of the microbial-chemical composite oil-displacing agent, the sodium lauroyl sarcosinate-diglucoside peptide composite oil-displacing agent is preferably prepared by the preparation method of the sodium lauroyl sarcosinate-diglucoside peptide composite oil-displacing agent.
The technical scheme provided by the invention researches the oil displacement by taking the synergistic effect of microorganisms and chemical agent oil into consideration, provides a brand-new microorganism-chemical compound oil displacement system, and provides a solution for improving the crude oil recovery ratio of an oil field.
Compared with the prior art, the invention has the following beneficial effects:
(1) the method is simple, has strong operability and applicability, and can meet the requirements of actual oil field production.
(2) The microbial-chemical compound oil displacement agent provided by the invention has the effects of degrading and reducing viscosity of the microbial flooding on crude oil and the effects of emulsifying and reducing viscosity of the chemical flooding, and can enlarge the swept volume and the dispersion efficiency of a chemical agent in an oil layer in a biological carrying mode, effectively improve the synergistic reaction efficiency of the microbes and the chemical agent and realize the purpose of 1+1> 2.
(3) The sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent provided by the invention can well realize coexistence with oil extraction functional bacteria, and provides favorable conditions for growth and propagation of the oil extraction functional bacteria.
(4) The sodium lauroyl sarcosinate-diglucoside peptide compound provided by the invention has good emulsifying property on crude oil, is a good surfactant for oil displacement, and can be modified on the surface of oil extraction functional bacteria.
(5) The microbial-chemical composite oil displacement agent prepared by the preparation method successfully modifies the sodium lauroyl sarcosinate-diglucoside compound on the surface of the oil extraction functional bacteria.
Drawings
FIG. 1 is an infrared image of the sodium lauroyl sarcosinate-diglucoside peptide compound provided in example 1.
FIG. 2 is a comparison graph of the emulsification stability characterization curves of the microbial-chemical composite oil-displacing agent obtained by compounding the sodium lauroyl sarcosinate-diglucoside peptide composite oil-displacing agent with the oil recovery functional bacteria, water, the oil recovery functional bacteria and sodium lauroyl sarcosinate which are emulsified with white oil respectively.
FIG. 3 is a comparison graph of the effect of reducing crude oil viscosity of the microbial-chemical composite oil displacement agent, the oil recovery functional bacteria and sodium lauroyl sarcosinate obtained when the sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent is compounded with the oil recovery functional bacteria.
FIG. 4 is a comparison graph of oil displacement sweep efficiency of a microbial-chemical composite oil displacement agent and a sodium lauroyl sarcosinate chemical oil displacement agent which are obtained by compounding a sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent and oil extraction functional bacteria.
FIG. 5 is a graph showing the comparison of the load efficiency of sodium lauroyl sarcosinate-diglucoside peptide compound, sodium lauroyl sarcosinate and functional bacteria for oil recovery.
FIG. 6 is a comparison graph of the emulsified particle size distribution of the microbial-chemical composite oil displacement agent, the oil recovery functional bacteria and the crude oil containing sodium lauroyl sarcosinate, which is obtained by compounding the sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent and the oil recovery functional bacteria.
FIG. 7 is a physical simulation diagram of an oil displacement device used for sweep efficiency verification.
Fig. 8 is a schematic diagram of a sweep efficiency validation core injection slug.
FIG. 9 is a schematic diagram of a core injection slug from a sweep efficiency validation control experiment.
Detailed Description
The technical solutions of the present invention will be described in detail below in order to clearly understand the technical features, objects, and advantages of the present invention, but the present invention is not limited to the practical scope of the present invention.
Example 1
The embodiment provides a microorganism-chemical compound oil displacement agent, which is prepared by the following steps:
(1) adding 200mL of culture medium inoculated with the strain into a 500mL triangular flask, placing the triangular flask in an air bath shaker for culture at 65 ℃ and 170r/min, when the bacteria grow to the late logarithmic phase, centrifuging at 8000r/min for 15min to collect cells, washing with dilute sulfuric acid (pH value is 2) to remove impurities, suspending the bacteria, preparing a suspension (1cm cuvette) with the light absorption value of 1 at the wavelength of 600nm, subpackaging 20mL of each part, centrifuging at 8000r/min for 10min, and collecting the bacteria at the lower layer;
(2) eluting the bacterial body with clear water at 40 deg.C for 12h, centrifuging, collecting the upper layer capsular solution, performing chromatographic separation, and purifying to obtain diglucoside peptide (in the chromatographic separation process, the retention time of the upper layer capsular solution is determined according to the retention time of the diglucoside peptide standard sample);
(3) placing a 50% diglucoside peptide solution in a water bath at 39 ℃, adding sodium lauroyl sarcosinate with the mass of 3 times that of the diglucoside peptide, and reacting for 12 hours to obtain a sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent;
(4) adding the sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent into a bacterial culture medium, transferring the oil extraction functional bacterial agent into the bacterial culture medium to form a mixture, wherein the volume ratio of the sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent added into the bacterial culture medium to the oil extraction functional bacterial agent is 10:1, and culturing and proliferating for 24-36h until the mixture in the culture medium has an absorbance value of 1 measured by a 1cm cuvette under the wavelength of 600nm to obtain the microorganism-chemical composite oil displacement agent.
