Composite microorganisms and microbial oil displacement agents, and methods and apparatus for microbial oil displacement

Abstract

The present application relates to the field of microbial oil displacement, and discloses a composite microorganism, a microbial oil displacement agent, application of the microbial oil displacement agent, a method for microbial oil displacement, and a device for microbial oil displacement.The composite microorganism comprises yeast, and bacillus and / or pseudomonas; the yeast is Candida viswanathii with a preservation number of CGMCC No.3456; and the ratio of the total viable count of bacillus and pseudomonas to the viable count of yeast is 2-8.The microbial oil displacement agent of the present application can significantly reduce the interfacial tension and has a good oil displacement effect.

Description

Technical Field

[0001] This invention relates to the field of microbial oil displacement, specifically to a composite microorganism, a microbial oil displacement agent and their applications, a method for microbial oil displacement, and an apparatus for microbial oil displacement. Background Technology

[0002] As conventional oil reservoirs gradually age, the development of low-permeability and ultra-low-permeability oilfields has become a crucial potential for the stable development of my country's onshore petroleum industry. The fracturing and acidizing measures used in water injection development of low-permeability and ultra-low-permeability reservoirs, as well as the application of chemical flooding technologies such as polymer flooding and composite flooding, result in complex composition of produced water, posing significant threats to the environment and formation. Traditional treatment methods such as sedimentation and filtration suffer from high costs, complex equipment processes, short service life, and secondary pollution. Microbial enhanced oil recovery (MEOR) technology, with its advantages of being environmentally friendly and requiring no special treatment of produced water, is widely used.

[0003] CN112374701A provides an oilfield produced water treatment process that uses oleophilic bacteria to remove oil. The biological agent contains Bacillus licheniformis, Bacillus brevis, and Pseudomonas spp. in a compound ratio of 2:1:1. This process decomposes the oil in the produced water into non-toxic carbon dioxide and water. The water is then filtered through a membrane to meet the water quality standards for oilfield injection. However, the microorganisms in the treated water are also filtered out and cannot be reused.

[0004] CN104312561A discloses the application of a composite microbial strain in tertiary oil recovery. It uses a 1:1:1 weight ratio of Halobacterium salinarium, Brevibacillus brevis, and Bacillus cereus mixed in a fermentation broth for tertiary oil recovery. The strain survives in high-salt environments (salt content above 12%), reducing crude oil viscosity by 15.4%, 26.4%, and 35.2%, respectively, and reducing oil-water interfacial tension by 91.3%, 76.5%, and 58.6%, respectively. This demonstrates significant benefits in reducing crude oil viscosity and oil-water interfacial tension, showing broad application value. However, the mixed fermentation broth requires large-scale fermentation and propagation in a factory and transportation to the test site, resulting in high costs.

[0005] Against the backdrop of the requirements for "low-cost, green and efficient" energy development, how to combine produced water microbial treatment with microbial flooding technology to enhance oil recovery, effectively utilize produced water resources, improve oilfield recovery, and achieve environmental protection both on the surface and underground, is of great strategic significance for the "high-quality, efficient and sustainable development" of oil reservoirs. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies, such as unsatisfactory oil displacement effects of biological agents, cumbersome fermentation processes of ground-based strains, long-distance transportation of bacterial solutions, and the inability to reinject produced water into the formation for oil displacement. This invention provides a composite microorganism, a microbial oil displacement agent, its application, a method for microbial oil displacement, and a device for microbial oil displacement. The microbial oil displacement agent can significantly reduce interfacial tension and has excellent oil displacement effects. The microbial oil displacement device can, on the one hand, utilize the growth and metabolism of the composite microorganisms to remove oil from the produced water, and on the other hand, utilize the produced water after proliferation and expansion to inject into the formation for oil displacement.

[0007] To achieve the above objectives, the first aspect of the present invention provides a composite microorganism, wherein the composite microorganism comprises yeast, and Bacillus and / or Pseudomonas;

[0008] The yeast strain is Candida viswanathii, with accession number CGMCC No. 3456.

[0009] The ratio of the total viable number of Bacillus and Pseudomonas to the viable number of yeast is 2-8.

[0010] A second aspect of the present invention provides a microbial oil displacement agent, wherein the microbial oil displacement agent comprises: a composite microorganism provided by the present invention, and the metabolites of the composite microorganism.

[0011] A third aspect of the present invention provides the application of the microbial flooding agent provided by the present invention in reducing interfacial tension or in reservoir flooding.

[0012] The fourth aspect of the present invention provides a method for microbial enhanced oil recovery, wherein the method includes: mixing the composite microorganisms provided by the present invention with oil-bearing produced water for expansion culture, and then contacting the expanded material with the oil reservoir to be enhanced.

[0013] A fifth aspect of the present invention provides an apparatus for microbial enhanced oil recovery, wherein the apparatus comprises:

[0014] The primary, secondary, and tertiary expansion tanks, connected in series, are used to expand the culture of complex microorganisms and remove oil impurities from oily produced water.

[0015] The inlet located upstream of the primary expansion tank is used to introduce oil-containing produced water.

[0016] The outlet located downstream of the three-stage expansion tank is used to draw out the expanded material and the extracted water after oil removal.

[0017] The beneficial effects of the present invention through the above technical solution are as follows:

[0018] The microbial oil displacement agent provided by this invention can significantly reduce interfacial tension and has a good oil displacement effect.

