Pseudomonas stutzeri, fungicide and application of pseudomonas stutzeri
The metabolism of Pseudomonas Schiseri produces glycolipid biosurfactants, especially rhamnolipids, which solves the problem of improving heterogeneity in the oil field reservoirs, and achieves the improvement of crude oil recovery and reservoir regulation. Effect.
Patent Information
- Application Number
- CN202411639663.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-15
- Publication Date
- 2025-01-10
AI Technical Summary
There are currently no reports on the improvement of the heterogeneity of reservoirs in oil fields due to the metabolism of Pseudomonas Schilli, and there is a lack of effective microbial oil recovery technology.
A Pseudomonas stutzeri 3T4 is provided, which can metabolize and produce glycolipid biosurfactants with foaming properties. The fermentation is optimized by fermentation medium such as sucrose and sodium nitrate as carbon and nitrogen sources. Rhamnolipid is produced under conditions, which is used to improve the heterogeneity of the reservoir in the oil field.
Pseudomonas Schieri 3T4 can effectively reduce crude oil viscosity, improve crude oil recovery, and improve reservoir heterogeneity by producing foam and improve oil recovery efficiency.
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Abstract
Description
Technical Field
[0001] The present application belongs to the technical field of microbial oil recovery, and in particular relates to a strain of Pseudomonas stutzeri, a bacterial agent and application thereof. Background Art
[0002] Microbial oil recovery refers to injecting appropriate bacteria, nutrients, fermentation liquid or activating native bacteria into the target reservoir, so that the strains can reproduce and metabolize in the reservoir to produce biogas or active substances such as biosurfactants, polymers, and organic solvents, and improve crude oil recovery by reducing oil-water interfacial tension, changing wettability, and changing permeability. Microorganisms produce foam in situ under reservoir conditions. Due to the Jamin effect, the foam can effectively block the high-permeability channel, has good profile control capabilities, improves sweep efficiency, and improves reservoir heterogeneity.
[0003] Currently, there is no report on the improvement of oil field reservoir heterogeneity by producing foam through the metabolism of Pseudomonas stutzeri. Summary of the invention
[0004] The purpose of the present application is to provide a strain of Pseudomonas stutzeri, a bacterial agent and applications thereof. The Pseudomonas stutzeri of the present application can metabolize to produce glycolipid biosurfactants with foaming properties, which can be used to improve the heterogeneity of oil field reservoirs.
[0005] The present application provides a strain of Pseudomonas stutzeri 3T4, which is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration, with the deposit number: CGMCC No.7535.
[0006] The present application also provides the culture or fermentation product of Pseudomonas stutzeri 3T4 described in the above scheme.
[0007] The present application also provides a bacterial agent, including the Pseudomonas stutzeri 3T4 described in the above scheme.
[0008] The present application also provides the use of the Pseudomonas stutzeri 3T4, the culture or fermentation product, or the bacterial agent described in the above scheme in producing glycolipid biosurfactants.
[0009] Preferably, the glycolipid biosurfactant comprises rhamnolipid.
[0010] The present application also provides a method for preparing glycolipid biosurfactant, comprising the following steps:
[0011] The Pseudomonas stutzeri 3T4 described in the above scheme is inoculated into a fermentation medium for fermentation to obtain a fermentation product containing glycolipid biosurfactant.
[0012] Preferably, the carbon source of the fermentation medium includes a water-soluble carbon source; the water-soluble carbon source includes sucrose; and the nitrogen source of the fermentation medium includes sodium nitrate.
[0013] Preferably, the fermentation is carried out under facultative anaerobic conditions.
[0014] The present application also provides a glycolipid biosurfactant prepared by the method described in the above scheme, wherein the glycolipid biosurfactant includes rhamnolipid.
[0015] The present application also provides the use of the Pseudomonas stutzeri 3T4, the culture or fermentation product, the bacterial agent, the method or the glycolipid biosurfactant described in the above scheme in reducing crude oil viscosity, increasing crude oil recovery or improving reservoir heterogeneity.
