WO2023041062A1 - 假单胞菌及其用途 - Google Patents

假单胞菌及其用途 Download PDF

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WO2023041062A1
WO2023041062A1 PCT/CN2022/119404 CN2022119404W WO2023041062A1 WO 2023041062 A1 WO2023041062 A1 WO 2023041062A1 CN 2022119404 W CN2022119404 W CN 2022119404W WO 2023041062 A1 WO2023041062 A1 WO 2023041062A1
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oil
pseudomonas
bacteria liquid
reservoir
water
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PCT/CN2022/119404
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French (fr)
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汪卫东
胡婧
曹嫣镔
钱钦
张雷
郭辽原
曹功泽
孙刚正
林军章
宋永亭
岳胜辉
袁长忠
吴晓玲
丁明山
高光军
王静
刘涛
冯云
李彩风
宋欣
陈子慧
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中国石油化工股份有限公司
中国石油化工股份有限公司胜利油田分公司石油工程技术研究院
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Priority to CA3232458A priority Critical patent/CA3232458A1/en
Publication of WO2023041062A1 publication Critical patent/WO2023041062A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K8/00—Compositions for drilling of boreholes or wells; Compositions for treating boreholes or wells, e.g. for completion or for remedial operations
    • C09K8/58—Compositions for enhanced recovery methods for obtaining hydrocarbons, i.e. for improving the mobility of the oil, e.g. displacing fluids
    • C09K8/582—Compositions for enhanced recovery methods for obtaining hydrocarbons, i.e. for improving the mobility of the oil, e.g. displacing fluids characterised by the use of bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/38—Pseudomonas

Definitions

  • the invention belongs to the fields of energy biotechnology and environmental biotechnology, and specifically relates to a strain of pseudomonas and its application.
  • Microbial oil recovery technology utilizes injection or activation of microbes with oil displacement function inside the reservoir, through microbial growth and metabolic activities and their metabolites interact with crude oil and reservoirs to enhance oil recovery.
  • This technology has carried out a lot of indoor research and field experiments around the world. Although it has shown good application prospects, it has not been widely applied in the field. Due to the complexity of reservoir development, it is necessary to improve oil washing efficiency and expand the swept volume at the same time to effectively enhance oil recovery.
  • the purpose of the present invention is to overcome the above problems existing in the prior art, and provide a pseudomonas that can expand the swept volume and improve the oil washing efficiency and its application.
  • the first aspect of the present invention provides a strain of Pseudomonas (Pseudomonas sp.), its preservation number is CGMCC No.22583.
  • the second aspect of the present invention provides the application of the aforementioned Pseudomonas and/or its metabolites in oil extraction.
  • the third aspect of the present invention provides a method for extracting oil, the method comprising: injecting the oil production bacteria liquid into the oil reservoir, and then extracting oil, wherein the oil production bacteria liquid contains the above-mentioned Pseudomonas bacteria , at least one of an extracellular metabolite of the Pseudomonas and an intracellular metabolite of the Pseudomonas.
  • Pseudomonas Pse-1 provided by the present invention has a growth temperature of 37-45° C., and its fermentation broth can be denatured from fluid without viscosity to surface active under medium-high temperature (60-95° C.) oil reservoir conditions It is a multi-functional oil recovery bacterial fluid with viscoelasticity, and has the functions of expanding the swept volume and improving the oil washing efficiency. After the multi-functional oil recovery bacterial fluid is denatured at the reservoir temperature of 60-95°C, the apparent viscosity of the bacterial fluid is ⁇ 50mPa ⁇ s. Liquid surface tension ⁇ 30mN/m, oil washing efficiency ⁇ 90%, and physical simulation experiment improves oil recovery by more than 20%;
  • the classification of the bacterial strain of the present invention is named: Pseudomonas sp. (Pseudomonas sp.), on May 24, 2021, was preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee (Address: 1 Beichen West Road, Chaoyang District, Beijing) No. 3, Institute of Microbiology, Chinese Academy of Sciences, postal code: 100101, the abbreviation of the depository unit is CGMCC), and the deposit number is CGMCC No.22583.
  • Fig. 1 is a phylogenetic tree diagram of a strain of Pseudomonas Pse-1 disclosed by the present invention
  • Fig. 2 is a colony morphology diagram of a strain of Pseudomonas Pse-1 disclosed by the present invention
  • Fig. 3 is the morphological figure after the bacterial cell staining of a Pseudomonas Pse-1 disclosed by the present invention
  • Figure 4 is a morphological diagram of the network polysaccharide structure in the bacterial liquid of a strain of Pseudomonas Pse-1 denatured at 60°C disclosed by the present invention
  • Fig. 5 is a schematic diagram of viscosity changes at 60° C. at different times of a Pseudomonas Pse-1 fermentation broth (50% dilution) disclosed by the present invention.
  • fertilization product refers to the whole product obtained without any separation operation after cultivating Pseudomonas, including Pseudomonas cells and metabolites of Pseudomonas product etc.
  • the present invention provides a strain of Pseudomonas, which is characterized in that its preservation number is CGMCC No.22583.
  • the sequence of the 16s rDNA of Pseudomonas of the present invention is as shown in SQE ID NO: 1, therefore, the present invention also relates to a strain of Pseudomonas, its preservation number is CGMCC No.22583 or its 16s rDNA sequence is as shown in SQE ID NO: 1.
  • the present invention also provides the application of the aforementioned Pseudomonas and/or its metabolites in oil extraction.
  • the present invention provides a method for oil extraction, which is characterized in that the method includes: injecting oil recovery bacteria liquid (or oil recovery agent) into the oil reservoir for oil recovery, wherein the oil recovery bacteria liquid (or oil recovery agent) contains The thallus and/or metabolites (including intracellular and/or extracellular metabolites) of the above-mentioned Pseudomonas.
  • the Pseudomonas of the present invention can produce metabolites (extracellular metabolites and/or intracellular metabolites, possibly including biosurfactants and/or thermosensitive viscoelastic biopolysaccharides) that are beneficial to oil recovery.
  • metabolites extracellular metabolites and/or intracellular metabolites, possibly including biosurfactants and/or thermosensitive viscoelastic biopolysaccharides
  • Different forms of oil recovery bacteria fluids containing the metabolites of Pseudomonas can be used, for example, the oil recovery bacteria fluid can contain thalline, the extracellular metabolites of Pseudomonas and thalline lysate (or thalline At least one of the intracellular metabolites released by cleavage).
  • the thallus of the Pseudomonas can be live or dead, as long as the metabolites produced during the growth (fermentation) of the thalline are at least partly retained in the oil recovery bacterial fluid, the It only needs to be able to contact with the oil reservoir to carry out oil displacement.
  • the metabolites produced by Pseudomonas that are beneficial to oil recovery include extracellular metabolites and intracellular metabolites. Therefore, the oil recovery bacteria fluid containing bacteria can be directly injected into the oil reservoir, and the bacteria will be cleaved and secreted in the oil reservoir. Internal metabolites and extracellular metabolites together promote oil recovery.
  • the temperature of medium-high temperature oil reservoir is relatively high, and bacteria cells will be cracked under the action of surfactant produced by extracellular metabolism after injection. High temperature reservoirs. Even so, it is also possible to lyse the bacterium in advance (such as thermal lysis), and inject the obtained lysate into the oil reservoir for oil recovery. It can be understood that this method of lysing in advance is especially suitable for low-temperature oil reservoirs ( ⁇ 60°C) ).
