WO2011050509A1 - 一种调控微生物采油的方法 - Google Patents

一种调控微生物采油的方法 Download PDF

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WO2011050509A1
WO2011050509A1 PCT/CN2009/001526 CN2009001526W WO2011050509A1 WO 2011050509 A1 WO2011050509 A1 WO 2011050509A1 CN 2009001526 W CN2009001526 W CN 2009001526W WO 2011050509 A1 WO2011050509 A1 WO 2011050509A1
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reservoir
microbial
microorganisms
microorganism
oil
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PCT/CN2009/001526
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English (en)
French (fr)
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牟伯中
刘金峰
杨世忠
刚洪泽
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华东理工大学
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Priority to US13/504,947 priority Critical patent/US20120214713A1/en
Publication of WO2011050509A1 publication Critical patent/WO2011050509A1/zh

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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
    • 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
    • C12N1/36—Adaptation or attenuation of cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously

Definitions

  • the present invention relates to crude oil exploration and production techniques, and more particularly to a method of regulating microbial oil recovery.
  • microbial flooding is a technology with wide adaptability and potential for enhanced oil recovery, and has broad application prospects.
  • the technology through the beneficial activities of microorganisms (degrading crude oil, etc.) and metabolites
  • microbial flooding is to adjust the microbial community structure of the reservoir, establish or optimize the biological environment of the oil displacement functional population, and make the oil displacement functional population in the reservoir environment a dominant population. 'Based on this, microbial flooding design and testing is more scientific and practical, thus achieving a significant increase in oil recovery. Purpose. .
  • An object of the present invention is to provide a method for regulating microbial oil recovery which is highly targeted, highly utilized, and capable of exerting its performance advantages in order to overcome the drawbacks of the prior art described above.
  • a method for regulating microbial oil recovery characterized in that the method comprises the following steps: (1) analyzing the microbial community structure in the produced fluid of the reservoir by molecular biological methods and/or Or detecting metabolites in the produced fluid; (2) adjusting the microorganisms to be injected into the reservoir and/or the corresponding nutrient system of the microorganism; (3) injecting the regulated microorganisms and/or the microorganisms into the reservoir via the water injection well Corresponding nutrient system; (4) Harvesting crude oil from corresponding beneficial production wells.
  • the molecular biology method described in the step (1) is to collect the water sample produced in the test reservoir, extract the microbial community genomic DNA, amplify the 16S rRNA gene, sequence the genomic library, and analyze the microbial community diversity by using the RFLP method.
  • the community structure of the microbial organisms in the reservoir is obtained, and the abundance of the functional microorganisms is analyzed by RT-PCR to obtain the compositional structure information of the microorganisms in the reservoir; the metabolites in the detected production fluid are analyzed by the produced fluid. Metabolite content or lipopeptide content gives metabolite information.
  • the microorganism prepared in step (2) and prepared for injecting into the reservoir and/or the corresponding nutrient system of the microorganism is determined according to the analysis result of step (1), if the concentration of a certain functional microorganism is higher than 1% of the concentration reached by the indoor culture.
  • the microorganism and the corresponding nutrient system need not be replenished into the reservoir; if the concentration of a certain functional microorganism is lower than 1% of the concentration reached in the indoor culture, and the concentration of the metabolite is higher than 0.1% of the concentration reached by the indoor culture, then Replenishing the reservoir with the nutrient system corresponding to the microorganism; if the concentration of a certain functional microorganism is lower than 1% of the concentration reached in the indoor culture, and the concentration of the metabolite is less than 0.1% of the concentration reached by the indoor culture, the reservoir is supplemented Note the microorganism and the corresponding nutrient system.
  • the microorganisms prepared for injecting into the reservoir include metabolically producing biosurfactants, One or two or more microorganisms that degrade hydrocarbon properties.
  • microorganisms prepared for injecting into the reservoir further include microorganisms capable of stimulating the metabolism of glycolipids or lipopeptide products by the microbial communities present in the reservoir.
  • the microorganism includes Bacillus subtilis, Clostridium acetobutylicum, Bacillus stearothermophilus, (?. uzenensis, Bacillus subtilis, Bacillus lentus, Pseudomonas aeruginosa, Enterobacter cloacae, Salmonella faecalis, fluorescent Pseudomonas, Streptomyces.
  • the microorganism includes Bacillus subtilis, Pseudomonas putida or Bacillus stearothermophilus.
  • the microbial corresponding nutrient system adjusts the mass ratio of the carbon source to the nitrogen source (5-25): 1, and stimulates the functional microorganism or metabolite to evolve into a dominant microorganism or a major metabolite.
  • the carbon source includes sucrose, glucose, starch, crude oil, and the nitrogen source includes peptone, ammonium chloride, and amine nitrate.
  • the method for injecting the adjusted microorganism into the oil reservoir and/or the corresponding nutrient system of the microorganism according to the step (3) is to inject the microbial fermentation liquid or the corresponding nutrient system of the microorganism into the test oil layer by the water injection well, or The microbial fermentation broth is uniformly mixed with the nutrient solution corresponding to the microorganism, and then injected into the test oil layer by the water injection well.
  • the present invention performs a plurality of operations on the test reservoir according to the method of "community structure analysis - regulation of injecting microorganisms and / or nutrient system - injecting microorganisms and / or nutrient system", which can promote microorganisms in the reservoir
  • the community evolved into a system that is beneficial to microbial oil recovery by injecting microorganisms as the dominant bacteria and other bacteria, and exerting synergistic oil displacement to further improve the oil displacement performance of the injected functional microorganisms.
  • the present invention can maximize the effects of injecting microorganisms and nutrient solution.
  • the method of the invention first analyzes the microbial community structure existing in the reservoir source, and then adjusts the microbial type and the nutrient system composition of the implant according to the design, thereby regulating the functional bacteria to become the dominant bacteria in the reservoir, thereby maximizing The role of the function of microorganisms in oil displacement.
  • microbial oil recovery tests are carried out in the absence of a comprehensive systematic understanding of the microbial community structure and activity of the reservoir.
  • the method of the invention has the advantages of high specificity, high utilization rate and sufficient utilization of the injected microorganisms and nutrient system. By taking advantage of its performance, it also contributes to the performance of the original microbial flooding in the reservoir, and is therefore a scientific and economical microbial flooding method. detailed description The invention will now be described in detail in connection with specific embodiments.
  • the oil-displacement bacteria include hydrocarbon-degrading bacteria TF2 and biosurfactant-producing bacteria HN1.
  • TF2 can grow with glucose and n-hexadecane as a carbon source. When culturing a mixed hydrocarbon of a positive hexadecanal to a twenty-two hydrazine as a carbon source, TF2 preferentially utilizes n-hexadecane.