[0016] The present application provides a strain of Pseudomonas stutzeri 3T4, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Committee, with a deposit number of CGMCC No.7535. The Pseudomonas stutzeri 3T4 of the present application can metabolize and produce glycolipid biosurfactants, which have good foaming properties. Physical simulation experiments show that Pseudomonas stutzeri 3T4 can well improve the heterogeneity of oil field reservoirs (cores) and improve crude oil recovery.
[0017] Biological Deposit Description
[0018] Pseudomonas stutzeri 3T4 was deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number: CGMCC No.7535, the deposit date was April 28, 2013, and the deposit address was No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.
[0020] Figure 1 This is the infrared spectrum of crude 3T4 metabolite;
[0021] Figure 2 is the HPLC spectrum of rhamnolipid;
[0022] Figure 3This is a diagram of the crude oil emulsification state after 7 days of mixed culture of 3T4 fermentation liquid and crude oil (from Jianghan Oilfield);
[0023] Figure 4 Schematic diagram of the core displacement experiment device; wherein, 1-micro constant flow pump, 2-gas cylinder, 3-six-way valve, 4-steel intermediate container, 5-valve, 6-flow meter, 7-core holder, 8-pressure sensor, 9-constant temperature box, 10-measuring cylinder, 11-computer, 12-hand pump;
[0024] Figure 5 This is the graph showing the change of the pressure in the core with the culture time during the in situ culture of 3T4 bacteria;
[0025] Figure 6 This is the relationship between the production rate and the injected PV for the first water flooding and subsequent water flooding when the core is not saturated with oil. DETAILED DESCRIPTION
[0026] The present application provides a strain of Pseudomonas stutzeri 3T4, which is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration, with the deposit number: CGMCC No.7535.
[0027] The Pseudomonas stutzeri 3T4 of the present application has excellent performance in producing surfactants, can metabolize and produce glycolipid biosurfactants, and has great application potential in the petroleum industry. In the present application, the Pseudomonas stutzeri 3T4 is isolated from sewage of Dagang Oilfield in Tianjin, China.
[0028] The present application also provides the culture or fermentation product of Pseudomonas stutzeri 3T4 described in the above scheme.
[0029] In the present application, the culture or fermentation product includes in vitro metabolites of Pseudomonas stutzeri 3T4, and preferably also includes the bacterial cells of Pseudomonas stutzeri 3T4.
[0030] The present application also provides a bacterial agent, including the Pseudomonas stutzeri 3T4 described in the above scheme.
[0031] In the present application, the bacterial agent includes a solid bacterial agent or a liquid bacterial agent.
[0032] The present application also provides the use of the Pseudomonas stutzeri 3T4, the culture or the bacterial agent described in the above scheme in producing glycolipid biosurfactants.
[0033] In the present application, the glycolipid biosurfactant preferably includes rhamnolipid.
[0034] The present application also provides a method for preparing glycolipid biosurfactants, comprising the following steps: inoculating Pseudomonas stutzeri 3T4 into a fermentation medium, performing fermentation, and obtaining a fermentation product containing glycolipid biosurfactants.
[0035] Before inoculating Pseudomonas stutzeri 3T4 into the fermentation medium, the present application preferably further comprises inoculating Pseudomonas stutzeri 3T4 into an activation medium for activation culture.
[0036] In the present application, the activation culture medium is preferably a broth culture medium; the broth culture medium preferably uses water as a solvent and includes the following components in the following concentrations: 3 g / L beef extract, 10 g / L peptone and 5 g / L NaCl.
[0037] In the present application, the carbon source of the fermentation medium preferably includes a water-soluble carbon source, and the Pseudomonas stutzeri 3T4 of the present application can utilize the water-soluble carbon source metabolism to produce glycolipid surfactants; the water-soluble carbon source preferably includes sucrose; the concentration of sucrose in the fermentation medium is preferably 20-60 g / L, more preferably 40 g / L; the nitrogen source of the fermentation medium preferably includes sodium nitrate; the concentration of sodium nitrate in the fermentation medium is preferably 2-6 g / L, more preferably 4 g / L.