[0019] Microbial enhanced oil recovery (DEOR) is performed in the microbial enhanced oil recovery device provided by this invention. By adding composite microorganisms to the device, on the one hand, the growth and metabolism of microorganisms can remove oil-containing impurities from the produced water of the reservoir; on the other hand, the water after microbial culture treatment is injected into the formation for oil recovery. This not only purifies the water quality to meet the oilfield reinjection standards, but also eliminates the cumbersome process of industrial fermentation of single strains on the ground and the inconvenience of long-distance transportation of microbial agents. In other words, by using the device and method of this invention to remove oil from the produced water containing oil, and then reinjecting it into the reservoir to be recovered for oil recovery, the oil recovery rate of the reservoir is improved while recycling water resources. This achieves multiple benefits and has advantages such as low cost, simple operation, and environmental friendliness, and has broad prospects for promotion and application. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of a microbial oil recovery device according to a preferred embodiment of the present invention.

[0021] Explanation of reference numerals in the attached figures

[0022] 1. Primary expansion tank 2. Secondary expansion tank 3. Tertiary expansion tank 4. Sedimentation tank

[0023] 5. Inlet 6. Outlet 7. Overflow 8. Packing material 9. Mixing tank

[0024] 10 Buffer tank 11 Dosing pump 12 Aeration pump Detailed Implementation

[0025] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0026] The first aspect of the present invention provides a composite microorganism, wherein the composite microorganism comprises yeast, and Bacillus and / or Pseudomonas;

[0027] The yeast strain is Candida viswanathii, with accession number CGMCC No. 3456.

[0028] The ratio of the total viable number of Bacillus and Pseudomonas to the viable number of yeast is 2-8, preferably 3.5-6.5.

[0029] According to the present invention, preferably, the Bacillus is selected from at least one of Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloliquefaciens, and more preferably from at least one of Bacillus subtilis with accession number CGMCC No. 21860, Bacillus licheniformis with accession number BNCC188083, and Bacillus amyloliquefaciens with accession number BNCC188065.

[0030] According to the present invention, preferably, the pseudomonad is selected from Pseudomonas aeruginosa and / or Pseudomonas stearothermiae, more preferably from Pseudomonas aeruginosa, and more preferably from Pseudomonas aeruginosa with accession number CGMCC No. 23170.

[0031] According to a preferred embodiment of the present invention, the composite microorganisms include Candida viswanathii (CGMCC No. 3456), Bacillus licheniformis (BNCC188083), and Pseudomonas aeruginosa (CGMCC No. 23170).

[0032] According to a preferred embodiment of the present invention, the composite microorganisms include Candida viswanathii (CGMCC No. 3456), Bacillus amyloliquefaciens (BNCC188065), and Pseudomonas aeruginosa (CGMCC No. 23170).

[0033] According to a preferred embodiment of the present invention, the composite microorganisms include Candida viswanathii (CGMCC No. 3456), Bacillus licheniformis (BNCC188083), Bacillus amyloliquefaciens (BNCC188065), and Pseudomonas aeruginosa (CGMCC No. 23170).

[0034] According to a preferred embodiment of the present invention, the composite microorganisms include Candida viswanathii (CGMCC No. 3456), Bacillus subtilis (CGMCC No. 21860), Bacillus licheniformis (BNCC188083), and Pseudomonas aeruginosa (CGMCC No. 23170).

[0035] According to a preferred embodiment of the present invention, the composite microorganisms include *Candida viswanathii* (CGMCC No. 3456), *Bacillus subtilis* (CGMCC No. 21860), *Bacillus amyloliquefaciens* (BNCC188065), and *Pseudomonas aeruginosa* (CGMCC No. 23170).

[0036] According to a preferred embodiment of the present invention, the composite microorganisms include *Candida viswanathii* (CGMCC No. 3456), *Bacillus subtilis* (CGMCC No. 21860), *Bacillus licheniformis* (BNCC188083), *Bacillus amyloliquefaciens* (BNCC188065), and *Pseudomonas aeruginosa* (CGMCC No. 23170).

[0037] According to a preferred embodiment of the present invention, the composite microorganisms comprise *Candida viswanathii* (CGMCC No. 3456), *Bacillus subtilis* (CGMCC No. 21860), and *Pseudomonas aeruginosa* (CGMCC No. 23170). The inventors have discovered that this specific combination of *Candida viswanathii* (CGMCC No. 3456) with *Bacillus subtilis* (CGMCC No. 21860) and *Pseudomonas aeruginosa* (CGMCC No. 23170) exhibits a better oil displacement effect.

[0038] *Candida viride* with accession number CGMCC No. 3456 has been published in CN101781624B. *Bacillus subtilis* with accession number CGMCC No. 21860 has been published in CN116200285A. *Bacillus licheniformis* was purchased from Beijing Beina Chuanglian Biotechnology Research Institute, accession number BNCC188083. *Bacillus amyloliquefaciens* was purchased from Beijing Beina Chuanglian Biotechnology Research Institute, accession number BNCC188065. *Pseudomonas aeruginosa* with accession number CGMCC No. 23170 has been published in CN116445316A.

[0039] According to the present invention, preferably, the ratio of viable counts of yeast, Bacillus, and Pseudomonas in the composite microorganisms is 1:(1-3):(1-5), more preferably 1:(1.5-2.5):(2-4). When the ratio of viable counts of yeast, Bacillus, and Pseudomonas meets this range, the oil displacement effect of the composite microorganisms can be further improved.

