CN107022486A - A kind of screening technique of anaerobic degradation petroleum hydrocarbon methanogen floras - Google Patents
A kind of screening technique of anaerobic degradation petroleum hydrocarbon methanogen floras Download PDFInfo
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- 239000003209 petroleum derivative Substances 0.000 title claims abstract description 50
- 238000006731 degradation reaction Methods 0.000 title claims abstract description 37
- 230000015556 catabolic process Effects 0.000 title claims abstract description 35
- 238000000034 method Methods 0.000 title claims abstract description 29
- 238000012216 screening Methods 0.000 title claims abstract description 25
- 239000007788 liquid Substances 0.000 claims abstract description 49
- 239000010802 sludge Substances 0.000 claims abstract description 45
- 238000005065 mining Methods 0.000 claims abstract description 26
- 238000004519 manufacturing process Methods 0.000 claims abstract description 24
- 230000001580 bacterial effect Effects 0.000 claims abstract description 19
- 239000000203 mixture Substances 0.000 claims abstract description 18
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 claims description 54
- 239000007789 gas Substances 0.000 claims description 33
- 239000000243 solution Substances 0.000 claims description 22
- 235000015097 nutrients Nutrition 0.000 claims description 20
- 238000005070 sampling Methods 0.000 claims description 18
- 238000006243 chemical reaction Methods 0.000 claims description 15
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 12
- 230000001954 sterilising effect Effects 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 150000002430 hydrocarbons Chemical class 0.000 claims description 10
- 230000004087 circulation Effects 0.000 claims description 9
- 229930195733 hydrocarbon Natural products 0.000 claims description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 8
- 239000004215 Carbon black (E152) Substances 0.000 claims description 8
- 239000000284 extract Substances 0.000 claims description 7
- 239000011521 glass Substances 0.000 claims description 6
- 238000007789 sealing Methods 0.000 claims description 6
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 6
- 235000011152 sodium sulphate Nutrition 0.000 claims description 6
- 238000000967 suction filtration Methods 0.000 claims description 6
- 238000004458 analytical method Methods 0.000 claims description 5
- 238000003935 denaturing gradient gel electrophoresis Methods 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 238000002347 injection Methods 0.000 claims description 4
- 239000007924 injection Substances 0.000 claims description 4
- 238000005259 measurement Methods 0.000 claims description 4
- 229910052757 nitrogen Inorganic materials 0.000 claims description 4
- 238000004659 sterilization and disinfection Methods 0.000 claims description 4
- 238000012360 testing method Methods 0.000 claims description 4
- 238000010792 warming Methods 0.000 claims description 4
- 150000001335 aliphatic alkanes Chemical class 0.000 claims description 3
- 238000000137 annealing Methods 0.000 claims description 3
- 238000003556 assay Methods 0.000 claims description 3
- VZGDMQKNWNREIO-UHFFFAOYSA-N carbon tetrachloride Substances ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 claims description 3
- 238000004925 denaturation Methods 0.000 claims description 3
- 230000036425 denaturation Effects 0.000 claims description 3
- 238000010790 dilution Methods 0.000 claims description 3
- 239000012895 dilution Substances 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 3
- 238000002309 gasification Methods 0.000 claims description 3
- 238000013517 stratification Methods 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 239000003921 oil Substances 0.000 claims 17
- 239000010779 crude oil Substances 0.000 claims 1
- 230000002035 prolonged effect Effects 0.000 claims 1
- 239000004575 stone Substances 0.000 claims 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 12
- 229910052799 carbon Inorganic materials 0.000 abstract description 12
- 150000003839 salts Chemical class 0.000 description 12
- 229940088594 vitamin Drugs 0.000 description 10
- 229930003231 vitamin Natural products 0.000 description 10
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- 150000003722 vitamin derivatives Chemical class 0.000 description 10
- 239000007864 aqueous solution Substances 0.000 description 9
- PLXBWHJQWKZRKG-UHFFFAOYSA-N Resazurin Chemical compound C1=CC(=O)C=C2OC3=CC(O)=CC=C3[N+]([O-])=C21 PLXBWHJQWKZRKG-UHFFFAOYSA-N 0.000 description 8
- 239000002609 medium Substances 0.000 description 8
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- 235000013619 trace mineral Nutrition 0.000 description 6
- 239000003208 petroleum Substances 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
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- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- AUNGANRZJHBGPY-UHFFFAOYSA-N D-Lyxoflavin Natural products OCC(O)C(O)C(O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-UHFFFAOYSA-N 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 239000007836 KH2PO4 Substances 0.000 description 2
