CN107267623B - A kind of Microbial Prospecting of Oil and Gas method - Google Patents

A kind of Microbial Prospecting of Oil and Gas method Download PDF

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CN107267623B
CN107267623B CN201710545896.5A CN201710545896A CN107267623B CN 107267623 B CN107267623 B CN 107267623B CN 201710545896 A CN201710545896 A CN 201710545896A CN 107267623 B CN107267623 B CN 107267623B
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罗楚平
鲍浩然
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Ying song oil energy technology (Beijing) Co., Ltd.
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鲍浩然
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Abstract

The present invention relates to a kind of microbial prospecting technology of oil ＆, including：(1) sampled point two-way array is designed；(2) sample is acquired；(3) culture of microorganism detection butane oxidation bacterial content；(4) the rudimentary hydrocarbon oxidizing bacteria content of fluorescence quantitative PCR detection；(5) candidate exploration area is selected；(6) candidate well location is determined.

Description

A kind of Microbial Prospecting of Oil and Gas method
Technical field
The invention belongs to geological exploration fields, and in particular to a kind of to determine candidate exploration area and candidate according to butane oxidation bacterium The technology of well location.It is more particularly to a kind of that microculture result and microbial molecules detection technique are combined exploration petroleum resources Method.
Background technology
Microbiological Prospecting of Oil and Gas is derived from the former Soviet Union.Early in nineteen thirty-seven, former Soviet Union geomicrobiology scholar Mogilewskii It was found that in the near surface soil caused by the bacterial reproduction hydrocarbon gas Growth season variation, and then propose oil and natural gas Microbial prospecting method, and in practice use indicator bacteria of the methane-oxidizing bacteria as underground gas reservoir.The late period fifties, the U.S. Geomicrobiology scholar doctor Hitzman develops a kind of oil gas microorganism prospecting technique MOST for Phillips oil companies (Microbial Oil Survey Technique) technology.The technology is detected using the characteristic of the high butanol of butane oxidation bacterium Hydrocarbon microseepage phenomenon.Since 1956, the novel earth's surface exploration skill of independently developed one of German doctor M.Wagner Art-microbial prospecting technology of oil ＆ MPOG (Microbial Prospectionfor Oiland Gas), the technology utilize first Alkoxide bacterium and butane oxidation bacterium detect hydrocarbon microseepage phenomenon.The beginning of the nineties, the technology are opened up using beginning from northwest Europe land It opens up into North Sea region.To the latter stage nineties, the physical-chemical and microbiology theoretical foundation of MPOG technologies, method and technology and Using into the stage of ripeness, modern microbial prospecting technology of oil ＆ is formd.China is since nineteen fifty-five, by Chinese Academy of Sciences bacterium The kind preservation committee (one of predecessor of institute of microbiology) has carried out gaseous state hydrocarbon oxidizing bacteria and oil gas field with Ministry of Petroleum Industry's cooperation The research of microbiology prospecting.
Microbial prospecting technology of oil ＆ experienced the development of over half a century, has carried out a large amount of field application, has achieved Good application effect, provide hydrocarbon enrichment positive evidence in terms of, illustrate the advantage that other technologies do not have, into The oil-gas exploration technology being widely recognized as one.
Existing microbial prospecting technology of oil ＆, mainly including following several：
1. microbiological petroleum technique for investigation (Microbial Oil Survey Technology, MOST)：By the U.S. Doctor Hitzman develops (Hitzman D O.Prospecting for petroleum deposits:United States Patent.1959,2,880142).The technology uses selective medium to be detected as hydrocarbon oxidizing bacteria and counts culture medium, this choosing Selecting property culture medium only has hydrocarbon oxidizing bacteria that could grow wherein, has murder by poisoning and inhibiting effect to non-hydrocarbon oxidizing bacteria, so overcoming This interference of the domestication of microorganism, obtained result are relatively more reliable.The technology is mainly in conventional microbiological cultural method On carried out certain improvement so that turn out come hydrocarbon oxidizing bacteria numerical value it is more reliable.
2. microbial prospecting technology of oil ＆ MPOG (Microbial Prospectionfor Oiland Gas, MPOG)：Moral State doctor Wanger develops, detection method mainly (Wagner Manfred, Wagner based on microbial cell culture Martin,Joachim Piske,et al.Case histories of microbial prospection for oil and gas onshore and offshore in north-west Europe//Schumacher D,Le Schack L A.AAPG Studies in Geology,Surface exploration case histories:applications of geochemistry,magnetic,and remote sensing.Tulsa,OK:AAPG,2002,48:453-479)。2000 Year, which is introduced to China, Pilot trial has been done in North China Oilfield by Changjiang University.In the MPOG technologies of China's practice Detection method is mainly：Culture counting, and comprehensive some other indexs are carried out to hydrocarbon oxidizing bacteria as carbon source using gaseous alkanes Finally obtain the evaluation index of hydrocarbon oxidizing bacteria --- MU values (Measurement Unit).
