Compound microorganism, microorganism oil-displacing agent, application of microorganism oil-displacing agent, microorganism oil-displacing method and microorganism oil-displacing device

Abstract

The invention relates to the field of microbial oil displacement, and discloses a compound microorganism, a microbial oil displacement agent and application thereof, a microbial oil displacement method and a microbial oil displacement device. The compound microorganisms comprise saccharomycetes, bacillus and / or pseudomonas; wherein the saccharomycetes are Candida viswanthalii of which the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.3456, and the saccharomycetes are Candida viswanthalii of which the preservation number is The ratio of the total viable count of the bacillus and the pseudomonas to the viable count of the saccharomycetes is 2-8. The microbial oil-displacing agent disclosed by the invention can be used for remarkably reducing interfacial tension and has a good oil-displacing effect.

Description

Technical Field

[0001] The invention relates to the field of microbial oil recovery, and in particular to a composite microorganism, a microbial oil recovery agent and applications thereof, a microbial oil recovery method and a microbial oil recovery device. Background Art

[0002] With the gradual aging of conventional oil reservoirs, the development of low-permeability and ultra-low-permeability oilfield reserves has become an important potential for the stable development of my country's onshore oil industry. The fracturing and acidification measures in the process of water injection development of low-permeability and ultra-low-permeability oil reservoirs, as well as the application of chemical flooding technologies such as polymer flooding and composite flooding, make the composition of oil-bearing produced water complex, which has great harm to the environment and formations. Traditional treatment such as sedimentation and filtration has the problems of high process cost, complex equipment process, short service life, and secondary pollution. Microbial oil recovery technology has the advantages of being environmentally friendly and no special treatment of produced water, and is widely used.

[0003] CN112374701A provides an oilfield produced water treatment process using an oleophilic bacteria group to remove oil. The biological bacterial agent contains Bacillus licheniformis, Bacillus brevis, and Pseudocycosporium in a compound ratio of 2:1:1. The oil in the produced water is decomposed into non-toxic carbon dioxide and water, and then the effluent is filtered through a membrane to meet the oilfield injection water quality standards. However, the microorganisms in the water after treatment are also filtered out and cannot be reused.

[0004] CN104312561A discloses a composite microbial strain for use in tertiary oil recovery. A mixed fermentation liquid of three bacteria, namely, Halobacterium salinarium, Brevibacillus brevis and Bacillus cereus, is used in tertiary oil recovery in a weight ratio of 1:1:1. The mixed fermentation liquid can survive in a high-salt oil recovery environment with a salt content higher than 12%. The crude oil viscosity can be reduced by 15.4%, 26.4% and 35.2% respectively, and the interfacial tension between oil and water can be reduced by 91.3%, 76.5% and 58.6% respectively. The mixed fermentation liquid has obvious beneficial effects of reducing the viscosity of crude oil and the interfacial tension between oil and water, and shows a wide range of application value. However, the mixed fermentation liquid needs to be fermented and expanded in large quantities in a factory and transported to a test site, which is costly.

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

[0006] The purpose of the present invention is to overcome the problems that the bio-bacterial agent oil recovery effect in the prior art is not ideal, the fermentation process of ground strains is cumbersome, the bacterial liquid needs to be transported over long distances, and the produced water after oil recovery cannot be injected back into the formation to recover oil. A composite microorganism and a microbial oil recovery agent and their application, a microbial oil recovery method and a microbial oil recovery device are provided. The microbial oil recovery agent can significantly reduce interfacial tension and has a good oil recovery effect. The microbial oil recovery device can, on the one hand, utilize the growth metabolism of the composite microorganism to remove oil in the produced water, and on the other hand, utilize the produced water after proliferation and expansion to inject into the formation to recover oil.

[0007] In order to achieve the above object, the present invention provides a composite microorganism in a first aspect, wherein the composite microorganism comprises yeast, and Bacillus and / or Pseudomonas;

[0008] Wherein, the yeast is Candida viswanathii with a preservation number of CGMCC No.3456;

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

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

[0011] The third aspect of the present invention provides the use of the microbial oil-displacing agent provided by the present invention in reducing interfacial tension or in oil recovery in oil reservoirs.

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

[0013] A fifth aspect of the present invention provides a device for microbial oil recovery, wherein the device comprises:

[0014] The first-stage expansion tank, the second-stage expansion tank and the third-stage expansion tank connected in series are used to expand the composite microorganisms and remove oil impurities in the oil-containing produced water;

[0015] A water inlet disposed upstream of the first-stage expansion tank for introducing oil-containing produced water;

[0016] The water outlet arranged at the downstream of the three-stage expansion tank is used to lead out the expanded material and the produced water after de-oiling.