[0040] A second aspect of this invention provides a microbial oil-displacing agent, comprising: a composite microorganism provided by this invention, and metabolites of the composite microorganism. The metabolites are produced during the growth and reproduction of the composite microorganism using a nutrient solution; wherein, based on a volume of 1L of the nutrient solution, the nutrient solution comprises 1-2g of molasses, 0.1-1g of peptone, 0.1-0.5g of ammonium sulfate, 0.1-0.5g of sodium dihydrogen phosphate, 0.01-0.05g of dipotassium hydrogen phosphate, and 0.1-0.5g of sodium citrate.

[0041] According to the present invention, preferably, the pH of the microbial oil displacement agent is 6-8.

[0042] A third aspect of the present invention provides the application of the microbial flooding agent provided by the present invention in reducing interfacial tension or in reservoir flooding; preferably in the application in low-permeability reservoir flooding.

[0043] The composite microorganisms and their metabolites in this invention can reduce interfacial tension and have a good oil displacement effect, especially in low-permeability reservoirs.

[0044] The permeability of low-permeability reservoirs is (0.1-50)×10 -3 μm 2 Based on actual production characteristics, low-permeability oil reservoirs can be further classified into general low-permeability oil reservoirs, ultra-low permeability oil reservoirs, and extra-low permeability oil reservoirs according to their average permeability, with an average permeability of (10-50)×10. -3 μm 2 (1-10)×10 -3 μm 2 and (0.1-1)×10 -3 μm 2 .

[0045] The fourth aspect of the present invention provides a method for microbial enhanced oil recovery, wherein the method includes: mixing the composite microorganisms provided by the present invention with oil-bearing produced water for expansion culture, and then contacting the expanded material with the oil reservoir to be enhanced.

[0046] According to the present invention, preferably, the contact conditions include a temperature of 28-42°C.

[0047] According to the present invention, preferably, the oil content in the oil-bearing produced water is not higher than 50 mg / L.

[0048] According to the present invention, preferably, the conditions for the expansion culture include: a temperature of 28-42°C, more preferably 30-37°C; and a dissolved oxygen content (DO) of 2 mg / L or higher.

[0049] According to the present invention, preferably, the expansion time is such that the viable count in the expanded material is 1×10⁻⁶. 8 CFU / mL or higher.

[0050] According to the present invention, preferably, the expansion culture is carried out in the presence of a nutrient solution; wherein, based on a volume of 1L of the nutrient solution, the nutrient solution comprises 1-2g of molasses, 0.1-1g of peptone, 0.1-0.5g of ammonium sulfate, 0.1-0.5g of sodium dihydrogen phosphate, 0.01-0.05g of dipotassium hydrogen phosphate and 0.1-0.5g of sodium citrate.

[0051] According to the present invention, preferably, the amount of the expanded composite microorganisms added is such that the viable count in the material after contact is 1×10⁻⁶. 8 CFU / mL or higher.

[0052] A fifth aspect of the present invention provides an apparatus for microbial enhanced oil recovery, wherein the apparatus comprises:

[0053] The primary, secondary, and tertiary expansion tanks, connected in series, are used to expand the culture of complex microorganisms and remove oil impurities from oily produced water.

[0054] The inlet located upstream of the primary expansion tank is used to introduce oil-containing produced water.

[0055] The outlet located downstream of the three-stage expansion tank is used to draw out the expanded material and the extracted water after oil removal.

[0056] A composite microorganism is added to the microbial enhanced oil recovery (DEOR) device, and oil-bearing produced water (incoming water) is introduced from the inlet upstream of the primary expansion tank. The composite microorganisms are mixed with the oil-bearing produced water for expansion, and the growth and metabolism of the microorganisms remove oil impurities from the produced water. The expanded material is then drawn out from the outlet downstream of the tertiary expansion tank and injected into the formation for oil displacement. The expanded material contains produced water with oil impurities removed, expanded composite microorganisms, composite microbial metabolites, and nutrients. The produced water with oil impurities removed meets oilfield reinjection standards. Injecting the expanded material into the formation for oil displacement improves reservoir recovery while recycling water resources.

[0057] The device of the present invention includes a primary expansion tank, a secondary expansion tank, and a tertiary expansion tank, which can expand microorganisms to a suitable number and effectively remove crude oil impurities from the water.

[0058] According to the present invention, preferably, each of the primary expansion tank, the secondary expansion tank, and the tertiary expansion tank is independently equipped with packing material for microbial attachment and growth. The packing material can be one or more of aldehyde-modified fiber soft material, suspended ball packing material, and fiber ball packing material.

[0059] According to the present invention, preferably, each of the primary expansion tank, the secondary expansion tank and the tertiary expansion tank is independently equipped with an aeration device to provide oxygen required for microbial growth.

[0060] Preferably, the inlet is located at the lower part of the primary expansion tank, according to the needs of treating oilfield produced water.

[0061] According to the present invention, preferably, the overflow outlets of the primary expansion tank and the tertiary expansion tank are located at the upper part of the tank body, and the overflow outlet of the secondary expansion tank is located at the lower part of the tank body, so that the liquid flow direction is three-dimensional S-shaped. This design can ensure sufficient hydraulic retention time to meet the needs of microbial growth and reproduction.

[0062] According to the present invention, preferably, the device is further configured with a sedimentation tank, which is located downstream of the tertiary expansion tank and is used to settle solid impurities in the water.

[0063] According to the present invention, preferably, the outlet is located on the side wall of the sedimentation tank.