  • the degraded TF2 was identified as G. subtilis G subterraneus Str. 34T.
  • HN1 bacteria are uniform in size, light in color and high in frequency of movement. The colony is fine, the surface is flat, the color is pale yellow, opaque, the surface is dry and rough and wrinkled, the colony edge is irregular, and the single colony is small and easy to provoke.
  • the HN1 was identified as Bacillus subtilis.
  • the carbon source in the nutrient system is sucrose (1%) or crude oil (1%) or a mixture of sucrose and crude oil (0.5%+0.5%).
  • the nitrogen source is peptone (0.25%) or chlorine.
  • Ammonium (0.2%), supplemented with yeast extract (0.2%) and K 2 HPO 4 (0.08%), NaH 2 PO 4 (0.04%) > MgSO 4 '7H 2 O (0.02%), CaCl 2 '23 ⁇ 4O (0.01%), NaCl (0.02%) o were cultured at 37° (:, 55 °C, 65 °C, 4 times, respectively, to investigate the regulation of microbial metabolism and community structure by different carbon sources and nitrogen sources. effect.
  • the microbial population had the highest concentration, and the main bacteria were HN1.
  • the mixed carbon source of sucrose and crude oil showed good emulsification effect regardless of whether the peptone was nitrogen source or ammonium chloride was nitrogen source.
  • the sucrose was carbon source and peptone.
  • the content of lipopeptide in the nitrogen source system reached 180 mg/L, and the surface tension was reduced by 29%.
  • the microbial community concentration is slightly lower, the main bacteria is TF2, and the better emulsification effect is obtained by using crude oil as carbon source and peptone as nitrogen source, while sucrose and crude oil mixed carbon source and peptone are nitrogen source.
  • the lipopeptide content reached 320 mg L and the surface tension was reduced by 25%.
  • the microbial population had the lowest concentration, and the strains in the baseline developed and became the main bacteria of the microbial system.
  • the mixed carbon source of sucrose and crude oil and the ammonium chloride as the nitrogen source had better emulsifying effect.
  • sucrose is a carbon source and peptone is a nitrogen source
  • the lipopeptide content reaches 200 mg/L, and the surface tension is reduced by 30%.
  • the same nutrient system composed of different carbon sources and nitrogen sources is used to culture the same
  • the bacterial population, the main bacteria of the system after the culture is different, the yield of the surfactant in the obtained culture system is different, and the surface tension is reduced. Therefore, according to different reservoir temperatures, the targeted selection of vegetative bodies can maximize the performance of the target microorganisms, thereby enhancing the effect of microbial oil recovery.
  • the optimal carbon and nitrogen sources for the production of glycolipids by Pseudomonas aeruginosa were soybean oil and sodium nitrate, respectively.
  • the optimum medium formula is: yeast extract 0.2 g/L, soybean oil 120 g L, NaN0 3 6.5 g/L, KH 2 P0 4 1.0 g/L, Na 2 HP0 4 ⁇ 12 ⁇ 2 0 1.0L, MgSO 4 ⁇ 7 ⁇ 2 0 0.1 g/L, FeS0 4 ⁇ 7 ⁇ 2 0 0.2 g/L.
  • the RFLP fingerprint analysis method was used to evaluate the bacterial diversity of water samples produced in a reservoir. The results showed that among the 74 operational classification units, the largest number of 4 accounted for 73.6% of the total number of clones, and the other 70 abundances were At a lower level, 57 have only one clone. The dominant flora in the reservoir environment is very obvious. The number of main strain types accounts for more than half of the total, and the largest number accounts for 47.7% of the total, indicating that the strain may be suitable for high temperature and high pressure environment conditions. .
  • the 16S rRNA gene library analysis method was used to analyze the diversity of bacteria and archaea communities in a high temperature water flooding reservoir environment on the domestic land using RFLP fingerprinting.
  • the types and amounts of bacteria obtained were: Gamme-Proteobacteria (85.7%), Thermotogales (6.8%), Epsilon-Proteobacteria (2.4%) > Low-G+C Gram-positive (2.1%), High-G+C Gram- Positive, Beta-Proteobacteria and Nitrospira (both ⁇ 1.0%).
  • there are many types of high temperature bacteria but the number of normal temperature bacteria such as Pseudomonas is relatively large.
  • the obtained archaea mainly belongs to the genus Arthrax, including: Methanobacteriales, Methanococcales > Methanomicrobiales and Methanosarcinales, of which Methanomicrobiales is the dominant flora.
  • a total of 28 sequence types are mainly divided into three categories: (1) Mesophilic melanogenic bacteria, including: Methanosarcina, Methanohalophilus, Methanocalculus, and Methanosaeta; (2) Thermophilic mesogenic bacteria, including: Methanothermobacter > Methanococcus and Methanoculleus; (3) not The type of archaea cultivated.
  • strain types have been previously discovered, but there are a few similar types of strains reported earlier, which may be new strain types.
  • Several strains of thermophilic methanogenic archaea have been previously detected in other reservoir environments, suggesting that they may be widely distributed in sorghum reservoir environments.
  • a typical high-temperature water flooding reservoir in the sea was selected and the microbial community diversity was studied by 16S rRNA sequence analysis.
  • the results showed that the bacterial types belonged mainly to Firmicutes, Thermotogae, Nitrospirae and Proteobacteria, while the archaeal types belonged mainly to the Methanothermobacter, Methanobacter, Methanobrevibacter and Methanococcus genus of methanogens. Only one clone belonged to ThermoproteL, and the diversity of bacteria was higher.
  • the archaea the dominant bacteria are: a few types of archaea-producing archaea, fermenting bacteria and sulfate-reducing bacteria, indicating that the microbial diversity of the reservoir environment is relatively low compared with other environments.
  • a strain closely related to Hydrocarboniphaga effUsa was found in the reservoir environment. This bacterium has the ability to decompose alkanes and aromatics and may be suitable for growth in a reservoir environment.
  • some sequence types are not found in the database. More than 97% of the strains are related.
  • the microbial flooding test area is a 3 injection 8 production well group of an oil field.
  • the average porosity of the reservoir in the test area is 28%, and the average air permeability is 0.7um 2 .
  • the oil layer temperature is 53 ° C, the formation crude oil viscosity is 21 mPa ⁇ s, the ground degassed crude oil density is 0.92 g/cm 3 , the wax content is 8.8%, the colloidal asphalt is 14.6%, and the freezing point is -8 ° C.
  • the geological reserve is 75xl0 4 t ; the formation water type is NaHC0 3 , and the salinity is 5528mg L.
  • the microbial flooding test was first injected into the bacterial liquid and nutrient solution 7426m 3 , of which 120m 3 was injected into the bacterial liquid (GX-043 : Pseudomonas putida, 60 m 3 ; GX-104: Bacillus subtilis, 40 m 3 ; GX-118 : Bacillus stearothermophilus, 20 m 3 ).
