CN106929419A - 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 31
- 238000000034 method Methods 0.000 title claims abstract description 28
- 238000012216 screening Methods 0.000 title claims abstract description 23
- 238000006731 degradation reaction Methods 0.000 title claims abstract description 18
- 230000015556 catabolic process Effects 0.000 title claims abstract description 16
- 239000007788 liquid Substances 0.000 claims abstract description 68
- 239000010802 sludge Substances 0.000 claims abstract description 50
- 235000015097 nutrients Nutrition 0.000 claims abstract description 34
- 238000004519 manufacturing process Methods 0.000 claims abstract description 28
- 238000005065 mining Methods 0.000 claims abstract description 24
- 239000000203 mixture Substances 0.000 claims abstract description 24
- 230000001580 bacterial effect Effects 0.000 claims abstract description 17
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 claims description 72
- 239000000243 solution Substances 0.000 claims description 33
- 239000007789 gas Substances 0.000 claims description 31
- 150000003839 salts Chemical class 0.000 claims description 27
- 239000002609 medium Substances 0.000 claims description 23
- 229940088594 vitamin Drugs 0.000 claims description 21
- 229930003231 vitamin Natural products 0.000 claims description 21
- 235000013343 vitamin Nutrition 0.000 claims description 21
- 239000011782 vitamin Substances 0.000 claims description 21
- 150000003722 vitamin derivatives Chemical class 0.000 claims description 21
- 239000007864 aqueous solution Substances 0.000 claims description 20
- 239000011573 trace mineral Substances 0.000 claims description 19
- 235000013619 trace mineral Nutrition 0.000 claims description 19
- PLXBWHJQWKZRKG-UHFFFAOYSA-N Resazurin Chemical compound C1=CC(=O)C=C2OC3=CC(O)=CC=C3[N+]([O-])=C21 PLXBWHJQWKZRKG-UHFFFAOYSA-N 0.000 claims description 17
- 238000005070 sampling Methods 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 230000001954 sterilising effect Effects 0.000 claims description 14
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 12
- 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 claims description 10
- 239000001963 growth medium Substances 0.000 claims description 10
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 claims description 10
- 238000010438 heat treatment Methods 0.000 claims description 9
- 238000007789 sealing Methods 0.000 claims description 9
- 229910052757 nitrogen Inorganic materials 0.000 claims description 6
- 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 claims description 5
- 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 claims description 5
- 239000007836 KH2PO4 Substances 0.000 claims description 5
- 229910021380 Manganese Chloride Inorganic materials 0.000 claims description 5
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 claims description 5
- 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 claims description 5
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 5
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 5
- 229930003451 Vitamin B1 Natural products 0.000 claims description 5
- 229930003471 Vitamin B2 Natural products 0.000 claims description 5
- 229960002685 biotin Drugs 0.000 claims description 5
- 235000020958 biotin Nutrition 0.000 claims description 5
- 239000011616 biotin Substances 0.000 claims description 5
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 5
- 239000004327 boric acid Substances 0.000 claims description 5
- 239000001110 calcium chloride Substances 0.000 claims description 5
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 5
- 238000001816 cooling Methods 0.000 claims description 5
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 5
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 5
- AGBQKNBQESQNJD-UHFFFAOYSA-M lipoate Chemical compound [O-]C(=O)CCCCC1CCSS1 AGBQKNBQESQNJD-UHFFFAOYSA-M 0.000 claims description 5
- 235000019136 lipoic acid Nutrition 0.000 claims description 5
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L magnesium chloride Substances [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 5
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 5
- 239000011565 manganese chloride Substances 0.000 claims description 5
- 235000002867 manganese chloride Nutrition 0.000 claims description 5
- 229940099607 manganese chloride Drugs 0.000 claims description 5
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 5
- SPIFDSWFDKNERT-UHFFFAOYSA-N nickel;hydrate Chemical compound O.[Ni] SPIFDSWFDKNERT-UHFFFAOYSA-N 0.000 claims description 5
- 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 claims description 5
- 229960002477 riboflavin Drugs 0.000 claims description 5
- 239000011734 sodium Substances 0.000 claims description 5
- 229910052708 sodium Inorganic materials 0.000 claims description 5
- 239000011684 sodium molybdate Substances 0.000 claims description 5
- 235000015393 sodium molybdate Nutrition 0.000 claims description 5
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 229960003495 thiamine Drugs 0.000 claims description 5
- 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 claims description 5
- 229960002663 thioctic acid Drugs 0.000 claims description 5
- 235000010374 vitamin B1 Nutrition 0.000 claims description 5
- 239000011691 vitamin B1 Substances 0.000 claims description 5
- 235000019164 vitamin B2 Nutrition 0.000 claims description 5
- 239000011716 vitamin B2 Substances 0.000 claims description 5
- 229940084600 vitamin b 12 0.1 mg Drugs 0.000 claims description 5
- 229940023356 vitamin b6 10 mg Drugs 0.000 claims description 5
- 239000011592 zinc chloride Substances 0.000 claims description 5
- 235000005074 zinc chloride Nutrition 0.000 claims description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 claims description 4
- VLSOAXRVHARBEQ-UHFFFAOYSA-N [4-fluoro-2-(hydroxymethyl)phenyl]methanol Chemical compound OCC1=CC=C(F)C=C1CO VLSOAXRVHARBEQ-UHFFFAOYSA-N 0.000 claims description 4
- 238000009835 boiling Methods 0.000 claims description 4
- 238000002347 injection Methods 0.000 claims description 4
- 239000007924 injection Substances 0.000 claims description 4
