CN102732504A - Method for extracting microorganism macrogenome from oil/gas pool environment - Google Patents
Method for extracting microorganism macrogenome from oil/gas pool environment Download PDFInfo
- Publication number
- CN102732504A CN102732504A CN2011100965256A CN201110096525A CN102732504A CN 102732504 A CN102732504 A CN 102732504A CN 2011100965256 A CN2011100965256 A CN 2011100965256A CN 201110096525 A CN201110096525 A CN 201110096525A CN 102732504 A CN102732504 A CN 102732504A
- Authority
- CN
- China
- Prior art keywords
- oil
- water
- sample
- adds
- mikrobe
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000000034 method Methods 0.000 title claims abstract description 45
- 244000005700 microbiome Species 0.000 title claims abstract description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 39
- 238000001914 filtration Methods 0.000 claims abstract description 15
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims abstract description 12
- 238000000605 extraction Methods 0.000 claims abstract description 12
- 230000008021 deposition Effects 0.000 claims abstract description 11
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 11
- 239000002689 soil Substances 0.000 claims abstract description 11
- 150000004676 glycans Chemical class 0.000 claims abstract description 5
- 239000012071 phase Substances 0.000 claims abstract description 5
- 229920001282 polysaccharide Polymers 0.000 claims abstract description 5
- 239000005017 polysaccharide Substances 0.000 claims abstract description 5
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 5
- 229920002521 macromolecule Polymers 0.000 claims abstract description 4
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 4
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 4
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 4
- 239000012074 organic phase Substances 0.000 claims abstract description 3
- 150000003839 salts Chemical class 0.000 claims abstract description 3
- 108010014251 Muramidase Proteins 0.000 claims abstract 3
- 102000016943 Muramidase Human genes 0.000 claims abstract 3
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 claims abstract 3
- 239000004325 lysozyme Substances 0.000 claims abstract 3
- 229960000274 lysozyme Drugs 0.000 claims abstract 3
- 235000010335 lysozyme Nutrition 0.000 claims abstract 3
- 239000003921 oil Substances 0.000 claims description 56
- 235000019198 oils Nutrition 0.000 claims description 51
- 239000007789 gas Substances 0.000 claims description 33
- 239000010779 crude oil Substances 0.000 claims description 28
- 239000006228 supernatant Substances 0.000 claims description 25
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 22
- 239000000203 mixture Substances 0.000 claims description 20
- 238000002156 mixing Methods 0.000 claims description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 13
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 12
- 239000012528 membrane Substances 0.000 claims description 10
- 108010064696 N,O-diacetylmuramidase Proteins 0.000 claims description 8
- 238000004458 analytical method Methods 0.000 claims description 8
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 7
- 239000000284 extract Substances 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- 239000011780 sodium chloride Substances 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 7
- 241000894006 Bacteria Species 0.000 claims description 6
- 230000007613 environmental effect Effects 0.000 claims description 6
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 claims description 6
- 238000003828 vacuum filtration Methods 0.000 claims description 6
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 5
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 5
- 108010067770 Endopeptidase K Proteins 0.000 claims description 5
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 5
- 238000012216 screening Methods 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 4
- 239000008367 deionised water Substances 0.000 claims description 4
- 229910021641 deionized water Inorganic materials 0.000 claims description 4
- 235000019476 oil-water mixture Nutrition 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims description 3
- 239000008346 aqueous phase Substances 0.000 claims description 3
- 238000010276 construction Methods 0.000 claims description 3
- 238000005336 cracking Methods 0.000 claims description 3
- 238000011331 genomic analysis Methods 0.000 claims description 3
- 239000012535 impurity Substances 0.000 claims description 3
- 238000010353 genetic engineering Methods 0.000 claims description 2
- 238000003809 water extraction Methods 0.000 claims description 2
- 239000007791 liquid phase Substances 0.000 claims 3
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 claims 2
- 239000000243 solution Substances 0.000 claims 2
- 241000233866 Fungi Species 0.000 claims 1
- 230000001476 alcoholic effect Effects 0.000 claims 1
- 230000000975 bioactive effect Effects 0.000 claims 1
- 238000004364 calculation method Methods 0.000 claims 1
- 235000009508 confectionery Nutrition 0.000 claims 1
- 230000009089 cytolysis Effects 0.000 claims 1
- 238000010790 dilution Methods 0.000 claims 1
- 239000012895 dilution Substances 0.000 claims 1
- 238000010230 functional analysis Methods 0.000 claims 1
- 239000003345 natural gas Substances 0.000 claims 1
- 238000001556 precipitation Methods 0.000 claims 1
- 238000012882 sequential analysis Methods 0.000 claims 1
- 230000008569 process Effects 0.000 abstract description 6
- 108091005804 Peptidases Proteins 0.000 abstract description 4
- 230000008901 benefit Effects 0.000 abstract description 4
- 238000001962 electrophoresis Methods 0.000 abstract description 2
- 239000003208 petroleum Substances 0.000 abstract description 2
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 abstract 2
- 239000004365 Protease Substances 0.000 abstract 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 abstract 1
- 230000015556 catabolic process Effects 0.000 abstract 1
- 238000006731 degradation reaction Methods 0.000 abstract 1
- 230000000813 microbial effect Effects 0.000 description 20
- 238000005516 engineering process Methods 0.000 description 8
- 238000011160 research Methods 0.000 description 7
- 239000011435 rock Substances 0.000 description 7
- 230000008859 change Effects 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 230000004060 metabolic process Effects 0.000 description 6
- 239000003643 water by type Substances 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- 108020004465 16S ribosomal RNA Proteins 0.000 description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 239000002207 metabolite Substances 0.000 description 4
- 229920000642 polymer Polymers 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 3