And (4) separating and purifying the sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent obtained in the step (3) by using a focusing chromatography method to obtain a sodium lauroyl sarcosinate-diglucoside peptide compound.
FIG. 1 is an infrared spectrum of the sodium lauroyl sarcosinate-diglucoside peptide compound provided in this example, which is clearly shown at 3391cm-1-3283cm-1Is an N-H stretching vibration absorption peak; 1653cm-1C is an O stretching vibration absorption peak; 1531cm-1The part is a C-C skeleton vibration absorption peak in aromatic hydrocarbon, 1404cm-1Is located at 1200cm of C-N telescopic vibration absorption peak-1、1137cm-1Is C-O telescopicAbsorption peak of vibration of 860cm-1-680cm-1The position is a C-H out-of-plane bending vibration absorption peak. These characteristic peaks indicate that synthesis under the conditions under which the product molecules synthesized were polymerized successfully to modify the diglucoside peptide with sodium lauroyl sarcosinate.
The relative molecular weights of the materials before and after the reaction were determined by the VPO method to further verify the success of the formation of the reaction product. The relative molecular mass of the sodium lauroyl sarcosinate is 291.6, the relative molecular mass of the diglucoside peptide is 131.0, and the relative molecular mass of the sodium lauroyl sarcosinate-diglucoside peptide compound is 376.8. Compared with sodium lauroyl sarcosinate, the relative molecular mass difference between the reaction product and the reaction product is 85.2, and the relative molecular mass of the side product in the reaction is similar to that of the diglucoside peptide, so that the diglucoside peptide can be further proved to successfully modify the sodium lauroyl sarcosinate.
Example 2
The embodiment provides a microorganism-chemical compound oil displacement agent, which is prepared by the following steps:
(1) adding 200mL of culture medium inoculated with the strain into a 500mL triangular flask, placing the triangular flask in an air bath shaker for culture at 65 ℃ and 170r/min, when the bacteria grow to the late logarithmic phase, centrifuging at 8000r/min for 15min to collect cells, washing with dilute sulfuric acid (pH value is 2) to remove impurities, suspending the bacteria, preparing a suspension (1cm cuvette) with the light absorption value of 1 at the wavelength of 600nm, subpackaging 20mL of each part, centrifuging at 8000r/min for 10min, and collecting the bacteria at the lower layer;
(2) eluting the bacterial body with clear water at 40 deg.C for 12h, centrifuging, collecting the upper layer capsular solution, performing chromatographic separation, and purifying to obtain diglucoside peptide (in the chromatographic separation process, the retention time of the upper layer capsular solution is determined according to the retention time of the diglucoside peptide standard sample);
(3) placing a 50% diglucoside peptide solution in a water bath at 37 ℃, adding sodium lauroyl sarcosinate with the mass of 2.45 times that of the diglucoside peptide, and reacting for 12 hours to obtain a sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent;
(4) adding the sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent into a bacterial culture medium, transferring into an oil extraction functional bacterial agent to form a mixture, wherein the volume ratio of the sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent added into the bacterial culture medium to the oil extraction functional bacterial agent is 10:1, and culturing and proliferating for 24-36h until the mixture in the culture medium has a light absorption value of 1 measured by a 1cm cuvette under the wavelength of 600nm to obtain the microorganism-chemical composite oil displacement agent.
Example 3
The embodiment provides a microorganism-chemical compound oil displacement agent, which is prepared by the following steps:
(1) adding 200mL of culture medium inoculated with the strain into a 500mL triangular flask, placing the triangular flask in an air bath shaker for culture at 65 ℃ and 170r/min, when the bacteria grow to the late logarithmic phase, centrifuging at 8000r/min for 15min to collect cells, washing with dilute sulfuric acid (pH value is 2) to remove impurities, suspending the bacteria, preparing a suspension (1cm cuvette) with the light absorption value of 1 at the wavelength of 600nm, subpackaging 20mL of each part, centrifuging at 8000r/min for 10min, and collecting the bacteria at the lower layer;
(2) eluting the bacterial body with clear water at 40 deg.C for 12h, centrifuging, collecting the upper layer capsular solution, performing chromatographic separation, and purifying to obtain diglucoside peptide (in the chromatographic separation process, the retention time of the upper layer capsular solution is determined according to the retention time of the diglucoside peptide standard sample);
(3) placing a 50% diglucoside peptide solution in a water bath at 40 ℃, adding sodium lauroyl sarcosinate with the mass of 4 times that of the diglucoside peptide, and reacting for 6 hours to obtain a sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent;
(4) adding the sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent into a bacterial culture medium, transferring the oil extraction functional bacterial agent into the bacterial culture medium to form a mixture, wherein the volume ratio of the sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent added into the bacterial culture medium to the oil extraction functional bacterial agent is 10:1, and culturing and proliferating for 24-36h until the mixture in the culture medium has an absorbance value of 1 measured by a 1cm cuvette under the wavelength of 600nm to obtain the microorganism-chemical composite oil displacement agent.