- 229910021380 Manganese Chloride Inorganic materials 0.000 description 2
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 2
- 229910021586 Nickel(II) chloride Inorganic materials 0.000 description 2
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 229930003451 Vitamin B1 Natural products 0.000 description 2
- 229930003471 Vitamin B2 Natural products 0.000 description 2
- VLSOAXRVHARBEQ-UHFFFAOYSA-N [4-fluoro-2-(hydroxymethyl)phenyl]methanol Chemical compound OCC1=CC=C(F)C=C1CO VLSOAXRVHARBEQ-UHFFFAOYSA-N 0.000 description 2
- ZLXPLDLEBORRPT-UHFFFAOYSA-M [NH4+].[Fe+].[O-]S([O-])(=O)=O Chemical compound [NH4+].[Fe+].[O-]S([O-])(=O)=O ZLXPLDLEBORRPT-UHFFFAOYSA-M 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 2
- 239000004327 boric acid Substances 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 2
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 2
- 230000007850 degeneration Effects 0.000 description 2
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- AGBQKNBQESQNJD-UHFFFAOYSA-M lipoate Chemical compound [O-]C(=O)CCCCC1CCSS1 AGBQKNBQESQNJD-UHFFFAOYSA-M 0.000 description 2
- 235000019136 lipoic acid Nutrition 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L magnesium chloride Substances [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 229910001629 magnesium chloride Inorganic materials 0.000 description 2
- 239000011565 manganese chloride Substances 0.000 description 2
- 235000002867 manganese chloride Nutrition 0.000 description 2
- 229940099607 manganese chloride Drugs 0.000 description 2
- 239000013028 medium composition Substances 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- SPIFDSWFDKNERT-UHFFFAOYSA-N nickel;hydrate Chemical compound O.[Ni] SPIFDSWFDKNERT-UHFFFAOYSA-N 0.000 description 2
- 239000003129 oil well Substances 0.000 description 2
- GNHOJBNSNUXZQA-UHFFFAOYSA-J potassium aluminium sulfate dodecahydrate Chemical compound O.O.O.O.O.O.O.O.O.O.O.O.[Al+3].[K+].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O GNHOJBNSNUXZQA-UHFFFAOYSA-J 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 229960002477 riboflavin Drugs 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000011684 sodium molybdate Substances 0.000 description 2
- 235000015393 sodium molybdate Nutrition 0.000 description 2
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 229960003495 thiamine Drugs 0.000 description 2
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 2
- 229960002663 thioctic acid Drugs 0.000 description 2
- 239000010913 used oil Substances 0.000 description 2
- 235000010374 vitamin B1 Nutrition 0.000 description 2
- 239000011691 vitamin B1 Substances 0.000 description 2
- 235000019164 vitamin B2 Nutrition 0.000 description 2
- 239000011716 vitamin B2 Substances 0.000 description 2
- 229940084600 vitamin b 12 0.1 mg Drugs 0.000 description 2
- 229940023356 vitamin b6 10 mg Drugs 0.000 description 2
- 239000011592 zinc chloride Substances 0.000 description 2
- 235000005074 zinc chloride Nutrition 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 241001148470 aerobic bacillus Species 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000004700 fetal blood Anatomy 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 238000003541 multi-stage reaction Methods 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 230000018612 quorum sensing Effects 0.000 description 1
- 235000012239 silicon dioxide Nutrition 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
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Classifications
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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
- C12N1/02—Separating microorganisms from their culture media
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
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- Medicinal Chemistry (AREA)
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- Virology (AREA)
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- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a kind of screening technique of anaerobic degradation petroleum hydrocarbon methanogen floras, the present invention is used as sample using oil field mining liquid and oily sludge, Anaerobic culturel is carried out to flora therein, in the case where being added without carbon source, flora can only be used as carbon source by the use of petroleum hydrocarbon, and then cultivate that filter out can be by the use of petroleum hydrocarbon as the flora of carbon source, while in order to obtain the decomposing petroleum hydrocarbon mixed bacterial that adaptability is stronger；In addition, the present invention uses oil field mining liquid and oily sludge as sample simultaneously, and sample of the gas production more than 0.4 μm of ol/d and 1.15 μm of ol/d is filtered out according to the different attribute of flora in oil field mining liquid and oily sludge respectively, and then be mixed, obtain being capable of the mixture of decomposing petroleum hydrocarbon mixed bacterial；Finally, the present invention also contemplates the degradation rate to petroleum hydrocarbon simultaneously, so that it is guaranteed that the mixed bacterial finally given has outstanding degradation capability.