  • GX-043 Pseudomonas putida, 60 m 3
  • GX-104 Bacillus subtilis, 40 m 3
  • GX-118 Bacillus stearothermophilus
  • the reporter fluorophore of the 5' end of the probe gene sequence is FAM
  • the quenching fluorophore of the 3' end of the probe gene sequence is TAMRA.
  • the carbon source is sucrose 0.5% + crude oil 0.5%
  • the nitrogen source is 0.2% ammonium chloride.
  • the growth and metabolism of the bacteria is strong.
  • the actual supplemental injection of nutrient system sucrose 0.5%, chlorine Ammonium 0.2%, yeast extract 0.2%, K 2 HP0 4 0.08%, NaH 2 PO 4 0.04%.
  • the fermentation broth is mixed with the bacterial liquid and then injected into the reservoir by the water injection well.
  • the microbial flooding test area is a 2 injection 5 well group in an oil field.
  • the average porosity of the reservoir in the test area is 22%, the average air permeability is 0.83um 2 , the oil layer temperature is 38°C, and the formation crude oil viscosity is 19mPa.s.
  • the gas crude oil density is 0.90g/cm 3 , the wax content is 20.2%, and the colloidal asphalt is 10.6%.
  • the geological reserve is 60xl0 4 t; the formation water type is NaHC0 3 , and the salinity is 7137mg/L.
  • First microbial flooding test bacteria solution injected nutrient solution 4320m 3, wherein the injection bacteria 320m 3.
  • the abundance of the injected bacteria was detected by RT-PCR at 18 months after the injection of the microorganism:
  • the reporter fluorophore of the 5' end of the probe gene sequence is FAM
  • the quenching fluorophore of the 3' end of the probe gene sequence is TAMRA.
  • the laboratory study the nutrient solution composed of: sucrose 1%, peptone 0.25%, further supplemented with yeast extract (0.2%), and K 2 HPO 4 (0.0 8% ), NaH 2 PO 4 (0.04%) o bacteria in The growth and metabolism under the following nutrient system is strong.
  • the concentration of functional microorganism DQ-003 (Bacillus subtilis, A b to) was restored to 2* 10 7 cells/ml, and the average daily oil production of the benefit well was increased from 1.2t to 1.9t, and the comprehensive water content was 95.7. % decreased by 95.0%, and the microbial oil production in this block increased the cumulative yield of crude oil by 605t.
  • a method for regulating microbial oil recovery comprising the following steps:
  • the concentration of a certain functional microorganism in the chamber is 2 X 10 8 cdl/ml, according to the analysis result of step (1), If the functional microbial concentration is higher than 1% of the indoor culture concentration, it is not necessary to replenish the bacteria to the reservoir; if the functional microbial concentration is lower than 1% of the indoor culture concentration, the bacteria are replenished into the reservoir. .
  • Microorganisms commonly used in oil recovery include Bacillus subtilis (eg B. subtilis, CGMCC 1.400), Clostridium acetobutylicum (eg C. acetobutylicum, CGMCC 1.244) Bacillus stearothermophilus (eg B. stearothermophilus, CGMCC 1.1923) > G. uzenensis (eg CGMCC 1.2674), Bacillus subtilis (eg G. subterraneus CGMCC 1.2673) Bacillus lentus (eg B. lentus, CGMCC 1.2013), Pseudomonas aeruginosa (eg P.
  • Bacillus subtilis eg B. subtilis, CGMCC 1.400
  • Clostridium acetobutylicum eg C. acetobutylicum, CGMCC 1.244
  • Bacillus stearothermophilus eg B. stearothermophilus, CGMCC 1.1923
  • Enterobacter cloacae eg: E. cloacae, CGMCC 1.2022
  • Halobacterium salina eg: salinarium, CGMCC1.1952
  • Pseudomonas fluorescens eg: P. fluo recommended ens, CGMCC1.1802
  • Streptomyces faecalis for example: P. putida, CGMCC 1.1820
  • the injection method is that the microbial fermentation liquid controls the pore volume by 0.01 ° / according to the test well group. Injecting, the bacterial liquid is injected into the test oil layer through the injection well.
  • a method for regulating oil recovery of a nutrient system corresponding to a microorganism comprising the steps of:
  • step (2) adjusting the composition of the nutrient system to be injected into the reservoir: obtaining metabolite information by analyzing the glycolipid content or the lipopeptide content in the produced liquid, and judging according to the analysis result of step (1), if the concentration of a certain functional microorganism is higher than 1% of the concentration reached in the indoor culture (the functional microbial indoor culture concentration is 2 X 10 6 cell/ml), it is not necessary to replenish the reservoir with the corresponding nutrient system; if the concentration of a certain functional microorganism is lower than 1% of the concentration reached in the indoor culture, the concentration of metabolites is higher than that of the indoor culture. At a concentration of 0.1%, the corresponding nutrient system of the bacteria is replenished into the reservoir.
  • sucrose as a carbon source
  • peptone as a nitrogen source
  • a method for regulating the recovery of microorganisms and their corresponding nutrient systems comprises the following steps:
  • step (1) if the concentration of a certain functional microorganism is higher than 1% of the indoor culture concentration (the indoor concentration of the functional microorganism is 2 X 10 6 cdl/ml), it is not necessary to replenish the bacteria into the reservoir. And corresponding nutrient system; if a functional microbial concentration is lower than 1% of the indoor culture concentration, and the metabolite concentration is higher than 0.1% of the indoor culture concentration, the corresponding nutrient system of the bacteria is supplemented into the reservoir; If the functional microbial concentration is less than 1% of the indoor culture concentration and the product concentration is less than 0.1% of the indoor culture concentration, the bacteria and the corresponding nutrient system are replenished into the reservoir.
  • Microorganisms commonly used in oil recovery include Bacillus subtilis (eg B. subtilis, CGMCC 1.400), Clostridium acetobutylicum (eg C. acetobutylicum, CGMCC 1.244) Bacillus stearothermophilus (eg B. stearothermophilus, CGMCC 1.1923) , G. uzenensis (eg: CGMCC 1.2674), Bacillus subtilis (eg G. subterraneus CGMCC 1.2673), Bacillus lentus (eg B. lentus, CGMCC 1.2013), Pseudomonas aeruginosa (eg P.
  • Bacillus subtilis eg B. subtilis, CGMCC 1.400
  • Clostridium acetobutylicum eg C. acetobutylicum, CGMCC 1.244
  • Bacillus stearothermophilus eg B. stearothermophilus, CGMCC 1.192
  • aeruginosa CGMCC 1.1785) Enterobacter cloacae (eg: E. cloacae, CGMCC 1.2022), Halobacterium salina (eg: /. salinarium, CGMCC 1.1952) > Pseudomonas fluorescens (eg: P. fluorescens, CGMCC 1.1802) Cytobacteria (for example: P. putida, CGMCC 1.1820), etc., but are not limited to the above species. Preferred are Bacillus subtilis, Pseudomonas putida, and Bacillus stearothermophilus.