  • the method of the present invention may also include the step of preparing oil recovery bacteria liquid: (i) inoculating Pseudomonas into nutrient medium for fermentation; optionally, (ii) subjecting the obtained fermentation product to lysing operation or The bacterial cells are separated from the fermentation product and the obtained bacterial cells are lysed.
  • the fermentation product obtained according to the step (i) contains bacteria and can be directly used in medium-high temperature oil reservoirs.
  • the lysate obtained according to step (ii) contains metabolites of Pseudomonas and is suitable for low temperature oil reservoirs.
  • the nutrient medium contains carbon source, nitrogen source and inorganic salt.
  • the initial pH value of the nutrient medium is preferably 7.5-8.
  • the content of carbon source in the nutrient medium is 20-50g/L, more preferably 23-47g/L.
  • the carbon source can be commonly used carbonaceous substances for cultivating Pseudomonas, preferably xylose and/or glycerol, more preferably xylose and glycerol.
  • the weight ratio of glycerol to xylose is 1-5 (such as 1, 1.2, 1.5, 1.8, 2, 2.5, 3, 3.5, 4, 4.5, 5 or any value between the above values).
  • the content of xylose in the nutrient medium is 8-12g/L.
  • the content of glycerol in the nutrient medium is 15-35g/L.
  • the content of nitrogen source in the nutrient medium is 0.5-2g/L.
  • the nitrogen source may be a common nitrogen-containing substance used for cultivating Pseudomonas, preferably yeast powder.
  • the content of inorganic salt in the nutrient medium is 3-12g/L, more preferably 3.3-10.8g/L.
  • the inorganic salts can be common inorganic salts used to cultivate Pseudomonas, including at least one of sodium salts, potassium salts, calcium salts and magnesium salts, such as sodium nitrate, potassium nitrate, dipotassium hydrogen phosphate , at least one of disodium hydrogen phosphate, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate and magnesium chloride, preferably sodium nitrate, dipotassium hydrogen phosphate, sodium chloride, calcium chloride and magnesium sulfate.
  • the nutrient medium contains xylose 8-12g/L, glycerol 15-35g/L, sodium nitrate 1-4g/L, dipotassium hydrogen phosphate 1-3g/L, Sodium chloride 1-3g/L, calcium chloride 0.05-0.1g/L, magnesium sulfate 0.3-0.7g/L, yeast powder 0.5-2g/L, initial pH value 7.5-8. More pseudomonas and/or their metabolites can be obtained by using the culture medium of this preferred embodiment, which is beneficial to further save energy and material consumption in oil recovery.
  • the fermentation can be carried out under conventional conditions for cultivating Pseudomonas, preferably, the conditions of the fermentation are such that the content of Pseudomonas in the fermentation broth is greater than or equal to 10 9 CFU/mL, or , the conditions of the fermentation are such that the surface tension of the fermented liquid is ⁇ 30mN/m and the apparent viscosity is ⁇ 50mPa ⁇ s after being heated at 60°C and above for 8h. More preferably, the fermentation conditions include: the inoculum size is 10 6 -10 8 CFU/mL.
  • the temperature of fermentation is preferably 37-45°C.
  • the time of fermentation is preferably 48-72h.
  • the ventilation rate of fermentation is preferably 1-2L/min.
  • a step of culturing seeds may also be included to amplify the Pseudomonas.
  • the seed culture medium used for seed cultivation may contain: glucose 2-4g/L, peptone 2-4g/L, yeast powder 2-4g/L.
  • the conditions for seed culture can be 20-30h at 37-45°C with shaking at 150-200r/min.
  • the fermentation product can be directly cracked as a whole, so that the bacteria in the fermentation product are cracked to obtain a bacteria lysate; it is also possible to first separate the bacteria from the fermentation product, and then crack the bacteria to obtain Bacterial lysates.
  • Lysis is mainly for the purpose of breaking the bacteria to release intracellular metabolites, which can be carried out by conventional methods in the field, such as high temperature lysis (such as 8-10 hours at 60-95°C), or repeated freezing and thawing Method, ultrasonic treatment, etc., can also be lysed with the help of lysate.
  • the surface tension of the oil recovery bacteria liquid at 25°C is ⁇ 30mN/m, preferably 24.5-28.5mN/m.
  • the apparent viscosity of the oil recovery bacteria liquid at 60° C. is ⁇ 50 mPa ⁇ s, preferably 65-150 mPa ⁇ s.
  • the surface tension can be measured with reference to the plate method in GB/T22237-2008.
  • the apparent viscosity can be measured using a Brookfield rotational viscometer (rotary model: No. 3 rotor, rotor speed: 60r/min) (refer to GB 1886.41-2015).
  • the fermentation product obtained in step (i) and the cell lysate obtained in step (ii) can be used for oil recovery after being diluted with a diluent.
  • the diluent is preferably tap water or formation water, more preferably formation water, especially formation water with a salinity of 1000-100000mg/L.
  • the method of injecting the oil recovery bacteria liquid into the oil reservoir for oil recovery may be oil well huff and puff.
  • the Pseudomonas of the present invention is beneficial to shorten the culture time of the bored well. Therefore, when using oil well stimulation, the culture time of the bored well can be 10-30d, preferably 15-30d.
  • the injection rate of the oil production bacteria liquid can be 10-20m 3 /h, and the oil well is opened for production after the well is cultured.
  • V total injection volume of oil production bacteria liquid, unit is m 3 ;
  • R treatment radius, unit is m
  • the method of injecting the oil production bacteria liquid into the oil reservoir for oil recovery can be water injection well displacement, and the oil production bacteria liquid is slug injected from the water injection well. Therefore, the method of injecting the oil production bacteria liquid into the oil reservoir for water well displacement can include :
  • the amount of the oil recovery bacteria liquid used is 14-50% of the total injected amount of the oil recovery bacteria liquid.
  • the total injection amount of oil production bacteria liquid is preferably 0.05-0.1PV (pore volume).
  • PV is the pore volume of the reservoir where the reservoir is located, and the pore volume is the total volume of effective pores in the reservoir, which is obtained by calculating the product of the reservoir volume and porosity.
  • the porosity can be measured according to the SY/T6298-1997 method after taking cores from the reservoir.
  • the injection rate of the oil production bacteria liquid in step (1) can be 5-20m 3 /h.
  • oil production bacteria fluid and water are injected from water injection wells.
  • the water injection amount in step (1) and step (3) is 14-50% of the total water injection amount.
  • the water injection ratio in step (1) and step (3) is preferably 1:0.05-0.7.
  • the total water injection amount is preferably 0.1-0.3 PV (pore volume).
  • the injection rate of the water injected in both step (1) and step (3) can be 10-50m 3 /h. Oil production bacteria fluid and water are injected from water injection wells.
  • step (2) the time for stopping injection is 1-2d.
  • the number of cycles performed is 2-7 times.
  • the temperature of the oil reservoir can fluctuate within a wide range.
  • the temperature of the oil reservoir is generally 60-95°C.
  • the temperature of the reservoir is generally ⁇ 60°C (eg, 30-59°C).
  • the viscosity of the dehydrated and degassed crude oil in the reservoir can be ⁇ 10000mPa ⁇ s (such as 1500-4000mPa ⁇ s) at 50°C. Crude oil viscosity can be measured at 50°C by using a rotational viscometer with reference to the SY/T0520-2008 method.