- 102000035195 Peptidases Human genes 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000002906 microbiologic effect Effects 0.000 description 3
- 235000019833 protease Nutrition 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 239000003876 biosurfactant Substances 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 229930195733 hydrocarbon Natural products 0.000 description 2
- 150000002430 hydrocarbons Chemical class 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 230000000050 nutritive effect Effects 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 239000004576 sand Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000000123 temperature gradient gel electrophoresis Methods 0.000 description 2
- 239000001993 wax Substances 0.000 description 2
- 239000004215 Carbon black (E152) Substances 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 150000004945 aromatic hydrocarbons Chemical class 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 235000011089 carbon dioxide Nutrition 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000013256 coordination polymer Substances 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000011544 gradient gel Substances 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 150000002431 hydrogen Chemical class 0.000 description 1
- 238000010921 in-depth analysis Methods 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 235000015110 jellies Nutrition 0.000 description 1
- 239000008274 jelly Substances 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 244000005706 microflora Species 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 235000015927 pasta Nutrition 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000010408 sweeping Methods 0.000 description 1
- 230000002110 toxicologic effect Effects 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
Images
Abstract
The invention provides a simple method for directly extracting microorganism macrogenome from an oil/gas pool environment sample, which comprises the following steps: an organic phase/water phase is extracted to obtain an microbe component in an oil sample/oil-water sample, vacuum filtering is carried out on the water phase enrichment microbes, cells are cracked by lysozyme, protease, sodium dodecyl sulfate (SDS) and the like, passes through the cetyl trimethyl ammonium bromide (CTAB) under high salt condition to remove organic macro-molecule components like protein and polysaccharide, extraction is carried out through nucleic acid and deposition is carried out, thereby the unbiased oil/gas pool microorganism macrogenome component can be obtained. The method of the invention has the advantages that the process is fast and simple, the cost is low, the whole extraction process can be completed in 10 hours; the obtained genome enables high purity, the ratio of OD260nm to OD280nm is between 1.7 and 2.0, the electrophoresis result detects that the integral genome possesses no degradation condition. The method of the invention aims at the oil/gas pool complex environments, and is suitable for extraction of other microbe genome in the soil and water body polluted by petroleum.
Description
Technical field
The present invention relates to the molecular ecology technical field, specifically by directly extracting the grand genomic simple and easy method of mikrobe in oil/gas reservoir complex environment.
Background technology
One of emphasis of microbe oil production/gas technology and oil/gas reservoir microbiological prospecting technology is to understand the Global Information of mikrobe in oil/gas reservoir complex environment.The limitation that is limited to present technique means; Have only about mikrobe of about 1% to can be artificial culture and understanding; Last century Mo for this reason; Researcher has been developed grand genome-based technologies, and grand genomics is through extracting all microbe genome DNAs, the structure genomic library in a certain environment and a kind of method of seeking and finding new functional gene and active metabolite being screened in the library.
Handelsman in 1998 etc. propose grand genomic notion first; And it is defined as: a kind of with all genomes of the mikrobe in the environmental sample as research object; With functional gene screening and sequencing analysis is research means; Be microbial diversity, population structure, evolutionary relationship, functionally active, cooperation relation each other, and and environment between relation be the new microbe research method of research direction, and used this noun of Meta-genomics for the first time.Its elementary tactics flow process is: the enrichment of sample and gene (group); Extract the genomic dna in the specific environment; Make up the macro genome DNA library; The screening goal gene; And the goal gene active result is expressed.Grand genomics has been avoided the process of mikrobe separation and Culture, has greatly expanded the space that utilizes of Microbial resources, is new developing direction of modern genetic engineering and research focus.
The primary prerequisite of grand genomics research is to obtain the microbial genome information that nature does not truly have preference, but because the complicacy of oil/gas reservoir environment, its operation is difficulty relatively.
Oil/gas reservoir is a kind of extreme ecotope of uniqueness, and it is containing abundant microbe groups; These mikrobes have multiple metabolic type; Through itself breeding growth and effect of meta-bolites in oil/gas reservoir; Can be at MEOR (Microbial Enhanced Oil Recovery; Microbial enhanced oil recovery) and among the MPOG (Microbiological Prospecting of Oil and Gas, microbial oil gas exploration) play a significant role.
MEOR is meant surface separation cultured microorganism bacterium liquid and nutritive medium is injected oil reservoir; Or inject nutritive medium merely and activate the mikrobe in the oil reservoir; Make its growth and breeding in oil reservoir, produce the meta-bolites that helps improving RF, to improve the oil production method of oil recovery rate; Be commonly called as microbe oil production, its mechanism is:
1. change the composition of crude oil, reduce its viscosity
Mikrobe is in the growth metabolism process, and with the carbon source of the normal paraffin in the crude oil as self growth, thereby the carbochain that changes crude oil is formed; On the other hand, the enzyme that discharges during microorganism growth can degrading crude oil, makes the fracture of crude oil carbochain, and high carbon chain crude oil becomes low carbon chain crude oil, and heavy constituent reduce, and light constituent increases, zero pour and viscosity reduction, thus play the effect of viscosity reduction, wax control.