  • the mass ratio of carbon source to nitrogen source in the reservoir is adjusted to 25: 1, and the stimulating functional microorganisms or metabolites evolve into dominant microorganisms or major metabolites.
  • the injection method is that the microbial fermentation liquid is injected according to 0.01% of the controlled pore volume of the test well group, and the nutrient liquid amount is injected according to 0.1% of the controlled pore volume of the test well group; if the nutrient solution and the bacterial liquid are simultaneously supplemented, the bacterial liquid is in the nutrient solution. After mixing and injecting; the bacterial liquid and the nutrient solution are injected into the test oil layer through the injection well.

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Description

一种调控微生物采油的方法 技术领域 本发明涉及原油勘探及开采技术， 尤其是涉及一种调控微生物采油的方 法。
背景技术 由于我国陆相储层地质条件复杂， 水驱以后仍有近三分之二的原油残留在 地下， 油田原油采收率普遍较低， 加之储量接替困难等严峻形势， 迫切需要研 究开发高效、 适应性强的提高原油采收率技术以满足社会对能源的需求。
研究表明， 微生物驱油是一项适应范围宽、 具有提高原油采收率潜力的技 术， 应用前景广阔。 该技术通过微生物的有益活动 （降解原油等） 和代谢产物
(生物表面活性剂等）来提高原油采收率。微生物驱油技术研究起始于 20世纪 20年代， 70年代世界石油危机推动了该技术的进展。近 35年来， 波兰、 美国、 前苏联、罗马尼亚等国家先后开展了 30余个微生物驱油矿场试验，见到较好的 试验效果。 微生物采油的现实有效性己经在矿场试验中得到证实， 但是， 试验 同时显示微生物提高采收率的幅度有限， 技术水平不高。 对油藏中的微生物缺 乏全面系统的认识是导致这种局面的原因之一。
研究证实， 长期水驱的油藏是一个复杂的生态系统， 其中孕育着物种多样 的微生物， 在整个生态系统中占有重要的位置。 但是， 由于分析手段的限制， 长期以来应用基于纯培养的方法只能认知油藏中很少一部分微生物（约 1-3%)， 绝大多数的微生物因无法培养而不能被认知， 这些微生物的群落结构与功能已 成为认识油藏微生物的一个盲区。
分子生物学手段、尤其是分子生态学的出现，克服了传统培养方法的缺陷， 已经应用于土壤、 活性污泥、 生物肥料等环境微生物生态的分析， 为系统认识 微生物生态提供了一个可行的手段。 将这种原理和手段应用于油藏环境微生物 群落结构的分析， 必将获得油藏微生物的崭新的、 系统的、 完整的认识。 事实 上， 微生物驱油就是要对油藏微生物群落结构进行调整， 建立或优化驱油功能 种群的生物环境， 使油藏环境中的驱油功能种群成为优势种群。 '以此为基础进 行微生物驱油设计及试验更具科学性和实践性， 从而达到大幅度提高采收率的 目的。 .
现有技术采用培养的方法分析油藏中微生物的群落结构， 所得结果只反映 出油藏微生物的很小的一部分， 无法获得油藏微生物群落及功能的全面系统的 认识， 在这种认识基础上， 通过注入营养体系、 或少数菌种来改变系统中群落 组成， 发挥驱油功能菌的作用， 这往往具有较大的盲目性和随机性， 从而很难 达到稳定提高采收率的目的。 发明内容 本发明的目的就是为了克服上述现有技术存在的缺陷而提供一种针对性 强、 利用率高、 能充分发挥其性能优点的调控微生物采油的方法。
本发明的目的可以通过以下技术方案来实现:一种调控微生物采油的方法， 其特征在于， 该方法包括如下步骤： （1 ) 采用分子生物学方法分析油藏产出液 中微生物群落结构和 /或检测产出液中代谢产物； （2)调节准备注入油藏中的微 生物和 /或该微生物对应的营养体系； （3 )经由注水井向油藏中注入调节后的微 生物和 /或该微生物对应的营养体系； （4) 由对应的受益采油井收获原油。
步骤（1 )所述的分子生物学方法是采集试验油藏产出水样， 提取微生物群 落基因组 DNA, 扩增 16S rRNA基因， 测序后构建基因组文库， 采用 RFLP方 法分析油藏微生物群落多样性， 由此获得油藏环境微生物的群落结构， 通过 RT-PCR, 分析功能微生物的丰度， 获得油藏中微生物的组成结构信息； 所述的 检测产出液中代谢产物是通过分析产出液中糖脂含量或脂肽含量获得代谢产物 信息。
步骤 （2) 所述的调节准备注入油藏中的微生物和 /或该微生物对应的营养 体系是根据步骤（1 )分析结果判断， 如果某种功能微生物浓度高于室内培养所 达到浓度的 1%， 则不需要向油藏中补注该微生物及对应的营养体系； 如果某 种功能微生物浓度低于室内培养所达到浓度的 1%、 代谢产物浓度高于室内培 养所达到浓度的 0.1%， 则向油藏中补注该微生物对应的营养体系； 如果某种功 能微生物浓度低于室内培养所达到浓度的 1%、 代谢产物浓度低于室内培养所 达到浓度的 0.1%， 则向油藏中补注该微生物和对应的营养体系。
所述的调节准备注入油藏中的微生物包括具有代谢产生生物表面活性剂、 降解烃性能的一种或两种以上的微生物。