  • the method of the present invention can expand the swept volume and improve the oil washing efficiency at the same time. Therefore, the present invention also relates to a method for simultaneously expanding the swept volume and improving the oil washing efficiency.
  • the method is characterized in that the method includes: injecting the oil production bacteria liquid into the oil reservoir Oil recovery is carried out, wherein the oil recovery bacteria liquid is as described above, and will not be repeated here.
  • the composition of the nutrient medium (nutrient solution) that adopts is: xylose 10g/L, glycerol 30g/L, sodium nitrate 2g/L, dipotassium hydrogen phosphate 2g/L, Sodium chloride 2g/L, calcium chloride 0.1g/L, magnesium sulfate 0.5g/L, yeast powder 1g/L, pH is 7.8, and the balance is water.
  • the composition of the seed liquid culture medium is: glucose 3g/L, peptone 3g/L, yeast powder 3g/L; the method for obtaining the seed liquid is: inoculate 1mL strain cryopreservation tube into 100mL seed liquid medium , under aerobic conditions at 37°C and cultured with shaking at about 170r/min for 24 hours to obtain seed liquid, the content of Pseudomonas Pse-1 in the seed liquid was 10 8 CFU/mL.
  • the method for obtaining the fermented liquid is as follows: the Pseudomonas Pse-1 seed liquid is inoculated in the nutrient medium according to the inoculation amount of 2 vol%, cultivated at 37° C. for 60 h, the stirring speed is 200 rpm, and the ventilation rate is 1.5 L/min to obtain the fermented liquid.
  • the content of Pseudomonas Pse-1 in the fermentation broth was 10 9 CFU/mL.
  • oil wells and blocks use conventional metering separators to measure the daily oil volume, which is also called the glass tube oil measuring method.
  • the water cut value of the produced fluid is obtained to calculate the daily oil production of the oil well or block.
  • the 16s rDNA was amplified and sequenced, and the bacterial species was identified. The steps were as follows: denaturation, PCR amplification, PCR product purification, and sequencing of the target fragment. The sequence of its 16s rDNA is shown in SQE ID NO:1. Carry out sequence alignment at NCBI according to sequencing result, similarity 96%, phylogenetic tree construction (Fig. 1), identification Pseudomonas Pse-1 of the present invention is a new strain, belongs to Pseudomonas, its classification Named: Pseudomonas sp.
  • the research results showed that the strain was short rod-shaped, with a single flagella, able to move, the colony was raised, the edge was uneven, the surface was smooth and wet in a fusion state, and there were sticky polysaccharides on the surface of the colony.
  • the cells are long rods with a size of (0.1-0.2) ⁇ (2-3) ⁇ m.
  • the morphology of the colonies is shown in Figure 2, and the morphology of the cells after staining is shown in Figure 3.
  • Physiological and biochemical characteristics of Pseudomonas Pse-1 aerobic, growth temperature 37-45°C, optimum growth temperature 37°C, growth pH range 5-10, optimum growth pH range 7.8-8, NaCl concentration tolerance The range is 0-10 wt%.
  • Performance parameters such as the surface tension of the fermentation broth of Pseudomonas Pse-1 under the reservoir conditions, the oil sand washing performance of the fermentation broth, the apparent viscosity, and the recovery factor value enhanced by the physical simulation experiment were evaluated.
  • the reservoir temperature is 60°C.
  • oil sand preparation and standard curve drawing were carried out. Weigh 3g of oil sand into a 50mL colorimetric tube and mark it as A.
  • the Pse-1 fermentation broth was heated at the reservoir temperature for 8h, and then diluted in different proportions.
  • the inoculation age refers to the time of cultivating when preparing the seed liquid:
  • the nutrient medium is xylose 10g/L, glycerol 30g/L, sodium nitrate 2g/L, dipotassium hydrogen phosphate 2g/L, sodium chloride 2g/L, calcium chloride 0.1g/L L, magnesium sulfate 0.5g/L, yeast powder 1g/L, pH is 7.8.
  • the nutrient medium is xylose 12g/L, glycerol 35g/L, sodium nitrate 4g/L, dipotassium hydrogen phosphate 3g/L, sodium chloride 3g/L, calcium chloride 0.1g/L L, magnesium sulfate 0.7g/L, yeast powder 2g/L, pH is 7.5, and the balance is water.
  • the nutrient medium is xylose 8g/L, glycerol 15g/L, sodium nitrate 1g/L, dipotassium hydrogen phosphate 1g/L, sodium chloride 1g/L, calcium chloride 0.05g/L L, magnesium sulfate 0.3g/L, yeast powder 0.5g/L, pH is 8, and the balance is water.
  • Example 3.1 the fermentation conditions of the Pseudomonas were as follows: inoculum size 2vol%, inoculum age 12h, initial pH 7.5, temperature 37°C, stirring speed 220rpm, ventilation rate 1L/min, fermentation time 72h.
  • Example 3.2 the fermentation conditions of the Pseudomonas were: inoculum size 3vol%, inoculum age 24h, initial pH 7.5, temperature 40°C, stirring speed 200rpm, ventilation rate 1.5L/min, fermentation time 60h.
  • Example 3.3 the fermentation conditions of the Pseudomonas were as follows: inoculum size 1vol%, inoculum age 12h, initial pH 8, temperature 45°C, stirring speed 160rpm, ventilation rate 2L/min, fermentation time 48h.
  • Oil well profile Formation temperature 65°C, formation water salinity 18712mg/L, oil layer effective thickness 6.7m, porosity 35%, permeability 567 ⁇ 10 -3 ⁇ m 2 , viscosity of dehydrated and degassed crude oil at 50°C 3764mPa ⁇ s.
  • the microbial single-well throughput is to inject the Pseudomonas Pse-1 fermentation broth into the formation through the oil well, and use the synergistic effect of biosurfactant and temperature-sensitive viscoelastic biopolysaccharide to improve single-well production capacity.
  • the specific implementation steps are as follows: injecting the prepared Pseudomonas Pse-1 fermentation liquid with a dilution rate of 30% into the formation through the oil well casing annulus at an injection rate of 15m 3 /h.
  • V injection volume, m 3 ;
  • the consumption of Pseudomonas Pse-1 fermentation broth with a dilution of 30% is 397m 3 ; use a pump truck to inject the prepared Pse-1 fermentation broth into the formation through the oil well casing annulus; the oil well is shut down and cultured for 30 days Then open the well for production.
  • Oil recovery was carried out by water flooding before implementation. After the well A1 is put into production, the date when the daily oil volume of the oil well is greater than the daily oil volume before the implementation is the starting date of the validity period, and the liquid production and oil production both increase compared with before the implementation, and the average daily oil increase of a single well reaches 2.8t , the accumulative increase in oil was 980t, the minimum viscosity of crude oil dropped to 762mPa ⁇ s, and the drop rate reached 80%. , the field test of Pseudomonas Pse-1 works well.
  • Oil well profile Formation temperature 60°C, formation water salinity 22140mg/L, oil layer effective thickness 5.1m, porosity 30%, permeability 467 ⁇ 10 -3 ⁇ m 2 , viscosity of dehydrated and degassed crude oil at 50°C 1875mPa ⁇ s.
  • the microbial single-well throughput is to inject the Pseudomonas Pse-1 fermentation broth into the formation through the oil well, and use the synergistic effect of biosurfactant and temperature-sensitive viscoelastic biopolysaccharide to improve single-well production capacity.