2. change the displacement of reservoir oil environment of crude oil, make it be easier to extraction ground
(1) produces tensio-active agent
The metabolism meeting of mikrobe produces tensio-active agent, and these bio-surfactants can reduce oil water interfacial tension, reduce the water displacing oil capillary force.In addition, bio-surfactant can change the wettability of oil/gas reservoir rock, and it is hydrophilic that it is become from oleophylic, and the oil film that is adsorbed on rock surface like this splits away off, and the oil reservoir ROS reduces, thereby improves oil recovery factor.
(2) produce biogas
Most of microbe all can produce gas in metabolic process, like carbonic acid gas, hydrogen, nitrogen etc., these gas dissolvings can reduce viscosity of crude in water, improves the crude oil flow ability.In addition, the existence of these gases also can increase sand pressure, reduces ROS indirectly.Simultaneously, the Jamin effect of bubble also can increase flow resistance, improves and injects ripples and efficient.
(3) produce acid and organic solvent
The metabolism of mikrobe produces low-molecular-weight relatively organic acid, and the part mineral acid is also arranged, and their ability dissolved carbon hydrochlorates increase rock porosity on the one hand, have improved rate of permeation; On the other hand, release of carbon dioxide has improved sand pressure, thereby reduces viscosity of crude, has improved the flow capacity of crude oil.The metabolic process of mikrobe also can produce organic solvents such as alcohol, ester, can change rock surface character and crude oil physical properties, makes the crude oil that is adsorbed on rock surface be released out, and is easy to extraction ground.
(4) form XC polymer
In the metabolic process of mikrobe, can produce XC polymer.These XC polymers can be at oil reservoir high permeability zones macropore plugging optionally, and the intake profile of adjustment water filling oil reservoir increases and injects water to sweep pasta long-pending.XC polymer can also increase the viscosity of water, reduces WOR, improves the oily efficient of sweeping of injecting water, thereby improves RF.
3. the direct effect of mikrobe
Mikrobe grows at rock surface, can occupy pore space and drives away crude oil.In addition, mikrobe can be adsorbed onto rock surface, and growth is pushed oil film open at last under oil film, and crude oil is discharged.
Microbial Prospecting of Oil and Gas (Microbial Prospection for Oil and Gas; MPOG) be a kind of method of carrying out oil-gas exploration, mainly study the correlationship of microbiological anomaly and underground deep oil-gas reservoir in the near surface pedosphere according to face of land microbial profile situation.The Application Areas of MPOG comprises: ⑴ unworked country oil and gas prediction, and it is according to being: the high value exceptions area of specified microorganisms is the reliability index of hydro carbons seepage in the stratum normally; ⑵ ripe exploratory area evaluating reservoir: microbial oil gas exploration technology is used at it and only is used for the prediction of hydrocarbon potential district in early days.But over nearly 10 years, this technology is obtaining breakthrough aspect the explanation of measuring achievement.U.S. geology microbial technique company takes the lead in this method is used among the evaluation to underground oil and gas reservoir distribution situation; Set up " mikrobe reservoir characteristics evaluation technique (MRC-Microbial Reservoir Characterization) ", this The Application of Technology scope has been extended to development field by Exploration Domain.
Because the restriction of traditional method, the indicator microoraganism of MPOG research at present mainly concentrates on a few bacterial strains such as methanogen, and has a large amount of microbe species that can be used for indicating in the geology soil probably; Therefore; The utilization quantitative PCR, the advantage that high-flux sequence detects can combine actual oil/gas reservoir situation; Carry out quantitative examinations extensive, many bacterial classifications, utilize the data conclusion of many indexs to increase the success ratio that MPOG predicts.Simultaneously, can the different microorganisms of different areas, different soil property, DIFFERENT DEPOSITIONAL ENVIRONMENTS be compared, filter out the new indication flora that is fit to the MPOG application.
Still do not have now both at home and abroad any about the grand genome of oil at MEOR, correlative study among MPOG and the MRC and application.Therefore, for obtaining mikrobe complete genome group workable and simple and practical high-efficiency method is provided, can have farthest obtained complete sample gene group information through this inventive method.
Summary of the invention
For overcoming the problems referred to above, the invention provides grand genomic method in a kind of extraction and the purifying oil/gas reservoir complex environment, this method can be directed against most oil/gas reservoir sample, simple to operate, economy, environmental friendliness, high-quality and high-efficiency.
Stripping oil of the present invention/gas reservoir environment macro is genomic to obtain comprising step:
(1) organic phase/water extraction obtains mikrobe component in oil sample/profit appearance.Specifically, in crude oil, add the sherwood oil/normal hexane mixture (1-3:1 v/v) of isopyknic 10-50% ethanol and half volume, fully shake up; Staticly make the layering of profit phase, extract lower floor's water with sebific duct.Fall a small amount of crude oil of aqueous phase and other impurity with filter paper filtering; (2) step 1 is obtained water through 0.22 μ m millipore filtration vacuum filtration, the enrichment thalline is on filter membrane; (3) step 2 gained microorganism cells is carried out cracking, specifically, filter membrane is shredded put into the 10mL centrifuge tube, add 100-500 μ L N,O-Diacetylmuramidase (10mg/mL is dissolved in 10mM Tris-HCl, pH8.0), 37 ℃ of water-bath 0.5-2 h; The SDS and the 10-50 μ L Proteinase K (20mg/mL) that add 50-300 μ L10%, 37 ℃ of water-bath 1-4 h; (4) remove step 3 through CTAB under the high salt condition and obtain protein and the organic macromolecule (like polysaccharide) in the component, specifically, the 5M NaCl that adds 100-500 μ L puts upside down mixing, adding 100-500 μ L 5%CTAB, 50-80 ℃ of water-bath 10-60min; (5) step 4 is obtained component and carry out nucleic acid extracting and deposition, specifically, cooling back sucking-off supernatant; Broken film with the washing of 100-500 μ L aseptic deionized water once; The sucking-off supernatant with the supernatant liquid mixing of twice sucking-off, adds the mixture of isopyknic chloroform: primary isoamyl alcohol=24:1; Put upside down mixing 0.5-5min, leave standstill 1-5min; The centrifugal 10-30min of 12000rpm, the sucking-off supernatant.The Virahol that adds 0.5-1 times of volume is placed 3h or placement is spent the night for-20 ℃; The centrifugal 10-30min of 12000rpm carefully outwells supernatant, and deposition adds 1-5mL 75% cold washing with alcohol once, and the volatilization of cold to ethanol totally adds the dissolving of 10-100 μ L aseptic deionized water.