所述的调节准备注入油藏中的微生物还包括能够刺激油藏中本源存在的微 生物群落代谢糖脂或脂肽产物的微生物。 '
所述的微生物包括枯草芽孢杆菌、 丙酮丁醇梭菌、 嗜热脂肪芽孢杆菌、 （?. uzenensis, 地下地杆菌、 迟缓芽孢杆菌、 铜绿假单胞菌、 阴沟肠杆菌、 盐生盐 杆菌、 荧光假单胞菌、 恶臭单胞菌。
所述的微生物包括枯草芽孢杆菌、 恶臭假单胞菌或嗜热脂肪芽孢杆菌。 所述的微生物对应的营养体系为调节其中的碳源与氮源的质量比例为 (5-25)： 1， 以刺激功能微生物或代谢产物演化为优势微生物或主要代谢产物。
所述的碳源包括蔗糖、 葡萄糖、 淀粉、 原油， 所述的氮源包括蛋白胨、 氯 化铵、 硝酸胺。
步骤 （3 ) 所述的向油藏中注入调节后的微生物和 /或该微生物对应的营养 体系注入方式为将微生物发酵液或该微生物对应的营养体系分别由注水井注入 到试验油层， 或将微生物发酵液与该微生物对应的营养液混合均匀后由注水井 注入到试验油层。
与现有技术相比， 本发明按照 "群落结构分析 -调节注入微生物和 /或营养 体系-注入微生物和 /或营养体系"方法， 对试验油藏进行多次操作， 可以促使 油藏中的微生物群落演变为以注入微生物为优势菌、 其他菌共生互利的有利于 微生物采油的体系， 发挥协同驱油作用， 进一步提高注入的功能微生物的驱油 性能。 由此可见， 本发明能够最大限度地发挥了注入微生物和营养液的作用。
本发明所述方法即先对油藏本源存在的微生物群落结构进行分析， 然后据 此设计调节注入的微生物类型和营养体系质构成， 由此调节功能菌成为油藏中 的优势菌， 进而最大限度地发挥功能微生物的驱油作用。 而现有技术是在对油 藏微生物群落结构和活性缺乏全面系统认识的情况下开展微生物采油试验的， 与此比较， 本发明方法具有注入微生物和营养体系针对性强、 利用率高、 能充 分发挥其性能的优点， 还有助于发挥油藏中本源微生物驱油的性能， 因此是一 '种既科学又经济有效的微生物驱油方法。 具体实施方式 下面结合具体实施例对本发明进行详细说明。
实施例 1
1 ) 不同营养体系及温度对微生物生长代谢的调节作用
体系构成： 某油田地层水 + 原油或蔗糖 + 驱油菌种 + 基线菌种 （地层 水中微生物和活性污泥中微生物） +营养体系。其中，驱油菌包括烃降解菌 TF2 和生物表面活性剂产生菌 HN1。 TF2能够以葡萄糖和正十六垸为碳源生长。 在 以正十六垸至正二十二垸混合烃为碳源培养时， TF2 优先利用正十六烷。 TF2 在温度 50°C〜65 °C、 pH= 6〜9、 矿化度 < 2% (NaCl) 条件下生长良好， 在 55 、 pH= 7、 矿化度 0.5% (NaCl) 时获得最佳生长。 经鉴定 降解菌 TF2为地 下地杆菌 G subterraneus Str. 34T。 HN1菌大小均一，颜色较淡，运动频率较高。 其菌落细密， 表面平坦， 颜色呈淡黄色， 不透明， 表面较干燥且粗糙有褶皱感， 菌落边缘不规则， 单个菌落较小， 易挑起。 经鉴定 HN1 为枯草芽孢杆菌 . subtilis) 营养体系中碳源为蔗糖（1%)或原油（1%)或蔗糖和原油混合物 (0.5%+0.5%), 氮源为蛋白胨 (0.25%)或氯化铵 (0.2%)， 另外补加酵母膏 (0.2%)和 K2HPO4(0.08%)、 NaH2PO4(0.04%)> MgSO4'7H2O(0.02%)、 CaCl2'2¾O(0.01%)、 NaCl(0.02%)o 分别在 37° (：、 55 °C、 65 °C下培养， 转接 4次， 分别考察不同碳 源、 氮源对微生物代谢性能和群落结构的调控作用。
调控培养结果， 即使由同样的体系出发， 经不同的碳源、 氮源及不同温度 的调控下， 体系的群落和功能均发生了明显的变化。
37 °C下， 微生物群系浓度最高， 主菌为 HN1， 蔗糖和原油混合碳源不论 蛋白胨为氮源或氯化铵为氮源均表现出较好的乳化效果， 在蔗糖为碳源、 蛋白 胨为氮源体系中脂肽含量达到 180mg/L， 表面张力降低 29%。
55°C下， 微生物群系浓度稍低， 主菌为 TF2， 以原油为碳源、 蛋白胨为氮 源获得了较好的乳化效果， 而蔗糖和原油混合碳源和蛋白胨为氮源的体系中脂 肽含量达到 320mg L， 表面张力降低 25%。
65 °C下， 微生物群系浓度最低， 基线中的菌种繁殖发育成为微生物群系 主菌， 蔗糖和原油混合碳源、 氯化铵为氮源时乳化效果较好。 在蔗糖为碳源、 蛋白胨为氮源体系中脂肽含量达到 200mg/L， 表面张力降低 30%。
由上可见， 在不同温度下， 采用不同碳源、 氮源构成的营养体系培养同一 菌群， 培养后体系的主菌不同， 所得培养体系中表面活性剂产量不同， 表面张 力降低情况不同。 因此根据不同油藏温度， 针对性地选择营养体 可以将目标 微生物的性能最大程度地发挥出来， 从而增强微生物采油的作用效果。
2) 铜绿假单胞菌最佳营养体系
经正交设计实验研究， 铜绿假单胞菌发酵生产糖脂的最佳碳源和氮源分别 为豆油和硝酸钠。最佳培养基配方为：酵母膏 0.2 g/L,豆油 120g L， NaN03 6.5 g/L, KH2P04 1.0 g/L, Na2HP04 ·12Η20 1.0L, MgSO4 ·7Η20 0.1 g/L, FeS04 ·7Η20 0.2 g/L。
3 ) 恶臭假单胞菌最佳营养体系
通过单因素实验和正交实验， 对恶臭假单胞菌发酵生产糖脂的营养体系进 行了研究， 最佳组成 (％): 碳源为蔗糖 0.5%+原油 0.5%、 氮源为氯^ ^安 0.2%， 酵母膏 0.2%， K2HP04 0.08% ^ NaH2P04 0.04%。
4) 油藏微生物群落结构分析
运用 RFLP指纹图谱分析方法评估了某油藏产出水样细菌多样性， 结果表 明， 在 74个操作分类单元中， 数量最多的 4个占克隆子总数的 73.6%， 另外 70个的丰度均处于较低水平， 有 57个仅含有 1个克隆子。 油藏环境中优势菌 群十分明显， 主要菌种类型的数量占到总量的一半以上， 其中数量最多的占到 总数的 47.7%， 表明该菌种可能比较适合油藏高温、 高压的环境条件。