[0064] According to the present invention, preferably, the device is further provided with a buffer tank along the liquid flow direction, the buffer tank being located upstream of the primary expansion tank, for buffering the oil-containing produced water (incoming water).

[0065] According to the present invention, preferably, the device is further provided with a liquid mixing tank, which is located upstream of the primary expansion tank along the liquid flow direction, and is used to mix the compound microorganisms and nutrients.

[0066] According to the present invention, preferably, a dosing pump is also provided between the liquid preparation tank and the primary expansion tank for introducing the mixture of compound microorganisms and nutrients into the primary expansion tank.

[0067] According to the present invention, preferably, the device is further configured with a compound microorganism dosing device and a nutrient dosing device, which are used to dosing compound microorganisms and nutrients, respectively.

[0068] To ensure sufficient microbial growth on the packing material in the expansion tanks, oily produced water can be injected into the system to connect the expansion tanks. The inlet and outlet of the system should then be closed. Next, the compound microorganisms and nutrients should be injected for expansion. There are no strict time limits for expansion; for example, the bacterial concentration in the expansion tanks can be increased to 1×10⁻⁶. 8 The concentration of CFU / mL or higher is sufficient. Nutrients can be added during the process, and those skilled in the art can add them as needed.

[0069] After expansion culture, the inlet and outlet of the device are opened to allow fresh oil-bearing produced water (incoming water) to flow in, while the material containing bacterial solution flows out and is injected into the oil reservoir for oil displacement. The hydraulic retention time can be selected within a wide range, such as 6-8 hours. During operation, compound microorganisms and nutrient solution can be added.

[0070] According to a preferred embodiment of the present invention, in Figure 1 The method for expanding and cultivating composite microorganisms and displacing oil from the reservoir in the microbial enhanced oil recovery apparatus shown includes the following steps:

[0071] S1. Fermentation broths were prepared for *Candida viswanathii* (CGMCC No. 3456), *Bacillus subtilis* (CGMCC No. 21860), and *Pseudomonas aeruginosa* (CGMCC No. 23170), respectively, such that the viable cell count in the fermentation broth of each of the three strains was independently 1 × 10⁻⁶. 8 The fermentation broths of the three bacteria with a concentration of CFU / mL or higher are mixed in the mixing tank 9. The ratio of viable cells of Candida aestivum, Bacillus subtilis and Pseudomonas aeruginosa in the mixed fermentation broth is 1:(1-3):(1-5), thus obtaining a compound microbial agent.

[0072] S2. Introduce oily produced water (oil content not exceeding 50 mg / L) through inlet 5 to fill the device with oily produced water. Add nutrient solution to mixing tank 9 and mix it with the fermentation broth of the three bacteria. Then, slowly pump the mixture into the primary expansion tank 1 by dosing pump 11 (based on the total volume of the liquid in the device, the amount of fermentation broth of the three bacteria is 0.5-1.5% by volume, and the amount of nutrient solution is 0.08-0.12% by volume). Close the inlet 5 and outlet 6 of the device, and turn on the aeration pumps 12 in the primary expansion tank 1, secondary expansion tank 2, and tertiary expansion tank 3 to provide oxygen for microbial growth. Expand for 10-15 days (temperature 28-42℃, blower air intake ensures oxygen content in the device is above 2.0 mg / L), allowing microorganisms to attach and multiply on the packing material 8 in the primary expansion tank 1, secondary expansion tank 2, and tertiary expansion tank 3. The expansion time should result in a viable bacteria count of 1×10⁻⁶ in the expanded material. 8 CFU / mL or higher.

[0073] S3, When the total concentration of viable bacteria in the device reaches 1×10⁻⁶ 8Once the flow rate reaches a certain level (e.g., cells / mL), the inlet 5 and outlet 6 of the device are opened. The oil-bearing produced water (fresh water) is buffered in the buffer tank 10 and then flows into the primary expansion tank 1 through the inlet 5. The water then overflows through the overflow outlet 7 in an S-shaped path, sequentially passing through the primary expansion tank 1, the secondary expansion tank 2, the tertiary expansion tank 3, and the sedimentation tank 4. Finally, it is injected into the oil reservoir through the outlet 6 according to the oilfield geological distribution. The hydraulic retention time in the expansion device is 6-8 hours.

[0074] The present invention will be described in detail below through embodiments.

[0075] 1. Method for determining the content of glycolipid surfactants produced by yeast:

[0076] (1) Determination of glucose standard curve:

[0077] ① To prepare a 10 mg / mL glucose solution: Accurately weigh 0.1 g of analytical grade anhydrous glucose that has been dried to constant weight in a 105℃ drying oven, transfer it to a 100 mL volumetric flask, and dilute to the mark with distilled water. Shake well. The glucose concentration at this point is 1000 mg / L. Add 0.5 mL, 1 mL, 2 mL, 3 mL, and 4 mL of this solution to the corresponding 100 mL volumetric flasks and dilute to the mark. The resulting concentrations are 5 mg / L, 10 mg / L, 20 mg / L, 30 mg / L, and 40 mg / L, respectively.

[0078] ② Preparation of anthrone reagent: Accurately weigh 0.2g of anthrone reagent and add it to 100mL of concentrated sulfuric acid. Mix well to obtain the anthrone reagent.

[0079] ③ Construction of the glucose standard curve: Accurately measure 4 mL of anthrone reagent and 1 mL of the prepared standard glucose solution into stoppered test tubes (simultaneously measure 1 mL of distilled water as a blank control). Quickly cool in an ice-water bath, then heat in a boiling water bath for 10 min, followed by further ice-water cooling. Once completely cooled, pipette 200 μl into a 96-well plate and measure the OD value at 620 nm using a microplate reader. Plot the standard curve based on the results.