[0038] In the present application, the fermentation medium preferably uses water as a solvent, and preferably includes the following components in concentrations: sucrose 20-60 g / L, NaNO3 2-6 g / L, Na2HPO4 3-7 g / L, KH2PO4 2-6 g / L, MgSO4 0.1-0.5 g / L, CaCl2 5-10 mg / L, EDTA-Na2 0.5-2 mg / L, FeSO4 0.1-0.5 mg / L, MnSO4 0.1-0.5 mg / L and ethanol with a volume concentration of 1%.
[0039] In the present application, the fermentation is preferably carried out under facultative anaerobic conditions; the fermentation time is preferably 2 to 4 days. In the present application, the pH of the fermentation is preferably 5 to 10; the temperature of the fermentation is preferably 25 to 40°C, more preferably 30 to 35°C; the salinity of the fermentation is preferably 1 to 30×10 3 mg / L; the reagent for adjusting the salinity of the fermentation is preferably KCl. After the fermentation, the present application preferably further comprises removing the bacterial cells in the fermentation product, and using the fermentation liquid (containing glycolipid biosurfactants) and the foam generated by the biogas in the fermentation product for profile adjustment (improving the heterogeneity of the oil field reservoir).
[0040] After obtaining the fermentation broth, the present application preferably further comprises extracting glycolipid biosurfactants from the fermentation broth. The present application has no particular limitation on the extraction method, and conventional methods in the art can be used.
[0041] In the specific implementation process of this application, the steps of extracting glycolipid biosurfactant from the fermentation broth are as follows:
[0042] The pH value of the fermentation liquid is adjusted to 2 with 6 mol / L hydrochloric acid, and the fermentation liquid is allowed to stand at 4°C for 24 hours to obtain an acidified liquid; the acidified liquid is centrifuged at 8000 r / min for 10 minutes at 20-30°C to obtain a first precipitate; the first precipitate is mixed with methanol, and a first extraction is performed for more than 3 times to obtain a first methanol extract; the first methanol extract is centrifuged at 8000 r / min for 10 minutes at 20-30°C to obtain a supernatant, and the supernatant is concentrated by rotary evaporation at 45°C to obtain a concentrate, and the concentrate is precipitated with distilled water. The method comprises the steps of: redissolving the solution to obtain a solution; adjusting the pH value of the solution to 7, filtering the solution, taking the supernatant, adjusting the pH value to 2, standing the solution at 4°C overnight, centrifuging the solution at 8000 r / min for 10 min at 20-30°C, and obtaining a second precipitate; mixing the second precipitate with methanol, and performing a second extraction three times to obtain a second methanol extract; centrifuging the second methanol extract, taking the supernatant, concentrating the supernatant under reduced pressure using a rotary evaporator to obtain a concentrate, and drying the concentrate in a constant temperature electric oven at 60°C until a constant weight is reached to obtain a glycolipid biosurfactant.
[0043] The present application also provides a glycolipid biosurfactant prepared by the method described in the above scheme, wherein the glycolipid biosurfactant includes rhamnolipid.
[0044] In the present application, the rhamnolipids preferably include monorhamnolipids and dirhamnolipids; the monorhamnolipids include Rha-C 12:2 , Rha-C8-C8 and Rha-C 10:1 -C8; the dirhamnolipid includes Rha-Rha-C 10 , Rha-Rha-C 12 , Rha-Rha-C 14 , Rha-Rha-C 10 -C 8:1 , Rha-Rha-C 10 -C8, Rha-Rha-C 10 -C 10 and Rha-Rha-C 10 -C 12 .
[0045] In the present application, the glycolipid biosurfactant has good physical properties, low surface tension and good foaming properties, can effectively reduce surface tension, and has good emulsification properties for petroleum and various hydrocarbons. It can be used in microbial oil recovery, effectively improving reservoir heterogeneity and increasing crude oil recovery.