Extraction object samples of the present invention can be crude oil sample, also includes, but not limited to WIW profit compound sample, oil-polluted soils, petroleum pollution water body, samples such as the water of natural air reservoir surrounding enviroment, soil simultaneously.To different samples; The mode of microbial enrichment is slightly different; As for profit appearance (oil-contg is less than 5%), main through filtering the collection water, it is resuspended then the former oil sample of collecting on the filter paper to be carried out the vortex concussion with the PBS damping fluid; Filter once more and collect water, merge water then and obtain the mikrobe component.Behind the mikrobe component after the acquisition enrichment, working method reaches unanimity.
Through 0.22 μ m millipore filtration vacuum filtration, but the environmental microorganism of enrichment 99.5% or more in theory, so can fully obtain all information of oil/gas reservoir mikrobe.
Add N,O-Diacetylmuramidase, be primarily aimed at the gram positive bacterium that is difficult to broken wall under the normal condition; Combine anionic detergent SDS and Proteinase K to handle simultaneously, can promote that under gentle relatively condition bacteria cell wall breaks, thereby discharge genome.SDS can also suppress the activity of intracellular DNase simultaneously, and DNA is provided stable environment.
Under hypersaline environment, CTAB can combine organic macromolecules such as protein and the polysaccharide in the cracking component, and the nucleic acid component then keeps dissolved state, thereby obtains to separate.
Mixture with isopyknic chloroform: primary isoamyl alcohol=24:1 carries out extracting, can further remove residual protein; Then through isopropanol precipitating, obtain complete whole grand genome.
The grand genome that this aspect method is obtained can be used for subsequent P CR reaction, and the library is set up, various molecular biology operations such as Southern.Method is simple, need not complex apparatus, and total overall reaction can be accomplished in 10 hours; Environmental friendliness is to environment and experimenter's toxicological harmless effect.
The main advantage of present method is following:
1 the present invention is primarily aimed at the extraction of grand genome composition in oil/gas reservoir complex environment; Microbe to screen compared with techniques with routine; The present invention has got around complicated microbial cultivation process; But take into account simultaneously culturing micro-organisms with cannot not culturing micro-organisms, therefore can reflect the truth of microbial profile in oil/gas reservoir complex environment to the full extent;
2 are started with by method provided by the present invention; Based on in-depth analysis to grand genome component; Can shorten the construction cycle of microbial strains and meta-bolites greatly, improve understanding, in mikrobe and functional molecular sieve screening process, accomplish to shoot the arrow at the target indigenous microorganism;
3 with regard to the present invention self, compares with conventional genome extraction process, exist with low cost, repeatable high, the sample that obtains does not have preference, the purpose mikrobe that is directed against is extensive, waits many advantages;
The crude oil component, wax, bituminous matter, the arene that exist in 4 oil/gas reservoir environment; And materials such as the soil ulmin of introducing because of construction, other environmental factorss etc., vegetable jelly polyose; Molecular biology operation to routine can cause considerable influence; The present invention is directed to above-mentioned knotty problem, on technology, carried out improving one by one, therefore improved the purification efficiency of grand genome composition to the full extent.
Description of drawings
The grand genome of Fig. 1 extracts the situation summary view; Line1 is embodiment 1 illustration among Fig. 1, and Line2 is embodiment 2 illustrations, and Line3-4 is embodiment 3 illustrations.Line1: PetroChina Company Limited.'s block crude oil sample microbial genome; Line2: PetroChina Company Limited.'s block profit sample microbial genome; Line3: PetroChina Company Limited.'s block face of land greasy filth sample microbial genome, Line4: PetroChina Company Limited.'s block polluted-water sample microbial genome.
16S rDNA sequence PCR fragment behind Fig. 2 purifying.Line1: come from PetroChina Company Limited.'s block crude oil sample, Line2: come from PetroChina Company Limited.'s block profit sample, Line3: come from PetroChina Company Limited.'s block face of land greasy filth sample, Line4: come from PetroChina Company Limited.'s block polluted-water sample.
Embodiment
Below with embodiment application method of the present invention is described.Scope shown in volumetric parameter is not limited to describe strengthens the extraction scale like need, can amplify in proportion.Should be understood that these instances only to be used to the present invention is described and be not used in the restriction the inventive method the scope of application.
The embodiment 1 grand genomic simple and easy method of mikrobe that abstracts from crude oil
1. get about 100mL crude oil and place glass beaker, 70 ℃ of water-baths 30 minutes.