[0080] (2) Determination of glycolipid content in yeast fermentation broth:

[0081] ① Take 0.5 ml of fermentation broth, add 1 mL of ethanol, shake to mix, centrifuge at 12000 r / min for 9 min, take 20 μL of supernatant, add it to an EP tube, then add 980 μL of distilled water and 4 mL of anthrone reagent, quickly place on ice, then heat in a boiling water bath for 8 min, cool in an ice bath, and measure the OD value (m1) of the reaction solution at 620 nm using a spectrophotometer (using the solution obtained by reacting distilled water with anthrone reagent as a control). Based on the ratio between the molecular weights of glycolipids and glucose, i.e., 1.91 g of glycolipids is equivalent to 1 g of glucose, the total glycolipid content can be calculated.

[0082] ② Take 0.5 ml of fermentation broth into an EP tube, add an equal volume of 1 ml ethyl acetate, shake to mix, centrifuge at 12000 r / min for 9 min, take 20 μL of the supernatant, add it to the EP tube, then add 980 μL of distilled water and 4 mL of anthrone reagent, quickly place on ice, then heat in a boiling water bath for 8 min, cool in an ice bath, and measure the OD value (m2) of the reaction solution at 620 nm using a spectrophotometer (using the solution obtained by reacting distilled water with anthrone reagent as a control). The glycolipid content in the yeast can be determined.

[0083] 2. Method for determining the oil removal rate of yeast:

[0084] ① The oil content in produced water from oilfields was determined using the national standard HJ 637-2018 "Determination of Petroleum and Animal Oils by Infrared Spectrophotometry".

[0085] ② Mix oilfield produced water with a certain oil content with 2% bacterial fermentation broth, and place it in a 50℃ constant temperature shaking incubator for 48 hours.

[0086] ③ The oil content in produced water from oilfields treated with microorganisms was determined using the national standard HJ 637-2018 "Determination of Petroleum and Animal Oils by Infrared Spectrophotometry".

[0087] Oil removal rate = (①Measured oil content - ②Measured oil content) / ①Measured oil content × 100%

[0088] 3. Method for determining the content of biopolymers produced by Bacillus:

[0089] The fermentation broth was centrifuged at 12,000 rpm for 10 min, and the resulting white precipitate was placed at 3°C ​​for 12 h. Then, it was centrifuged at 5,000 rpm for 30 min, the precipitate was collected, and the precipitate was suspended with a minimum amount of distilled water. The pH was adjusted to 7.0 with HaOH, at least a portion of the precipitate was dissolved, and the precipitate was dried and frozen at low temperature to obtain crude dry biopolymer, which was then weighed.

[0090] 4. Method for determining the emulsification rate (E24) of Pseudomonas fermentation broth:

[0091] Mix the fermentation broth of the strain with kerosene (2:3, V / V) and add it to a graduated colorimetric tube. Shake and stir thoroughly for a period of time, and let it stand at room temperature for 24 hours. After standing, measure the height of the emulsion layer and the total liquid, and calculate the emulsification coefficient of each sample.

[0092] Emulsification rate E24 = (Emulsion layer height / Total liquid height) × 100%

[0093] After the mixture was left to stand for a period of time, the originally homogeneous emulsion gradually separated into layers. The emulsion rate E24 of Pseudomonas was calculated by measuring the height of the emulsion layer and the total liquid.

[0094] Composition of nutrient solution: Based on a total volume of 1L, the nutrient solution contains 1g of molasses, 0.5g of peptone, 0.2g of ammonium sulfate, 0.3g of sodium dihydrogen phosphate, 0.02g of dipotassium hydrogen phosphate and 0.2g of sodium citrate.

[0095] Before the experiment, microorganisms that did not significantly inhibit Candida vesicularis or did not have an antagonistic relationship with it were screened out. Further screening was conducted to identify strains with relatively good performance, and experiments were performed based on these selected microorganisms. Table 1 shows the strains used in the following examples and their sources.

[0096] Table 1

[0097] 1 Candida viviparus CGMCC No. 3456 2 Bacillus subtilis CGMCC No. 21860 3 Pseudomonas aeruginosa CGMCC No. 23170 4 Acinetobacter CCTCC KB 20081402 5 Enterobacter cloacae CCTCC KB 20081555 6 Micrococcus luteus CGMCC 1.258 7 Bacillus licheniformis BNCC188083 8 Pseudomonas schrenckii CCTCC KB 20081295 9 Bacillus amyloliquefaciens BNCC188065 10 Bacillus subtilis CCTCC CB 20082425 11 Pseudomonas aeruginosa CCTCC AB 93066

[0098] Preparation Example 1

[0099] This preparation example illustrates the preparation of microbial fermentation broth.

[0100] Pure culture fermentation was performed on strains 1-11 in Table 1. Frozen strains were thawed in warm water and streaked onto sterile LB agar plates. These plates were then incubated upside down at 37°C for 24 hours. Single colonies were then picked and placed in Erlenmeyer flasks containing LB liquid medium, and incubated at 37°C with shaking for 48 hours at 180 rpm. After 48 hours, the cell concentration in the fermentation broth for each strain was measured. Based on the measured cell concentrations of each strain, the cell concentrations in the fermentation broths of strains 1-11 were adjusted with sterile water to ensure they were all 1×10⁻⁶. 8 CFU / mL.