[0046] The application of the Pseudomonas stutzeri 3T4, the culture, the bacterial agent, the method or the glycolipid biosurfactant described in the above scheme of the present application in reducing crude oil viscosity, improving crude oil recovery or improving reservoir heterogeneity.
[0047] In the present application, the reservoir preferably includes a reservoir core.
[0048] In order to further illustrate the present application, a strain of Pseudomonas stutzeri, a bacterial agent and applications thereof provided by the present application are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present application.
[0049] Example 1 Extraction and analysis of 3T4 metabolites
[0050] 1. Fermentation of 3T4
[0051] The Pseudomonas stutzeri 3T4 provided in this application was isolated from the sewage of Dagang Oilfield in Tianjin, China.
[0052] Separation culture medium used (g / L): L-asparagine 1g, KNO3 1g, KH2PO4 1g, sodium succinate 8.5g, FeCl3·6H2O 0.5g, CaCl2·2H2O 0.2g, MgSO4·7H2O 0.07g and 1ml / L BTB (pH indicator 1% bromoacetic acid dissolved in ethanol); pH 7.0-7.2. Optimum culture temperature: 30°C;
[0053] Broth medium used for activation and enrichment: 3 g / L beef extract, 10 g / L peptone, and 5 g / L NaCl;
[0054] Fermentation medium: sucrose 40 g / L, NaNO3 4 g / L, Na2HPO4 6 g / L, KH2PO4 4 g / L, MgSO4 0.1 g / L, CaCl2 6 mg / L, EDTA-Na2 1 mg / L, FeSO4 0.2 mg / L, MnSO4 0.2 mg / L and ethanol 1%, pH 7.0, sterilization at 115 °C for 30 min.
[0055] The slant-preserved Pseudomonas stutzeri 3T4 strain was activated by streaking on the plate with an inoculation loop, and cultured at 30°C for 20 hours. Then three rings (each inoculation loop contains more than 3 single colonies with obvious characteristics) of the strain were picked from the plate and inoculated into a first-level seed shake flask (250mL triangular flask, fermentation medium, liquid volume of 50mL), and cultured at 30°C, 180r / min for 18 hours. 2% of the volume was inoculated into a second-level shake flask (250mL conical flask, fermentation medium, liquid volume of 100mL), and cultured under the same conditions for 18 hours. 2% of the volume was inoculated into a fermentation shake flask (500mL conical flask, fermentation medium, liquid volume of 150mL), and cultured under the same conditions until the spore rate reached 100%.
[0056] 2. Extraction and identification of glycolipids produced by 3T4
[0057] Centrifuge 10L of fermentation broth at room temperature and 8000r / min for 10min, remove the precipitate, then adjust the pH to 2.0 with 6mol / L hydrochloric acid, and then store at 4℃ for 24h. Centrifuge the acidified solution at room temperature and 8000r / min for 10min, remove the supernatant, and then collect the precipitate and extract it with appropriate amount of methanol for more than 3 times. Centrifuge the methanol extract under the same conditions, take the supernatant and concentrate it by rotary evaporation (45℃), then dissolve it with appropriate amount of distilled water and adjust the pH to 7.0, filter it, take the supernatant and adjust the pH to 2.0 again, let it stand at 4℃ overnight, centrifuge it at 8000r / min for 10min, take the precipitate, collect the precipitate and extract it again with the same volume of methanol as the fermentation broth for 3 times, then centrifuge it at 8000r / min for 10min, take the supernatant and concentrate it under reduced pressure with a rotary evaporator, place it in a 60℃ constant temperature electric oven until it reaches constant weight, and finally measure the weight. The product obtained is a brown solid powder. The obtained brown solid powder and potassium bromide were ground in a mortar to prepare compressed tablets, which were analyzed by infrared spectroscopy and liquid chromatography-mass spectrometry.