2. the sherwood oil that adds equal-volume 30% (V/V) ethanol and half volume of crude oil: normal hexane (V/V=1:1) mixture fully shakes up.
3. staticly make the layering of profit phase, extract lower floor's water with sebific duct.Fall a small amount of crude oil of aqueous phase and other impurity with filter paper filtering.
4. filter back liquid with 0.22 μ m millipore filtration vacuum filtration, the enrichment thalline is on filter membrane.
5. filter membrane is shredded and puts into the 10mL centrifuge tube, add 360 μ L N,O-Diacetylmuramidases (10mg/mL is dissolved in 10mM Tris-HCl, pH8.0), 37 ℃ of water-bath 1h.
6. the SDS and the 25 μ L Proteinase Ks (20mg/mL) that add 115 μ L 10%, 37 ℃ of water-bath 2h.
7. the 5M NaCl that adds 400 μ L puts upside down mixing, adds 280 μ L 5%CTAB, 65 ℃ of water-bath 20min.
8. cool off back sucking-off supernatant, broken film washs once with 200 μ L aseptic deionized waters, and the sucking-off supernatant with the supernatant liquid mixing of twice sucking-off, adds the mixture of isopyknic chloroform: primary isoamyl alcohol=24:1, puts upside down mixing 1min, leaves standstill 2min.
9.12000rpm centrifugal 30min, the sucking-off supernatant.The Virahol that adds 0.6 times of volume is placed 3h or placement is spent the night for-20 ℃.
10. the centrifugal 20min of 12000rpm carefully outwells supernatant, and deposition adds cold 75% washing with alcohol of 1mL once, and the volatilization of cold to ethanol totally adds the 30 μ L aseptic deionized waters dissolving grand genome composition that obtains.
11. the genome composition that obtains is shown in Fig. 1 Line1, deposition condition is following 30 minutes of 75V.
1. get about 100mL oil-water mixture and place glass beaker, 70 ℃ of water-bath 30min.
2. use the filter paper filtering oil-water mixture, collect water.
3. the crude oil of staying the filter paper top mixes with the long-pending aseptic PBS of diploid, fully stirs, and collects water with filter paper filtering, triplicate.
4. merge and filter the back water, with 0.22 μ m millipore filtration vacuum filtration, the enrichment thalline is on filter membrane.
5. filter membrane is shredded and puts into the 10mL centrifuge tube, add 360 μ L N,O-Diacetylmuramidases (10mg/mL is dissolved in 10mM Tris-HCl, pH8.0), 37 ℃ of water-bath 1h.
6. the SDS and the 25 μ L Proteinase Ks (20mg/mL) that add 115 μ L10%, 37 ℃ of water-bath 2h.
7. the 5M NaCl that adds 400 μ L puts upside down mixing, adds 280 μ L 5%CTAB, 65 ℃ of water-bath 20min.
8. cool off back sucking-off supernatant, broken film washs once with 200 μ L aseptic deionized waters, and the sucking-off supernatant with the supernatant liquid mixing of twice sucking-off, adds the mixture of isopyknic chloroform: primary isoamyl alcohol=24:1, puts upside down mixing 1min, leaves standstill 2min.
9.12000rpm centrifugal 30min, the sucking-off supernatant.The Virahol that adds 0.6 times of volume is placed 3h or placement is spent the night for-20 ℃.
10. the centrifugal 20min of 12000rpm carefully outwells supernatant, and deposition adds cold 75% washing with alcohol of 1mL once, and the volatilization of cold to ethanol totally adds the 30 μ L aseptic deionized waters dissolving grand genome composition that obtains.
11. the genome composition that obtains is shown in Fig. 1 Line2, deposition condition is following 30 minutes of 75V.
1. get about 10g oil-containing soil and place glass beaker, add 100mL PBS, repeat to stir 70 ℃ of water-bath 30min.
2. use the filter paper filtering mixture, collect water.
3. crude oil and the soil of staying the filter paper top dissolve with the aseptic PBS of 100mL, fully stir, and collect water with filter paper filtering, triplicate.
4. merge and filter the back water, with 0.22 μ m millipore filtration vacuum filtration, the enrichment thalline is on filter membrane.
5. filter membrane is shredded and puts into the 10mL centrifuge tube, add 360 μ L N,O-Diacetylmuramidases (10mg/mL is dissolved in 10mM Tris-HCl, pH8.0), 37 ℃ of water-bath 1h.
6. the SDS and the 25 μ L Proteinase Ks (20mg/mL) that add 115 μ L 10%, 37 ℃ of water-bath 2h.
7. the 5M NaCl that adds 400 μ L puts upside down mixing, adds 280 μ L 5%CTAB, 65 ℃ of water-bath 20min.
8. cool off back sucking-off supernatant, broken film washs once with 200 μ L aseptic deionized waters, and the sucking-off supernatant with the supernatant liquid mixing of twice sucking-off, adds the mixture of isopyknic chloroform: primary isoamyl alcohol=24:1, puts upside down mixing 1min, leaves standstill 2min.
9.12000rpm centrifugal 30min, the sucking-off supernatant.The Virahol that adds 0.6 times of volume is placed 3h or placement is spent the night for-20 ℃.
10. the centrifugal 20min of 12000rpm carefully outwells supernatant, and deposition adds cold 75% washing with alcohol of 1mL once, and the volatilization of cold to ethanol totally adds the 30 μ L aseptic deionized waters dissolving grand genome composition that obtains.