[0101] Example 1: Screening of composite microorganisms

[0102] Fermentation broths from strains 1-11 obtained in Preparation Example 1 were collected. These broths were used or mixed as described in Table 2, and stirred thoroughly to prepare a microbial oil-displacing agent. The interfacial tension of each strain was then measured, and their magnitudes were compared. Here, v represents volume. Unless otherwise specified, *Bacillus subtilis* is identified as *Bacillus subtilis* CGMCC No. 21860, and *Pseudomonas aeruginosa* as *Pseudomonas aeruginosa* CGMCC No. 23170.

[0103] Method for determining interfacial tension: A rotating drop interfacial tension meter was used to measure the interfacial tension values ​​of each group of oil-displacing bacteria at 50℃ under the condition of 1% volume concentration of dehydrated and degassed crude oil.

[0104] Table 2

[0105]

[0106]

[0107]

[0108] The results above show that the lowest interfacial tension value of the fermentation broth of a single strain is that of yeast, at 0.8626 mN / m, followed by Bacillus subtilis and Pseudomonas aeruginosa, with interfacial tensions of 1.5248 mN / m and 1.3654 mN / m, respectively.

[0109] Yeast and other bacterial solutions were compounded one by one, resulting in combinations 20 and 25. The interfacial tension values ​​of Candida albicans compounded with Bacillus subtilis and Pseudomonas aeruginosa were 0.0114 and 0.0564 mN / m, respectively, while the values ​​of other combinations were higher. This indicates that combination 20 has the strongest ability to reduce the oil-water interfacial tension. Therefore, the microbial oil displacement agent prepared from Candida albicans CGMCC No. 3456, Bacillus subtilis CGMCC No. 21860, and Pseudomonas aeruginosa CGMCC No. 23170 at a viable cell ratio of 1:(1.5-2.5):(2-4) can achieve a better synergistic effect.

[0110] Example 2

[0111] This example illustrates an indoor core displacement simulation test of a composite microbial enhanced oil recovery system:

[0112] Microbial inoculum or composite microbial inoculum was prepared according to the method described in Example 1, and an indoor core displacement simulation test was conducted. The test apparatus used was a gas injection core evaluation system produced by Zhongshi Dashi Instrument Technology Co., Ltd.

[0113] The experimental steps are as follows:

[0114] (1) Core saturated water:

[0115] ① Take the test core, dry it in a 60℃ constant temperature oven until constant weight, and weigh it m0;

[0116] ② Place the core in a vacuum pump, pressurize it with saturated water overnight, and weigh it m1.

[0117] ③ The pore volume (PV) can be calculated as m1-m0;

[0118] (2) Core saturated oil:

[0119] ①Place the experimental oil sample and water sample into the corresponding intermediate containers, turn on the equipment to heat, and set the temperature to 50℃;

[0120] ② Open the core displacement equipment operation interface, enter the basic data such as core length, diameter, pore volume, gas phase permeability, place the core in the middle of the core holder, connect the pipeline, adjust the flow rate, run the instrument to purge and check the pipeline sealing.

[0121] ③ Adjust the flow rate, open the switch of the intermediate container containing the oil sample, place the measuring cylinder at the outlet of the core holder, turn on the switch of the equipment operation interface, and start the experiment;

[0122] ④ Inject 3-5 PV, stop injecting, close the valve of the intermediate container containing the oil sample, measure the total outflow m2, and calculate the saturation.

[0123] (3) Water drive experiment:

[0124] ① Open the valve of the intermediate container filled with water, disconnect the inlet pipe, and when liquid comes out of the inlet, stop the pump, reconnect the inlet pipe, and start the experiment;

[0125] ② Inject 3-5 PV volumes until the water content at the outlet is 98%, then stop the injection, close the valve of the intermediate container containing the water sample, and measure the oil output, pressure difference, and flow rate corresponding to each PV volume. The corresponding core displacement efficiency can then be calculated.

[0126] (4) Microbial activation water drive experiment:

[0127] ①Pour the prepared microbial fermentation broth into the intermediate container, open the valve, disconnect the inlet pipeline, and when liquid comes out of the inlet, stop the pump, reconnect the inlet pipeline, and start the experiment;

[0128] ② Inject 3-5 PV volumes, stop the injection, close the valve of the intermediate container containing the water sample, and measure the oil output, pressure difference and flow rate corresponding to each PV volume. The corresponding core displacement efficiency can be calculated.

[0129] (5) Subsequent water drive:

[0130] ① Open the valve of the intermediate container filled with water, disconnect the inlet pipe, and when liquid comes out of the inlet, stop the pump, reconnect the inlet pipe, and start the experiment;

[0131] ② Inject 3-5 PV volumes, stop the injection, close the valve of the intermediate container containing the water sample, and measure the oil output, pressure difference, and flow rate corresponding to each PV volume. The corresponding core displacement efficiency can be calculated, and the results are shown in Table 3.