Example 4
The embodiment provides a microorganism-chemical compound oil displacement agent, which is prepared by the following steps:
(1) adding 200mL of culture medium inoculated with the strain into a 500mL triangular flask, placing the triangular flask in an air bath shaker for culture at 65 ℃ and 170r/min, when the bacteria grow to the late logarithmic phase, centrifuging at 8000r/min for 15min to collect cells, washing with dilute sulfuric acid (pH value is 2) to remove impurities, suspending the bacteria, preparing a suspension (1cm cuvette) with the light absorption value of 1 at the wavelength of 600nm, subpackaging 20mL of each part, centrifuging at 8000r/min for 10min, and collecting the bacteria at the lower layer;
(2) eluting the bacterial body with clear water at 40 deg.C for 12h, centrifuging, collecting the upper layer capsular solution, performing chromatographic separation, and purifying to obtain diglucoside peptide (in the chromatographic separation process, the retention time of the upper layer capsular solution is determined according to the retention time of the diglucoside peptide standard sample);
(3) placing a 50% diglucoside peptide solution in a water bath at 38 ℃, adding sodium lauroyl sarcosinate with the mass of 2.8 times that of the diglucoside peptide, and reacting for 10 hours to obtain a sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent;
(4) adding the sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent into a bacterial culture medium, transferring the oil extraction functional bacterial agent into the bacterial culture medium to form a mixture, wherein the volume ratio of the sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent added into the bacterial culture medium to the oil extraction functional bacterial agent is 10:1, and culturing and proliferating for 24-36h until the mixture in the culture medium has an absorbance value of 1 measured by a 1cm cuvette under the wavelength of 600nm to obtain the microorganism-chemical composite oil displacement agent.
Example 5
The embodiment provides a microorganism-chemical compound oil displacement agent, which is prepared by the following steps:
(1) adding 200mL of culture medium inoculated with the strain into a 500mL triangular flask, placing the triangular flask in an air bath shaker for culture at 65 ℃ and 170r/min, when the bacteria grow to the late logarithmic phase, centrifuging at 8000r/min for 15min to collect cells, washing with dilute sulfuric acid (pH value is 2) to remove impurities, suspending the bacteria, preparing a suspension (1cm cuvette) with the light absorption value of 1 at the wavelength of 600nm, subpackaging 20mL of each part, centrifuging at 8000r/min for 10min, and collecting the bacteria at the lower layer;
(2) eluting the bacterial body with clear water at 40 deg.C for 12h, centrifuging, collecting the upper layer capsular solution, performing chromatographic separation, and purifying to obtain diglucoside peptide (in the chromatographic separation process, the retention time of the upper layer capsular solution is determined according to the retention time of the diglucoside peptide standard sample);
(3) placing a 50% diglucoside peptide solution in a water bath at 40 ℃, adding sodium lauroyl sarcosinate with the mass of 3.5 times that of the diglucoside peptide, and reacting for 9 hours to obtain a sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent;
(4) adding the sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent into a bacterial culture medium, transferring the oil extraction functional bacterial agent into the bacterial culture medium to form a mixture, wherein the volume ratio of the sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent added into the bacterial culture medium to the oil extraction functional bacterial agent is 10:1, and culturing and proliferating for 24-36h until the mixture in the culture medium has an absorbance value of 1 measured by a 1cm cuvette under the wavelength of 600nm to obtain the microorganism-chemical composite oil displacement agent.
Example 6
The embodiment provides a microorganism-chemical compound oil displacement agent, which is prepared by the following steps:
(1) adding 200mL of culture medium inoculated with the strain into a 500mL triangular flask, placing the triangular flask in an air bath shaker for culture at 65 ℃ and 170r/min, when the bacteria grow to the late logarithmic phase, centrifuging at 8000r/min for 15min to collect cells, washing with dilute sulfuric acid (pH value is 2) to remove impurities, suspending the bacteria, preparing a suspension (1cm cuvette) with the light absorption value of 1 at the wavelength of 600nm, subpackaging 20mL of each part, centrifuging at 8000r/min for 10min, and collecting the bacteria at the lower layer;
(2) eluting the bacterial body with clear water at 40 deg.C for 12h, centrifuging, collecting the upper layer capsular solution, performing chromatographic separation, and purifying to obtain diglucoside peptide (in the chromatographic separation process, the retention time of the upper layer capsular solution is determined according to the retention time of the diglucoside peptide standard sample);
(3) placing a 50% diglucoside peptide solution in a water bath at 39 ℃, adding sodium lauroyl sarcosinate with the mass of 3.6 times that of the diglucoside peptide, and reacting for 10 hours to obtain a sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent;
(4) adding the sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent into a bacterial culture medium, transferring the oil extraction functional bacterial agent into the bacterial culture medium to form a mixture, wherein the volume ratio of the sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent added into the bacterial culture medium to the oil extraction functional bacterial agent is 10:1, and culturing and proliferating for 24-36h until the mixture in the culture medium has an absorbance value of 1 measured by a 1cm cuvette under the wavelength of 600nm to obtain the microorganism-chemical composite oil displacement agent.
Verification experiment
Experimental groups:
the microorganism-chemical compound oil displacement agent obtained in the example 1 is purified, and the specific operation is as follows: centrifuging the microorganism-chemical compound oil-displacing agent obtained in example 1 at 8000r/min for 15min, collecting the cells at the lower layer, washing with dilute sulfuric acid (pH 2) to remove impurities, suspending the thalli to prepare a suspension (1cm cuvette) with the light absorption value of 1 at the wavelength of 600nm, transferring the suspension to a liquid culture medium again according to the volume ratio of the suspension to the liquid culture medium of 1:10, and culturing the suspension in a constant-temperature incubator at 37 ℃ for 48 hours to obtain the microorganism-chemical compound oil-displacing agent of the experimental group.
