WO2021077888A1 - Procédé de préparation et procédé d'utilisation pour la réduction de la viscosité d'huile lourde et inoculum microbien mixte de dégradation - Google Patents

Procédé de préparation et procédé d'utilisation pour la réduction de la viscosité d'huile lourde et inoculum microbien mixte de dégradation Download PDF

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WO2021077888A1
WO2021077888A1 PCT/CN2020/110934 CN2020110934W WO2021077888A1 WO 2021077888 A1 WO2021077888 A1 WO 2021077888A1 CN 2020110934 W CN2020110934 W CN 2020110934W WO 2021077888 A1 WO2021077888 A1 WO 2021077888A1
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heavy oil
strain
degradation
acinetobacter
bacteria
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PCT/CN2020/110934
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Chinese (zh)
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张秀霞
辛瑞
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中国石油大学(华东)
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    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
    • B09C—RECLAMATION OF CONTAMINATED SOIL
    • B09C1/00—Reclamation of contaminated soil
    • B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C02F3/343—Biological treatment of water, waste water, or sewage characterised by the microorganisms used for digestion of grease, fat, oil
    • 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
    • C12N1/36—Adaptation or attenuation of cells
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/30—Organic compounds
    • C02F2101/32—Hydrocarbons, e.g. oil

Definitions

  • the invention relates to a preparation method and an application method of a thick oil viscosity-reducing and degrading mixed bacterial agent, which belongs to the fields of environmental protection and pollution prevention and treatment.
  • the hydrophobicity of crude oil is one of the main factors limiting its degradation rate.
  • the high viscosity of heavy oil makes it difficult for microorganisms to degrade and utilize it.
  • the degradation of heavy oil by microorganisms is mainly carried out at the oil-water interface.
  • the emulsification of oil can greatly increase the degree of oil dispersion, increase the contact surface area between strains and oil droplets, and promote the absorption and degradation of petroleum hydrocarbons by microorganisms.
  • the direct addition of surfactants can only change the spatial structure of heavy oil macromolecules, but cannot change its chemical composition.
  • microorganisms can not only pass the glycolipids produced in the metabolic process Biosurfactants, lipopeptides, phospholipids, etc., reduce the interfacial tension of oil and water and emulsify crude oil, and can improve the biochemical activity of the heavy components of crude oil, thereby improving the viscosity of crude oil.
  • the hydrocarbons are converted into low-molecular hydrocarbons, thereby reducing the macromolecular components in the heavy oil, reducing its average molecular weight, and reducing the viscosity more thoroughly.
  • Studies have shown that certain bacteria can promote the emulsification of crude oil through the production of biosurfactants and passively diffuse into the cells, thereby being trapped and degraded by biological enzymes, thereby increasing the degradation rate of crude oil.
  • CN 109763803 A discloses a Pseudomonas rhamnolipo-producing QFP and its application in heavy oil exploitation, including a facultative anaerobic pseudomonas (Pseudomonas sp.) strain QFP.
  • the application method is as follows: transfer the Pseudomonas QFP stored in the glycerol tube to the LB liquid medium, shake culture at 20-30°C for 12 hours to obtain the seed liquid, and inoculate the seed liquid at a volume ratio of 0.5%.
  • the surfactant fermentation medium continuously aerated culture at 20-30°C for 96h, and the fermentation broth is obtained after the fermentation is completed.
  • the rhamnolipid produced by the invention has strong surfactant performance and emulsifying ability, can significantly reduce the viscosity of heavy oil, and can inhibit the growth of other bacteria.
  • the bacteria can be applied to the repair of petroleum hydrocarbon polluted environment and oil reservoir environment It has the potential to improve oil recovery, and the produced rhamnolipids have important application value in medicine, environmental protection and energy extraction.
  • CN 109576191 A discloses a composite microbial inoculum for the development of heavy oil and its preparation method and application. It is proposed for the problem that the existing microbial oil extraction technology is complicated in preparation and that a single strain is not resistant to environmental impact.
  • the compound microbial inoculum of the present invention is made by mixing Bacillus pallisi, Pantoea, and thermophilic anaerobic bacillus after fermentation. The beneficial effect is to avoid the defect that single-function strains cannot tolerate environmental impacts, and the difference between the three strains The functional expression is not inhibited, the preparation method is simple, and the cost is low.
  • CN 104109646 A discloses a visbreaker suitable for heavy oil wells with different mineralization degrees and its application.
  • the visbreaker is composed of Bacillus licheniformis, Bacillus subtilis and Bacillus thuringiensis, at a temperature of 30-70°C, After culturing for 24 hours under the conditions of pH 5-10 and salinity 1000-12000mg/L and 0-30MPa, the cell concentration of each strain can reach 10 8 -10 9 cells/mL, and the reproductive ability is strong.
  • the bacterial agent can reduce the viscosity of crude oil by degrading heavy components of crude oil, metabolizing biological surfactants, biogas, etc., so as to ensure stable production and increased production of heavy oil wells.
  • the method of use includes: taking the slime reducing bacteria to ferment to produce the fermentation bacteria liquid; using the fermentation bacteria liquid to prepare the injection bacteria liquid system; squeeze the injected bacteria liquid system into the target oil layer.