3. microbial geochemistry exploration engineering (MGCE), by big belly hundred million, safe geomicrobiology technology (Beijing) Co., Ltd opens Hair (the microbial geochemistries exploration engineering such as Hao Chun and its application Oil Exploration in China in South China Sea Deep Water exploration, 2015, 20(5):54-61).The technology is that some adjustment have been carried out on the basis of MOST detections, is also based on microbial cell culture side What the detection of method was realized.
4. butane oxidation bacterium quantitative technique (CN201510400854.3)：It is extremely with total bacterium of butane oxidation bacterium and viable bacteria The method that index carries out oil exploration, reservoir characterization and Abnormity judgement, by quantitative detection oil exploration area and/or reservoir region Total bacterium of butane oxidation bacterium and viable bacteria obtain the total bacteria count amount of butane oxidation bacterium or same in square topsoil and/or deposit When obtain butane oxidation bacterium total bacterium exception and number of viable.
Existing microbial prospecting technology of oil ＆ using single microbial culture technique or microbial molecules quantitative technique as It is main, by relative quantification or the test format of a kind of hydrocarbon oxidizing bacteria of part absolute quantitation, realize the knowledge to microbiological anomaly Not.
Although specifically, microbial culture technique relative maturity, information content is low, limited resolution, it is impossible to completely anti- Reflect response process and responsiveness that microorganism is enriched with hydrocarbon, it is difficult to the quantity of the hydrocarbon oxidizing bacteria in the actual response region；In addition, When equal microbiological anomaly region area size or bacterium colony group's number phase, can not area further discriminate between two identical micro- lifes The grade and quality of object abnormal area.It, cannot be really anti-although molecular method quantification technology has a distinct increment in accuracy of detection Should all hydrocarbon alkoxide bacterium situation, it is difficult to fully assess the response characteristic that microorganism leaks hydrocarbon；In addition, molecular method quantification technology Of high cost, extensive sampling practice and extension difficulty is larger.
Invention content
In view of the drawbacks of the prior art, microorganism is leaked response characteristic and Molecular Detection means by the present invention to lower paraffin hydrocarbon It is combined, a kind of hydrocarbon oxidizing bacteria broad covered area is provided, takes into account microbial drug tolerance and molecular labeling, cost be controllable, positioning is accurate True Exploration of Oil And Gas method.
Specifically, the present invention provides a kind of Microbial Prospecting of Oil and Gas method, it is characterised in that includes the following steps：It is wrapped It includes：(1) sampled point two-way array is designed；(2) sample is acquired；(3) culture of microorganism detection butane oxidation bacterial content；(4) it is glimmering Fluorescent Quantitative PCR detects rudimentary hydrocarbon oxidizing bacteria content；(5) candidate exploration area is selected；(6) candidate well location is determined.
Microbial Prospecting of Oil and Gas method of the present invention, it is characterised in that including：
(1) sampled point two-way array in exploration area is set, sampled point is determined with reference to exploration area landform and two-way array Coordinate；
(2) sample is acquired, the sampling quantity of sample is 10-100g, and sampling depth 20-60cm, the sample acquired is 0 To -20 DEG C of storage and transport；
(3) using selective medium by culture of microorganism detect butane oxidation bacterium content, drawing isoline, really Determine butane oxidation bacterium culture exceptions area；
(4) real-time fluorescence quantitative PCR detection is carried out to the sample of butane oxidation bacterium culture exceptions area, the real-time fluorescence is determined Amount PCR is detected for rudimentary hydrocarbon oxidizing bacteria specific conservative area, and the rudimentary hydrocarbon oxidizing bacteria includes methane-oxidizing bacteria, ethane Oxidation bacteria and oxidation of propane bacterium；
(5) according to real-time fluorescence quantitative PCR as a result, the candidate exploration area of selection；
(6) on the basis of microculture result, fluorescent quantitative PCR result, with reference to surface temperature, Profiling for Soil Lithology, soil The factors such as earth pH value, Soil salinity, surface vegetation determine well location candidate point.
Microbial Prospecting of Oil and Gas method of the present invention, it is characterised in that survey area inner plane matrix in step (1) Mesh-density is 200-500m × 100-250m.