11. the genome composition that obtains is shown in Fig. 1 Line3, and 4, respectively by obtaining in mud appearance and the water sample; Deposition condition is following 30 minutes of 75V.
With by method was obtained among the embodiment 1-3 grand genome component as template; The template add-on can suitably be diluted according to actual abundance; Reference value is to add the grand genome component of 10-100ng in the single PCR system, copy to hang down like the sequence of required amplification and then can suitably improve.
2. use bacterial 16 S rDNA sequence universal primer: 8F (5 '-TTTGATCCTGGCTCAG-3 ') and 1492R (5 '-GGTTACCTTGTTACGACTT-3 ').
3. the PCR reaction system is 25 μ L, and reaction conditions is 94 ℃ of sex change 4min; Next carry out 30 circulating reactions: 94 ℃ of sex change 45 s, 50 ℃ of annealing 45 s, 72 ℃ are extended 90 s; 72 ℃ are extended 10 min more then, at last in 4 ℃ of preservations.
4. obtain the purified back of 16S rDNA sequence PCR product and be connected in commercialization or the self-control T carrier, will connect product and transform DH5 α competent cell, coat corresponding resistant panel and make up the library through the TA clone, and screening positive clone.
5. select the 16S rDNA partial sequence that positive colony measure to insert, and with GenBank in known array carry out homology relatively, confirm the kind of corresponding mikrobe.
6. obtaining grand genome component can be other diversity analysis technique means equally microflora's Back ground Information is provided; The downstream correlation technique comprises; But be not limited to; RDNA amplified fragments restriction endonuclease analysis (ARDRA), deformation gradient gel electrophoresis (DGGE), TGGE (TGGE) chain conformation polymorphism analysis (SS-CP), end limit property fragment length polymorphism are analyzed (T-PFLP), the random amplification fragment length polymorphism is analyzed (RAPD), AFLP analysis (AFLP), etc.
The method of the invention is quick and easy, and is with low cost, and whole leaching process can be accomplished within 10 hours; Institute's genome purity that obtains is high, OD
260nm: OD
280nmRatio is between 1.7-2.0, and electrophoresis result detects the whole no degraded situation of genome.The inventive method is not only to oil/gas reservoir complex environment, also is applicable to the extraction of microbial genome in soil that other are petroleum-polluted and the water body.
Claims (8)
1. one kind is extracted the grand genomic method of mikrobe from oil/gas reservoir environment, it is characterized in that: comprise step:
(1) organic phase/water extraction obtains mikrobe component in the liquid phase sample; Specifically, in liquid phase sample, add and the ethanol of the isopyknic 10-50% of sample (V/V) and the sherwood oil/normal hexane mixture of sample half volume, sherwood oil/normal hexane (V/V)=1-3:1 fully shakes up; Staticly make the layering of profit phase, extract lower floor's water with sebific duct; Fall aqueous phase crude oil and other impurity with filter paper filtering;
(2) with water that step 1) obtains warp 0.22 μ m millipore filtration vacuum filtration, the enrichment thalline is on filter membrane;
(3) to step 2) the gained microorganism cells carries out cracking, and specifically, filter membrane shredded put into the 10mL centrifuge tube; Add 100-500 μ L lysozyme soln; Lysozyme concentration is that N,O-Diacetylmuramidase 5-20mg/mL is dissolved in 10mM Tris-HCl, pH8.0,37 ℃ of water-bath 0.5-2h; The SDS and the 10-50 μ L concentration 20mg/mL Proteinase K that add 50-300 μ L 10% (W/V), 37 ℃ of water-bath 1-4h;
(4) remove step 3) through CTAB under the high salt condition and obtain protein and organic macromolecule (like polysaccharide) in the component; Specifically, the 5M NaCl that in the whole solution of step (3), adds 100-500 μ L puts upside down mixing, adds 100-500 μ L 5% CTAB (cetyl trimethylammonium bromide), 50-80 ℃ of water-bath 10-60min;
(5) component that step 4) obtains is carried out nucleic acid extracting and deposition; Specifically, be cooled to sucking-off supernatant after the room temperature, broken film with the washing of 100-500 μ L aseptic deionized water once; The sucking-off supernatant with the supernatant liquid mixing of twice sucking-off, adds the mixture of isopyknic chloroform: primary isoamyl alcohol=24:1; Put upside down mixing 0.5-5min, leave standstill 1-5min; The centrifugal 10-3min of 12000rpm, the sucking-off supernatant; The Virahol that adds 0.5-1 times of volume is placed 3h or placement is spent the night for-20 ℃; The centrifugal 10-30min of 12000rpm outwells supernatant, and deposition adds cold 75% washing with alcohol of 1-5mL once, and the volatilization of cold to ethanol totally adds the 10-100 μ L aseptic deionized water dissolving grand genome composition that obtains.
2. the method for claim 1; It is characterized in that: the said liquid phase sample of step (1) comprises, the water in crude oil sample, oil-water mixture, water sample, oil-polluted soils or water body or plant and the Sweet natural gas lab environment, with the pedotheque of saline water dilution.
3. the method for claim 1 is characterized in that: step (1) but the said mikrobe that is extracted in the sample to be comprised had both comprised culturing micro-organisms, also comprising cannot not culturing micro-organisms
.
4. the method for claim 1 is characterized in that: the mikrobe that step (1) is said to be extracted in the sample to be comprised comprises, a kind of in Gram-negative bacteria, gram-positive microorganism, archeobacteria, the fungi or more than two kinds.