  • the microbial agent has achieved a good effect in oil well field tests under the conditions of salinity 6000 ⁇ 83000mg/L, pH7 ⁇ 8.6, and 40 ⁇ 60°C. The viscosity of crude oil is reduced by 50% ⁇
  • CN 104450543 A discloses an original microbial repair composite preparation and an application method thereof; the composite preparation is composed of four parts: heavy oil degrading bacteria, nutrients, composite surfactant penetrant, and oxidation promoting activator, in which heavy oil is degraded
  • the flora is composed of aerobic rod-shaped bacteria (Lactobacillus sp.) and anaerobic rod-shaped spore bacteria (bacillus sp.) screened and isolated from heavy oil soil; spray the compound formulation on the contaminated soil after 2-3 Within months, the degradation rate of heavy oil contaminated soil reached 50-80%.
  • the present invention provides a method for preparing a thick oil viscosity reduction and viscosity reduction mixed bacteria agent.
  • the mixed bacterial agent can not only realize high-efficiency degradation of heavy oil, but also can obviously improve the viscosity of heavy oil.
  • a method for preparing a viscous-reducing and degrading mixed bacterial agent for heavy oil includes the following steps:
  • the first step is to prepare seed culture medium
  • the second step is to inoculate the following single bacteria into the seed medium respectively, and obtain the seed liquid of each single bacteria after culturing.
  • the single bacteria include Acinetobacter sp.strain ZX-15, and Achromobacter pulmonis strain PI3-03 , Acinetobacter sp. RS206 and Chrobactrum anthropi strain CON21;
  • the seed liquid of each single bacteria is mixed in proportion to obtain the viscous-reducing and degrading mixed bacteria agent of heavy oil.
  • the above-mentioned single bacteria are all existing strains, which can be searched in the NCBI nucleic acid database by number, such as: Acinetobacter sp.strain ZX-15 in the NCBI database with the accession number MF148465.1, Achromobacter pulmonis
  • the accession number of strain PI3-03 in the NCBI database is MK396599.1
  • the accession number of Acinetobacter sp.RS206 in the NCBI database is EU912468.1
  • the accession number of chrobactrum anthropi strain CON21 in the NCBI database is MK167392 .1.
  • preparation of the seed culture medium in the first step includes the following steps:
  • R1 Dissolve 5.0g beef extract, 10.0g peptone, and 5.0g sodium chloride in 1000mL deionized water, stir well to obtain mixed liquid I;
  • R2 To the mixed solution I, add 0.1g sodium molybdate, 0.05g aluminum nitrate, 0.04g zinc chloride, 0.06g copper sulfate, 0.03g manganese chloride, 0.09g ferrous sulfate, 0.10g magnesium nitrate and 0.06 g of potassium chloride, and then fully mixed to obtain the seed culture medium.
  • the mixing volume ratio of the seed liquid of Acinetobacter sp.strain ZX-15, Achromobacter pulmonis strain PI3-03, Acinetobacter sp. RS206 and Chrobactrum anthropi strain CON21 It is (1-2):(1-2):(1-2):(1-2), especially 2:2:1:1.
  • Acinetobacter sp.strain ZX-15 Achromobacter pulmonis strain PI3-03, Acinetobacter sp. RS206 and Chrobactrum anthropi strain CON21 were contaminated by heavy oil from the heavy oil mining area.
  • the specific steps are as follows:
  • the preparation method of the enrichment/degradation medium is: adding 5.0g sodium chloride, 1.0g ammonium sulfate, 0.25g magnesium sulfate heptahydrate, 2.0g sodium nitrate, 4.0g potassium dihydrogen phosphate, 7.6g Dipotassium hydrogen phosphate is dissolved in 1000 mL of deionized water, and then a suitable amount of heavy oil is added.
  • step S3 includes the following steps:
  • T1 Inoculate a single strain in an inorganic salt medium containing heavy oil, and culture it on a constant temperature shaker at 160r/min and 37°C for 8 days;
  • T2 Extract the residual oil in the culture medium to obtain the petroleum hydrocarbon degradation rate, and select the two strains with the highest petroleum hydrocarbon degradation rate;
  • the measurement of the surface tension of the bacterial liquid includes the following steps:
  • M1 Inoculate a single strain into the fermentation medium, and culture it for 3 days at 160r/min and 37°C;
  • M2 Centrifuge the fermentation broth at 8000r/min for 10 minutes to remove the cell bodies in the fermentation broth
  • M3 The surface tension of the fermentation broth is measured, and the two strains with the smallest surface tension of the fermentation broth are selected as high-efficiency viscous oil-lowering bacteria.
  • the 16sRNA sequencing in step S4 is to extract the genomic DNA of the strain and perform electrophoresis detection, use universal primers for gene amplification, then perform electrophoresis PCR product identification, perform sequencing, homology comparison, and evolutionary tree analysis to determine
  • the two strains with the highest degradation rate of petroleum hydrocarbons are Acinetobacter sp.strain ZX-15 and Achromobacter pulmonis strain PI3-03.
  • the two strains with the best effect on reducing viscosity of heavy oil are Acinetobacter sp. RS206 and chrobactrum anthropi strain CON21.
  • the application method of the thick oil viscosity reducing and degrading mixed bacterial agent obtained by the above preparation method is given. It is applied to the viscosity reduction and degradation of heavy oil pollution.
  • the application temperature is controlled at 25-40°C, and the best is 35°C; pH control is applied It is 7-8, the best is 7.5; the application of inoculum volume is controlled at 3-5% by volume.
  • the present invention has the following advantages:
  • the sieving and culturing process of the mixed inoculum related to the present invention all use conventional culture medium, no special culture environment is required, and the preparation of the mixed inoculum is simpler.
  • the mixed bacterial agent involved in the present invention is obtained by separation, enrichment and purification from heavy oil contaminated soil.
  • the strain itself comes from heavy oil soil and belongs to the original microbial degrading bacteria. Therefore, it is more suitable for heavy components and colloidal asphalt.