[0017] Through the above technical solution, the beneficial effects of the present invention are:

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

[0019] In the microbial oil recovery device provided by the present invention, microbial oil recovery is carried out. By adding composite microorganisms into the microbial oil recovery device, on the one hand, the growth and metabolism of microorganisms can be used to remove oil-containing impurities in the produced water of the oil reservoir; on the other hand, the water treated by microbial expansion is injected into the formation for oil recovery, which not only purifies the water quality to meet the oil field reinjection standard, but also avoids the tedious process of industrial fermentation of single strains on the ground and the inconvenience of long-distance transportation of microbial agent products; that is, the oil in the oil-containing produced water is removed by the device and method of the present invention, and then it is reinjected into the oil reservoir to be recovered for oil recovery, which improves the recovery rate of the oil reservoir while recycling water resources, achieving multiple goals at one stroke, and has the advantages of low cost, simple operation, green environmental protection, etc., and has broad prospects for promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0021] Description of Reference Numerals

[0022] 1. Primary expansion pool 2. Secondary expansion pool 3. Third expansion pool 4. Sedimentation pool

[0023] 5 water inlet 6 water outlet 7 overflow 8 filler 9 liquid distribution tank

[0024] 10 buffer tank 11 dosing pump 12 aeration pump DETAILED DESCRIPTION

[0025] The endpoints and any values ​​of the ranges disclosed in this article 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 each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

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

[0027] Wherein, the yeast is Candida viswanathii with a preservation number of CGMCC No.3456;

[0028] The ratio of the total viable count of Bacillus and Pseudomonas to the viable count 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, preferably selected from at least one of Bacillus subtilis with a preservation number of CGMCC No. 21860, Bacillus licheniformis with a preservation number of BNCC188083 and Bacillus amyloliquefaciens with a preservation number of BNCC188065.

[0030] According to the present invention, preferably, the Pseudomonas is selected from Pseudomonas aeruginosa and / or Pseudomonas stutzeri, preferably selected from Pseudomonas aeruginosa, and more preferably Pseudomonas aeruginosa with a preservation number of CGMCC No.23170.

[0031] According to a preferred embodiment of the present invention, the composite microorganism comprises Candida viswanathii with a preservation number of CGMCC No.3456, Bacillus licheniformis with a preservation number of BNCC188083 and Pseudomonas aeruginosa with a preservation number of CGMCC No.23170.

[0032] According to a preferred embodiment of the present invention, the composite microorganism comprises Candida viswanathii with a preservation number of CGMCC No.3456, Bacillus amyloliquefaciens with a preservation number of BNCC188065 and Pseudomonas aeruginosa with a preservation number of CGMCC No.23170.

[0033] According to a preferred embodiment of the present invention, the composite microorganism comprises Candida viswanathii with a preservation number of CGMCC No.3456, Bacillus licheniformis with a preservation number of BNCC188083, Bacillus amyloliquefaciens with a preservation number of BNCC188065 and Pseudomonas aeruginosa with a preservation number of CGMCC No.23170.

[0034] According to a preferred embodiment of the present invention, the composite microorganism comprises Candida viswanathii with a preservation number of CGMCC No.3456, Bacillus subtilis with a preservation number of CGMCC No.21860, Bacillus licheniformis with a number of BNCC188083 and Pseudomonas aeruginosa with a preservation number of CGMCC No.23170.

[0035] According to a preferred embodiment of the present invention, the composite microorganism comprises Candida viswanathii with a preservation number of CGMCC No.3456, Bacillus subtilis with a preservation number of CGMCC No.21860, Bacillus amyloliquefaciens with a number of BNCC188065 and Pseudomonas aeruginosa with a preservation number of CGMCC No.23170.

[0036] According to a preferred embodiment of the present invention, the composite microorganism comprises Candida viswanathii with a preservation number of CGMCC No.3456, Bacillus subtilis with a preservation number of CGMCC No.21860, Bacillus licheniformis with a preservation number of BNCC188083, Bacillus amyloliquefaciens with a preservation number of BNCC188065 and Pseudomonas aeruginosa with a preservation number of CGMCC No.23170.

[0037] According to a most preferred embodiment of the present invention, the composite microorganism comprises Candida viswanathii with a preservation number of CGMCC No. 3456, Bacillus subtilis with a preservation number of CGMCC No. 21860, and Pseudomonas aeruginosa with a preservation number of CGMCC No. 23170. The inventors found that the specific combination of Candida viswanathii with a preservation number of CGMCC No. 3456, Bacillus subtilis with a preservation number of CGMCC No. 21860, and Pseudomonas aeruginosa with a preservation number of CGMCC No. 23170 has a better oil displacement effect.

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

[0039] According to the present invention, preferably, in the composite microorganism, the ratio of the number of live bacteria of yeast, bacillus and pseudomonas is 1:(1-3):(1-5), preferably 1:(1.5-2.5):(2-4). When the ratio of the number of live bacteria of yeast, bacillus and pseudomonas meets this range, the oil displacement effect of the composite microorganism can be further improved.