  • the prepared Pseudomonas Pse-1 fermented liquid with a dilution rate of 50% is injected into the formation through the annular space of the oil well casing, and the injection rate is 10m 3 /h.
  • V injection volume, m 3 ;
  • the dosage of 50% Pseudomonas Pse-1 fermentation liquid is 208m 3 ; the prepared Pse-1 fermentation liquid is injected into the formation through the oil well casing annulus with a pump truck; the oil well is shut down and cultivated for 20 days Then open the well for production.
  • Oil recovery was carried out by water flooding before implementation. After the well B1 is put into production, the date when the daily oil volume of the oil well is greater than the daily oil volume before the implementation is the starting date of the validity period.
  • the liquid production and oil production both increase compared with before the implementation, and the average daily oil increase of a single well reaches 3.7t.
  • the accumulative increase of oil was 1166t, and the minimum viscosity of crude oil dropped to 283mPa ⁇ s, a decrease of 84.9%.
  • the daily oil volume of a single well is less than the daily oil volume before the implementation, the expiration date of the validity period is taken as the expiration date, and the validity period reaches 315d, and the output-input ratio is 6.
  • the field test works well.
  • Oil well profile formation temperature 65°C, formation water salinity 24134mg/L, oil layer effective thickness 4.7m, porosity 0.32, permeability 653 ⁇ 10 -3 ⁇ m 2 , total pore volume of the well group 8.75 ⁇ 10 4 m 3 , dehydration
  • the viscosity of degassed crude oil at 50°C is 2719mPa ⁇ s, and the well spacing is 230m.
  • the microbial oil flooding is to slug inject the Pseudomonas fermentation liquid from the water injection well, and use the metabolites of the Pseudomonas fermentation liquid Pse-1 to increase the productivity of the well group's oil wells.
  • the microbial flooding adopts the method of alternate slug injection, the total injection volume of oil production bacteria liquid is 0.05PV, totaling 4350m 3 , and the total water injection volume is 0.1PV, totaling 8700m3 .
  • Oil recovery was carried out by water flooding before implementation.
  • the well group has injected 7 rounds in total. After a single round of injection, the peak daily oil volume is greater than the daily oil volume before the implementation as the starting date of the validity period.
  • the daily oil increase reached 6.3t, and the cumulative oil increase was 13,261t.
  • the lowest viscosity of crude oil dropped to 316mPa ⁇ s, a drop of 88%.
  • the output-to-input ratio is about 4.2, and the field test effect is good.
  • Oil well profile formation temperature is 72°C, formation water salinity is 19473mg/L, oil layer effective thickness is 5.7m, porosity is 35%, permeability is 429 ⁇ 10 -3 ⁇ m 2 , total pore volume of the well group is 3.4 ⁇ 10 4 m 3 ,
  • the viscosity of dehydrated and degassed crude oil at 50°C is 3271mPa ⁇ s, and the well spacing is 270m.
  • the microbial flooding adopts the method of alternate slug injection, the total injection volume of oil production bacteria liquid is 0.1PV, totaling 3400m 3 , and the total water injection volume is 0.15PV, totaling 5100m3 .
  • Oil recovery was carried out by water flooding before implementation.
  • the well group has been injected for 6 rounds in total.