Description
Technical field
Technical field is utilized the invention belongs to Residual petroleum, and in particular to a kind of anaerobic degradation petroleum hydrocarbon methanogen floras
Screening technique.
Background technology
Deep development is carried out to old filed, improve oil recovery has turned into the central task that current old filed is developed.It is existing
The subject matter for being improved the technology presence of recovery ratio is that increase rate is limited, and substantial amounts of Residual petroleum is detained underground.And how
Turn into an important topic again using the oil largely remained in oil reservoir.By microbial degradation oil methane phase, so that right
Residual petroleum is utilized, as an effective way.Microbiological anaerobic degraded oil methane phase process is that a variety of floras are participated in
Multi-step reaction, in whole degradation process, influence reaction rate factor it is very many.
Experiment in oil reservoir also demonstrate that in organic matter (particularly Residual petroleum) degradation process, it is recognized that degraded terminal
Product is methane, and it is basis that methane is produced that electron acceptor, which exhausts, specifically the ancient bacterium of methane phase and other functional floras association
Same-action, finally makes Residual petroleum be degraded and produces methane.And geological research is also indicated that, for thousands of years, oil reservoir
Inside is spontaneous always to carry out microbial degradation oil methane phase, because the anaerobic environment of earth interior is conducive to this process
Carry out.
There are some researches show：Micropopulation in oil reservoir is by micro- aerobic flora and amphimicrobian type and strictly anaerobic type
Bacterium composition, hydro carbons provides the basic condition that they survive, and its possible cause is the exploitation with oil reservoir, can largely be injected
Water and other materials, this changes the original environment of oil reservoir.
At present, also without a kind of simple and effective, the method appearance of screening anaerobic degradation petroleum hydrocarbon methanogen floras.
The content of the invention
In order to solve the above-mentioned technical problem, the invention provides a kind of screening side of anaerobic degradation petroleum hydrocarbon methanogen floras
Method, the present invention carries out Anaerobic culturel to flora therein, is being added without carbon source using oil field mining liquid and oily sludge as sample
In the case of, flora can only be by the use of petroleum hydrocarbon as carbon source, and then cultivates to filter out the bacterium of carbon source can be used as by the use of petroleum hydrocarbon
Group, while in order to obtain the decomposing petroleum hydrocarbon mixed bacterial that adaptability is stronger；In addition, the present invention simultaneously using oil field mining liquid and
Oily sludge is as sample, and it is big according to the different attribute of flora in oil field mining liquid and oily sludge to filter out gas production respectively
In 0.4 μm of ol/d and 1.15 μm of ol/d sample, and then it is mixed, obtains being capable of the mixing of decomposing petroleum hydrocarbon mixed bacterial
Thing；Finally, the present invention also contemplates degradation rate to petroleum hydrocarbon simultaneously, so that it is guaranteed that the mixed bacterial finally given have it is outstanding
Degradation capability.
The technical scheme is that, a kind of screening technique of anaerobic degradation petroleum hydrocarbon methanogen floras, including following step
Suddenly：
S1, sampling：Oil field mining liquid and oily sludge are gathered from the different zones in oil field respectively, sample is used as；
S2, Anaerobic culturel：By the oil field collection liquid and oily sludge respectively according to anaerobic condition culture；
S3, cultivation results are determined：After a period of time, the methane production and petroleum hydrocarbon of measurement oil field mining liquid and oily sludge
Degradation rate；
S4, strain mixing：Gas production is more than gas production in 0.4 μm of ol/d and oily sludge in screening oil field collection liquid respectively
Two maximum samples of more than 1.15 μm ol/d petrochina alkane degradations rates, take nutrient solution respectively as optimal oil field collection liquid culture
Liquid and optimal oily sludge nutrient solution, and optimal oil field collection liquid nutrient solution and optimal oily sludge nutrient solution are mixed,
Obtain the mixture containing decomposing petroleum hydrocarbon mixed bacterial.
The screening technique carries out Anaerobic culturel using oil field mining liquid and oily sludge as sample to flora therein,
In the case where being added without carbon source, flora can only be by the use of petroleum hydrocarbon as carbon source, and then cultivates to filter out and can utilize petroleum hydrocarbon
As the flora of carbon source, while in order to obtain the decomposing petroleum hydrocarbon mixed bacterial that adaptability is stronger；In addition, the present invention is used simultaneously
Oil field mining liquid and oily sludge are sieved respectively as sample, and according to the different attribute of flora in oil field mining liquid and oily sludge
Sample of the gas production more than 0.4 μm of ol/d and 1.15 μm of ol/d is selected, and then is mixed, obtaining being capable of decomposing petroleum hydrocarbon mixing
The mixture of flora；Finally, the present invention also contemplates the degradation rate to petroleum hydrocarbon simultaneously, so that it is guaranteed that the Mixed Microbes finally given
Group has outstanding degradation capability.