[0040] The second aspect of the present invention provides a microbial oil-displacing agent, wherein the microbial oil-displacing agent comprises: the composite microorganism provided by the present invention, and the metabolites of the composite microorganism. The metabolites are produced in the process of the composite microorganism growing and reproducing using a nutrient; wherein, based on the volume of the nutrient being 1L, the nutrient 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-displacing agent is 6-8.

[0042] The third aspect of the present invention provides the use of the microbial oil-displacing agent provided by the present invention in reducing interfacial tension or in oil recovery in oil reservoirs; preferably, the use of the microbial oil-displacing agent in low permeability oil reservoirs.

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

[0044] The permeability of low permeability reservoirs is (0.1-50)×10 -3 μm 2 According to the actual production characteristics, low permeability oil layers can be further divided into general low permeability oil layers, ultra-low permeability oil layers and ultra-low permeability oil layers according to the average permeability of the oil layers. The average permeability of the oil layers is (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 oil recovery, wherein the method comprises: mixing the composite microorganism provided by the present invention with oil-containing produced water for expansion, and then contacting the expanded material with the oil reservoir to be flooded.

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

[0047] According to the present invention, preferably, the oil content in the oil-containing 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, preferably 30-37°C; and a dissolved oxygen content DO of 2 mg / L or more.

[0049] According to the present invention, preferably, the expansion time is such that the number of viable bacteria in the material after expansion is 1×10 8 CFU / mL and above.

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

[0051] According to the present invention, preferably, the amount of the composite microorganism added after the expansion is such that the number of viable bacteria in the contacted material is 1×10 8 CFU / mL and above.

[0052] A fifth aspect of the present invention provides a device for microbial oil recovery, wherein the device comprises:

[0053] The first-stage expansion tank, the second-stage expansion tank and the third-stage expansion tank connected in series are used to expand the composite microorganisms and remove oil impurities in the oil-containing produced water;

[0054] A water inlet disposed upstream of the first-stage expansion tank for introducing oil-containing produced water;

[0055] The water outlet arranged at the downstream of the three-stage expansion tank is used to lead out the expanded material and the produced water after de-oiling.

[0056] The composite microorganisms are added to the microbial oil recovery device, and the oil-containing produced water (incoming water) is introduced from the water inlet upstream of the first-stage expansion pool, so that the composite microorganisms and the oil-containing produced water are mixed for expansion. The oil impurities in the oil-containing produced water can be removed by the growth metabolism of the microorganisms, and then the expanded materials are drawn out from the outlet downstream of the third-stage expansion pool and injected into the formation for oil recovery. Among them, the expanded materials contain the produced water after the oil impurities are removed, the expanded composite microorganisms, the metabolites of the composite microorganisms and the nutrients. The produced water after the oil impurities are removed meets the oil field reinjection standard. The expanded materials are injected into the formation for oil recovery, which improves the recovery rate of the oil reservoir while recycling water resources.

[0057] The device of the present invention comprises a primary culture pool, a secondary culture pool and a tertiary culture pool, which can culture microorganisms to a suitable number and fully remove crude oil impurities in water.

[0058] According to the present invention, preferably, the first-stage expansion culture tank, the second-stage expansion culture tank and the third-stage expansion culture tank are each independently equipped with a filler for microorganisms to attach and grow, wherein the filler may be one or more of aldehyded cellulose soft material, suspended ball filler and fiber ball filler.

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

[0060] According to the need of treating oilfield produced water, preferably, the water inlet is located at the lower part of the first-level expansion tank.

[0061] According to the present invention, preferably, the overflow ports of the first-stage expansion culture tank and the third-stage expansion culture tank are respectively located at the upper part of the tank body, and the overflow port of the second-stage expansion culture tank is located at the lower part of the tank body, so that the liquid flow direction is a three-dimensional S-shape. Such a 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 also equipped with a sedimentation tank, which is located downstream of the three-stage expansion tank and is used to precipitate solid impurities in the water body.

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

[0064] According to the present invention, preferably, the device is also equipped with a buffer tank, which is located upstream of the primary expansion tank along the liquid flow direction and is used to buffer the oil-containing produced water (incoming water).

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

[0066] According to the present invention, preferably, a dosing pump is further arranged between the liquid preparation tank and the primary culture expansion tank for introducing a mixed solution of composite microorganisms and nutrients into the primary culture expansion tank.

[0067] According to the present invention, preferably, the device is also equipped with a composite microorganism dosing device and a nutrient dosing device, which are used to add composite microorganisms and nutrients, respectively.