[0058] Infrared spectroscopy analysis:
[0059] The infrared spectrum of crude metabolites of 3T4 bacteria is shown in Figure 1 At 3397.128cm -1 The broad absorption peak at 2927.348 cm indicates the presence of -OH groups. -1 、2856.633cm -1 and 1384.363cm -1 The absorption bands near represent the absorption peaks of -CH2- and -CH3- groups of the aliphatic chain. -1 The carbonyl stretching band is found at 1654.902 cm, which is the characteristic peak of ester compounds and corresponds to the C=O group. -1The absorption peak at 1119.279 cm -1 and 1052.895cm -1 The strong absorption peak at 807.783 cm indicates the presence of ester groups, corresponding to the COC group. -1 The absorption peak at 595.024 cm -1 The absorption peak near represents -(CH2)n-(n=6) group. Compared with previous literature (see [Lan, G. et al., 2015. Rhamnolipid production from waste cooking oil using Pseudomonas SWP-4. Biochem. Eng. J., 101: 44-54.] and [ M., G., M.and I., 2012. Production and characterization of rhamnolipids from Pseudomonas aeruginosa san-ai. J. Serb. Chem. Soc., 77(1): 27-42.]), these main chemical structural groups conform to the characteristics of rhamnolipids. Therefore, it is inferred that the glycolipid biosurfactant produced by the metabolism of strain 3T4 is rhamnolipid.
[0060] Liquid chromatography-mass spectrometry analysis:
[0061] The components of rhamnolipids show strong [MH] - The molecular ion peak has an m / z range of about 300 to 700, and its precursor β-hydroxycarboxylic acid (C8 to C 12 ) has an m / z range of about 150 to 400. Therefore, the HPLC m / z scanning range of 100 to 1000 was selected to scan the rhamnolipids, and then the specific components were inferred by combining the existing molecular library with the measured m / z. The results are as follows Figure 2 shown. Figure 2 The results show that there are 10 peaks in the residence time of rhamnolipids. By comparing with the existing rhamnolipid molecular library, 10 rhamnolipid components with specific structures were inferred. The detailed results are shown in Table 1. The rhamnolipids produced by 3T4 contain two types of rhamnolipids: monorhamnolipids and disrhamnolipids, of which 3 monorhamnolipids and 7 disrhamnolipids are the dominant components, and the types of disrhamnolipids are more than those of monorhamnolipids.
[0062] Table 1 Structure, molecular weight, m / z and ion fragments of rhamnolipids
[0063]
[0064]
[0065] Example 2 Optimization of 3T4 rhamnolipid production fermentation system
[0066] Seed liquid: inoculate the seed liquid into the enrichment medium (basic composition includes: beef extract 3g / L, peptone 10g / L, NaCl 5g / L), and culture to a concentration of 10 8 ~10 9 cells / mL, as the seed solution, for the study of this example.
[0067] 1. Carbon source optimization
[0068] Prepare the culture medium: measure 200mL of distilled water, weigh NaNO3 4g / L, Na2HPO4 6g / L, KH2PO4 4g / L, MgSO4 0.1g / L, CaCl2 6mg / L, EDTA-Na2 1mg / L, FeSO4 0.2mg / L, MnSO4 0.2mg / L, ethanol 1%, and adjust the pH to 7.0. Divide into 20mL portions in 5 50mL glass bottles. Add the following five carbon sources (content 40g / L): glucose (No. 1), sucrose (No. 2), maltose (No. 3), molasses (No. 4), sodium acetate (No. 5), respectively, and seal completely with butyl rubber caps and aluminum seals. After sterilization, inoculate 5% seed solution, culture facultatively anaerobically at 35°C, shake culture at 150r / min for 72h, and measure the surface tension, foam volume and OD of the fermentation liquid. 600 , and the results are as follows:
[0069] Table 2 Effects of different carbon sources on rhamnolipid production by strain 3T4
[0070] Carbon source Surface tension (mN / m) Foam volume (mL) <![CDATA[OD 600 ]]> 1 30.2 9.500 0.665 2 31.6 10.607 0.797 3 38.8 6.367 1.001 4 41.6 3.127 0.350 5 53.7 0 0.049
[0071] Observe the measured surface tension and foam volume data. The surface tension of the bacterial solution with carbon source culture medium No. 1 and No. 2 is not much different, but the foam volume produced by the bacterial solution with carbon source culture medium No. 2 is 11.65% more, and the dry weight of the bacteria is 970mg / L. Considering the reduction of surface tension and foaming volume of the fermentation broth, sucrose was selected as the best carbon source for fermentation. Next, the amount of sucrose was optimized, and the surface tension reduction and foaming volume at different concentrations were observed. It was found that when the amount of sucrose was 20-60g / L, the surface tension was low and the foaming performance was good. Therefore, 20-60g / L of sucrose was selected as the best carbon source for rhamnolipid production by strain 3T4.