Microbial Prospecting of Oil and Gas method of the present invention, it is characterised in that sample amount is 50g in step (2), is sampled Depth is 40cm, and the sample acquired is in -20 DEG C of storage and transport.
Microbial Prospecting of Oil and Gas method of the present invention, it is characterised in that by the way that cultivation is selected to detect fourth in step (3) The content of alkoxide bacterium first uses the fluid nutrient medium using butane as sole carbon source to be enriched with, then using butanol as sole carbon source Solid plate pass through plate dilution method detect sample in butane oxidation bacterium content.
Microbial Prospecting of Oil and Gas method of the present invention, it is characterised in that described using butane as the enrichment method of sole carbon source It is as follows：It weighs 1g pedotheques on aseptic operating platform to be placed in the 150mL triangular flasks for filling 50mL fluid nutrient mediums, to culture Butagas is filled with below base fluid face makes butagas in culture solution reach saturation, and the gas compartment in triangular flask is made to be full of butane Gas, sealing, 28 DEG C of shake culture 7d of 180rpm.
Microbial Prospecting of Oil and Gas method of the present invention, it is characterised in that described using butanol as the solid plate of sole carbon source In contain 5% butanol as carbon source.
Microbial Prospecting of Oil and Gas method of the present invention, it is characterised in that the culture medium includes K2HPO41.75g KH2PO40.96g, NaNO32.0g, MgCl20.1g, (NH4)2SO4 0.1g, micro- 0.2mL, pH 7.2；It is wherein every It rises in trace element and contains CaCl25.54g ZnCl25.3g, MnCl25.06g FeSO44.99g, (NH4)6Mo7O24 1.10g CuSO41.57g CoCl21.61g。
Microbial Prospecting of Oil and Gas method of the present invention, it is characterised in that fluorescence quantitative PCR detection is directed in step (4) Target gene is low alkoxylated enzyme gene.
Microbial Prospecting of Oil and Gas method of the present invention, it is characterised in that each lower hydrocarbon oxygen in retrieval GenBank databases Change low alkoxylated enzyme gene sequence in bacterium, carry out multiple alignment, fluorescence quantification PCR primer is designed for conserved region sequence.
Low alkoxylated enzyme gene of the present invention includes paraffin hydrocarbon mono oxygenase gene, alkane dioxygenase gene, described Paraffin hydrocarbon mono oxygenase gene methane monooxygenase enzyme gene, ethane monooxygenase gene, propane monooxygenase gene.
Compared with prior art, the present invention achieves following beneficial technique effect：
(1) information content is comprehensive.Traditional microbial cell cultural method is the sxemiquantitative pair can cultivate hydrocarbon oxidizing bacteria, and Molecular biology for detection is to carry out quantitative technology to the gene for being metabolized lower paraffin hydrocarbon in molecule rank.Microculture and Molecular Detection is combined, and detection range not only includes obtains hydrocarbon oxidizing bacteria strain library by microbial culture method, but also Include the functional gene that pertinent literature is mentioned, therefore in terms of information content more comprehensively, so as to improve exploration areas and The precision of candidate well location and accuracy.
(2) almost all of volatile alkane is related to.The exploitation method of the application not only considers the fourth of simple function Alkoxide bacterium or methane-oxidizing bacteria.Culture of microorganism is related to the response to butane, and Molecular Detection method is related to other volatilizable The response of property lower paraffin hydrocarbon.In addition, culture of microorganism of the present invention employs butane enrichment, butanol detection, compared to existing Butane cultivation or butanol cultivation ensure response of the microorganism to butane, and facilitate solid plate detection.
(3) it positions stage by stage, realizes the mutual supplement with each other's advantages of culture of microorganism and molecular detecting method.First with micro- life Object cultural method differentiates butane oxidation bacterium abundance in work area, identifies the construction of microbiological anomaly development, then to knowing The microbiological anomaly not gone out carries out accurate quantification with molecular detection technology to the absolute quantity of hydrocarbon oxidizing bacteria.It is this to divide index in batches Detection mode, drastically reduce the sample size of Molecular Detection, dropped on the basis of accuracy of detection and detection information amount is met Low testing cost.
Description of the drawings
Fig. 1：Exploration area sample plane matrix design figure.
Fig. 2：The butane oxidation bacterium evaluation index isogram that culture of microorganism obtains.
Fig. 3：Three microculture exceptions areas of conventional microbiological cultivation identification.