[0068] In order to allow a sufficient amount of microorganisms to grow on the fillers in the expansion pool, the expansion pools can be connected by injecting oil-containing produced water into the device, the water inlet and outlet of the device are closed, and then the composite microorganisms and nutrients are injected into them for expansion. There is no special restriction on the expansion time. For example, the bacterial concentration in the expansion pool can reach 1×10 8 CFU / mL or above is sufficient. Nutrients can be added during this process, and those skilled in the art can add them as needed.

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

[0070] According to a preferred embodiment of the present invention, Figure 1 The method for culturing composite microorganisms in the microbial oil recovery device and recovering oil from the oil reservoir to be recovered comprises the following steps:

[0071] S1. Fermentation broths of Candida viswanathii with a preservation number of CGMCC No. 3456, Bacillus subtilis with a preservation number of CGMCC No. 21860, and Pseudomonas aeruginosa with a preservation number of CGMCC No. 23170 are prepared respectively, so that the number of viable bacteria in the fermentation broths of the three bacteria is independently 1×10 8 CFU / mL or more, the fermentation broths of the three bacteria are mixed in a liquid preparation tank 9, and the ratio of the number of live bacteria of Candida vesica, Bacillus subtilis and Pseudomonas aeruginosa in the mixed fermentation broth is 1:(1-3):(1-5), thereby preparing a composite microbial agent.

[0072] S2. Introduce oil-containing produced water (the oil content is not higher than 50 mg / L) from the water inlet 5 to fill the device with oil-containing produced water, put the nutrient solution into the liquid preparation tank 9, mix it with the fermentation liquid of the three bacteria, and then slowly pump it into the first-level expansion culture tank 1 by the dosing pump 11 (based on the total volume of the feed liquid in the device, the amount of the fermentation liquid of the three bacteria is 0.5-1.5% by volume, and the amount of the nutrient solution is 0.08-0.12% by volume). Close the water inlet 5 and the water outlet 6 of the device, turn on the aeration pump 12 in the first-stage expansion culture tank 1, the second-stage expansion culture tank 2 and the third-stage expansion culture tank 3 to provide oxygen required for the growth of microorganisms, and expand the culture for 10-15 days (the temperature is 28-42°C, and the air intake of the fan ensures that the oxygen content in the device is above 2.0 mg / L), so that the microorganisms grow and reproduce on the fillers 8 in the first-stage expansion culture tank 1, the second-stage expansion culture tank 2 and the third-stage expansion culture tank 3. The expansion time is such that the number of viable bacteria in the material after expansion is 1×10 8 CFU / mL and above.

[0073] S3, wait until the total concentration of live bacteria in the device reaches 1×10 8After the oil content exceeds 100 / mL, the water inlet 5 and the water outlet 6 of the device are opened, and the oil-containing produced water (fresh water) is buffered in the buffer tank 10 and flows from the water inlet 5 into the primary expansion tank 1, and the water flows in an S-shaped route through the overflow port 7 to pass through the primary expansion tank 1, the secondary expansion tank 2, the tertiary expansion tank 3 and the sedimentation tank 4 in sequence, and is injected into the oil reservoir through the water outlet 6 according to the oil field geological injection. The hydraulic retention time in the expansion device is 6-8h.

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

[0075] 1. Determination of the content of glycolipid surfactants produced by yeast:

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

[0077] ①Prepare 10mg / mL glucose solution: Accurately weigh 0.1g of analytical pure anhydrous glucose that has been dried to constant weight in a 105℃ drying oven, transfer to a 100ml volumetric flask, dilute to volume with distilled water, and shake well. The glucose concentration is now 1000mg / L. Take 0.5mL, 1mL, 2mL, 3mL, and 4mL and add them to the corresponding 100mL volumetric flasks to dilute to volume. The resulting concentrations are 5mg / L, 10mg / L, 20mg / L, 30mg / L, and 40mg / L, respectively.

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

[0079] ③ Drawing of glucose standard curve: Accurately measure 4 mL of anthrone reagent and 1 mL of the standard glucose solution prepared above into a stoppered test tube (measure 1 mL of distilled water as a blank control experiment at the same time), quickly cool in an ice water bath, then heat in a boiling water bath for 10 min and continue to cool in an ice bath. After cooling thoroughly, pipette 200 μl into a 96-well plate and measure the OD value at 620 nm in an ELISA reader. Draw a standard curve based on the results.

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

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

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

[0083] 2. Determination method of yeast oil removal rate:

[0084] ① The national standard HJ 637-2018 "Infrared spectrophotometric method for the determination of petroleum and animal and vegetable oils" is used to detect the oil content in oilfield produced water.

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

[0086] ③ The national standard HJ 637-2018 "Infrared spectrophotometric method for the determination of petroleum and animal and vegetable oils" was used to detect the oil content in oilfield produced water treated with microorganisms.