采用 16S rRNA基因文库分析方法， 并联合使用 RFLP指紋图谱法分析国 内陆上某高温水驱油藏环境中的细菌和古菌群落多样性。 得到的细菌种类和数 量 为 ： Gamme-Proteobacteria ( 85.7% ) 、 Thermotogales ( 6.8% ) 、 Epsilon-Proteobacteria ( 2.4% ) > Low-G+C Gram-positive ( 2.1% ) , High-G+C Gram-positive、 Beta-Proteobacteria和 Nitrospira (均 <1.0%)。 其中高温菌种类 型比较多， 但 Pseudomonas等常温菌的数量却比较多。 得到的古菌主要属于产 甲焼古菌， 包括： Methanobacteriales、 Methanococcales > Methanomicrobiales禾口 Methanosarcinales, 其中 Methanomicrobiales是优势菌群。 总共 28个序列类型 主要分为三类： （ 1 ) 嗜温性产甲垸菌， 主要包括： Methanosarcina、 Methanohalophilus、 Methanocalculus和 Methanosaeta等； (2)嗜热性产甲院菌， 主要包括： Methanothermobacter > Methanococcus 禾口 Methanoculleus; (3 ) 未 培养的古菌类型。 其中大部分菌种类型都是以前发现过的， 但是也有少数跟早 先报道的菌种类型相似性较低， 可能是新的菌种类型。 检测到的几株嗜热产甲 烷古菌以前也在其他油藏环境中发现过， 表明它们可能广泛分布于各地的高溘 油藏环境。 研究发现该油田油藏中氢营养型产甲垸菌和乙酸营养型产甲垸菌同 时存在。
选取国内典型的海上某高温水驱油藏， 用 16S rRNA序列分析法研究了其 中的微生物群落多样性。结果表明，细菌类型主要属于 Firmicutes、Thermotogae、 Nitrospirae 和 Proteobacteria， 而古菌类型主要属于产甲烷菌类群的 Methanothermobacter、 Methanobacter、 Methanobrevibacter和 Methanococcus等 属， 只有一个克隆属于 ThermoproteL 其中细菌的多样性要高于古菌， 优势菌 群为： 产甲垸古菌、 发酵菌和硫酸盐还原菌等少数几个类型， 表明该油藏环境 的微生物多样性相较其他环境还是比较低的。 首次在油藏环境中发现了与 Hydrocarboniphaga effUsa亲缘关系很近的菌种， 这种细菌具有分解烷烃和芳烃 能力， 可能比较适于在油藏环境中生长。 另外， 尚有部分序列类型在数据库中 找不到亲缘关系 97%以上的菌种。
5 ) 同时注入营养体系和微生物发酵液提高微生物采油技术应用效果 微生物驱试验区为某油田 3注 8采井组， 试验区储层平均孔隙度为 28%， 平均空气渗透率为 0.7um2， 油层温度 53°C， 地层原油粘度 21mPa.s， 地面脱气 原油密度为 0.92g/cm3， 含蜡量 8.8%， 含胶质沥青 14.6%， 凝固点 -8°C。 地质 储量 75xl04t; 地层水水型为 NaHC03， 矿化度为 5528mg L 。
微生物驱试验首次注入菌液及营养液 7426m3， 其中， 注入菌液 120m3 (GX-043 : 恶臭假单胞菌， 60 m3; GX-104: 地下地杆菌， 40 m3; GX-118: 嗜 热脂肪芽孢杆菌， 20 m3)。注入微生物后 26个月时， 油井产量接近注入微生物 前的水平。 采用 RT-PCR技术检测注入菌丰度：
嗜热月旨肪芽孢杆菌 B. Stearothermophilus
上游引物 5'- CCCTGACAACCCAAGAGATT -3'
下游引物 5'- ATCTCACGACACGAGCTGAC -3'
荧光探针基因序列
5'- AACCATGCACCACCTGTCACCC -3' 地下地杆菌 G. subterraneus
上游引物 5'- CCCTGACAACCCAAGAGATT -3'
下游引物 5'- ATCTCACGACACGAGCTGAC -3'
荧光探针基因序列 5'- AACCATGCACCACCTGTCACCC -3'
恶臭单胞菌 p. putida
上游引物 5'- GTCAGCTCGTGTCGTGAGAT -3'
下游弓 I物 5'- CTCCTTAGAGTGCCCACCAT -3'
荧光探针基因序列 5'- CCCGTAACGAGCGCAACCCT -3'
探针基因序列 5'端标记的报告荧光基团是 FAM，探针基因序列 3'端标记的 淬灭荧光基团是 TAMRA。 结果发现与见效高峰时比较， GX-043 浓度由 107cell/ml 降低到 105cell/ml， 群落群系结构已发生明显改变， 注入的功能微生 物的相对比例 GX-043: GX-104: GX-118由 7: 4： 2变为 5: 5： 3； 检测此时 油井产出液糖脂含量仅为室内培养时的 0.06%。 因此向试验油藏中同时补充 GX-043发酵液 54 m3及对应的营养液 540m3。根据室内研究，碳源为蔗糖 0.5%+ 原油 0.5%、 氮源为氯化铵 0.2%时该菌生长代谢旺盛， 考虑到油藏中存在原油， 故实际补充注入营养体系： 蔗糖 0.5%， 氯化铵 0.2%， 酵母膏 0.2%， K2HP04 0.08%、 NaH2PO4 0.04%。 将发酵液与菌液混合均勾后由注水井注入油藏。
补注营养液和主菌发酵液后， GX-043 丰度逐渐提高， 三个菌比例恢复到 接近开始时的水平。 受益油井平均单井日产油由 2.2t上升到 4.7t， 综合含水由 93.7%降低 90.2%, 该区块微生物采油累计增产原油 3400t。
6) 单独注入营养体系提高微生物采油技术应用效果