Control 1:
(1) adding 200mL of culture medium inoculated with the oil recovery functional bacterial strain into a 500mL triangular flask, placing the triangular flask in an air bath shaking table for culturing at 65 ℃ and 170r/min, when the oil recovery functional bacterial strain grows to the late logarithmic phase, centrifuging at 8000r/min for 15min to collect cells, washing with dilute sulfuric acid (pH value is 2) to remove impurities, suspending thalli, and preparing an oil recovery functional bacterial suspension (1cm cuvette) with the light absorption value of 1 at the wavelength of 600 nm;
(2) mixing 5 per mill of sodium lauroyl sarcosinate solution with the oil extraction functional bacteria suspension according to the mass ratio of 1:1, and carrying out water bath reaction at 40 ℃ for 10 hours to obtain a second oil extraction functional bacteria suspension;
(3) and eluting impurities from the second oil extraction functional bacteria suspension and purifying, wherein the specific operations are as follows: discarding the supernatant of the second oil recovery functional bacteria suspension obtained in the step 2), centrifuging for 10min at 8000r/min, collecting bacteria at the lower layer, washing with dilute sulfuric acid (pH value is 2) to remove impurities, suspending bacteria, preparing suspension (1cm cuvette) with the light absorption value of 1 at the wavelength of 600nm, transferring the suspension to a liquid culture medium again according to the volume ratio of the suspension to the liquid culture medium of 1:10, and culturing the bacteria again, specifically, culturing for 48 hours in a 37 ℃ constant temperature incubator to obtain a control sample 1.
Control 2:
a 5% o aqueous solution of lauroyl sarcosine sodium was prepared as a control 2.
Control 3:
preparing oil extraction functional bacteria suspension with the light absorption value of 1 measured by a cuvette of 1cm under the wavelength of 600nm to serve as a control sample 3.
Verification experiment 1-verification of emulsifying Properties
The chemical compound oil displacement agent is one of the main technologies for improving the crude oil recovery rate of the oil field at present, and the oil displacement mechanism is complex. For the oil displacement effect of the oil displacement agent, the emulsion stability of the emulsifier is generally measured to evaluate the emulsion performance of the surfactant oil displacement system, namely an emulsion method, wherein the larger the volume of an emulsion layer is, the better the stability is, the better the oil displacement effect is.
The experimental procedure was as follows:
5ml of the experimental group microorganism-chemical compound oil displacement agent is placed in a beaker, 5ml of white oil is added, and the temperature is kept for 30 min. After taking out, stirring for 10min (shaft rotation speed 1000r/min) by using a high-speed dispersion emulsifying instrument, pouring into a measuring cylinder, standing for 72h, and recording the change of the volume of an emulsifying layer in the 72-h period.
5ml of control 1 was placed in a beaker and 5ml of white oil was added and incubated for 30 min. After being taken out, the mixture is stirred for 10min (the rotating speed of the shaft is 1000r/min) by a high-speed dispersion emulsifying instrument, poured into a measuring cylinder and kept stand for 72h, and the change of the volume of an emulsifying layer in the 72h period is recorded to be used as a control experiment 1.
5ml of control 2 was placed in a beaker and 5ml of white oil was added and incubated for 30 min. After being taken out, the mixture is stirred for 10min (the rotating speed of the shaft is 1000r/min) by a high-speed dispersion emulsifying instrument, poured into a measuring cylinder and kept stand for 72h, and the change of the volume of an emulsifying layer in the 72h period is recorded to be used as a control experiment 2.
5ml of water was placed in a beaker and 5ml of white oil was added and the temperature was maintained for 30 min. After being taken out, the mixture is stirred for 10min (the rotating speed of the shaft is 1000r/min) by a high-speed dispersion emulsifying instrument, poured into a measuring cylinder and kept stand for 72h, and the change of the volume of an emulsifying layer in the 72h period is recorded to be used as a control experiment 3.
The results of the experiment are shown in FIG. 2. Fig. 2 is a comparison graph of emulsion stability characterization curves of the emulsification of the microbial-chemical composite oil displacement agent (experimental group, composite agent for short in fig. 2), water, oil recovery functional bacteria (biological agent for short in fig. 1 and fig. 2), and sodium lauroyl sarcosinate (chemical agent for short in fig. 2) which are obtained by compounding the sodium lauroyl sarcosinate-diglucoside composite oil displacement agent and the oil recovery functional bacteria, and white oil respectively. The reduction rate is faster within 8 hours after the microorganism-chemical compound oil displacement agent and the white oil are stirred and emulsified by a high-speed dispersion emulsifier, and the microorganism-chemical compound oil displacement agent and the white oil are basically unchanged after 40 hours and keep stable. The water and the white oil generate a small amount of unstable emulsion layer due to the mechanical stirring of the high-speed dispersion emulsifier, and the emulsion layer is basically absent and is obvious in layering within 2 hours. The control 2 (chemical oil displacement agent) and white oil were stirred in a high-speed dispersion emulsifier to produce a small amount of stable emulsion layer. The control sample 1 (microbial oil displacement agent) and the white oil generate a small amount of emulsion layer after being stirred by a high-speed dispersion emulsifier, and the emulsion layer disappears after 8 hours and does not exist stably any more.