Reuse the mixture or therefrom extract flora, carry out the anaerobic degradation of petroleum hydrocarbon to produce methane.
To ensure sample quality and diversity, it is preferred that in step S1, the oil density of the oil field mining liquid is
0.87-0.93g/cm3, viscosity of crude is 61.6-169.3mPa s, gathers after the oil field mining liquid, is protected using nitrogen-sealed
Deposit；Take away from below ground 30cm by the oily sludge that the oil content of oil pollution is 5.8-11.3%, detest after sampling at -5 DEG C
Oxygen is preserved.
Different zones in oil field gather oil field mining liquid or oily sludge, and by different samples respectively according to anaerobism bar
Part culture；Anaerobic culturel is：30-40g oil field mining liquid or oily sludge, and 60mL inorganic salts enriched mediums is taken to be filled into
In 120mL aseptic bottles, enrichment culture terminates until that can detect stable methane production, culture in blake bottle at 30 DEG C.
In the present invention, the composition and content of the inorganic salts enriched medium used are：KH2PO45.0g,
K2HPO45.0g, NH4Cl 5.0g, NaCl 1.0g, MgCl22.0g, CaCl20.1g, dusty yeast 0.5g, L-cysteine hydrochloride
0.5g, iron ammonium sulfate 0.5g and resazurin 1.0mg, add water and are settled to 1L.
The need for the inorganic salts enriched medium disclosure satisfy that flora growth, it is ensured that the normal growth of flora.
To make the more preferable of flora growth, it is preferred that also include the vitamin aqueous solution in the inorganic salts enriched medium
The composition and concentration of vitamin are in 5mL, the vitamin aqueous solution：Biotin 2.0mg/L, FA 2.0mg/L,
Vitamin B6 10mg/L, vitamin B1 5.0mg/L, vitamin B2 5.0mg/L, vitamin B12 0.1mg/L, lipoic acid
5.0mg/L。
Further, trace element water-soluble liquid 5mL, the micro- water are also included in the inorganic salts enriched medium
Composition and concentration in solution are：NTA 1.5g/L, cobalt chloride 0.1g/L, anhydrous Manganese chloride 0.1g/L, protochloride
Iron 0.1g/L, CoCL2 6H2O 0.18g/L, zinc chloride 0.1g/L, cupric sulfate pentahydrate 0.01g/L, Potassium aluminum sulfate dodecahydrate
0.02g/L, boric acid 0.01g/L, two water sodium molybdate 0.01g/L, six water nickel chloride 0.025g/L.
The compound method of the inorganic salts enriched medium is：Will be in culture medium except resazurin, the vitamin aqueous solution and micro-
Other components mixing outside the secondary element aqueous solution, heating is constantly passed through nitrogen while boiling, until culture medium is become by pink colour
For colourless, stopping heating, after after system cooling, the vitamin aqueous solution and trace element water-soluble liquid are added, is added finally after sterilizing
Nine water vulcanized sodium that mass ratio is 0.03%, final mass are than the sodium acid carbonate for 0.02%, and resazurin, sealing preserve.
Wherein, resazurin needs matching while using.
Because the viscosity and concentration of oily sludge are larger, for ease of follow-up Anaerobic culturel, it is preferred that to the oil-containing
Sludge is carried out before Anaerobic culturel, and the inorganic salts enriched medium that 10 times of weight is added into the oily sludge carries out primary training
Support 6-8 hours, primary culture takes supernatant to proceed Anaerobic culturel after terminating.
In the present invention, the methane production of generation is measured using chromatograph, wherein FID temperature is 200 DEG C, gasification
The temperature of injector is 150 DEG C；The initial temperature of chromatographic column is 35 DEG C, is warming up to after keeping 15min with 5 DEG C/min programming rate
200 DEG C, and 5min is kept, gas is collected above sample using gas collector during sampling, is then taken again with sealing injection device
Sample and sample introduction, sampling volume are 0.3mL.
In the present invention, the assay method of petroleum hydrocarbon degradation rate is：Oil is surveyed using infrared spectroscopy to the petroleum hydrocarbon before and after degraded
Instrument is tested, and oil hydrocarbon content and change before and after degraded is conversed, so as to obtain petroleum hydrocarbon degradation rate.