Specific embodiment
Below in conjunction with specific embodiment, the present invention is further explained.These embodiments are merely to illustrate the present invention and do not have to In limiting the scope of the invention.Test method without specific conditions in the following example usually according to normal condition or is pressed According to the condition proposed by manufacturer.Unless otherwise defined, all professional and scientific terms used in text and this field are ripe It is identical to practice meaning known to personnel.In addition, any method similar or impartial to described content and material all can be applied to In the method for the present invention.The preferred methods and materials described herein are for illustrative purposes only.
In the examples where no specific technique or condition is specified, according to the described technology of document in the art or condition, Or it is carried out according to product description.Reagents or instruments used without specified manufacturer, be can be by regular channel commercially available from The conventional products of acquisition.
Embodiment
For a land-based area new prospecting area.Main geological tasks：On the basis of geological prospecting early period, to what is identified Advantageous trap microbe oil-gas exploration technology carries out evaluation of oil and gas bearing property, preferably exploration targets, gives full play to integrated exploration Advantage, exploration targets is carried out to bore preceding risk assessment, be exploration well well location preferably with lay, it is found that it is reliable oil-gas reservoir provides Foundation, and the deployment of evaluation well, oil-gas reservoir extension and the rolling prospecting for the trap exploration later stage provide technical support.Specific operation Step is as follows：
1. conceptual design：This work area is based on survey area geologic setting and previous research, for exploration situation The problem of and difficult point carry out technological service scheme customization.This work area is trap exploration, according to the different layers position identified Trap design sampling point spacing, determining mesh-density be 200-500m × 100-250m.Sampled point 195.Two-way array is set Meter figure is as shown in Figure 1.
2. according to designing scheme, mapping worker calculates the coordinate of each website.
3. according to the topographical features of survey area and mankind's activity situation, the sampling depth of sample is determined.Determining sampling Depth 40cm.
4. the index detected as needed determines the collection capacity of sample.Determining sampling quantity is 10g.
5. the index detected as needed determines the storage and transport mode of sample, by -20 DEG C of preservations of sample, refrigeration fortune It is defeated.
6. detecting the total amount of butane oxidation bacterium by dystrophic culture medium with conventional microbiological cell culture method, fourth is obtained The evaluation index of alkoxide bacterium.Specifically, the fluid nutrient medium using butane as sole carbon source is first used to be enriched with, then with fourth The content that alcohol passes through butane oxidation bacterium in plate dilution method detection sample for the solid plate of sole carbon source.Specific data are shown in Table 1.
It is as follows as the enrichment method of sole carbon source using butane：It 1g pedotheques is weighed on aseptic operating platform is placed in and fill In the 150mL triangular flasks of 50mL fluid nutrient mediums, butagas is filled with below culture medium liquid level reaches butagas in culture solution Saturation, and make the gas compartment in triangular flask full of butagas, sealing, 28 DEG C of shake culture 7d of 180rpm.
Using butanol as the solid plate of sole carbon source in contain 5% butanol as carbon source.
The dystrophic culture medium includes K2HPO41.75g KH2PO40.96g, NaNO32.0g, MgCl2 0.1g, (NH4)2SO4 0.1g, micro- 0.2mL, pH 7.2；Contain CaCl in wherein every liter of trace element25.54g ZnCl25.3g, MnCl25.06g FeSO44.99g, (NH4)6Mo7O241.10g CuSO41.57g CoCl2 1.61g。
7. the evaluation index of the butane oxidation bacterium of acquisition and sample point coordinate are painted on geologic map, and painted with geology software Isopleth, principium identification microbiological anomaly is made.See Fig. 2.
8. three exceptions areas are had identified in conventional microbiological cultural method, respectively No. I exceptions area, No. II exception Area, No. III exceptions area.See Fig. 3.
9. No. I exceptions area, No. II exceptions area, the sample of No. III exceptions area carries out fluorescence quantitative PCR detection, and hydrocarbon is aoxidized The absolute quantity of bacterium carries out accurate quantification.
Low alkoxylated enzyme gene sequence in each rudimentary hydrocarbon oxidizing bacteria, carries out multiple alignment in retrieval GenBank databases, Fluorescence quantification PCR primer is designed for conserved region sequence, is quantified using SYBR Green I fluorescent dyes.It designs and synthesizes Primer sequence it is as follows：
p1：5’-ggngactgggacttctgg-3’
p2:5’-ccggmgcaacgtcyttacc-3’
For abnormal point 14 in No. I exceptions area, No. II exceptions area abnormal point 19, No. III exceptions area abnormal point 7 is common Fluorescence quantitative PCR detection is carried out to 40 samples.Tables of data is shown in Table 2.