  • the peak daily oil volume after a single round of injection is greater than the daily oil volume before the implementation as the starting date of the validity period.
  • the daily oil increase reached 5.5t, and the cumulative oil increase was 8316t.
  • the lowest viscosity of crude oil dropped to 215mPa ⁇ s, a drop of 93%.
  • the ratio of output to input is 4.7, and the effect of the field test is good.

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Abstract

假单胞菌及其用途，该假单胞菌的保藏编号为CGMCC No.22583。上述假单胞菌的生长温度为37-45℃，能够在中高温油藏条件下从没有粘度的流体变性为同时具有表面活性及粘弹性的多功能采油菌液，同时具有扩大波及体积及提高洗油效率的功能，在物理模拟实验中提高采收率20％以上。

Description

假单胞菌及其用途
相关申请的交叉引用
本申请要求2021年09月18日提交的中国专利申请202111101957.1的权益，该申请的内容通过引用被合并于本文。
技术领域
本发明属于能源生物技术和环境生物技术领域，具体涉及一株假单胞菌及其用途。
背景技术
大部分油田在应用常规采油技术开发后，仍有部分的原油滞留在油藏中无法采出，但已进入高含水开发阶段。如何提高高含水开发阶段油田的采收率是油田持续稳产的关键。
微生物采油技术利用注入或激活油藏内部具有驱油功能的微生物，通过微生物生长代谢活动及其产生的代谢产物与原油和储层相互作用，提高原油采收率。该技术在全球范围内都开展了大量的室内研究和现场试验，虽然展现出良好的应用前景，但一直未实现现场大规模推广应用。由于油藏开发具有复杂性，需要同时提高洗油效率和扩大波及体积才能有效提高原油采收率，现有微生物采油技术主要包括：激活内源微生物或注入外源微生物，内源微生物菌群的复杂性使其很难实现目标菌群的准确激活和调控，而外源微生物又存在功能单一的局限性，无法有效解决油藏内部的多 种矛盾，因此，开发能够同时扩大波及体积(表观粘度大于50mPa·s)及提高洗油效率的多功能微生物菌种是提高微生物采油技术实施效果的关键。赵明等(一株采油用假单胞菌的筛选评价及现场应用，山东化工，2021年第50卷，公开日为2021年8月8日)获得了一株假单胞菌NGD，但其发酵液仅具备洗油功能且洗油效率最高为77％。
发明内容
本发明的目的是为了克服现有技术存在的以上问题，提供一种能够扩大波及体积和提高洗油效率的假单胞菌及其用途。
本发明第一方面提供了一株假单胞菌(Pseudomonas sp.)，其保藏编号为CGMCC No.22583。
本发明第二方面提供了如前所述的假单胞菌和/或其代谢产物在石油开采中的应用。
本发明第三方面提供了一种开采石油的方法，该方法包括：将采油菌液注入油藏，然后采出石油，其中，所述采油菌液含有如上所述的假单胞菌的菌体、所述假单胞菌的胞外代谢产物和所述假单胞菌的胞内代谢产物中的至少一种。
本发明至少获得了如下有益的技术效果：
(1)本发明提供的假单胞菌Pse-1，生长温度为37-45℃，其发酵液能够在中高温(60-95℃)油藏条件下从没有粘度的流体变性为具有表面活性及粘弹性的多功能采油菌液，同时具有扩大波及体积及提高洗油效率的功能，多功能采油菌液在60-95℃油藏温度下变性后菌液表观粘度≥50mPa·s， 菌液表面张力≤30mN/m，洗油效率≥90％，物理模拟实验提高采收率大于20％；
(2)该假单胞菌Pse-1的发酵液应用于微生物吞吐时，平均单井日产油量增幅大于50％，吨剂增油量达到20t以上，有效期250d以上，产出投入比＞5；
(3)该假单胞菌Pse-1的发酵液应用于微生物驱油，井组最终提高采收率达到8％以上，吨剂增油量达到15t以上，有效期350d以上，产出投入比＞3。
生物保藏
本发明的菌株的分类命名为：假单胞菌(Pseudomonas sp.)，于2021年5月24日被保藏在中国微生物菌种保藏管理委员会普通微生物中心(地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，邮政编码：100101，保藏单位的缩写为CGMCC)，保藏编号为CGMCC No.22583。
附图说明
附图是用来提供对本发明的进一步理解，并且构成说明书的一部分，与下面的具体实施方式一起用于解释本发明，但并不构成对本发明的限制。在附图中：
图1为本发明公开的一株假单胞菌Pse-1的系统发育树图；
图2为本发明公开的一株假单胞菌Pse-1的菌落形态图；
图3为本发明公开的一株假单胞菌Pse-1的菌体染色后的形态图；
图4为本发明公开的一株假单胞菌Pse-1在60℃变性后菌液中的网络状多糖结构的形态图；
图5为本发明公开的一株假单胞菌Pse-1发酵液(稀释度50％)在60℃下不同时间的粘度变化示意图。
具体实施方式
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值，这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说，各个范围的端点值之间、各个范围的端点值和单独的点值之间，以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围，这些数值范围应被视为在本文中具体公开。
本发明中，在未作相反说明的情况下，“发酵产物”是指培养假单胞菌后未进行任何分离操作而获得的产物整体，含有假单胞菌菌体和假单胞菌的代谢产物等。
第一方面，本发明提供了一株假单胞菌，其特征在于，其保藏编号为CGMCC No.22583。
本发明的假单胞菌的16s rDNA的序列如SQE ID NO：1所示，因此，本发明还涉及一株假单胞菌，其保藏编号为CGMCC No.22583或其16s rDNA的序列如SQE ID NO：1所示。
第二方面，本发明还提供了如上所述的假单胞菌和/或其代谢产物在石油开采中的应用。
第三方面本发明提供了一种开采石油的方法，其特征在于，该方法包 括：将采油菌液(或采油剂)注入油藏进行采油，其中，所述采油菌液(或采油剂)含有如上所述的假单胞菌的菌体和/或代谢产物(包括胞内和/或胞外代谢产物)。
本发明中，本发明的假单胞菌能够产生有利于采油的代谢产物(胞外代谢产物和/或胞内代谢产物，可能包括生物表面活性剂和/或温敏性粘弹生物多糖)。可以使用不同形式的含所述假单胞菌代谢产物的采油菌液，例如，所述采油菌液可以含有菌体、所述假单胞菌胞外代谢产物及菌体裂解物(或菌体裂解释放的胞内代谢产物)中的至少一种。
本发明中，所述假单胞菌的菌体可以为活菌体，也可以为死菌体，只要菌体生长(发酵)过程中产生的代谢产物至少部分保留在采油菌液中，在使用时能够与油藏接触进行驱油即可。假单胞菌产生的有利于采油的代谢产物包括胞外代谢产物及胞内代谢产物，因此，可以直接将含菌体的采油菌液直接注入油藏，菌体在油藏中裂解分泌出胞内代谢产物和胞外代谢产物一起促进采油。中高温油藏的油藏温度较高，菌体注入后会在胞外代谢产生的表面活性剂的作用下发生裂解，因此这种直接注入含菌体的采油菌液的采油方式尤其适合于中高温油藏。虽然如此，也可以将菌体预先进行裂解(如加热裂解)，将得到的裂解物注入油藏进行采油，可以理解的是，这种预先进行裂解的方式尤其适合于低温油藏(＜60℃)。因此，本发明的方法还可以包括制备采油菌液的步骤：(i)将假单胞菌接种至营养培养基中进行发酵；任选地，(ii)将所得发酵产物进行菌体裂解操作或从发酵产物分离菌体并将所得菌体进行裂解。按照步骤(i)获得的发酵产物含有菌体，可以直接用于中高温油藏。按照步骤(ii)获得的裂解产物中含 有假单胞菌的代谢产物，适用于低温油藏。