Verification experiment 2-viscosity reduction performance verification
The chemical compound oil displacement agent is one of the main technologies for improving the crude oil recovery rate of the oil field at present, and the oil displacement mechanism is complex. For the oil displacement effect of the oil displacement agent, the viscosity reduction performance of the oil displacement agent is generally evaluated by measuring the viscosity of the crude oil added with the oil displacement agent, namely, a viscosity reduction method, and the more obvious the viscosity reduction effect is, the better the oil displacement effect is.
The experimental procedure was as follows:
and (3) placing the experimental group microorganism-chemical compound oil displacement agent in a beaker, adding crude oil with the volume ratio of 1:1, and preserving heat for 30 min. Taking out, stirring with a mechanical stirrer for 5min (shaft rotation speed of 500r/min), mixing thoroughly, and measuring viscosity with a viscometer.
The control 1 was placed in a beaker and added with crude oil in a volume ratio of 1:1 and incubated for 30 min. After being taken out, the mixture was stirred for 5min (shaft rotation speed 500r/min) by a mechanical stirrer, and after being sufficiently mixed, the viscosity was measured by a viscometer to prepare a control experiment 1.
Adding the control sample 2 into the crude oil according to the volume ratio of 1:1, and preserving the temperature for 30 min. After being taken out, the mixture is stirred for 5min (the shaft rotating speed is 500r/min) by a mechanical stirrer, and the viscosity is measured by a viscometer after the mixture is fully mixed so as to be used as a control experiment 2.
The crude oil was kept at the temperature for 30min, and the viscosity was measured with a viscometer to make a control experiment 3.
See fig. 3 for experimental results. Fig. 3 is a comparison graph of the effect of reducing the crude oil viscosity of the microbial-chemical composite oil displacement agent (experimental group, composite agent for short in fig. 3) obtained by compounding the sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent and the oil extraction functional bacteria, the oil extraction functional bacteria (reference 1, biological agent for short in fig. 3), and the sodium lauroyl sarcosinate (reference 2, chemical agent for short in fig. 3). As can be seen from fig. 3, the biological oil-displacing agent of the control sample 1 has the worst viscosity-reducing effect on crude oil, the chemical oil-displacing agent of the control sample 2 has relatively good viscosity-reducing effect, and the viscosity-reducing effect of the microbial-chemical composite oil-displacing agent is the best and can be reduced to about 55% of the original crude oil viscosity.
Validation experiment 3-load efficiency validation
The loading efficiency is one of indexes for evaluating the capability of microorganisms for loading the chemical oil-displacing agent. The higher the load efficiency, the larger the amount of the chemical agent carried by the microorganism, and the better the oil displacement effect in the underground. The oil displacement efficiency of the microorganism-loaded chemical oil displacement agent is generally measured by an ultraviolet-visible spectrophotometer.
The experimental procedure was as follows:
the experimental group microorganism-chemical compound oil displacement agent is placed in a cuvette, and the loading efficiency of the cuvette is measured by an ultraviolet-visible spectrophotometer. The measurements were made every half hour until the observation time reached 24h, and the experimental results are shown in the graph of the experimental group in fig. 5.
The control 1 was placed in a cuvette and its loading efficiency was measured by an ultraviolet-visible spectrometer. The measurement is carried out every half hour until the observation time reaches 24 hours, so as to make a control experiment 1, and the experimental result is shown in a graph of a control group in fig. 5.
See fig. 5 for experimental results. Fig. 5 is a curve of the load efficiency of the experimental group and the comparative sample 1 changing with time, which shows that the load efficiency of the load efficiency curve of the sodium lauroyl sarcosinate-diglucoside peptide compound oil displacement agent compounded with the oil recovery functional bacteria changing with time does not decrease greatly, changes very slowly and has a high value. The load efficiency of the sodium lauroyl sarcosinate-diglucoside peptide compound in the microbial-chemical composite oil displacement agent obtained by compounding the sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent and the oil extraction functional bacteria is high, and the compound is not easy to fall off. And the load efficiency curve of the contrast experiment shows that the load efficiency is gradually reduced, the amplitude is obvious, and the numerical value is low, which indicates that the load efficiency of sodium lauroyl sarcosinate in the oil displacement agent obtained by compounding sodium lauroyl sarcosinate and oil extraction functional bacteria is not high and the sodium lauroyl sarcosinate is easy to fall off.
Validation experiment 4-validation of emulsified particle size:
the emulsion particles are in polydispersion, and the size and distribution of the particle size have an influence on the viscosity and stability of the emulsion. In the oil displacement effect of the oil displacement agent, the emulsified particle size is one of important indexes. The size and distribution of emulsified particle size after the oil displacement agent is added are usually determined by centrifugal sedimentation. The smaller the particle size, the more stable the emulsion and the better the oil displacement effect.
The experimental steps are as follows:
and (3) placing the experimental group microorganism-chemical compound oil displacement agent in a beaker, adding white oil with the volume ratio of 1:1, and preserving heat for 30 min. Taking out, stirring with a high-speed dispersion emulsifying instrument for 10min (shaft rotation speed 1000r/min), pouring into a measuring cylinder, standing for 72h, taking out the emulsion part, and measuring the emulsion particle size distribution diagram with a Joyce-Loebl disc type centrifuge.