10. with reference to microorganism, the data such as geophysics and petroleum geology, multidisciplinary, multi-field technical research shows three The microbiological anomaly of a exceptions area is rated No. I exceptions area>No. II exceptions area>No. III exceptions area.
This integrated evaluating method can realize the high-resolution microbial prospecting evaluation of microbiological anomaly construction, for circle It closes and effectively support is preferably provided with well location suggestion.
Table 1：The butane oxidation bacterium evaluation index of each point position in two-way array
Table 2：Each point position molecular method quantification testing result in three exceptions areas
Above description is not limitation of the present invention, and the present invention is also not limited to the example above.The art it is common Technical staff is in the essential scope of the present invention, the variations, modifications, additions or substitutions made, should also belong to the protection of the present invention Range, protection scope of the present invention are subject to claims.

Claims (7)

  1. A kind of 1. Microbial Prospecting of Oil and Gas method, it is characterised in that include the following steps：
    (1) sampled point two-way array in exploration area is set, the coordinate of sampled point is determined with reference to exploration area landform and two-way array；
    (2) sample is acquired, the sampling quantity of sample is 10-100g, and sampling depth 20-60cm, the sample acquired is 0 to -20 DEG C storage and transport；
    (3) content of butane oxidation bacterium is detected by culture of microorganism using selective medium, drawing isoline determines fourth Alkoxide bacterium cultivates exceptions area, by the way that cultivation is selected to detect the content of butane oxidation bacterium, first uses using butane as sole carbon source Fluid nutrient medium be enriched with, then using butanol as sole carbon source solid plate pass through plate dilution method detect sample in butane The content of oxidation bacteria, the selective medium include K2HPO41.75g KH2PO40.96g, NaNO32.0g, MgCl2 0.1g, (NH4)2SO4 0.1g, micro- 0.2mL, pH 7.2；Contain CaCl in wherein every liter of trace element25.54g ZnCl25.3g, MnCl25.06g FeSO44.99g, (NH4)6Mo7O241.10g CuSO41.57g CoCl21.61g；
    (4) real-time fluorescence quantitative PCR detection, the real time fluorescent quantitative are carried out to the sample of butane oxidation bacterium culture exceptions area PCR for rudimentary hydrocarbon oxidizing bacteria specific conservative area or functional areas be detected, using SYBR Green I fluorescent dyes into Row is quantitative, and the rudimentary hydrocarbon oxidizing bacteria includes methane-oxidizing bacteria, ethane oxidation bacterium and oxidation of propane bacterium；
    (5) according to real-time fluorescence quantitative PCR as a result, the candidate exploration area of selection；
    (6) on the basis of microculture result, fluorescent quantitative PCR result, with reference to surface temperature, Profiling for Soil Lithology, soil pH The factors such as value, Soil salinity, surface vegetation determine well location candidate point；
    Wherein：Without using the content of culture of microorganism detection methane-oxidizing bacteria, ethane oxidation bacterium and oxidation of propane bacterium, and not Use the content of real-time fluorescence quantitative PCR detection butane oxidation bacterium.
  2. 2. Microbial Prospecting of Oil and Gas method as described in claim 1, it is characterised in that survey area inner plane matrix in step (1) Mesh-density be 200-500m × 100-250m.
  3. 3. Microbial Prospecting of Oil and Gas method as described in claim 1, it is characterised in that in step (2) sampling depth be 40cm, institute The sample of acquisition is in -20 DEG C of storage and transport.
  4. 4. Microbial Prospecting of Oil and Gas method as described in claim 1, it is characterised in that described using butane as the enrichment of sole carbon source Method is as follows：1g pedotheques are weighed on aseptic operating platform to be placed in the 150mL triangular flasks for filling 50mL fluid nutrient mediums, to Butagas is filled with below culture medium liquid level makes butagas in culture solution reach saturation, and the gas compartment in triangular flask is made to be full of fourth Alkane gas, sealing, 28 DEG C of shake culture 7d of 180rpm.
  5. 5. Microbial Prospecting of Oil and Gas method as described in claim 1, it is characterised in that described using butanol as the solid of sole carbon source Contain 5% butanol in tablet as carbon source.
  6. 6. Microbial Prospecting of Oil and Gas method as described in claim 1, it is characterised in that fluorescence quantitative PCR detection needle in step (4) To target gene be low alkoxylated enzyme gene.
  7. 7. Microbial Prospecting of Oil and Gas method as claimed in claim 6, it is characterised in that each lower alkyl in retrieval GenBank databases Oxidase gene sequences carry out multiple alignment, and fluorescence quantification PCR primer is designed for conserved region sequence.
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