步骤(i)中，所述营养培养基含有碳源、氮源和无机盐。所述营养培养基的初始pH值优选为7.5-8。
优选地，所述营养培养基中碳源的含量为20-50g/L，更优选为23-47g/L。所述碳源可以常见的用于培养假单胞菌的含碳物质，优选为木糖和/或甘油，更优选为木糖和甘油。进一步优选地，甘油和木糖的重量比值为1-5(如1、1.2、1.5、1.8、2、2.5、3、3.5、4、4.5、5或上述数值之间的任意值)。最优选地，所述营养培养基中木糖的含量为8-12g/L。最优选地，所述营养培养基中甘油的含量为15-35g/L。
优选地，所述营养培养基中氮源的含量为0.5-2g/L。所述氮源可以为常见的用于培养假单胞菌的含氮物质，优选为酵母粉。
优选地，所述营养培养基中无机盐的含量为3-12g/L，更优选为3.3-10.8g/L。所述无机盐可以为常见的用于培养假单胞菌的无机盐，包括钠盐、钾盐、钙盐和镁盐中的至少一种，例如可以为硝酸钠、硝酸钾、磷酸氢二钾、磷酸氢二钠、氯化钠、氯化钾、氯化钙、硫酸镁和氯化镁中的至少一种，优选为硝酸钠、磷酸氢二钾、氯化钠、氯化钙和硫酸镁。
根据本发明一种特别优选的实施方式，所述营养培养基含有木糖8-12g/L、甘油15-35g/L、硝酸钠1-4g/L、磷酸氢二钾1-3g/L、氯化钠1-3g/L、氯化钙0.05-0.1g/L、硫酸镁0.3-0.7g/L、酵母粉0.5-2g/L、初始pH值为7.5-8。采用该优选实施方式的培养基可以获得更多的假单胞菌和/或其代谢产物，从而有利于进一步节约采油的能耗或物耗。
步骤(i)中，所述发酵可以在常规的培养假单胞菌的条件下进行，优 选地，所述发酵的条件使得发酵液中假单胞菌的含量大于等于10 9CFU/mL，或者，所述发酵的条件使得发酵液在60℃及以上温度加热8h后的表面张力≤30mN/m，表观粘度≥50mPa·s。更优选地，所述发酵的条件包括：接种量为10 6-10 8CFU/mL。发酵的温度优选为37-45℃。发酵的时间优选为48-72h。发酵的通气量优选为1-2L/min。
本发明中，在进行步骤(i)的发酵之前，一般还可以包括进行种子培养的步骤，以使假单胞菌扩增。种子培养使用的种子培养基可以含有：葡萄糖2-4g/L，蛋白胨2-4g/L，酵母粉2-4g/L。种子培养的条件可以在37-45℃下150-200r/min震荡培养20-30h。
步骤(ii)中，可以直接将发酵产物整体进行裂解操作，从而使发酵产物中的菌体发生裂解，得到菌体裂解物；也可以先从发酵产物中分离出菌体，再裂解菌体得到菌体裂解物。裂解主要是为了使菌体破裂从而释放胞内代谢产物，可以采用本领域常规的方法进行，例如可以采用高温裂解法(如在60-95℃下处理8-10h)，也可以采用反复冻融法、超声波处理法等，还可以借助裂解液进行裂解。
本发明中，所述采油菌液在25℃下的表面张力≤30mN/m，优选为24.5-28.5mN/m。所述采油菌液在60℃下的表观粘度≥50mPa·s，优选为65-150mPa·s。其中，表面张力可以参照GB/T22237-2008中的平板法测得。表观粘度可以使用Brook field旋转黏度计(转子型号：3号转子，转子转速：60r/min)测得(可以参照GB 1886.41-2015)。步骤(i)得到的发酵产物与步骤(ii)得到的菌体裂解物可以用稀释剂稀释后再用于采油，稀释剂的用量优选使得采油菌液的表面张力和/或表观粘度满足以上要求，为了 满足以上要求，步骤(i)得到的发酵产物和/或步骤(ii)得到的菌体裂解物的稀释度(＝稀释剂体积/(发酵产物体积+稀释剂体积)×100％)一般不高于50％。所述稀释剂优选为自来水或地层水，更优选为地层水，特别是矿化度为1000-100000mg/L的地层水。
本发明中，将采油菌液注入油藏进行采油的方式可以为油井吞吐。本发明的假单胞菌有利于缩短闷井培养时间，因此，采用油井吞吐时，闷井培养的时间可以为10-30d，优选为15-30d。采油菌液的注入速度可以为10-20m 3/h，油井闷井培养后开井生产。
本发明中，对油井吞吐采油菌液的总注入量没有特别的要求，优选地，采油菌液的总注入量V＝πR 2Hфβ，其中：
V—采油菌液的总注入量，单位为m 3；
R—处理半径，单位为m；
H—有效厚度，单位为m；
ф—孔隙度；
β—用量系数，取值为1-1.5。
本发明中，将采油菌液注入油藏进行采油的方式可以为注水井驱替，采油菌液从注水井段塞式注入，因此，将采油菌液注入油藏进行水井驱替的方式可以包括：
(1)依次将采油菌液与水注入油藏；
(2)停注；
(3)将水注入油藏；
(4)循环进行步骤(1)-(3)至少两次。
优选地，步骤(1)中采油菌液的用量为采油菌液总注入量的14-50％。采油菌液的总注入量优选为0.05-0.1PV(孔隙体积)。“PV”为油藏所在储层的孔隙体积，孔隙体积为储层内有效孔隙的总容积，通过计算储层体积与孔隙度的乘积获得。其中，孔隙度可以通过油藏取芯后按照SY/T6298-1997方法测得。对于井距为200-300m的井组，步骤(1)中采油菌液的注入速度可以为5-20m 3/h。一般地，采油菌液和水均从注水井注入。
优选地，步骤(1)和步骤(3)中的注水量为总注水量的14-50％。步骤(1)和步骤(3)中的注水量比例优选为1：0.05-0.7。总注水量优选为0.1-0.3PV(孔隙体积)。对于井距为200-300m的井组，步骤(1)和步骤(3)中注入水的注入速度均可以为10-50m 3/h。采油菌液和水均从注水井注入。
优选地，步骤(2)中，停注的时间为1-2d。
优选地，步骤(4)中，循环进行的次数为2-7次。
本发明中，所述油藏的温度可以在较宽范围内波动。当所述油藏为中高温油藏时，所述油藏的温度一般为60-95℃。当所述油藏为低温油藏时，所述油藏的温度一般＜60℃(如30-59℃)。油藏脱水脱气原油在50℃条件下的粘度可以≤10000mPa·s(如1500-4000mPa·s)。原油粘度可以通过使用旋转粘度计参照SY/T0520-2008方法在50℃条件下测得。
本发明的方法能够同时扩大波及体积与提高洗油效率，因此，本发明还涉及一种同时扩大波及体积与提高洗油效率的方法，其特征在于，该方法包括：将采油菌液注入油藏进行采油，其中，所述采油菌液如上所述， 在此不再赘述。
以下将通过实施例对本发明进行详细描述。
以下实施例1、2、4-7中，采用的营养培养基(营养液)的组成为：木糖10g/L、甘油30g/L、硝酸钠2g/L、磷酸氢二钾2g/L、氯化钠2g/L、氯化钙0.1g/L、硫酸镁0.5g/L、酵母粉1g/L，pH为7.8，余量为水。
种子液培养基(营养液)的组成为：葡萄糖3g/L，蛋白胨3g/L，酵母粉3g/L；种子液的获得方法为：将1mL菌种冻存管接种到100mL种子液培养基中，37℃好氧条件下170r/min左右震荡培养24h，获得种子液，种子液中假单胞菌Pse-1的含量为10 8CFU/mL。
发酵液的获得方法为：将假单胞菌Pse-1种子液按2vol％接种量接种于营养培养基中，37℃下培养60h，搅拌速度200rpm，通气量1.5L/min，获得发酵液，发酵液中假单胞菌Pse-1的含量为10 9CFU/mL。
实施前和实施后油井及区块利用常规的计量分离器来计量日油量，通常也叫玻璃管量油法，它是根据连通管平衡原理，采用定容积的方法测量液体体积，结合取样测得的产出液含水值，计算出油井或区块的日油量。
实施例1
本发明的假单胞菌Pse-1的获取与鉴定
一、假单胞菌Pse-1的获取
从胜利油田的某油井的产出液中分离得到一株假单胞菌，简称Pse-1。
二、假单胞菌Pse-1的鉴定
对于Pse-1进行16s rDNA扩增并测序，进行菌种鉴定，步骤如下：变 性、PCR扩增、PCR产物纯化、目的片段测序。其16s rDNA的序列如SQE ID NO：1所示。根据测序结果在NCBI进行序列比对，相似度96％，系统发育树构建(图1)，鉴定本发明的假单胞菌Pse-1为一新菌种，属于假单胞菌属，其分类命名为：假单胞菌(Pseudomonas sp.)。