[0132] Table 3

[0133] bacterial agent concentration 1% by volume 1% by volume 1% by volume 1% by volume Core number A-1 A-2 A-3 A-4 Porosity (%) 22.58 23.58 24.35 26.21 <![CDATA[Permeability (10 -3 μm 2 )]]> 25.07 25.12 24.13 22.57 Core length (cm) 29.98 30 29.9 29.8 Oil saturation (%) 64.79 62.11 64.79 65.23 Water drive efficiency (%) 51 53.33 50.6 51.57 Microbial chemoradiation efficiency (%) 15.87 14.17 19.6 24.62 Subsequent water drive efficiency (%) 1.98 1.05 2.63 2.35 Overall improvement in oil displacement efficiency (%) 17.85 15.22 22.23 26.97

[0134] Table 3 shows that the oil displacement efficiencies of *Candida virescens* CGMCC No. 3456, *Bacillus subtilis* CGMCC No. 21860, *Pseudomonas aeruginosa* CGMCC No. 23170, and combination 20 were 15.87%, 14.17%, 19.6%, and 24.62%, respectively. Combination 20 also showed the highest overall improvement in oil displacement efficiency at 26.97%. Therefore, the indoor core displacement test also confirmed that the compound microbial agent has a higher oil displacement efficiency.

[0135] Example 3

[0136] The oil removal rate, glycolipid content, biopolymer content, and emulsification rate (E24) of the produced water treatment and oil displacement microbial agents (Candida viride CGMCC No. 3456), biopolymer-producing microbial agents (Bacillus subtilis CGMCC No. 21860), and hydrocarbon-degrading and oil-displacing microbial agents (Pseudomonas aeruginosa CGMCC No. 23170) were determined. The results showed that Candida viride CGMCC No. 3456 achieved an oil removal rate of 91% and a glycolipid content of 10.5 g / L; Bacillus subtilis CGMCC No. 21860 produced a biopolymer glucuronic acid content of 5.1 g / L; and the E24 of the fermentation broth from Pseudomonas aeruginosa CGMCC No. 23170 was 75%.

[0137] Based on the existing water injection system at the oilfield site, such as Figure 1 The propagation device shown (processing capacity 500m³) 3 The compound microbial propagation is carried out in a system that utilizes a series-connected steel buffer tank, a primary propagation tank, a secondary propagation tank, a tertiary propagation tank, and a sedimentation tank. It is also equipped with a compound microbial agent dosing device and a nutrient agent dosing device. Each of the three propagation tanks is independently equipped with packing material for microbial attachment and growth, and each also has an independent aeration device to provide the oxygen required for microbial growth. The inlet of the primary propagation tank is located at the bottom, and the outlet is located on the side wall of the sedimentation tank. The overflow outlets of the primary and tertiary propagation tanks are located at the top of the tank, and the overflow outlet of the secondary propagation tank is also located at the top, resulting in a three-dimensional S-shaped flow direction.

[0138] Expansion Cultivation: Oily produced water is introduced through the inlet to fill the device. The fermentation broth of the three bacteria in combination 20 is mixed in a biological stirring tank (the ratio of viable bacteria of yeast, Bacillus subtilis, and Pseudomonas aeruginosa in the mixed fermentation broth is 1:2:3), and nutrient solution is added. Then, it is slowly pumped into the primary expansion cultivation tank by a dosing pump (based on the total volume of the material in the device, the amount of fermentation broth of the three bacteria is 1% by volume, and the amount of nutrient solution is 0.1% by volume). The inlet and outlet of the device are closed, and the aeration devices in the primary, secondary, and tertiary expansion cultivation tanks are turned on to provide the oxygen required for microbial growth. Expansion cultivation is carried out for 10 days to allow microorganisms to grow and reproduce on the packing material in the primary, secondary, and tertiary expansion cultivation tanks. The viable bacteria count in the expanded material is ≥1×10⁻⁶. 8 CFU / mL.

[0139] Normal operation: Open the inlet and outlet of the unit. Oil-bearing produced water (fresh water) is buffered in the buffer tank and then flows into the primary expansion tank through the inlet. The water overflows through the overflow outlet in an S-shaped path, passing through the primary expansion tank, secondary expansion tank, tertiary expansion tank, and sedimentation tank in sequence. Finally, it is injected into the oil reservoir through the outlet according to the oilfield geological distribution. The hydraulic retention time in the expansion unit is 8 hours.

[0140] Technical requirements:

[0141] ① The oil content of the incoming water is ≤50.0mg / L, the suspended solids content is ≤50.0mg / L, and the influent flow rate is stable.

[0142] ② The temperature of the reaction tank is controlled at 35℃.

[0143] ③ Replenish the bacteria once a week (500kg fermentation liquid), and replenish the nutrient solution once every 3 days (1000kg, containing 1kg molasses, 0.5kg peptone, 0.2kg ammonium sulfate, 0.3kg sodium dihydrogen phosphate, 0.02kg dipotassium hydrogen phosphate, and 0.2kg sodium citrate).

[0144] ④ The air intake of the blower should ensure that the oxygen content in the reaction tank is above 2.0 mg / L.

[0145] ⑤ The water inlet regulating tank, biochemical reaction tank, sewage sedimentation tank, and purified water tank should be discharged for 3-5 minutes every day.

[0146] ⑥ No bactericides or sterilization devices shall be added to the produced water treatment process.

[0147] ⑦ The bacterial concentration in each stage of the expansion culture tank was tested weekly. The testing method was based on GB4789.2 - Food Microbiology Examination: Determination of Total Colony Count. The bacterial concentration results for each stage of the expansion culture tank are shown in Table 4 (unit: CFU / mL).