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

[0088] 3. Determination of the content of biopolymer produced by Bacillus:

[0089] The fermentation liquid was centrifuged at 12000rpm for 10min, and the formed white precipitate was placed at 3°C ​​for 12h, and then centrifuged at 5000rpm for 30min. The precipitate was collected and suspended with a minimum amount of distilled water, and the pH value was adjusted to 7.0 with HaOH. At least a part of the precipitate was dissolved, and it was low-temperature dried and frozen to obtain a crude dry biopolymer, which was weighed.

[0090] 4. Emulsification rate E24 determination method of Pseudomonas fermentation broth:

[0091] Take the fermentation liquid of the strain and mix it with kerosene (2:3, V / V), add it into a colorimetric tube with a scale, shake it thoroughly for a period of time, and let it stand at room temperature for 24 hours; after standing, measure the emulsion layer and the total liquid height, and calculate the emulsification coefficient of each sample.

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

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

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

[0095] Before the experiment, microorganisms that do not significantly inhibit Candida visnatus or have no antagonistic relationship with it were screened out, and strains with relatively good performance were further screened out, and experiments were conducted based on the screened microorganisms. Table 1 shows the strains used in the following examples and their sources.

[0096] Table 1

[0097] serial number strain Source or accession number 1 Candida vesica 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 stutzeri 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 is used to illustrate the preparation of microbial fermentation broth.

[0100] The strains 1-11 in Table 1 were subjected to pure culture fermentation. The frozen strains were revived with warm water and then streaked on sterile LB solid plates. They were placed in a 37°C constant temperature incubator and inverted for 24 hours. Single colonies were then picked out and placed in a triangular flask filled with LB liquid culture medium. The plates were placed in a 37°C constant temperature shaker and shaken for 48 hours at 180 rpm. After 48 hours, the bacterial concentration in the fermentation broth of each strain was detected. According to the measured bacterial concentration of each strain, the bacterial concentration in the fermentation broth of strains 1-11 was adjusted with sterile water to 1×10 8 CFU / mL.

[0101] Example 1: Screening of composite microorganisms

[0102] The fermentation broth of strains 1-11 obtained by fermentation in Preparation Example 1 was collected, and the fermentation broth of the above strains was used or mixed and prepared in the manner described in Table 2, and stirred evenly to prepare a microbial oil displacement agent, and then the interfacial tension thereof was measured respectively, and the magnitude of the interfacial tension thereof was compared. Wherein, v represents volume. In the absence of a mark, Bacillus subtilis is Bacillus subtilis CGMCC No. 21860, and Pseudomonas aeruginosa is Pseudomonas aeruginosa CGMCC No. 23170.

[0103] Method for measuring interfacial tension: Using a spinning drop interfacial tension meter, dehydrated and degassed crude oil was selected to measure the interfacial tension value of each group of oil-displacing bacterial agents at 1% volume concentration at 50°C.

[0104] Table 2

[0105]

[0106]

[0107]

[0108] From the above results, it can be seen that the interfacial tension value of the fermentation broth of a single strain is the lowest for yeast, which is 0.8626mN / m, followed by Bacillus subtilis and Pseudomonas aeruginosa, with interfacial tensions of 1.5248mN / m and 1.3654mN / m respectively.

[0109] The interfacial tension values ​​of the yeast and other bacterial solutions were compounded one by one, and the interfacial tension values ​​of the combinations 20 and 25, Candida, Bacillus subtilis, and Pseudomonas aeruginosa were 0.0114 and 0.0564 mN / m, respectively, and the other combinations were all higher than this value. It can be seen that the combination 20 has the strongest ability to reduce the oil-water interfacial tension. This shows that the microbial oil displacement agent prepared by Candida visweiss CGMCC No.3456, Bacillus subtilis CGMCC No.21860 and Pseudomonas aeruginosa CGMCC No.23170 with a live cell count ratio of 1: (1.5-2.5): (2-4) can achieve a better synergistic effect.

[0110] Example 2

[0111] This example is used to illustrate the indoor core displacement simulation test of the composite microbial oil recovery system:

[0112] A microbial culture liquid or a composite microbial culture liquid was prepared in the manner described in Example 1, and an indoor core displacement simulation test was conducted. The test device used a gas injection core evaluation system produced by Zhongshi Dashiyi Technology Co., Ltd.:

[0113] The experimental steps are as follows:

[0114] (1) Core saturated water:

[0115] ① Take the test core, place it in a 60℃ constant temperature box and dry it to constant weight, and weigh its weight m0;

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

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

[0118] (2) Core saturated with oil:

[0119] ①Put the experimental oil sample and water sample into the corresponding intermediate container respectively, turn on the equipment for heating, and set the temperature to 50℃;

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

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

[0122] ④Inject 3-5 PV, stop injecting, turn off the valve of the intermediate container containing the oil sample, measure the total water output m2, and calculate the saturation;