SQE ID NO：1：
Figure PCTCN2022119404-appb-000001
Figure PCTCN2022119404-appb-000002
参照《Bergey’s Mannual of Systematic Bacteriology》的实验方法观察：菌体大小和形态，有无鞭毛和芽孢，生长温度，需氧情况，菌落形态。
研究结果表明，该菌株菌体呈短杆状、有单根鞭毛、能运动、菌落隆起，边缘不平整，表面光滑湿润呈融合状态，菌落表面有粘性拉丝多糖。细胞呈长杆状，大小为(0.1-0.2)×(2-3)μm。菌落形态如图2所示，菌体染色后的形态如图3所示。
假单胞菌Pse-1的生理生化特性：好氧，生长温度37-45℃，最适生长温度37℃，生长pH范围5-10，最佳生长pH范围7.8-8，NaCl的浓度耐受范围为0-10wt％。
实施例2
对假单胞菌Pse-1的油藏条件下发酵液表面张力、发酵液洗油砂性能、表观粘度，物理模拟实验提高的采收率值等性能参数进行评价。其中，油藏温度为60℃。
假单胞菌Pse-1的性能评价方法：
(1)油藏条件下不同稀释倍数发酵液表面张力及表观粘度的测定
Pse-1发酵液用地层水(矿化度为18712mg/L)进行不同程度的稀释(稀释度＝地层水体积/(发酵液体积+地层水体积)×100％)，在油藏温度下加热8h，然后检测不同稀释度下发酵液的在25℃下的表面张力和在60℃下表观粘度(表面张力测试参照GB/T22237-2008的平板法测试标准，表观粘度使用Brook field旋转黏度计(转子型号：3号转子，转子转速：60r/min) 测得(参照GB 1886.41-2015中的黏度测试方法)，稀释度50％下发酵液的显微镜观察结果见图4，稀释度50％下发酵液加热不同时间的粘度变化如图5所示，加热8h时测得的结果如下表1所示。
表1
稀释度 表面张力(mN/m) 表观粘度(mPa·s) 洗油效率(％)
10％ 24.7 150 96.4
20％ 25.2 121 95.2
30％ 25.4 107 93.1
40％ 26.7 86 92.4
50％ 28.3 65 91.5
60％ 35.7 24 84.2
(2)发酵液洗油砂性能的评价
按照标准《Q/SH1020 1518-2013》进行油砂制备和标准曲线绘制。称取3g油砂于50mL比色管中，标记为A。
将Pse-1发酵液在油藏温度下加热8h，然后进行不同比例的稀释。
将上述处理并稀释好的Pse-1发酵液10mL加入比色管，加盖，将比色管放置于60℃恒温水浴中，每隔15min将比色管取出轻轻摇动10次，再置于水浴中，共放置1h后取出。
小心将上层液体倾倒出去，用蒸馏水反复冲洗比色管内残余的菌液直至洗出液呈透明状态为止，小心倾去上层液体。
另取50mL比色管一支，标记为B，置于比色管架上，将漏斗插入比色管内。
把比色管A置于漏斗正上方，口向下倾斜，用装满蒸馏水的洗瓶冲洗A的底部，小心将A中油砂全部转移到B中，并倾去上层洗液。
将带油砂的比色管放入105℃的烘箱内烘干4h，取出放入干燥器中放至室温。将带油砂比色管加入适量沸程60-90℃的石油醚充分摇动，并稀释至刻度。吸取石油醚溶液，在分管光度计上测吸光度，并在标准曲线上查出比色管内残余的含油量。洗油效率X＝(1-W 1/KW 0)×100％，其中W 1为比色管内油砂的残余含油量，K为油砂含油的质量分数，W 0为所称油砂的质量。
稀释度40％条件下的洗油效率X＝(1-0.0046/0.02×3.0067)×100％＝92.4％，最终评价不同稀释度下发酵液洗油效率，结果如上表1所示。
(3)物理模拟实验提高采收率值的评价
①岩心准备：装填岩心和灭菌，测定空气渗透率；
②抽真空、饱和模拟地层水，测定岩心的PV(孔隙体积)；
③饱和原油，岩心老化7d，计算束缚水饱和度；
④一次水驱，水驱至采出水含水98％以上，计算一次水驱采收率；
⑤注入稀释度50％的假单胞菌Pse-1发酵液约0.3PV，关闭注入端和产出端，在油藏温度下变性约9h；
⑥二次水驱，水驱至产出液含水100％，计算假单胞菌Pse-1提高采收率值，达到23％。
以上实验可以看出假单胞菌Pse-1的性能：≤50％稀释度下发酵液表面张力低于30mN/m，变性后粘度大于50mPa·s，洗油效率大于90％；物理模拟实验提高采收率(相比单纯水驱)为21％。
实施例3
假单胞菌Pse-1作为采油菌液的制备。
按照如上所述的方法先获得种子液，再将种子液接种于营养培养基中培养获得发酵液，接种龄是指制备种子液时培养的时间：
在实施例3.1中，所述营养培养基为木糖10g/L、甘油30g/L、硝酸钠2g/L、磷酸氢二钾2g/L、氯化钠2g/L、氯化钙0.1g/L、硫酸镁0.5g/L、酵母粉1g/L，pH为7.8。
在实施例3.2中，所述营养培养基为木糖12g/L、甘油35g/L、硝酸钠4g/L、磷酸氢二钾3g/L、氯化钠3g/L、氯化钙0.1g/L、硫酸镁0.7g/L、酵母粉2g/L，pH为7.5，余量为水。
在实施例3.3中，所述营养培养基为木糖8g/L、甘油15g/L、硝酸钠1g/L、磷酸氢二钾1g/L、氯化钠1g/L、氯化钙0.05g/L、硫酸镁0.3g/L、酵母粉0.5g/L，pH为8，余量为水。
在实施例3.1中，所述假单胞菌的发酵条件为：接种量2vol％，接种龄12h，初始pH7.5，温度37℃，搅拌速度220rpm，通气量1L/min，发酵时间72h。
在实施例3.2中，所述假单胞菌的发酵条件为：接种量3vol％，接种龄24h，初始pH7.5，温度40℃，搅拌速度200rpm，通气量1.5L/min，发酵时间60h。
在实施例3.3中，所述假单胞菌的发酵条件为：接种量1vol％，接种龄12h，初始pH8，温度45℃，搅拌速度160rpm，通气量2L/min，发酵时间48h。
以上发酵液在稀释度50％条件下的性能参数如下表2所示。
表2
Figure PCTCN2022119404-appb-000003
实施例4
本发明提供的假单胞菌Pse-1发酵液在胜利油田某区块稠油油井A1中的应用。
油井概况：地层温度65℃，地层水矿化度18712mg/L，油层有效厚度6.7m，孔隙度35％，渗透率567×10 -3μm 2，脱水脱气原油在50℃下的粘度3764mPa·s。
上述假单胞菌Pse-1发酵液在微生物单井吞吐中的应用。所述微生物单井吞吐是通过油井向地层注入假单胞菌Pse-1发酵液，利用生物表面活性剂和温敏性粘弹生物多糖的协同作用提高单井产能。
具体实施步骤为：将配制好的稀释度30％的假单胞菌Pse-1发酵液经油井油套环空注入地层，注入速度为15m 3/h。
根据油井的油层有效厚度、孔隙度、处理半径计算出发酵液的注入量。
注入总量公式：V＝3.14R 2Hфβ，其中
V—注入用量，m 3；
R—处理半径，7m；
H—有效厚度(6.7m)；
ф—孔隙度(取0.35)；
β—用量系数(取1.1)，
根据公式计算得到稀释度30％的假单胞菌Pse-1发酵液的用量为397m 3；用泵车将配制好的Pse-1发酵液经油井油套环空注入地层；油井关井培养30d后开井生产。
试验结果：实施前采用水驱的方式进行采油。该井A1开井生产后，当油井日油量大于实施前日油量对应的日期为有效期起始日，产液和产油量相比于实施前均上升，单井平均日增油达到2.8t，累计增油980t，原油粘度最低降至762mPa·s，降幅达到了80％，当单井日油量小于实施前日油量的时间作为有效期截止日，有效期达到了350d，产出投入比为5.7，假单胞菌Pse-1的现场试验效果良好。
实施例5
本发明提供的假单胞菌Pse-1发酵菌液在胜利油田某区块稠油油井B1中的应用。
油井概况：地层温度60℃，地层水矿化度22140mg/L，油层有效厚度5.1m，孔隙度30％，渗透率467×10 -3μm 2，脱水脱气原油在50℃下的粘度1875mPa·s。