5. the method for claim 1 is characterized in that: totally add and volume calculation by each solution, in the used extraction system of step (1); The alcoholic acid final concentration is 4-20%; The sherwood oil final concentration is 10-15%, and the normal hexane final concentration is 5-10%, and above concentration is volume ratio.
6. the method for claim 1, it is characterized in that: in step (3) lysis, N,O-Diacetylmuramidase, SDS, Proteinase K are united use, and the final concentration when wherein N,O-Diacetylmuramidase is handled is 5-20mg/mL; SDS uses final concentration to be 1-3%, and Proteinase K uses final concentration to be 0.2-2 mg/mL.
7. the method for claim 1, it is characterized in that: albumen and the used NaCl of polysaccharide precipitation step (4) and CTAB final concentration are respectively 1-4 M and 0.5-2.0%.
8. the method for claim 1; It is characterized in that: the subsequent use of the grand genome sample that obtains comprises, oil/gas reservoir environmental microorganism community diversity analysis, population structure analysis, evolutionary relationship, functionally active and each other cooperation relation molecular ecology, the oil/grand genomic library construction of gas reservoir indigenous microorganism, based on the functionally active molecular screening of functional analysis and sequential analysis or the structure of bioactive molecule genetic engineering bacterium.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110096525.6A CN102732504B (en) | 2011-04-15 | 2011-04-15 | Method for extracting microorganism macrogenome from oil/gas pool environment |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110096525.6A CN102732504B (en) | 2011-04-15 | 2011-04-15 | Method for extracting microorganism macrogenome from oil/gas pool environment |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102732504A true CN102732504A (en) | 2012-10-17 |
| CN102732504B CN102732504B (en) | 2014-05-28 |
Family
ID=46988807
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110096525.6A Expired - Fee Related CN102732504B (en) | 2011-04-15 | 2011-04-15 | Method for extracting microorganism macrogenome from oil/gas pool environment |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102732504B (en) |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103333883A (en) * | 2013-07-11 | 2013-10-02 | 内蒙古科技大学 | Method for efficiently extracting underground water microbial DNA for PCR amplification |
| CN103667256A (en) * | 2013-11-19 | 2014-03-26 | 克拉玛依市金山石油化工有限公司 | Method for extracting total DNA (deoxyribonucleic acid) of environmental microorganism in oilfield sewage by adopting freezing method |
| CN104087573A (en) * | 2014-06-18 | 2014-10-08 | 福建农林大学 | Extraction method for microbial genome DNA in water culture solution |
| CN105821033A (en) * | 2016-05-11 | 2016-08-03 | 清华大学 | Extracting method of cellulose degradation flora metagenome |
| CN105986025A (en) * | 2015-03-03 | 2016-10-05 | 核工业北京地质研究院 | Research method for relation between microorganisms and uranium mineralization in sandstone type uranium ore deposit |
| CN106754895A (en) * | 2017-03-01 | 2017-05-31 | 中国石油大学(北京) | Extract the method and kit of crude oil total dna |
| WO2017209990A1 (en) | 2016-05-31 | 2017-12-07 | Exxonmobil Upstream Research Company | METHODS FOR lSOLATING NUCLEIC ACIDS FROM SAMPLES |
| CN110438119A (en) * | 2019-08-23 | 2019-11-12 | 北京润世能源技术有限公司 | A kind of extracting method of crude oil microbe genome DNA |
| CN110511262A (en) * | 2019-08-01 | 2019-11-29 | 中海石油环保服务（天津）有限公司 | The extracting solution and extracting method of macro protein group in a kind of extraction oil sludge and sand |
| US10570735B2 (en) | 2016-07-01 | 2020-02-25 | Exxonmobil Upstream Research Comapny | Methods to determine conditions of a hydrocarbon reservoir |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1718738A (en) * | 2005-06-28 | 2006-01-11 | 国家海洋局第三海洋研究所 | Method of simultaneously extracting microorganism macrogenome DNA and total DNA from sea precipitate |
-
2011
- 2011-04-15 CN CN201110096525.6A patent/CN102732504B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1718738A (en) * | 2005-06-28 | 2006-01-11 | 国家海洋局第三海洋研究所 | Method of simultaneously extracting microorganism macrogenome DNA and total DNA from sea precipitate |
Non-Patent Citations (3)
| Title |
|---|
| 包木太等: "采油微生物代谢产物分析 ", 《油田化学》, vol. 19, no. 2, 25 June 2002 (2002-06-25), pages 188 - 192 * |
| 包木太等: "采油微生物代谢产物分析", 《油田化学》 * |
| 姜岩等: "生物技术在油田地面处理系统中的应用研究进展", 《化工进展》 * |
Cited By (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103333883B (en) * | 2013-07-11 | 2015-09-23 | 内蒙古科技大学 | A kind of high efficiency extraction is used for the method for the groundwater microbial DNA of pcr amplification |
| CN103333883A (en) * | 2013-07-11 | 2013-10-02 | 内蒙古科技大学 | Method for efficiently extracting underground water microbial DNA for PCR amplification |
| CN103667256A (en) * | 2013-11-19 | 2014-03-26 | 克拉玛依市金山石油化工有限公司 | Method for extracting total DNA (deoxyribonucleic acid) of environmental microorganism in oilfield sewage by adopting freezing method |
| CN103667256B (en) * | 2013-11-19 | 2016-01-20 | 克拉玛依市金山石油化工有限公司 | Freezing method extracts the method for environmental microorganism STb gene in oilfield sewage |