微生物驱试验区为某油田 2注 5采井组， 试验区储层平均孔隙度为 22% , 平均空气渗透率为 0.83um2， 油层.温度 38°C， 地层原油粘度 19mPa.s， 地面脱气 原油密度为 0.90g/cm3， 含蜡量 20.2% , 含胶质沥青 10.6%。 地质储量 60xl04t; 地层水水型为 NaHC03， 矿化度为 7137mg/L 。
微生物驱试验首次注入菌液及营养液 4320m3， 其中， 注入菌液 320m3。 注 入微生物后 18个月时采用 RT-PCR技术检测注入菌丰度：
枯草芽孢杆菌 B. subtilis
上游引物 5'-GTGTCTCAGTCCCAGTGTGG -3' 下游引物 5 '- GCGC ATTAGCTAGTTGGTGA -3 '
荧光探针基因序列 5' - ACGGCTCACCAAGGCAACGA -3'
总细菌
上游弓 I物 5 '- AGAGTTTGATCCTGGCTCAG -3 '
下游引物 5'- TACGGYTACCTTGTTACGACTT -3'
探针基因序列 5'端标记的报告荧光基团是 FAM，探针基因序列 3'端标记的 淬灭荧光基团是 TAMRA。 分析发现与见效高峰时比较， 群落群系结构已发生 改变，但注入的功能微生物 DQ-003(枯草芽孢杆菌， B. ^b^to)丰度变化不大（由 最高时的 9.1%降低到 7.8%) ;浓度仍然维持在 7* 106cell/ml，检测此时油井产出 液脂肽含量仅为室内培养时的 0.2%。 因此向试验油藏中补充 DQ-003对应的营 养液 650m3。 根据室内研究， 营养液组成： 蔗糖 1%、 蛋白胨 0.25%， 另外补加 酵母膏 (0.2%)和 K2HPO4(0.08%)、 NaH2PO4(0.04%)o 该菌在以下营养体系下生 长代谢旺盛。
补注营养液后， 功能微生物 DQ-003(枯草芽孢杆菌， A b to)浓度恢复到 2* 107cells/ml, 受益油井平均单井日产油由 1.2t上升到 1.9t， 综合含水由 95.7% 降低 95.0%， 该区块微生物采油累计增产原油 605t。
实施例 2
一种调控微生物采油的方法， 具体包括如下步骤：
( 1 ) 采用 16S rDNA文库、 PCR-DGGE、 RT-PCR等分子生物学方法分析 油藏产出液中微生物群落结构：
采集试验油藏产出水样， 采用《油藏微生物群落多样性的分子分析》 （华东 理工大学博士论文， 李辉， 2007年）公开的方法提取微生物群落基因组 DNA, 扩增 16S rRNA基因， 测序后构建基因组文库， 采用！^LP方法分析油藏微生 物群落多样性，由此获得油藏环境微生物的群落结构。采用《实时荧光定量 PCR 法检测环境假单胞菌属细菌丰度》 (赵传鹏等， 东南大学学报， 第 36卷第 1期， 2006年） 公开的方法， 通过 RT-PCR, 分析功能微生物的丰度。 通过以上方法 获得油藏中微生物的组成结构信息。
(2 ) 调节准备注入油藏中的微生物组成
某种功能微生物室内培养浓度为 2 X 108cdl/ml， 根据步骤 （1 ) 分析结果， 如果该种功能微生物浓度高于室内培养浓度的 1%， 不需要向油藏中补注该菌; 如果某种功能微生物浓度低于室内培养浓度的 1%， 则向油藏中补注该菌。
采油常用的微生物包括枯草芽孢杆菌 (例如： B. subtilis, CGMCC1.400), 丙 酮丁醇梭菌 (例如： C. acetobutylicum, CGMCC1.244) 嗜热脂肪芽孢杆菌 (例如： B. stearothermophilus， CGMCC 1.1923)> G. uzenensis (例如： CGMCC 1.2674) 、 地下地杆菌 (例如： G. subterraneus CGMCC 1.2673) 迟缓芽孢杆菌 (例如： B. lentus, CGMCC1.2013)、 铜绿假单胞菌 (例如： P. aeruginosa, CGMCC 1.1785) 阴沟肠杆菌 (例如： E. cloacae, CGMCC 1.2022) >盐生盐杆菌 (例如： ： salinarium, CGMCC1.1952), 荧光假单胞菌 (例如： P. fluo薦 ens, CGMCC1.1802), 恶臭单 胞菌 (例如： P. putida, CGMCC 1.1820)等， 但不局限于上述菌种。 优选枯草芽 孢杆菌、 恶臭假单胞菌、 嗜热脂肪芽孢杆菌。
( 3 ) 经由注水井向油藏中注入调节后的微生物或营养体系
注入方式为微生物发酵液按试验井组控制孔隙体积的 0.01°/。注入， 菌液经 由注水井注入到试验油层。
(4) 由对应的受益采油井收获原油
按照油田开发正常工作制度， 不改变任何采油工艺参数生产， 直接由受益 采油井收获原油。
实施例 3
一种调控微生物对应的营养体系采油的方法， 该方法包括如下步骤：
( 1 ) 检测产出液中代谢产物：
按照文献 "微生物发酵制备鼠李糖最佳条件的研究" （李祖义等， 《生物 工程学报》， 1999年 01期）公开的方法分析油井产出液中糖脂含量， 利用文献 "微生物发酵液中脂肽类生物表面活性剂的测定 "（陈涛等， 《油田化学》， 2004 年 04期）公开的方法分析脂肽含量， 由此获得油藏中功能微生物代谢产物的信 息。 根据代谢产物的变化分析获得产生该代谢产物的微生物的丰度和活性等信
Λ m∑!、 ο
(2 )调节准备注入油藏中的营养体系的组成：通过分析产出液中糖脂含量 或脂肽含量获得代谢产物信息， 根据步骤（1 )分析结果判断， 如果某种功能微 生物浓度高于室内培养所达到浓度的 1% (该种功能微生物室内培养浓度为 2 X 106cell/ml), 则不需要向油藏中补注该菌对应的营养体系； 如果某种功能微 生物浓度低于室内培养所达到浓度的 1%、 代谢产物浓度高于室内培养所达到 浓度的 0.1%， 则向油藏中补注该菌对应的营养体系。 以蔗糖为碳源， 以蛋白胨 为氮源， 调节油藏中的碳源与氮源的质量比例为 5: 1 , 以刺激功能微生物或代 谢产物演化为优势微生物或主要代谢产物。
(3 )经由注水井向油藏中注入调节后的营养体系：营养液量按试验井组控 制孔隙体积的 0.1%注入， 营养液经由注水井注入到试验油层。
(4) 由对应的受益采油井收获原油。.