Infrared spectrometric oil detector test process is：Nutrient solution is transferred in separatory funnel first, be then acidified to pH≤
2, use CCl4Washing three times, is added after NaCl vibrations, stratification, is removed layer extract and is added the glass core containing sodium sulphate
In funnel, and use vavuum pump suction filtration；Remove layer extract to be added in the same glass sand core funnel containing sodium sulphate, be used in combination
Filtrate in sand core funnel, is then transferred in volumetric flask, uses CCl by vavuum pump suction filtration4After constant volume dilution, infrared spectroscopy is used
Oil content analyzer determines oil hydrocarbon content.
In order to obtain the hereditary information of strain in flora, it is preferred that in step S4, after sample is mixed, to containing
The mixture of decomposing petroleum hydrocarbon mixed bacterial carries out PCR-DGGE analyses, to obtain the hereditary information of wherein flora.
Enter performing PCR reaction in the PCR-DGGE using 341F-GC and 534R as primer sets.
The primer sequence of the 341F-GC is：
5′-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGCCTACGGGAGGCAGCAG-3′；
The primer sequence of the 534R is：5′-ATTACCGCGGCTGCTGG-3′；
The reaction condition of PCR reaction is：First in 94 DEG C of pre-degeneration 5min；Then 30 circulations, each circulation are carried out
Include 94 DEG C of denaturation 1min, 55 DEG C of annealing 1min, 72 DEG C of extension 1min, after the completion of 30 circulations, last 72 DEG C of extensions successively
10min。
The reaction system of PCR reaction is：The μ L of 2 × Taq PCR MasterMix 10, the μ L of template DNA 0.5,
10nmol/L preceding primer 341F-GC 0.5 μ L, 10nmol/L the μ L of rear primer 534R 0.5 and the μ L of distilled water 8.5.
In order to verify that the gas finally produced produces for the flora in oil field collection liquid or oily sludge, it is preferred that described
Screening technique is also verified including the use of sterilizing group to cultivation results：Trained using the sample after sterilizing according to the anaerobic condition
Support, if the sample after final sterilization is produced after anaerobic condition culture without methane, prove methane gas by the flora in sample
Produce.If if the sample after final sterilization has methane generation after anaerobic condition culture, proving methane gas not entirely by sample
Flora in product is produced, and now needs to use again, repeats above step, mixed containing decomposing petroleum hydrocarbon mixed bacterial to obtain
Compound.
Beneficial effects of the present invention are：The screening technique is using oil field mining liquid and oily sludge as sample, to wherein
Flora carry out Anaerobic culturel, in the case where being added without carbon source, flora can only be using petroleum hydrocarbon as carbon source, and then cultivates and sieve
Selecting can be by the use of petroleum hydrocarbon as the flora of carbon source, while in order to obtain the decomposing petroleum hydrocarbon mixed bacterial that adaptability is stronger；
In addition, the present invention uses oil field mining liquid and oily sludge as sample simultaneously, and according in oil field mining liquid and oily sludge
The different attribute of flora filters out sample of the gas production more than 0.4 μm of ol/d and 1.15 μm of ol/d respectively, and then is mixed, and obtains
To the mixture for being capable of decomposing petroleum hydrocarbon mixed bacterial；Finally, the present invention also contemplates the degradation rate to petroleum hydrocarbon simultaneously, so that
Ensure that the mixed bacterial finally given has outstanding degradation capability.
Embodiment
To make the object, technical solutions and advantages of the present invention clearer, technical scheme will be carried out below
Detailed description.Obviously, described embodiment is only a part of embodiment of the invention, rather than whole embodiments.Base
Embodiment in the present invention, those of ordinary skill in the art are resulting on the premise of creative work is not made to be owned
Other embodiment, belongs to the scope that the present invention is protected.
Embodiment 1
A kind of screening technique of anaerobic degradation petroleum hydrocarbon methanogen floras, comprises the following steps：
S1, sampling：Oil field mining liquid and oily sludge are gathered from the different zones in oil field respectively, sample is used as；
In this step, six mouthfuls of oil well produced liquids investigation sampling of certain oil field block is gathered, several Plastic Drums are filled (about
5L), nitrogen-sealed is passed through before sampling, laboratory, Cord blood is transported back rapidly after sampling, numbering is YH1-YH6；Take 4 parts away from
Below ground 30cm is by the oily sludge that the oil content of oil pollution is 5.8-11.3%, and anaerobism is preserved at -5 DEG C after sampling,
Numbering is TH1-TH6 respectively；Wherein, YH1-YH6 physicochemical property is as shown in table 1.