| CN104087573A (en) * | 2014-06-18 | 2014-10-08 | 福建农林大学 | Extraction method for microbial genome DNA in water culture solution |
| CN104087573B (en) * | 2014-06-18 | 2016-06-08 | 福建农林大学 | The extracting method of microbe genome DNA in a kind of water planting liquid |
| CN105986025A (en) * | 2015-03-03 | 2016-10-05 | 核工业北京地质研究院 | Research method for relation between microorganisms and uranium mineralization in sandstone type uranium ore deposit |
| CN105821033A (en) * | 2016-05-11 | 2016-08-03 | 清华大学 | Extracting method of cellulose degradation flora metagenome |
| CN105821033B (en) * | 2016-05-11 | 2019-06-11 | 清华大学 | A kind of extracting method of the macro genome of cellulose degradation flora |
| WO2017209990A1 (en) | 2016-05-31 | 2017-12-07 | Exxonmobil Upstream Research Company | METHODS FOR lSOLATING NUCLEIC ACIDS FROM SAMPLES |
| US10724108B2 (en) | 2016-05-31 | 2020-07-28 | Exxonmobil Upstream Research Company | Methods for isolating nucleic acids from samples |
| US10570735B2 (en) | 2016-07-01 | 2020-02-25 | Exxonmobil Upstream Research Comapny | Methods to determine conditions of a hydrocarbon reservoir |
| US10895666B2 (en) | 2016-07-01 | 2021-01-19 | Exxonmobil Upstream Research Company | Methods for identifying hydrocarbon reservoirs |
| US10663618B2 (en) | 2016-07-01 | 2020-05-26 | Exxonmobil Upstream Research Company | Methods to determine conditions of a hydrocarbon reservoir |
| CN106754895A (en) * | 2017-03-01 | 2017-05-31 | 中国石油大学(北京) | Extract the method and kit of crude oil total dna |
| CN106754895B (en) * | 2017-03-01 | 2019-08-13 | 中国石油大学(北京) | Extract the method and kit of crude oil total dna |
| CN110511262A (en) * | 2019-08-01 | 2019-11-29 | 中海石油环保服务（天津）有限公司 | The extracting solution and extracting method of macro protein group in a kind of extraction oil sludge and sand |
| CN110511262B (en) * | 2019-08-01 | 2022-11-15 | 中海石油环保服务（天津）有限公司 | Extracting solution for extracting macro-proteome in oil sludge sand and extracting method |
| CN110438119A (en) * | 2019-08-23 | 2019-11-12 | 北京润世能源技术有限公司 | A kind of extracting method of crude oil microbe genome DNA |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102732504B (en) | 2014-05-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102732504B (en) | Method for extracting microorganism macrogenome from oil/gas pool environment | |
| CN102174509B (en) | Extraction and purification method of total plant endophyte genome DNA for colony analysis | |
| CN101880630B (en) | Method for increasing oil recovery ratio by utilizing symbiotic reproduction and complex metabolism and microbial preparation | |
| CN104388328B (en) | Degrade bacterial strain and its acquisition methods, the application of 5 rings and 6 ring polycyclic aromatic hydrocarbons | |
| CN105861388A (en) | Oil field thermophilic degradation long chain hydrocarbon bacillus HNMC 11117 and application thereof | |
| CN104830708A (en) | Crude oil degrading bacteria strain and application thereof | |
| CN105950514B (en) | One plant of microbe oil production bacterium W-Y3 and its application | |
| CN102154143A (en) | Marine aerobic denitrifying halomonas strain HGMN422 and application thereof | |
| CN104593298A (en) | Novel thermophilic and salt-resistant strain capable of degrading raw oil and generating emulsifying agent and application thereof | |
| CN109777747B (en) | Oil sludge petroleum degrading strain and culture method and application thereof | |
| CN102952846B (en) | Oil-reservoir endogenous microbe in-situ recovery method | |
| Pankratov et al. | Evaluation of the phylogenetic diversity of prokaryotic microorganisms in Sphagnum peat bogs by means of fluorescence in situ hybridization (FISH) | |
| CN113667616A (en) | Iron reducing bacterium DH4 strain and application thereof | |
| CN103789300B (en) | A kind of extracting method of epoxy propane saponified wastewater active sludge macro genome DNA | |
| CN106754895A (en) | Extract the method and kit of crude oil total dna | |
| CN110079479A (en) | One plant of rattan Flavimonas W-Y14 and its application | |
| CN106085902B (en) | One plant of microbe oil production bacterium W-Y1 and its application | |
| Xingbiao et al. | Influences of microbial community structures and diversity changes by nutrients injection in Shengli oilfield, China | |
| CN109234201A (en) | One plant of microbe oil production bacterium W-Y10 and its application | |
| Okoro et al. | Application of thermotolerant petroleum microbes at reservoir conditions for enhanced oil recovery | |
| CN101131082A (en) | Underground microbial oil production method | |
| CN107585880A (en) | A kind of method that high salt phenol wastewater is handled using gamboge coccus enzyme preparation enhancement microbiological | |
| CN103642798A (en) | Method for simultaneously extracting microbial genome deoxyribonucleic acid (DNA) and total ribonucleic acid (RNA) in mining area environmental sample | |
| CN109112091B (en) | Enterobacter cloacae FY-0701 and construction method and application thereof | |
| CN109385383A (en) | One plant of salt tolerant is dwelt salt pan bacterium W-Y11 and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140528 |