[0123] (3) Water flooding experiment:

[0124] ①Open the valve of the intermediate container filled with water, disassemble the inlet pipeline, and when liquid comes out from the inlet, stop the pump, connect the inlet pipeline, and start the experiment;

[0125] ② Inject 3-5 PVs until the water content at the outlet reaches 98%, stop injecting, turn off the valve of the intermediate container containing the water sample, measure the oil output, pressure difference and flow value corresponding to each PV, and calculate the corresponding core displacement efficiency;

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

[0127] ①Put the prepared microbial fermentation liquid into the intermediate container, open the valve, disassemble the inlet pipeline, and when liquid comes out from the inlet, stop the pump, connect the inlet pipeline, and start the experiment;

[0128] ②Inject 3-5 PV, stop injection, turn off the valve of the intermediate container containing the water sample, measure the oil output, pressure difference and flow value corresponding to each PV, and calculate the corresponding core displacement efficiency;

[0129] (5) Subsequent water flooding:

[0130] ①Open the valve of the intermediate container filled with water, disassemble the inlet pipeline, and when liquid comes out from the inlet, stop the pump, connect the inlet pipeline, and start the experiment;

[0131] ② Inject 3-5 PV, stop injection, turn off the valve of the intermediate container containing the water sample, measure the oil output, pressure difference and flow value corresponding to each PV, and calculate the corresponding core displacement efficiency. The results are shown in Table 3.

[0132] Table 3

[0133] parameter Yeast Bacillus subtilis Pseudomonas aeruginosa Combination 20 Bacterial agent concentration 1 volume% 1 volume% 1 volume% 1 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 flooding efficiency (%) 51 53.33 50.6 51.57 Microbial flooding efficiency (%) 15.87 14.17 19.6 24.62 Subsequent water flooding efficiency (%) 1.98 1.05 2.63 2.35 Comprehensive improvement of oil recovery efficiency (%) 17.85 15.22 22.23 26.97

[0134] As shown in Table 3, the oil displacement efficiencies of Candida vesica CGMCC No. 3456, Bacillus subtilis CGMCC No. 21860, Pseudomonas aeruginosa CGMCC No. 23170 and combination 20 are 15.87%, 14.17%, 19.6% and 24.62% respectively, and the combination 20 also has the highest comprehensive improvement in oil displacement efficiency, which is 26.97%. Therefore, the indoor core displacement test also confirms that the composite bacterial agent has a higher oil displacement efficiency.

[0135] Example 3

[0136] The oil removal rate, glycolipid content, biopolymer content and emulsification rate E24 index of the microbial agent capable of producing water treatment and oil recovery (Candida visweiss CGMCC No. 3456), the microbial agent producing biopolymer (Bacillus subtilis CGMCC No. 21860) and the microbial agent for hydrocarbon recovery and oil recovery (Pseudomonas aeruginosa CGMCC No. 23170) were respectively measured. It was found that the oil removal rate of Candida visweiss CGMCC No. 3456 was 91%, the glycolipid content was 10.5 g / L, the content of glucuronic acid of the biopolymer produced by Bacillus subtilis CGMCC No. 21860 was 5.1 g / L, and the E24 of the fermentation liquid of Pseudomonas aeruginosa CGMCC No. 23170 was 75%.

[0137] Based on the original water injection system at the oil field, Figure 1 The expansion device shown (processing capacity 500m 3 / day) for the expansion and cultivation of composite microorganisms, the expansion and cultivation device includes a steel buffer tank, a first-stage expansion and cultivation tank, a second-stage expansion and cultivation tank, a third-stage expansion and cultivation tank and a sedimentation tank connected in series, and is also equipped with a composite bacterial agent dosing device and a nutrient agent dosing device. Fillers are independently installed in the three expansion and cultivation tanks for the attachment and growth of microorganisms, and aeration devices are also independently installed to provide oxygen required for the growth of microorganisms. The water inlet in the first-stage expansion and cultivation tank is located at the lower part of the first-stage expansion and cultivation tank, the water outlet is located at the side wall of the sedimentation tank, the overflow ports of the first-stage expansion and cultivation tank and the third-stage expansion and cultivation tank are respectively located at the upper part of the tank body, and the overflow port of the second-stage expansion and cultivation tank is located at the upper part of the tank body, so that the liquid flow direction is three-dimensional S-shaped.