[0072] 2. Nitrogen source optimization
[0073] Measure 200mL of distilled water, weigh 40g / L sucrose, 6g / L Na2HPO4, 4g / L KH2PO4, 0.1g / L MgSO4, 6mg / L CaCl2, 1mg / L EDTA-Na2, 0.2mg / L FeSO4, 0.2mg / L MnSO4, 1% ethanol, and adjust the pH to 7.0. Divide into 20mL portions in 7 50mL glass bottles. Add the following seven nitrogen sources (content 4g / L): ammonium chloride (No. 1), urea (No. 2), corn steep liquor powder (No. 3), tryptone (No. 4), sodium glutamate (No. 5), sodium nitrate (No. 6), ammonium nitrate (No. 7), and seal completely with butyl rubber caps and aluminum seals. After sterilization, inoculate 5% seed solution, culture facultatively anaerobically at 35°C, shake culture at 150r / min for 72h, and measure the surface tension, foam volume and OD of the fermentation liquid. 600 , and the results are as follows:
[0074] Table 3 Effects of different nitrogen sources on rhamnolipid production by strain 3T4
[0075] Nitrogen source Surface tension (mN / m) Foam volume (mL) <![CDATA[OD 600 ]]> 1 44.8 0 0.091 2 40.9 4.307 0.101 3 46.8 0 0.247 4 46.4 0 0.269 5 35.3 6.293 0.11 6 27.7 21.320 0.427 7 29.0 15.333 0.965
[0076] Observing the measured surface tension and foam volume data, the surface tension of the bacterial solution added with carbon source medium No. 6 and No. 7 was not much different, but the foam volume produced by the bacterial solution of carbon source medium No. 6 was 39.05% more. Considering the reduction of surface tension and foaming volume of the fermentation broth, sodium nitrate was selected as the best nitrogen source for fermentation. Next, the amount of sodium nitrate was optimized, and the surface tension reduction and foaming volume at different concentrations were observed. It was found that when the amount of sodium nitrate was 2-6g / L, the surface tension was low and the foaming performance was good. Therefore, 2-6g / L sodium nitrate was selected as the best nitrogen source for rhamnolipid production by strain 3T4.
[0077] Example 3 Crude oil viscosity reduction test
[0078] 1. Preparation of bacterial solution for crude oil viscosity reduction
[0079] Prepare 1000 mL of surfactant bacteria fermentation medium and sterilize it with high pressure steam at 115°C for 30 min. Activate the 3T4 strain and transfer it to the fermentation medium and culture it in a shaking incubator at 35°C and 150 r / min for 72 h.