And (3) placing the control sample solution 3 in a beaker, adding white oil with the volume ratio of 1:1, and preserving the temperature for 30 min. After being taken out, the mixture is stirred for 10min (the rotating speed of a shaft is 1000r/min) by a high-speed dispersion emulsifying instrument, poured into a measuring cylinder and kept stand for 72h, and then the emulsion part is taken out, and a particle size distribution diagram of the emulsion is measured by a Joyce-Loebl disk centrifuge so as to be used as a control experiment 1.
Adding white oil into the control sample 2 according to the volume ratio of 1:1, and keeping the temperature for 30 min. Stirring for 10min (shaft rotation speed 1000r/min) by a high-speed dispersion emulsifying instrument, pouring into a measuring cylinder, standing for 72h, taking out an emulsion part, and measuring the particle size distribution diagram of the emulsion by a Joyce-Loebl disk centrifuge to prepare a control experiment 2.
See fig. 6 for experimental results. Fig. 6 is an emulsified particle size distribution diagram obtained after white oil is emulsified by a microorganism-chemical compound oil displacement agent (compound in experimental group and short for fig. 6), a chemical oil displacement agent (chemical in comparison sample 2 and short for fig. 6) and a biological oil displacement agent (biological in comparison sample 3 and short for fig. 6) which are obtained by compounding the sodium lauroyl sarcosinate-diglucoside compound oil displacement agent and the oil extraction functional bacteria. As shown in fig. 6, no matter the maximum value, the minimum value or the average value, the emulsified particle size after the action of the chemical oil-displacing agent is the largest, and the emulsified particle size after the action of the biological oil-displacing agent is the second, compared with the particle sizes of the action sum of the chemical oil-displacing agent and the biological oil-displacing agent, it can be obviously found that the emulsified particle size after the action of the microorganism-chemical compound oil-displacing agent is smaller than the other two particle sizes.
Validation experiment 5-sweep efficiency validation
The sweep efficiency is one of the main judgment standards for improving the crude oil recovery rate of the oil field at present, and for the oil field developed by water injection, the oil layer recovery rate is mainly determined by the sweep efficiency. Sweep efficiency is typically measured by a biological indicator of the produced fluid passing through the outlet end of the displacement simulator.
This time, a simulation apparatus diagram as shown in fig. 7 is used, which includes a Brine (Brine) storage apparatus 1, a distilled Water (Water) storage apparatus 2, an injection pump 3, a Pressure gauge (Pressure gauge)4, a biochemical oil displacement agent storage tank (tank)5, a reserve tank 6, a crude oil storage tank (oil tank)7, a Core holder (Core holder)8, a Confining Pressure pump (defining pump)9, a Back-Pressure pump (Back-Pressure pump)10, and a collector (Produced fluid collector) 11. The device comprises a brine storage device 1, a valve control device, a biochemical oil displacement agent storage tank 5, a six-way first interface, a pipeline, a brine storage device and a pipeline, wherein the brine storage device 1 is connected with an injection pump 3, an outlet of the injection pump is connected with a pressure gauge 4, the other end of the pressure gauge 4 is respectively connected with the lower end interface of the biochemical oil displacement agent storage tank 5 and the first interface of the six-way through a pipeline controlled by the valve, an upper end interface of a crude oil storage tank is connected with the second interface of the six-way through, and another pressure gauge 4 is arranged on a pipeline connecting the upper end interface of the crude oil storage tank with the second interface of the six-way; the distilled water storage device 2 is connected with another injection pump 3, the outlet of the injection pump is connected with another pressure gauge 4, the other end of the pressure gauge is respectively connected with the lower end interfaces of the crude oil storage tank 7 and the standby storage tank 6 through pipelines controlled by a valve, and the upper end interfaces of the crude oil storage tank 7 and the standby storage tank 6 are respectively connected with the third interface and the fourth interface of the six-way valve through pipelines controlled by a valve; the 6 th interface of the circulation is connected with an inlet of a core holder 8, an outlet of the core holder 8 is respectively connected with a back pressure pump 10 and a collector 11, and a confining pressure pump 9 is connected with the core holder 8 for applying confining pressure.
The experimental steps are as follows:
(1) carrying out saturated oil on the rock core in the rock core holder 8, and then carrying out water drive; and (3) finishing water flooding until the water content of the produced oil reaches 98%, and then injecting an oil displacement slug, which specifically comprises the following steps: injecting the experimental group microorganism-chemical compound oil displacement agent in a mode of two slug injection, wherein the total injection amount is 0.5 PV. As shown in fig. 8, 0.2PV saline is injected first, and then 0.25PV of the experimental group microorganism-chemical compound oil displacement agent, 0.1PV saline, 0.25PV of the experimental group microorganism-chemical compound oil displacement agent, and 0.2PV saline are injected in sequence.
(2) And after the oil displacement slug is injected, starting a subsequent water flooding process, sampling every 0.1PV from the outlet end of the core holder 8 in the subsequent water flooding process, and performing colony coating and infrared characterization to obtain a concentration change curve of the composite oil displacement agent in the flooding fluid along with increase of the PV number of the flooding fluid.
Control experiment:
(1) carrying out saturated oil on the rock core in the rock core holder 8, and then carrying out water drive; and (3) finishing water flooding until the water content of the produced oil reaches 98%, and then injecting an oil displacement slug, which specifically comprises the following steps: the control 2 (chemical oil-displacing agent) is injected in a two-slug injection mode, the total injection amount is 0.5PV, and as shown in FIG. 9, 0.2PV saline is injected first, and then 0.25PV saline for the control 2 (chemical oil-displacing agent), 0.1PV saline, 0.25PV saline for the control 2 (chemical oil-displacing agent) and 0.2PV saline are injected in sequence.