[0138] Expansion culture: Introduce oil-containing produced water from the water inlet to fill the device with oil-containing produced water. Mix the fermentation liquid of the three bacteria in combination 20 in a biological stirring tank (the ratio of the number of live bacteria of yeast, Bacillus subtilis and Pseudomonas aeruginosa in the mixed fermentation liquid is 1:2:3), add nutrient solution, and then slowly pump it into the first-level expansion culture tank by the dosing pump (based on the total volume of the liquid in the device, the amount of fermentation liquid of the three bacteria is 1 volume%, and the amount of nutrient solution is 0.1 volume%). Close the water inlet and outlet of the device, open the aeration device in the first expansion culture tank, the second expansion culture tank and the third expansion culture tank to provide the oxygen required for the growth of microorganisms, expand the culture for 10 days, so that the microorganisms grow and reproduce on the fillers in the first expansion culture tank, the second expansion culture tank and the third expansion culture tank, and the number of live bacteria in the material after expansion culture is ≥1×10 8 CFU / mL.

[0139] Normal operation: Open the water inlet and outlet of the device, and the oil-containing produced water (fresh water) flows from the water inlet into the primary expansion tank after being buffered in the buffer tank. The water flows in an S-shaped route through the overflow port and passes through the primary expansion tank, the secondary expansion tank, the tertiary expansion tank and the sedimentation tank in sequence, and is injected into the reservoir through the water outlet in accordance with the geological allocation of the oil field. The hydraulic retention time in the expansion device is 8h.

[0140] Technical requirements:

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

[0142] ②The reaction pool temperature is controlled at 35℃.

[0143] ③ Replenish the bacteria once every 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 fan should ensure that the oxygen content in the reaction tank is above 2.0 mg / L.

[0145] ⑤ The incoming water regulating tank, biochemical reaction tank, sewage sedimentation tank, and purified water tank discharge sewage for 3-5 minutes every day.

[0146] ⑥ No bactericide shall be added or bactericidal device shall be installed in the produced water treatment process.

[0147] ⑦ Test the concentration of bacteria in each level of expansion culture pool once a week. The test method refers to GB4789.2-Determination of total colony count in food microbiology. The test results of bacteria concentration in each level of expansion culture pool are shown in Table 4 (unit: CFU / mL).

[0148] Table 4

[0149] time Incoming water First-level expansion pool Secondary expansion pool Three-level expansion pool Water 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] It can be seen from the results in Table 4 that the number of viable bacteria in the effluent material reached 1×10 8 CFU / mL or above, so the expansion culture device and method can play the role of expanding the added microorganisms.

[0151] Example 4

[0152] The indoor core displacement simulation experiment was carried out according to the method and device of Example 2, except that the effluent from the expansion pool of Example 3 was collected for the indoor core displacement experiment. Three effluent samples from the expansion pool were taken in parallel and recorded as effluent 1, effluent 2 and effluent 3, respectively. The parameters and core displacement efficiency 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 can indeed improve the oil recovery efficiency of crude oil after verification by indoor microbial displacement experiments.

[0157] Comparative Example 1

[0158] The reservoir is flooded according to the method and device of Example 3, except that the device is two groups of expansion culture pools connected in series. Specifically, the expansion culture device includes a steel buffer pool, a primary expansion culture pool, a secondary expansion culture pool and a sedimentation tank connected in series, and is also equipped with a composite bacterial agent dosing device and a nutrient agent dosing device. Fillers are independently installed in the two expansion culture pools for microorganisms to attach and grow, and aeration devices are also independently installed to provide oxygen required for microbial growth. The water inlet in the primary expansion culture pool is located at the lower part of the primary expansion culture pool, the water outlet is located at the side wall of the sedimentation tank, the overflow port of the primary expansion culture pool is located at the upper part of the pool body, and the overflow port of the secondary expansion culture pool is located at the lower part of the pool body, so that the liquid flow direction is three-dimensional S-shaped. The three-stage expansion culture pool is reduced to a secondary expansion culture pool, and the crude oil impurities in the bacterial concentration in the effluent are shown in Table 6.

[0159] Table 6

[0160]

[0161] The results in Table 6 show that the effluent after the secondary expansion culture did not meet the oilfield water quality treatment standards (oil content ≤ 10.0 mg / L, number of viable bacteria in the effluent material ≥ 1 × 10 8CFU / mL). The primary expansion tank, the secondary expansion tank, and the tertiary expansion tank in the expansion device of the present invention are mainly used for the growth and reproduction of microorganisms and the treatment of crude oil pollutants in the incoming water. The three groups of expansion tanks are connected in series and are indispensable. If one group of expansion devices is arbitrarily reduced, the growth standard of bacterial concentration and the treatment standard of oilfield water quality cannot be achieved.

[0162] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A composite microorganism, characterized in that: The composite microorganisms include yeast, and bacillus and / or pseudomonas; Wherein, the yeast is Candida viswanathii with a preservation number of CGMCC No.3456; The ratio of the total viable counts of Bacillus and Pseudomonas to the viable counts of yeast was 2-8.