[0080] 2. Crude oil viscosity reduction test
[0081] 3T4 fermentation liquid and crude oils of different viscosities were poured into bottles at a volume ratio of 2:1, placed in a constant temperature shaker at 35°C for 7 days, centrifuged with a high-speed centrifuge, and the viscosity of the crude oil was measured using a viscometer at room temperature. The crude oil came from the Changqing Oilfield (No. 1) and Jianghan Oilfield (No. 2, 3) blocks, and the following results were obtained:
[0082] Table 4 Data of crude oil before and after viscosity reduction
[0083] Crude oil source Viscosity before viscosity reduction (cP) Viscosity after viscosity reduction (cP) Emulsion viscosity (cP) 1 30.60 14.58 10.06 2 2778 484 90.6 3 8970 1008 167.8
[0084] From the data in Table 4 and Figure 3 It can be seen that 3T4 fermentation liquid has a better effect on reducing the viscosity of heavy oil, with a viscosity reduction rate of 88.76%. The on-site formation water and fermentation medium were used as blank controls, and the experimental data showed that the viscosity reduction rates were 0 and 5.34%, respectively. The experimental results show that rhamnolipid can be used as an emulsifier to reduce the viscosity of crude oil and disperse the emulsified crude oil into oil droplets with small inner diameters.
[0085] Example 4 Profile Control Simulation Test
[0086] 1. Preparation of bacterial solution for profile control
[0087] Prepare 150mL of surfactant bacteria fermentation medium and 3L of inorganic salt medium. Sterilize with high pressure steam at 115℃ for 30min. Activate the 3T4 strain and transfer it to the fermentation medium. Culture it in a shaking incubator at 35℃ and 150r / min for facultative anaerobism. After 24h, add the fermentation liquid to 3L of optimized fermentation medium at a volume percentage of 10%, place it in a sterilized intermediate container and culture it in a facultative anaerobism at 35℃ for 48h.
[0088] 2. Core profile control physical model test
[0089] according to Figure 4 Connect the displacement process, which consists of a micro-constant flow pump (gas cylinder for pressure source of constant pressure displacement), intermediate container, core holder, constant temperature box, hand pump (confining pressure and back pressure), CO2 gas cylinder and other instruments. The above parts are connected with pipelines and valves. Install a pressure gauge to display the pressure value as required, and put the components that need to maintain the experimental temperature into the constant temperature box.
[0090] Three artificial cores, 1#, 2# and 3#, with gas permeabilities of 103, 505 and 1070 mD respectively, were selected, 5PV of 3T4 bacterial solution was injected, the wells were shut in for 3 to 4 days, and the effects of improving heterogeneity were measured respectively.
[0091] (1) Core saturated water
[0092] Three artificial cores were dried, weighed and placed in a wide-mouth bottle. Vacuum was evacuated with a vacuum pump for 24 hours, and simulated formation water was added to make the cores completely immersed in water. The vacuum pump was turned on until the vacuum gauge read 0, and then the cores were taken out and weighed with wet weight to calculate the pore volume of the cores.
[0093] (2) Water drive
[0094] Inject 5PV simulated formation water into the core at an injection rate of 2mL / min, record the pressure data at the inlet and outlet, and calculate the absolute permeability K of the core using the Darcy equation. W1 .
[0095] (3) Microbial repellent
[0096] 5 PV of 3T4 fermentation liquid was injected into the core at an injection rate of 2 mL / min, all valves at the inlet and outlet were closed, and the temperature of the thermostat was adjusted to 35 ° C. The changes in the pressure in the core were recorded in real time during the in situ cultivation of microorganisms. After 3 to 4 days of cultivation, only the valve at the inlet was opened.
[0097] (4) Subsequent water flooding
[0098] Inject 6PV of simulated formation water into the core at an injection rate of 2mL / min, record the pressure data at the inlet and outlet, and calculate the effective permeability K of the core using the Darcy equation. W2 Calculate the relative permeability K rw Evaluate the change of core permeability, K rw It's K W2 With K W1 ratio.
[0099] The cores used for producing biosurfactant bacteria 3T4 are 1#, 2# and 3#. The physical property test results of the three artificial cores are shown in Table 5. The pressure changes in the three cores during the 3-4-day in-situ culture after microbial flooding are as follows: Figure 5 As shown in the figure, the relationship between the subsequent water flooding production rate and the injected PV is as follows: Figure 6 As shown, the relative permeability K rw The data are shown in Table 6.