(2) After the flooding slug is injected, a subsequent water flooding process is started, in the subsequent water flooding process, sampling is performed every 0.1PV from the outlet end of the core holder 8, colony coating and infrared characterization are performed, and a concentration change curve of the chemical flooding agent in the flooding fluid along with increase of the PV number of the flooding fluid is obtained.
Compared with the control experiment, the earlier the trace shadow of the microorganism-chemical composite oil displacement agent appears in the displacement fluid, which indicates that the better the diffusion effect of the microorganism-chemical composite oil displacement agent is. FIG. 4 is a drawing showingAnd a comparison graph of oil displacement sweep efficiency of the microbial-chemical composite oil displacement agent (the concentration of the composite agent is abbreviated in an experimental group and a figure 4) and the sodium lauroyl sarcosine chemical oil displacement agent (the concentration of the chemical agent is abbreviated in a comparison sample 2 and a figure 4) which are obtained by compounding the sodium lauroyl sarcosine-diglucoside composite oil displacement agent and the oil extraction functional bacteria. In the control experiment, no effective concentration of sodium lauroyl sarcosinate is found in the first 0.2PV sample of the subsequent water flooding, which indicates that the sodium lauroyl sarcosinate has no diffusion capacity and only stays near the injection slug; in contrast, in the experiment of injecting the microbial-chemical composite oil-displacing agent, in the subsequent water-flooding process, the first sampling is carried out, the trace of the microbial-chemical composite oil-displacing agent is found in 0.1PV, and the biological concentration is 10 after the flat plate coating1And each ml indicates that the microorganism-chemical complexing agent has the self-migration capability compared with the chemical agent. Observing from the overall core layout, the concentration of the chemical agent is compounded with the injection slug to form two peak values; and the spreading efficiency of the microbial-chemical complexing agent is obviously improved, and the whole body presents a peak value and covers the whole rock core. The concentration of the chemical agent is compounded with the injection slug to form two peak values, the oil displacement effect is improved by chemical flooding injection, and the oil displacement efficiency is reduced after the chemical flooding comes out of the rock core. The integral sweep efficiency of the microbial-chemical complexing agent presents a peak value, which shows that the movement range of the microbial-chemical complexing agent covers the whole rock core and does not come out of the rock core along with the injection of the later water drive, and the sweep efficiency is obviously improved compared with the chemical drive.
Verification experiment 6-verification of colony growth condition when sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent and oil extraction functional bacteria are compounded
The experimental procedure was as follows:
diluting operation:
(1) 6 test tubes each containing 9ml of water were sterilized and numbered in the order of 10-1 to 10-6.
(2) 1ml of the microbial-chemical complex oil-displacing agent prepared in example 1 was aspirated by a pipette tube, and the solution was diluted in a test tube No. 10-1.
(3) 1ml of the dilution was aspirated from test tube No. 10-1 and injected into test tube No. 10-2. And so on until the dilution of the last test tube is completed.
Coating operation:
(1) and (3) dropwise adding a small amount of diluent in a No. 10-6 test tube onto the surface of the oil-extracting microorganism solid culture medium.
(2) Igniting the applicator dipped with a small amount of alcohol on flame, and cooling for 8-10s after the alcohol is burnt out.
(3) The diluted solution was uniformly spread on the surface of the medium by using a spreader.
And observing the growth condition of the coated microorganism-chemical compound oil-displacing agent, wherein the bacterial colony of the oil extraction functional bacteria grows well after the microorganism-chemical compound oil-displacing agent prepared in the example 1 is added into the oil extraction microorganism solid culture medium.
In the verification experiment, the chemical oil-displacing agent remained after the dilution step of the microbial-chemical composite oil-displacing agent obtained in example 1 is only the chemical oil-displacing agent successfully modified on the surface of the oil-extracting functional bacteria (because of 6 times dilution, the concentration of the chemical agent is 10 at this time-6Default absence) during subsequent propagation culture.
Claims (27)
1. A sodium lauroyl sarcosinate-diglucoside peptide compound, wherein the chemical structural formula is as follows:
2. a sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent comprises a sodium lauroyl sarcosinate-diglucoside peptide compound obtained by reacting sodium lauroyl sarcosinate with diglucoside peptide;
wherein the sodium lauroyl sarcosinate-diglucoside peptide compound is the sodium lauroyl sarcosinate-diglucoside peptide compound according to claim 1.
3. The preparation method of the sodium lauroyl sarcosinate-diglucoside peptide complex oil displacement agent according to claim 2, wherein the preparation method comprises the following steps:
adding sodium lauroyl sarcosinate into a diglucoside peptide solution, and reacting to obtain the sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent, wherein the molar ratio of the diglucoside peptide to the sodium lauroyl sarcosinate is 1: 1-1.8.
4. The method of claim 3, wherein the reaction is carried out at 37-40 ℃ for 6-12 h.
5. The preparation method according to claim 3, wherein the concentration by mass of the diglucoside peptide solution is 40-60%.