[0148] Table 4

[0149] Week 1 <![CDATA[1.2×10 3 ]]> <![CDATA[2.6×10 6 ]]> <![CDATA[8.2×10 6 ]]> <![CDATA[1.5×10 8 ]]> <![CDATA[2.8×10 8 ]]> Week 2 <![CDATA[2.3×10 3 ]]> <![CDATA[3.8×10 5 ]]> <![CDATA[2.4×10 7 ]]> <![CDATA[2.7×10 8 ]]> <![CDATA[3.1×10 8 ]]> Week 3 <![CDATA[1.8×10 3 ]]> <![CDATA[4.1×10 5 ]]> <![CDATA[6×10 6 ]]> <![CDATA[3.5×10 8 ]]> <![CDATA[4.9×10 8 ]]> Week 4 <![CDATA[8.8×10 2 ]]> <![CDATA[2.2×10 4 ]]> <![CDATA[3.4×10 5 ]]> <![CDATA[8.9×10 7 ]]> <![CDATA[1.2×10 8 ]]>

[0150] As shown in Table 4, the viable bacteria count in the effluent material reached 1×10⁻⁶. 8 With a concentration of CFU / mL or higher, this propagation device and method can effectively propagate the added microorganisms.

[0151] Example 4

[0152] An indoor core displacement simulation experiment was conducted according to the method and apparatus of Example 2, except that the effluent from the expansion tank of Example 3 was collected for the indoor core displacement experiment. Three parallel effluent samples from the expansion tank were taken and labeled as effluent 1, effluent 2, and effluent 3, respectively. The parameters and core displacement efficiencies of effluent 1, effluent 2, and effluent 3 are shown in Table 5.

[0153] Table 5

[0154]

[0155]

[0156] The results in Table 5 show that the effluent after expansion culture, as verified by indoor microbial displacement experiments, can indeed improve the oil displacement efficiency of crude oil.

[0157] Comparative Example 1

[0158] Oil displacement was performed on the reservoir using the method and apparatus of Example 3, except that the apparatus consisted of two sets of expansion tanks connected in series. Specifically, the expansion device included a steel buffer tank, a primary expansion tank, a secondary expansion tank, and a sedimentation tank connected in series, and was also equipped with a compound microbial agent dosing device and a nutrient agent dosing device. Each of the two expansion tanks was independently equipped with packing material for microbial attachment and growth, and each was also independently equipped with an aeration device to provide the oxygen required for microbial growth. The inlet of the primary expansion tank was located at the bottom of the tank, and the outlet was located on the side wall of the sedimentation tank. The overflow outlet of the primary expansion tank was located at the top of the tank, and the overflow outlet of the secondary expansion tank was located at the bottom of the tank, resulting in a three-dimensional S-shaped flow direction. The crude oil impurities in the effluent after reducing the expansion tank from three stages to two stages are shown in Table 6.

[0159] Table 6

[0160]

[0161] As shown in Table 6, the effluent after secondary expansion culture did not meet the oilfield water quality treatment standards (oil content ≤10.0 mg / L, viable bacteria count in effluent ≥1×10⁻⁶). 8(CFU / mL). The primary, secondary, and tertiary expansion tanks in the expansion device of this invention are mainly used for the addition of microorganisms for growth and reproduction, as well as for the treatment of crude oil contaminants in the incoming water. The three sets of expansion tanks are connected in series and none can be omitted. Reducing any one set of expansion devices will prevent the achievement of the bacterial concentration growth standard and the oilfield water quality treatment standard.

[0162] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A composite microorganism, characterized in that, The composite microorganisms include yeast, as well as Bacillus and Pseudomonas; The yeast strain mentioned is *Candida virescens*, with accession number CGMCC No. 3456. Candida viswanathii ; The Bacillus species mentioned is Bacillus subtilis with accession number CGMCC No. 21860; The Pseudomonas aeruginosa is Pseudomonas aeruginosa with accession number CGMCC No. 23170. In the composite microorganism, the ratio of viable yeast, Bacillus and Pseudomonas is 1:(1.5-2.5):(2-4).

2. A microbial oil displacement agent, characterized in that, The microbial oil displacement agent comprises: the composite microorganism as described in claim 1, and the metabolites of the composite microorganism.

3. The microbial oil displacement agent according to claim 2, characterized in that, The metabolites are produced during the growth and reproduction of the composite microorganisms using the nutrient solution; wherein, based on a volume of 1L of the nutrient solution, the nutrient solution contains 1-2g of molasses, 0.1-1g of peptone, 0.1-0.5g of ammonium sulfate, 0.1-0.5g of sodium dihydrogen phosphate, 0.01-0.05g of dipotassium hydrogen phosphate and 0.1-0.5g of sodium citrate; And / or, the pH of the microbial oil displacement agent is 6-8.

4. The application of the microbial flooding agent according to claim 2 or 3 in reducing the interfacial tension of crude oil or in reservoir flooding.

5. The application according to claim 4, characterized in that, The application described is in oil displacement in low-permeability reservoirs.

6. A method for microbial enhanced oil recovery, characterized in that, The method includes: mixing the composite microorganisms described in claim 1 with oil-bearing produced water for expansion culture, and then contacting the expanded material with the oil reservoir to be displaced.

7. The method according to claim 6, characterized in that, The contact conditions include a temperature of 28-42°C; And / or, the oil content in the oil-bearing produced water is not higher than 50 mg / L.

8. The method according to claim 6, characterized in that, The conditions for the expansion culture include: a temperature of 28-42℃; and a dissolved oxygen (DO) content of 2 mg / L or higher. And / or, the expansion time is such that the viable count in the expanded material is 1×10⁻⁶. 8 CFU / mL or higher.

9. The method according to claim 8, characterized in that, The conditions for the expansion cultivation include a temperature of 30-37℃.

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