上述假单胞菌Pse-1发酵液在微生物单井吞吐中的应用。所述微生物单井吞吐是通过油井向地层注入假单胞菌Pse-1发酵液，利用生物表面活性剂和温敏性粘弹生物多糖的协同作用提高单井产能。
具体实施步骤为：将配制好的稀释度为50％的假单胞菌Pse-1发酵液 采油菌液经油井油套环空注入地层，注入速度为10m 3/h。
根据油井的油层有效厚度、孔隙度、处理半径计算出发酵液的注入量。
注入总量公式：V＝3.14R 2Hфβ，其中
V—注入用量，m 3；
R—处理半径，6m；
H—有效厚度(5.1m)；
ф—孔隙度(取0.3)；
β—用量系数(取1.2)，
根据公式计算得到稀释度为50％假单胞菌Pse-1发酵液的用量为208m 3；用泵车将配制好的Pse-1发酵液经油井油套环空注入地层；油井关井培养20d后开井生产。
试验结果：实施前采用水驱的方式进行采油。该井B1开井生产后，当油井日油量大于实施前日油量对应的日期为有效期起始日，产液和产油量相比实施前均上升，单井平均日增油达到3.7t，累计增油1166t，原油粘度最低降至283mPa·s，降幅达到了84.9％，当单井日油量小于实施前日油量的时间作为有效期截止日，有效期达到了315d，产出投入比为6，现场试验效果良好。
实施例6
本发明提供的假单胞菌Pse-1发酵液在胜利油田某区块C1中的应用，该井组1注5采。
油井概况：地层温度65℃，地层水矿化度24134mg/L，油层有效厚度 4.7m，孔隙度0.32，渗透率653×10 -3μm 2，井组总孔隙体积8.75×10 4m 3，脱水脱气原油在50℃下的粘度2719mPa·s，井距为230m。
上述假单胞菌Pse-1发酵液在微生物驱油中的应用。所述微生物驱油是将假单胞菌发酵液从注水井段塞式注入，利用假单胞菌发酵液Pse-1的代谢产物提高井组油井的产能。
所述微生物驱采用段塞交替注入的方式，采油菌液总注入量为0.05PV，共4350m 3，总注水量为0.1PV，共8700m 3。
具体步骤包括：
(1)依次将采油菌液与水注入油藏，采油菌液注入量621m 3，注入速度为20m 3/d，注入水828m 3，注水速度30m 3/d；
(2)停注1d；
(3)注水414m 3，注水速度m 3/d，完成一个周期的注入。
试验结果：实施前采用水驱的方式进行采油。该井组总共注入7轮次，单轮次注入后峰值日油量大于实施前日油量作为有效期起始日，注入后所有油井产液和产油量相比于实施前均上升，单井平均日增油达到6.3t，累计增油13261t，原油粘度最低降至316mPa·s，降幅达到了88％，单轮次注入后峰值日油小于实施前日油量作为有效期截止日，有效期达到了421d，产出投入比约为4.2，现场试验效果良好。
实施例7
本发明提供的假单胞菌Pse-1发酵液在胜利油田某区块D1中的应用，该井组1注4采。
油井概况：地层温度72℃，地层水矿化度19473mg/L，油层有效厚度5.7m，孔隙度35％，渗透率429×10 -3μm 2，井组总孔隙体积3.4×10 4m 3，脱水脱气原油在50℃下的粘度3271mPa·s，井距为270m。
所述微生物驱采用段塞交替注入的方式，采油菌液总注入量为0.1PV，共3400m 3，总注水量为0.15PV，共5100m 3。
具体步骤包括：
(1)依次将采油菌液与水注入油藏，采油菌液注入量595m 3，注入速度为20m 3/d，注入水531m 3，注水速度40m 3/d；
(2)停注2d；
(3)注水318m 3，注水速度50m 3/d，完成一个周期的注入。
试验结果：实施前采用水驱的方式进行采油。该井组总共注入6轮次，单轮次注入后峰值日油量大于实施前日油量作为有效期起始日，注入后所有油井产液和产油量相比于实施前均上升，单井平均日增油达到5.5t，累计增油8316t，原油粘度最低降至215mPa·s，降幅达到了93％，单轮次注入后峰值日油小于实施前日油量作为有效期截止日，有效期达到了378d，产出投入比为4.7，现场试验效果良好。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于此。在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，包括各个技术特征以任何其它的合适方式进行组合，这些简单变型和组合同样应当视为本发明所公开的内容，均属于本发明的保护范围。

Claims (15)

  1. 一株假单胞菌(Pseudomonas sp.)，其特征在于，其保藏编号为CGMCC No.22583。
  2. 权利要求1所述的假单胞菌和/或其代谢产物在石油开采中的应用。
  3. 一种开采石油的方法，其特征在于，该方法包括：将采油菌液注入油藏进行采油，其中，所述采油菌液含有权利要求1所述的假单胞菌的菌体、所述假单胞菌的胞外代谢产物和所述假单胞菌的胞内代谢产物中的至少一种。
  4. 根据权利要求3所述的方法，其中，所述采油菌液在25℃下的表面张力≤30mN/m，优选为24.5-28.5mN/m；在60℃下的表观粘度≥50mPa·s，优选为65-150mPa·s。
  5. 根据权利要求3或4所述的方法，其中，所述方法还包括制备采油菌液的步骤：(i)将假单胞菌接种至营养培养基中进行发酵；任选地，(ii)将所得发酵产物进行菌体裂解操作，或者，从发酵产物分离菌体并将所得菌体进行裂解。
  6. 根据权利要求5所述的方法，其中，所述营养培养基含有碳源、氮源和无机盐，所述营养培养基的初始pH值为7.5-8。
  7. 根据权利要求6所述的方法，其中，所述营养培养基中碳源的含量为20-50g/L；
    和/或，所述营养培养基中氮源的含量为0.5-2g/L；
    和/或，所述营养培养基中无机盐的含量为3-12g/L。
  8. 根据权利要求6或7所述的方法，其中，所述碳源为木糖和/或甘油；
    和/或，所述氮源为酵母粉；
    和/或，所述无机盐包括钠盐、钾盐、钙盐和镁盐中的至少一种。
  9. 根据权利要求5-8中任意一项所述的方法，其中，所述营养培养基含有木糖8-12g/L、甘油15-35g/L、硝酸钠1-4g/L、磷酸氢二钾1-3g/L、氯化钠1-3g/L、氯化钙0.05-0.1g/L、硫酸镁0.3-0.7g/L、酵母粉0.5-2g/L、初始pH值为7.5-8。
  10. 根据权利要求5-9中任意一项所述的方法，其中，所述发酵的条件包括：接种量为10 6-10 8CFU/mL，发酵的温度为37-45℃，发酵的时间为48-72h。
  11. 根据权利要求3-10中任意一项所述的方法，其中，将采油菌液注入油藏进行采油的方式为油井吞吐，油井吞吐的闷井培养时间为15-30d。
  12. 根据权利要求11所述的方法，其中，所述采油菌液的总注入量V＝πR 2Hфβ，其中：
    V—采油菌液的总注入量，单位为m 3；
    R—处理半径，单位为m；
    H—有效厚度，单位为m；
    ф—孔隙度；
    β—用量系数，取值为1-1.5。
  13. 根据权利要求3-10中任意一项所述的方法，其中，将采油菌液注入油藏进行采油的方式为水井驱替，包括：
    (1)依次将采油菌液与水注入油藏，该步骤中采油菌液的用量为采油菌液总注入量的14-50％；
    (2)停注；
    (3)将水注入油藏，步骤(1)中的注水量和步骤(3)中的注水量之间的比值为1:0.05-0.7；
    (4)循环进行步骤(1)-(3)至少两次。
  14. 根据权利要求13所述的方法，其中，采油菌液的总注入量为0.05-0.1PV，水的总注入量为0.1-0.3PV，其中，PV为油藏所在储层的孔隙体积；
    和/或，步骤(4)中，循环进行的次数为2-7次。
  15. 根据权利要求3-14中任意一项所述的方法，其中，所述油藏的50℃原油脱水脱气粘度≤10000mPa·s。
PCT/CN2022/119404 2021-09-18 2022-09-16 假单胞菌及其用途 WO2023041062A1 (zh)

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