实施例 4
一种调控微生物及其对应的营养体系采油的方法， 包括如下步骤：
( 1 ) 采用 16S rDNA文库、 PCR-DGGE、 RT-PCR等分子生物学方法分析 油藏产出液中微生物群落结构和利用仪器分析方法检测产出液中生物表面活性 剂、 有机酸等微生物代谢产物。
采集试验油藏产出水样， 采用《油藏微生物群落多样性的分子分析》 (华东 理工大学博士论文， 李辉， 2007年）公开的方法提取微生物群落基因组 DNA, 扩增 16S rRNA基因， 测序后构建基因组文库， 分析微生物的系统发育信息， 构建进化树， 采用 RFLP方法分析油藏微生物群落多样性， 由此获得油藏环境 微生物的群落结构。 采用《油藏微生物群落多样性的分子分析》 (华东理工大学 博士论文， 李辉， 2007年） 公开的方法利用 PCR-DGGE指紋图谱法分析油藏 环境烃降解基因 （alkB) 的多样性； 采用《实时荧光定量 PCR法检测环境假单 胞菌属细菌丰度》 (赵传鹏等， 东南大学学报， 第 36卷第 1期， 2006年） 公开 的方法， 通过 RT-PCR, 分析功能微生物的丰度； 采用显微镜-血球计数板计数 的方法分析细菌浓度。 通过以上方法获得油藏中微生物的组成结构信息。
按照文献 "微生物发酵制备鼠李糖最佳条件的研究" （李祖义等， 《生物 工程学报》， 1999年 01期）公开的方法分析油井产出液中糖脂含量， 利用文献 "微生物发酵液中脂肽类生物表面活性剂的测定 "（陈涛等， 《油田化学》， 2004 年 04期）公开的方法分析脂肽含量， 由此获得油藏中功能微生物代谢产物的信 息。 根据代谢产物的变化分析获得产生该代谢产物的微生物的丰度和活性等信 ( 2 ) 调节准备注入油藏中的微生物和营养体系的组成
根据步骤 （1 ) 分析结果， 如果某种功能微生物浓度高于室内培养浓度的 1% (该种功能微生物室内培养浓度为 2 X 106cdl/ml ) ， 不需要向油藏中补注该 菌及对应的营养体系； 如果某种功能微生物浓度低于室内培养浓度的 1%、 代 谢产物浓度高于室内培养浓度的 0.1%， 则向油藏中补注该菌对应的营养体系； 如果某种功能微生物浓度低于室内培养浓度的 1%、 产物浓度低于室内培养浓 度的 0.1%， 则向油藏中补注该菌和对应的营养体系。
采油常用的微生物包括枯草芽孢杆菌 (例如： B. subtilis, CGMCC 1.400), 丙 酮丁醇梭菌 (例如： C. acetobutylicum, CGMCC 1.244) 嗜热脂肪芽孢杆菌 (；例如： B. stearothermophilus， CGMCC 1.1923), G. uzenensis (例如： CGMCC 1.2674) 、 地下地杆菌 (例如： G. subterraneus CGMCC 1.2673)、 迟缓芽孢杆菌 (例如： B. lentus, CGMCC1.2013)、 铜绿假单胞菌 (例如： P. aeruginosa, CGMCC 1.1785) 阴沟肠杆菌 (例如： E. cloacae, CGMCC 1.2022),盐生盐杆菌 (例如： / . salinarium, CGMCC 1.1952) > 荧光假单胞菌 (例如： P. fluorescens, CGMCC 1.1802) 恶臭单 胞菌 (例如： P. putida, CGMCC 1.1820)等， 但不局限于上述菌种。 优选枯草芽 孢杆菌、 恶臭假单胞菌、 嗜热脂肪芽孢杆菌。
以葡萄糖为碳源， 以蛋白胨为氮源， 调节油藏中的碳源与氮源的质量比例 为 25 : 1， 以剌激功能微生物或代谢产物演化为优势微生物或主要代谢产物。
(3 ) 经由注水井向油藏中注入调节后的微生物或营养体系
注入方式为微生物发酵液按试验井组控制孔隙体积的 0.01%注入， 营养液 量按试验井组控制孔隙体积的 0.1%注入； 如果同时补充营养液和菌液， 则将菌 液在营养液中混合均匀后注入； 菌液和营养液均经由注水井注入到试验油层。
(4) 由对应的受益采油井收获原油
按照油田开发正常工作制度， 不改变任何采油工艺参数生产， 直接由受益 采油井收获原油。
n

Claims

权 利 要 求
1. 一种调控微生物采油的方法， 其特征在于， 该方法包括如下步骤： （1 ) 采用分子生物学方法分析油藏产出液中微生物群落结构和 /或检测产出液中代 谢产物；（2)调节准备注入油藏中的微生物和 /或该微生物对应的营养体系； (3 ) 经由注水井向油藏中注入调节后的微生物和 /或该微生物对应的营养体系； （4) 由对应的受益采油井收获原油。
2. 根据权利要求 1所述的一种调控微生物采油的方法， 其特征在于， 步骤
( 1 )所述的分子生物学方法是采集试验油藏产出水样，提取微生物群落基因组 DNA, 扩增 16S rRNA基因， 测序后构建基因组文库， 采用 RFLP方法分析油 藏微生物群落多样性， 由此获得油藏环境微生物的群落结构， 通过 RT-PCR, 分析功能微生物的丰度， 获得油藏中微生物的组成结构信息； 所述的检测产出 液中代谢产物是通过分析产出液中糖脂含量或脂肽含量获得代谢产物信息。
3. 根据权利要求 1所述的一种调控微生物采油的方法， 其特征在于， 步骤
(2) 所述的调节准备注入油藏中的微生物和 /或该微生物对应的营养体系是根 据步骤（1 )分析结果判断， 如果某种功能微生物浓度高于室内培养所达到浓度 的 1%， 则不需要向油藏中补注该微生物及对应的营养体系； 如果某种功能微 生物浓度低于室内培养所达到浓度的 1%、 代谢产物浓度高于室内培养所达到 浓度的 0.1%， 则向油藏中补注该微生物对应的营养体系； 如果某种功能微生物 浓度低于室内培养所达到浓度的 1%、 代谢产物浓度低于室内培养所达到浓度 的 0.1%， 则向油藏中补注该微生物和对应的营养体系。
4. 根据权利要求 3所述的一种调控微生物采油的方法， 其特征在于， 所述 的调节准备注入油藏中的微生物包括具有代谢产生生物表面活性剂、 降解烃性 能的一种或两种以上的微生物。
5. 根据权利要求 3所述的一种调控微生物采油的方法， 其特征在于， 所述 的调节准备注入油藏中的微生物还包括能够刺激油藏中本源存在的微生物群落 代谢糖脂或脂肽产物的微生物。
6. 根据权利要求 4或 5所述的一种调控微生物采油的方法， 其特征在于， 所述的微生物包括枯草芽孢杆菌、 丙酮丁醇梭菌、 嗜热脂肪芽孢杆菌、 uzenensis, 地下地杆菌、 迟缓芽孢杆菌、 铜绿假单胞菌、 阴沟肠杆菌、 盐生盐 杆菌、 荧光假单胞菌、 恶臭单胞菌。
7. 根据权利要求 6所述的一种调控微生物采油的方法， 其特征在于， 所述 的微生物包括枯草芽孢杆菌、 恶臭假单胞菌或嗜热脂肪芽孢杆菌。
8. 根据权利要求 1或 3所述的一种调控微生物采油的方法， 其特征在于， 所述的微生物对应的营养体系为调节其中的碳源与氮源的质量比例为 （5-25 ): 1， 以刺激功能微生物或代谢产物演化为优势微生物或主要代谢产物。
9. 根据权利要求 8所述的一种调控微生物采油的方法， 其特征在于， 所述 的碳源包括蔗糖、 葡萄糖、 淀粉、 原油， 所述的氮源包括蛋白胨、 氯化铵、 硝 酸胺。
10. 根据权利要求 1所述的一种调控微生物采油的方法， 其特征在于， 步 骤 （3 ) 所述的向油藏中注入调节后的微生物和 /或该微生物对应的营养体系注 入方式为将微生物发酵液或该微生物对应的营养体系分别由注水井注入到试验 油层， 或将微生物发酵液与该微生物对应的营养液混合均匀后由注水井注入到 试验油层。
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