Table 1：YH1-YH6 physicochemical property
Note 1：YH represents Anaerobic culturel oil field mining liquid, and numbering below is sampling ground oil well numbering
S2, Anaerobic culturel：By the oil field collection liquid and oily sludge respectively according to anaerobic condition culture；
By different samples respectively according to anaerobic condition culture, Anaerobic culturel is：Take 30-40g oil field mining liquid or contain
Greasy dirt, and 60mL inorganic salts enriched mediums are filled into 120mL aseptic bottles, and enrichment culture is until energy in blake bottle at 30 DEG C
Stable methane production is detected, culture terminates.
The composition and content of the inorganic salts enriched medium used be：KH2PO45.0g, K2HPO45.0g, NH4Cl
5.0g, NaCl 1.0g, MgCl22.0g, CaCl20.1g, dusty yeast 0.5g, L-cysteine hydrochloride 0.5g, iron ammonium sulfate
0.5g and resazurin 1.0mg, adds water and is settled to 1L.
Also include vitamin in vitamin aqueous solution 5mL, the vitamin aqueous solution in the inorganic salts enriched medium
Composition and concentration be：Biotin 2.0mg/L, FA 2.0mg/L, vitamin B6 10mg/L, vitamin B1
5.0mg/L, vitamin B2 5.0mg/L, vitamin B12 0.1mg/L, lipoic acid 5.0mg/L.
Also include in the inorganic salts enriched medium in trace element water-soluble liquid 5mL, the trace element water-soluble liquid
Composition and concentration are：NTA 1.5g/L, cobalt chloride 0.1g/L, anhydrous Manganese chloride 0.1g/L, frerrous chloride 0.1g/L,
CoCL2 6H2O 0.18g/L, zinc chloride 0.1g/L, cupric sulfate pentahydrate 0.01g/L, Potassium aluminum sulfate dodecahydrate 0.02g/L, boric acid
0.01g/L, two water sodium molybdate 0.01g/L, six water nickel chloride 0.025g/L.
Other components in culture medium in addition to resazurin, the vitamin aqueous solution and trace element water-soluble liquid are mixed, plus
Nitrogen constantly is passed through while heat is boiled, until culture medium is become colorless by pink colour, stops heating, after after system cooling, is added
The vitamin aqueous solution and trace element water-soluble liquid, add final mass than the nine water vulcanized sodium for 0.03%, final matter after sterilizing
Amount is than the sodium acid carbonate for 0.02%, and resazurin, sealing preserve.Wherein, resazurin needs matching while using.
Because the viscosity and concentration of oily sludge are larger, for ease of follow-up Anaerobic culturel, enter to the oily sludge
Before row Anaerobic culturel, the inorganic salts enriched medium that 10 times of weight is added into the oily sludge carries out primary culture 6-8
Hour, primary culture takes supernatant to proceed Anaerobic culturel after terminating.
S3, cultivation results are determined：After culture 400 days, the methane production and oil of measurement oil field mining liquid and oily sludge
Alkane degradation rate；
In the present invention, the methane production of generation is measured using chromatograph, wherein FID temperature is 200 DEG C, gasification
The temperature of injector is 150 DEG C；The initial temperature of chromatographic column is 35 DEG C, is warming up to after keeping 15min with 5 DEG C/min programming rate
200 DEG C, and 5min is kept, gas is collected above sample using gas collector during sampling, is then taken again with sealing injection device
Sample and sample introduction, sampling volume are 0.3mL.
Specifically, the concrete operations of the aerogenesis measure of methane are in the present embodiment：
Gas component：Agilent gas chromatograph.Detector：FID 200℃；Gasify 150 DEG C of injector；Chromatographic column：
PONA elastic quartzs capillary column (50m × 0.2mm × 0.5 μm)；Column temperature：35 DEG C of initial temperature, 15min；5 DEG C/min is warming up to 200 DEG C,
5min.Gas first is collected from top with gas collector during sampling, is then sampled again with sealing injection device, sampling volume：
0.3mL, is diluted to various concentrations with high pure nitrogen by calibrating gas, is analyzed under these conditions, the area normalization of amendment
Standard measure gas content.
In the present embodiment, the gas production of each sample is as shown in Table 2 below
Table 2：The gas production result table of each sample
| Sample number into spectrum | YH1 | YH2 | YH3 | YH4 | YH5 | YH6 |
| Gas production (μm ol) | 70.5 | 99.2 | 61.7 | 108.3 | 246.9 | 0 |
| Sample number into spectrum | TH1 | TH2 | TH3 | TH4 | TH5 | TH6 |
| Gas production (μm ol) | 68.5 | 466 | 25.6 | 92.5 | 60.7 | 30.8 |
In the present embodiment, the assay method of petroleum hydrocarbon degradation rate is：Petroleum hydrocarbon before and after degraded is surveyed using infrared spectroscopy
Oily instrument test, converses oil hydrocarbon content and change before and after degraded, so as to obtain petroleum hydrocarbon degradation rate.