  • the high heavy oil environment has good adaptability and high efficiency.
  • the thick oil viscosity-reducing and degrading mixed bacterial agent disclosed in the present invention is a kind of artificially screened and constructed mixed bacterial agent, which has obvious advantages in biodegradation of pollutants compared with a natural mixed bacterial system with incomplete composition and a single strain.
  • the bacterial composition of the bacterial agent is clear, the preparation process is simple, the degradation efficiency is high, the viscosity reduction effect is good, and it has a good application prospect.
  • the viscosity-reducing and degrading mixed microbial agent for heavy oil disclosed in the present invention can simultaneously reduce the viscosity of heavy oil and efficiently degrade petroleum hydrocarbons, and has more comprehensive functions than the existing microbial technology.
  • Figure 1 is a schematic diagram of the surface tension of the bacterial liquid and the degradation rate of petroleum hydrocarbons at different culture temperatures in Example 2;
  • Example 2 is a schematic diagram of the surface tension of the bacterial liquid and the degradation rate of petroleum hydrocarbons under different pH in Example 2;
  • Example 3 is a schematic diagram of the surface tension of the bacterial liquid and the degradation rate of petroleum hydrocarbons under different inoculation amounts in Example 2;
  • Example 4 is a schematic diagram of the surface tension and petroleum hydrocarbon degradation rate of the bacterial liquid with different mixing ratios in Example 3.
  • Acinetobacter sp.strain ZX-15, Achromobacter pulmonis strain PI3-03, Acinetobacter sp. RS206 and Chrobactrum anthropi strain CON21 were inoculated into the seed culture medium at 37°C, Under the condition of 160r/min, the seed liquid was obtained by shaking at a constant temperature for 24 hours, and the seed liquid was mixed in a volume ratio of 2:2:1:1 to obtain a mixed microbial agent.
  • the seed culture medium preparation step dissolve 5.0 g beef extract, 10.0 g peptone, and 5.0 g sodium chloride in 1000 mL of deionized water, stir thoroughly to obtain mixed liquid I, and add trace elements to the mixed liquid I 0.1g sodium molybdate, 0.05g aluminum nitrate, 0.04g zinc chloride, 0.06g copper sulfate, 0.03g manganese chloride, 0.09g ferrous sulfate, 0.10g magnesium nitrate and 0.06g potassium chloride, and then mix them thoroughly.
  • the preparation method of the enrichment/degradation medium is as follows: 5.0g sodium chloride, 1.0g ammonium sulfate, 0.25g magnesium sulfate heptahydrate, 2.0g sodium nitrate, 4.0g potassium dihydrogen phosphate, 7.6g dipotassium hydrogen phosphate Dissolve in 1000mL deionized water, then add appropriate amount of heavy oil.
  • Acinetobacter sp.strain ZX-15, Achromobacter pulmonis strain PI3-03, Acinetobacter sp. RS206 and Chrobactrum anthropi strain CON21 were respectively inoculated into the seed culture medium at 37°C, 160r/ Under the condition of constant temperature and shaking for 24 hours, the seed liquid was obtained, and their seed liquids were mixed according to the volume ratio of 2:2:1:1, and inoculated into the degradation medium, and the experiments under the following degradation conditions were carried out.
  • the degradation medium is 1.0 g of ammonium sulfate, 0.25 g of magnesium sulfate heptahydrate, 2.0 g of sodium nitrate, 4.0 g of potassium dihydrogen phosphate, and 7.6 g of dipotassium hydrogen phosphate dissolved in 1000 mL of deionized water.
  • Degradation conditions pH 7.5, heavy oil 2g/L, inoculation amount 5% (V/V), sodium chloride 4g/L; temperature: 20°C, 25°C, 30°C, 35°C, 40°C, 45°C; After 10 days of cultivation, the degradation rate of petroleum hydrocarbons was measured by ultraviolet spectrophotometry, and the surface tension of the bacterial liquid was measured by a surface tension meter.
  • Degradation conditions temperature 37°C, heavy oil 2g/L, inoculation amount 5% (V/V), sodium chloride 4g/L; pH: 3, 5, 7, 7.5, 8, 9, 11, 12; culture for 10 days Then use ultraviolet spectrophotometry to measure the degradation rate of petroleum hydrocarbons, and use a surface tension meter to measure the surface tension of the bacterial liquid.
  • the mixed bacteria had a high degradation rate at pH 7-8, and the optimum pH was 7.5. At this time, the degradation rate reached the maximum 42.14%, and the surface tension of the bacterial liquid also reached the minimum 44.1mN/m.
  • Degradation conditions temperature 37°C, pH 7.5, heavy oil 2g/L, sodium chloride 4g/L; inoculation amount: 1%, 2%, 5%, 10%, 15%, 20%; after 10 days of culture, use UV
  • the degradation rate of petroleum hydrocarbons was measured by spectrophotometry, and the surface tension of the bacterial liquid was measured with a surface tension meter.
  • Acinetobacter sp.strain ZX-15, Achromobacter pulmonis strain PI3-03, Acinetobacter sp. RS206 and Chrobactrum anthropi strain CON21 were inoculated into the seed culture medium at 37°C, 160r/min Under the condition of constant temperature shaking for 24 hours, the seed liquid was obtained, and the seed liquid was mixed according to the following proportions according to the volume ratio:
  • Example 2 and Example 3 It can be seen from Example 2 and Example 3 that the degradation and viscosity reduction effect of the four strains after mixing is significantly greater than that of a single strain.
  • the highest degradation rate of heavy oil after the strains is mixed can reach 43.84%, while the highest degradation rate of a single strain for 10 days is 28.96%, the degradation effect is increased by 34%, and the lowest surface tension can reach 38.7mN/m, while the single strain is cultured in heavy oil.
  • the lowest surface tension of 10d in the base is about 54.1mN/m.
  • the combination of strains has a synergistic effect, which not only enhances the degradability of heavy oil, but also enhances the effect of heavy oil emulsification and dispersion, and the dual effects work together to improve the fluidity of heavy oil.