6. The preparation method according to claim 3 or 5, wherein the diglucoside peptide solution is formed by dissolving diglucoside peptide in water, wherein the diglucoside peptide is prepared by the following steps:
1) culturing the bacterial strain inoculated into the culture medium in an air bath shaker until the bacteria grow to the late logarithmic phase, centrifuging, and collecting the bacteria; wherein the strain is any strain of which the metabolite contains the diglucoside peptide;
2) washing the collected thallus with dilute sulfuric acid to remove impurities, and suspending the thallus to prepare a suspension;
3) subpackaging the suspension, centrifuging and collecting lower layer bacterial bodies;
4) eluting the collected lower layer bacterial body, centrifuging, taking the upper layer capsular solution, and carrying out chromatographic separation and purification to obtain the diglucoside peptide.
7. The method according to claim 6, wherein the centrifugation in step 1) is performed at 6000-10000 rpm for 10-20 min.
8. The method of claim 7, wherein the centrifugation in step 1) is at 8000 rpm for 15 min.
9. The method according to claim 6, wherein the suspension in step 2) has an absorbance of 1 as measured at a wavelength of 600nm using a 1cm cuvette.
10. The method according to claim 6, wherein the centrifugation in step 3) is performed at 8000 rpm and 6000-.
11. The method of claim 10, wherein the centrifugation in step 3) is at 8000 rpm for 10 min.
12. The preparation method according to claim 6, wherein the elution is performed with clear water at 38-40 ℃ for 10-12 h.
13. The method of claim 12, wherein the elution is with clear water at 40 ℃ for 12 h.
14. The method according to claim 6, wherein the culturing in step 1) is carried out at 63-68 ℃ and 180 r/min.
15. The method according to claim 14, wherein the culturing is performed at 65 ℃ and 170r/min in step 1).
16. The method according to claim 6, wherein the pH of the dilute sulfuric acid in the step 2) is 2 to 3.
17. The method according to claim 16, wherein the dilute sulfuric acid has a pH of 2 in step 2).
18. A microorganism-chemical compound oil-displacing agent, wherein the microorganism-chemical compound oil-displacing agent is prepared by compounding the sodium lauroyl sarcosinate-diglucoside peptide compound oil-displacing agent of claim 2 and oil extraction functional bacteria.
19. The microbial-chemical composite oil-displacing agent according to claim 18, wherein the sodium lauroyl sarcosinate-diglucoside peptide composite oil-displacing agent is produced by the production method according to any one of claims 3 to 17.
20. The microorganism-chemical composite oil displacement agent according to claim 18 or 19, wherein the sodium lauroyl sarcosinate-diglucoside peptide compound in the sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent is modified on the surface of the oil recovery functional bacteria.
21. The microbe-chemical composite oil-displacing agent according to claim 18 or 19, which is obtained by the following production method:
1) adding 200mL of culture medium inoculated with the strain into a 500mL triangular flask, placing the triangular flask in an air bath shaker for culture at 65 ℃ and 170r/min, when the strain grows to the late logarithmic phase, centrifuging at 8000r/min for 15min to collect cells, washing with dilute sulfuric acid with the pH value of 2 to remove impurities, suspending the strain, preparing a suspension with the light absorption value of 1 measured by a 1cm cuvette at the wavelength of 600nm, subpackaging 20mL of each suspension, centrifuging at 8000 rpm for 10min, and collecting the lower-layer bacterial cells; wherein the strain is any strain of which the metabolite contains the diglucoside peptide;
2) eluting the bacterial body with clear water at 40 deg.C for 12h, centrifuging, collecting the upper layer capsular solution, performing chromatographic separation, and purifying to obtain diglucoside peptide;
3) placing a diglucoside peptide solution with the mass concentration of 40-60% in a water bath at 39 ℃, adding sodium lauroyl sarcosinate with the mass of 3 times that of the diglucoside peptide, and reacting for 12 hours to obtain the sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent;
4) adding the sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent obtained in the step 3) into a bacterial culture medium, transferring into an oil extraction functional bacterial agent to form a mixture, wherein the volume ratio of the sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent to the oil extraction functional bacterial agent is 10:1-5:1, and culturing and proliferating for 24-36h until the solution is subjected to light absorption value measurement of 1 by using a 1cm cuvette under the wavelength of 600nm to obtain the microorganism-chemical composite oil displacement agent.
22. A method for preparing the microorganism-chemical composite oil-displacing agent of any one of claims 18 to 21, wherein the method comprises:
adding the sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent of claim 2 into a bacteria culture medium, transferring oil recovery functional bacteria to form a mixture, and culturing and proliferating to obtain the microorganism-chemical composite oil displacement agent.
23. The production method according to claim 22, wherein the culture proliferation is culture proliferation until an absorbance of the solution measured at a wavelength of 600nm in a 1cm cuvette is 1.
24. The method according to claim 22, wherein the culture is propagated for 24 to 36 hours.
25. The preparation method of claim 22, wherein the oil-producing functional bacteria is transferred in the form of an oil-producing functional bacteria microbial inoculum, wherein the volume ratio of the sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent to the oil-producing functional bacteria microbial inoculum is 10:1-5: 1.
26. The preparation method of claim 25, wherein the volume ratio of the sodium lauroyl sarcosinate-diglucoside peptide composite oil displacement agent to the oil recovery functional bacteria agent is 10: 1.
27. The preparation method of claim 22, wherein the sodium lauroyl sarcosinate-diglucoside peptide complex oil displacement agent is prepared by the preparation method of any one of claims 3 to 17.
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