Infrared spectrometric oil detector test process is：Nutrient solution is transferred in separatory funnel first, be then acidified to pH≤
2, use CCl4Washing three times, is added after NaCl vibrations, stratification, is removed layer extract and is added the glass core containing sodium sulphate
In funnel, and use vavuum pump suction filtration；Remove layer extract to be added in the same glass sand core funnel containing sodium sulphate, be used in combination
Filtrate in sand core funnel, is then transferred in volumetric flask, uses CCl by vavuum pump suction filtration4After constant volume dilution, infrared spectroscopy is used
Oil content analyzer determines oil hydrocarbon content.
Through measurement, the petroleum hydrocarbon degradation rate of each sample is as shown in Table 3 below
Table 3：The gas production result table of each sample
| Sample number into spectrum | YH1 | YH2 | YH3 | YH4 | YH5 | YH6 |
| Degradation rate (%) | 10.1 | 15.4 | 9.1 | 14.3 | 20.1 | 0 |
| Sample number into spectrum | TH1 | TH2 | TH3 | TH4 | TH5 | TH6 |
| Degradation rate (%) | 13.4 | 23.3 | 6.9 | 11.1 | 11.2 | 5.4 |
S4：Strain is mixed：Gas production is more than gas production in 0.4 μm of ol/d and oily sludge and is more than in screening oil field collection liquid
1.15 μm of ol/d sample, takes nutrient solution respectively as optimal oil field collection liquid nutrient solution and optimal oily sludge nutrient solution, and
Optimal oil field collection liquid nutrient solution and optimal oily sludge nutrient solution are mixed, that is, obtained containing decomposing petroleum hydrocarbon Mixed Microbes
The mixture of group.
It can be learnt from table 2, the gas production of YH5 and TH2 not only methane is maximum, while the degradation rate of petroleum hydrocarbon is also most
Height, meets the requirements, so YH5 and TH2 is respectively as optimal oil field collection liquid nutrient solution and optimal oily sludge nutrient solution, will
YH5 and TH2 nutrient solution is mixed, that is, obtains the mixture containing decomposing petroleum hydrocarbon mixed bacterial.
It is that the present embodiment uses Anaerobic culturel the reason for possible it should be noted that not detecting methane generation in YH6, and
Aerobic bacteria may be contained in YH6, the present embodiment does not carry out sterilization treatment to YH6.
In order to verify that the gas finally produced produces for the flora in oil field collection liquid or oily sludge, the screening technique
Also cultivation results are verified including the use of sterilizing group：Using the sample after sterilizing according to the anaerobic condition culture, as a result
It was found that, either oil field collection liquid or oily sludge, the amount of its methane finally produced is 0, thus, it is possible to draw, this reality
Apply in example, methane gas is produced by the flora in sample, and using the method in the present embodiment, obtaining being capable of degraded oil
The mixture of hydrocarbon mixed bacterial.
Embodiment 2
The present embodiment is that the mixture containing decomposing petroleum hydrocarbon mixed bacterial that embodiment 1 is obtained is entered using PCR-DGGE
Row analysis, to obtain the hereditary information of wherein flora.
The primer sequence of the 341F-GC is：
5′-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGCCTACGGGAGGCAGCAG-3′；
The primer sequence of the 534R is：5′-ATTACCGCGGCTGCTGG-3′；
The reaction condition of PCR reaction is：First in 94 DEG C of pre-degeneration 5min；Then 30 circulations, each circulation are carried out
Include 94 DEG C of denaturation 1min, 55 DEG C of annealing 1min, 72 DEG C of extension 1min, after the completion of 30 circulations, last 72 DEG C of extensions successively
10min。
The reaction system of PCR reaction is：The μ L of 2 × Taq PCR MasterMix 10, the μ L of template DNA 0.5,
10nmol/L preceding primer 341F-GC 0.5 μ L, 10hmol/L the μ L of rear primer 534R 0.5 and the μ L of distilled water 8.5.
As a result it is as shown in table 4
Table 4：YH5 and TH2 bacterial quorum sensing initial analyses
According to information above, not only initial analysis has gone out the substantially composition of flora, gives when configuring artificial flora
Technological guidance.