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Abstract

La présente invention concerne un procédé de préparation et un procédé d'utilisation pour la réduction de la viscosité d'huile lourde et un inoculum microbien mixte de dégradation. L'inoculum microbien mixte comprend les souches Acinetobacter sp. ZX-15, Achromobacter pulmonis PI3-03, Acinetobacter sp. RS206 et Ochrobactrum anthropi CON21, les quatre souches étant obtenues par criblage manuel, les deux premières souches étant des bactéries de dégradation d'hydrocarbures de pétrole à haut rendement, et les deux dernières souches étant des bactéries de réduction de viscosité d'huile lourde à haut rendement. L'inoculum microbien mixte de réduction et de dégradation de viscosité d'huile lourde possède une efficacité de dégradation élevée pour l'huile lourde, un bon effet de réduction de la viscosité et de bonnes perspectives d'utilisation en ce qui concerne la gestion de l'environnement.
PCT/CN2020/110934 2019-10-22 2020-08-25 Procédé de préparation et procédé d'utilisation pour la réduction de la viscosité d'huile lourde et inoculum microbien mixte de dégradation WO2021077888A1 (fr)

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CN109576191B (zh) * 2019-01-09 2021-11-19 安徽霖德生物科技有限公司 一种稠油开发的复合微生物菌剂及其制备方法和应用
CN110669692B (zh) * 2019-10-22 2021-07-23 中国石油大学(华东) 一种稠油降粘降解混合菌剂的制备方法及应用方法
CN115161218B (zh) * 2022-04-25 2023-05-09 江苏开放大学（江苏城市职业学院） 一种能降解农业枯枝落叶废弃物的细菌菌株、其筛选方法及应用

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