[0100] Table 5 Results of physical properties of experimental cores
[0101] Core number Length (cm) Height (cm) <![CDATA[Permeability K g (mD)]]> Porosity(%) 1# 6.510 2.53 103 11.79 2# 6.272 2.53 505 20.67 3# 6.250 2.53 1070 28.58
[0102] Table 6 Core permeability data after the first water flooding and subsequent water flooding
[0103] Core number <![CDATA[K w1 (mD)]]> <![CDATA[K w2 (mD)]]> <![CDATA[K rw ]]> 1# 43.4 32.8 0.756 2# 292.5 178.6 0.611 3# 658.2 331.5 0.504
[0104] Depend on Figure 5It can be seen that the pressure in the core tends to stabilize as the culture time increases during the culture process, and the pressure increase in the low permeability core (1#) is 10kPa higher than that in the high permeability core (2#, 3#). Under the same culture conditions, the pressure in the core injected with the culture medium inoculated with 3T4 bacteria increased, while the pressure in the core of the blank control (culture medium not inoculated with 3T4 bacteria) did not change. This result shows that the 3T4 bacteria in the core generated foam in situ, and the foam began to be generated 16 to 48 hours after the start of the culture. The foam blocked the pore throat channel, causing the pressure in the core to increase. Figure 6 It can be seen that the production rate of the subsequent water flooding is significantly lower than that of the first water flooding. This is because after the culture fluid is injected into the core, the 3T4 bacteria reproduce and grow in the large pore throats of the core and produce foam. The foam is preferentially moved to the high permeability channel through water flooding. At this time, the production rate will continue to increase until the foam occupies the high permeability channel and then rapidly decreases. The foam forms an effective blockage in the core, causing water to flow to the low permeability channel. As shown in Tables 5 and 6, the absolute permeability K of the core with in situ culture of 3T4 bacteria is w2 Reduced to the original permeability K w1 50-76% of the blank core K w2 After the displacement, the bacterial cell concentration of the effluent collected in the cylinder was 2.2×10 7 These results indicate that the foam generated in situ by Pseudomonas stutzeri can effectively reduce the permeability of highly permeable porous media, play a role in profile control, and improve the heterogeneity of the reservoir.
[0105] Although the above-mentioned embodiment provides a detailed description of the present application, it is only a part of the embodiments of the present application, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present application.
Claims
1. A strain of Pseudomonas stutzeri 3T4, characterized in that: It is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number: CGMCC No.7535.
2. The culture or fermentation product of Pseudomonas stutzeri 3T4 according to claim 1.
3. A bacterial agent, characterized in that: The present invention comprises the Pseudomonas stutzeri 3T4 as described in claim 1.
4. Use of the Pseudomonas stutzeri 3T4 according to claim 1, the culture or fermentation product according to claim 2, or the bacterial agent according to claim 3 in producing glycolipid biosurfactants.
5. The use according to claim 4, characterized in that: The glycolipid biosurfactant includes rhamnolipid.
6. A method for preparing glycolipid biosurfactant, characterized in that: The following steps are involved: The Pseudomonas stutzeri 3T4 according to claim 1 is inoculated into a fermentation medium for fermentation to obtain a fermentation product containing a glycolipid biosurfactant.
7. The method according to claim 6, characterized in that The carbon source of the fermentation medium includes a water-soluble carbon source; the water-soluble carbon source includes sucrose; and the nitrogen source of the fermentation medium includes sodium nitrate.
8. The method according to claim 6, characterized in that The fermentation was carried out under facultative anaerobic conditions.
9. The glycolipid biosurfactant prepared by the method according to any one of claims 6 to 8, characterized in that: The glycolipid biosurfactant includes rhamnolipid.
10. Use of the Pseudomonas stutzeri 3T4 according to claim 1, the culture or fermentation product according to claim 2, the bacterial agent according to claim 3, the method according to any one of claims 6 to 8, or the glycolipid biosurfactant according to claim 9 in reducing crude oil viscosity, improving crude oil recovery, or improving reservoir heterogeneity.