The foregoing is only a specific embodiment of the invention, but protection scope of the present invention is not limited thereto, any
Those familiar with the art the invention discloses technical scope in, change or replacement can be readily occurred in, should all be contained
Cover within protection scope of the present invention.Therefore, protection scope of the present invention should be based on the protection scope of the described claims.
Claims (10)
1. a kind of screening technique of anaerobic degradation petroleum hydrocarbon methanogen floras, it is characterised in that comprise the following steps：
S1, sampling：Oil field mining liquid and oily sludge are gathered from the different zones in oil field respectively, sample is used as；
S2, Anaerobic culturel：By the oil field collection liquid and oily sludge respectively according to anaerobic condition culture；
S3, cultivation results are determined：After a period of time, the methane production and petroleum hydrocarbon degradation of measurement oil field mining liquid and oily sludge
Rate；
S4：Strain is mixed：Gas production is more than more than gas production in 0.4 μm of ol/d and oily sludge in screening oil field collection liquid respectively
Two maximum samples of 1.15 μm of ol/d petrochina alkane degradations rates, take nutrient solution respectively as optimal oil field collection liquid nutrient solution and
Optimal oily sludge nutrient solution, and optimal oil field collection liquid nutrient solution and optimal oily sludge nutrient solution are mixed, produce
To the mixture containing decomposing petroleum hydrocarbon mixed bacterial.
2. screening technique according to claim 1, it is characterised in that in step S1, the crude oil of the oil field mining liquid is close
Spend for 0.87-0.93g/cm3, viscosity of crude is 61.6-169.3mPa s, is gathered after the oil field mining liquid, close using nitrogen
Envelope is preserved；Take away from below ground 30cm by the oily sludge that the oil content of oil pollution is 5.8-11.3%, at -5 DEG C after sampling
Lower anaerobism is preserved.
3. screening technique according to claim 1, it is characterised in that surveyed using chromatograph to the methane production of generation
Fixed, wherein FID temperature is 200 DEG C, and the temperature of gasification injector is 150 DEG C；The initial temperature of chromatographic column is 35 DEG C, keeps 15min
200 DEG C are warming up to 5 DEG C/min programming rate afterwards, and keeps 5min, is received during sampling using gas collector above sample
Collect gas, then sampled and sample introduction with sealing injection device again, sampling volume is 0.3mL.
4. screening technique according to claim 1, it is characterised in that the assay method of petroleum hydrocarbon degradation rate is：To degraded
Front and rear petroleum hydrocarbon is tested using infrared spectrometric oil detector, oil hydrocarbon content and change before and after degraded is conversed, so as to obtain stone
Petroleum hydrocarbon degradation rate.
5. screening technique according to claim 4, it is characterised in that infrared spectrometric oil detector test process is：First will
Nutrient solution is transferred in separatory funnel, is then acidified to pH≤2, uses CCl4Washing three times, adds NaCl vibrations, stratification
Afterwards, remove layer extract to add in the glass sand core funnel containing sodium sulphate, and use vavuum pump suction filtration；Layer extract is removed to add
Into the same glass sand core funnel containing sodium sulphate, and vavuum pump suction filtration is used, then shift the filtrate in sand core funnel
Into volumetric flask, CCl is used4After constant volume dilution, oil hydrocarbon content is determined with infrared spectrometric oil detector.
6. screening technique according to claim 1, it is characterised in that after sample is mixed, to containing degraded oil
The mixture of hydrocarbon mixed bacterial carries out PCR-DGGE analyses, to obtain the hereditary information of wherein flora.
7. screening technique according to claim 6, it is characterised in that 341F-GC and 534R is used in the PCR-DGGE
Enter performing PCR reaction as primer sets.
8. screening technique according to claim 7, it is characterised in that the reaction condition of the PCR reactions is：First at 94 DEG C
Pre-degeneration 5min；Then 30 circulations are carried out, each circulation includes 94 DEG C of denaturation 1min, 55 DEG C of annealing 1min successively, and 72 DEG C are prolonged
1min is stretched, after the completion of 30 circulations, last 72 DEG C of extensions 10min.
9. screening technique according to claim 8, it is characterised in that the reaction system of the PCR reactions is：2×Taq
Draw after the μ L of PCR MasterMix 10, template DNA 0.5 μ L, 10nmol/L preceding primer 341F-GC 0.5 μ L, 10nmol/L
The μ L of the thing 534R 0.5 and μ L of distilled water 8.5.
10. screening technique according to claim 1, it is characterised in that also cultivation results are carried out including the use of sterilizing group
Checking：Using the sample after sterilizing according to the anaerobic condition culture, if the sample after final sterilization is after anaerobic condition culture
Produced without methane, then prove that methane gas is produced by the flora in sample.
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