2. The composite microorganism according to claim 1, characterized in that The Bacillus is selected from at least one of Bacillus subtilis, Bacillus licheniformis and Bacillus amyloliquefaciens, preferably selected from at least one of Bacillus subtilis with a preservation number of CGMCC No. 21860, Bacillus licheniformis with a preservation number of BNCC188083 and Bacillus amyloliquefaciens with a preservation number of BNCC188065; And / or, the Pseudomonas is selected from Pseudomonas aeruginosa and / or Pseudomonas stutzeri, preferably selected from Pseudomonas aeruginosa, more preferably Pseudomonas aeruginosa with a preservation number of CGMCC No.23170.

3. The composite microorganism according to claim 1 or 2, characterized in that The composite microorganism comprises Candida vesica with a preservation number of CGMCC No.3456, Bacillus subtilis with a preservation number of CGMCC No.21860 and Pseudomonas aeruginosa with a preservation number of CGMCC No.23170.

4. The composite microorganism according to any one of claims 1 to 3, characterized in that In the composite microorganism, the ratio of the number of live yeast, bacillus and pseudomonas is 1:(1-3):(1-5), preferably 1:(1.5-2.5):(2-4).

5. A microbial oil-displacing agent, characterized in that: The microbial oil-displacing agent comprises: the composite microorganism according to any one of claims 1 to 4, and a metabolite of the composite microorganism; Preferably, the metabolites are produced in the process of the composite microorganism growing and reproducing using the nutrient agent; wherein, based on the volume of the nutrient agent being 1 L, the nutrient agent comprises 1-2 g of molasses, 0.1-1 g of peptone, 0.1-0.5 g of ammonium sulfate, 0.1-0.5 g of sodium dihydrogen phosphate, 0.01-0.05 g of dipotassium hydrogen phosphate and 0.1-0.5 g of sodium citrate; Preferably, the pH of the microbial oil-displacing agent is 6-8.

6. Use of the microbial oil-displacing agent according to claim 5 in reducing interfacial tension or in oil recovery from oil reservoirs; preferably in oil recovery from low permeability oil reservoirs.

7. A method for microbial oil recovery, characterized in that: The method comprises: mixing the composite microorganisms described in any one of claims 1 to 4 with oil-containing produced water for expansion, and then contacting the expanded material with an oil reservoir to be driven; Preferably, the contacting conditions include: a temperature of 28-42°C; Preferably, the oil content in the oil-containing produced water is not higher than 50 mg / L.

8. The method according to claim 7, characterized in that The conditions for the expansion culture include: a temperature of 28-42°C, preferably 30-37°C; a dissolved oxygen content DO of more than 2 mg / L; Preferably, the culture time is such that the number of viable bacteria in the material after culture is 1×10 8 CFU / mL and above.

9. A device for microbial oil recovery, wherein: The device comprises: The first-stage expansion tank, the second-stage expansion tank and the third-stage expansion tank connected in series are used to expand the composite microorganisms and remove oil impurities in the oil-containing produced water; A water inlet disposed upstream of the first-stage expansion tank for introducing oil-containing produced water; The water outlet arranged at the downstream of the three-stage expansion tank is used to lead out the expanded material and the produced water after de-oiling.

10. The device according to claim 9, wherein: The first-stage expansion culture tank, the second-stage expansion culture tank and the third-stage expansion culture tank are each independently equipped with a filler for microorganisms to attach and grow; Preferably, the first-stage expansion culture tank, the second-stage expansion culture tank and the third-stage expansion culture tank are each independently equipped with an aeration device for providing oxygen required for the growth of microorganisms.

11. The device according to claim 9 or 10, wherein the water inlet is located at the lower part of the primary culture tank; Preferably, the overflow ports of the first-stage expansion culture tank and the third-stage expansion culture tank are respectively located at the upper part of the tank body, and the overflow port of the second-stage expansion culture tank is located at the lower part of the tank body, so that the liquid flow direction is three-dimensional S-shaped; Preferably, the device is further equipped with a sedimentation tank, which is located downstream of the three-stage expansion tank and is used to precipitate solid impurities in the water body; Preferably, the water outlet is located on the side wall of the sedimentation tank.

12. The device according to any one of claims 9 to 11, wherein: The device is also equipped with a buffer tank, which is located upstream of the primary expansion tank along the liquid flow direction and is used to buffer the oil-containing produced water; Preferably, the device is further equipped with a liquid preparation tank, which is located upstream of the primary expansion tank along the liquid flow direction and is used to mix the composite microorganisms and the nutrient agent; Preferably, a dosing pump is further arranged between the liquid preparation tank and the primary expansion culture tank, for introducing a mixed liquid of composite microorganisms and nutrients into the primary expansion culture tank; Preferably, the device is also equipped with a composite microorganism dosing device and a nutrient dosing device, which are used to add composite microorganisms and nutrients, respectively.

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