- 238000010792 warming Methods 0.000 claims description 4
- 238000005259 measurement Methods 0.000 claims description 3
- 239000000047 product Substances 0.000 claims description 3
- 238000004659 sterilization and disinfection Methods 0.000 claims description 3
- ZLXPLDLEBORRPT-UHFFFAOYSA-M [NH4+].[Fe+].[O-]S([O-])(=O)=O Chemical compound [NH4+].[Fe+].[O-]S([O-])(=O)=O ZLXPLDLEBORRPT-UHFFFAOYSA-M 0.000 claims description 2
- 229930003761 Vitamin B9 Natural products 0.000 claims 1
- 238000005660 chlorination reaction Methods 0.000 claims 1
- 239000010941 cobalt Substances 0.000 claims 1
- 229910017052 cobalt Inorganic materials 0.000 claims 1
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 claims 1
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 claims 1
- 238000002360 preparation method Methods 0.000 claims 1
- 235000019159 vitamin B9 Nutrition 0.000 claims 1
- 239000011727 vitamin B9 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
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 239000003208 petroleum Substances 0.000 description 5
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 4
- 229910021586 Nickel(II) chloride Inorganic materials 0.000 description 4
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 4
- 229930195733 hydrocarbon Natural products 0.000 description 4
- 150000002430 hydrocarbons Chemical class 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 239000004215 Carbon black (E152) Substances 0.000 description 3
- 238000002309 gasification Methods 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000011790 ferrous sulphate Substances 0.000 description 2
- 235000003891 ferrous sulphate Nutrition 0.000 description 2
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 2
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 239000003129 oil well Substances 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 239000005864 Sulphur Substances 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
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 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
- 239000000463 material Substances 0.000 description 1
- 239000013028 medium composition 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
- 235000012239 silicon dioxide Nutrition 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000012549 training Methods 0.000 description 1
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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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a kind of screening technique of anaerobic degradation petroleum hydrocarbon methanogen floras, the present invention is using oil field mining liquid and oily sludge as sample, Anaerobic culturel is carried out to flora therein, in the case where carbon source is added without, flora can only be by the use of petroleum hydrocarbon as carbon source, and then cultivate that filter out can be by the use of petroleum hydrocarbon as the flora of carbon source, while the decomposing petroleum hydrocarbon mixed bacterial stronger in order to obtain adaptability；In addition, the present invention uses oil field mining liquid and oily sludge as sample simultaneously, and gas production is filtered out more than 0.4 μm of ol/d and 1.15 μm of sample of ol/d 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 mixed proportion of optimal oil field collection liquid nutrient solution and optimal oily sludge nutrient solution simultaneously, so that it is guaranteed that the mixed bacterial for finally giving 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, improving oil recovery has turned into the central task of current old filed exploitation.It is existing
The subject matter for being improved the technology presence of recovery ratio is limited increase rate, 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 various 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 electron acceptor exhaust be methane produce basis, specifically methane phase Gu bacterium and other functional floras assist
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, with the exploitation of oil reservoir, largely to inject
Water and other materials, this changes the original environment of oil reservoir.
At present, also the method appearance of anaerobic degradation petroleum hydrocarbon methanogen floras is screened without a kind of simple and effective.
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 using oil field mining liquid and oily sludge as sample to flora therein, is being added without carbon source
In the case of, flora by the use of petroleum hydrocarbon as carbon source, and then can only cultivate that filter out can be by the use of petroleum hydrocarbon as the bacterium of carbon source
Group, while the decomposing petroleum hydrocarbon mixed bacterial stronger in order to obtain adaptability；In addition, the present invention simultaneously using oil field mining liquid and
Oily sludge is used as sample, and it is big to filter out gas production respectively according to the different attribute of flora in oil field mining liquid and oily sludge
In 0.4 μm of ol/d and 1.15 μm of sample of ol/d, and then it is mixed, obtains being capable of the mixing of decomposing petroleum hydrocarbon mixed bacterial
Thing；Finally, the present invention also contemplates the mixed proportion of optimal oil field collection liquid nutrient solution and optimal oily sludge nutrient solution simultaneously,
So that it is guaranteed that the mixed bacterial for finally giving has 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, as sample；
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 of measurement oil field mining liquid and oily sludge；
S4：Strain mixes：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
1.15 μm of samples of ol/d, 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, by optimal oil field collection liquid nutrient solution and most
Excellent oily sludge nutrient solution is mixed by 1: 1,1: 2,1: 3,1: 5 and 2: 1,3: 1,5: 1 of volume ratio respectively, used as sample point
Not according to anaerobic condition culture, methane production is measured, filter out methane yield maximum nutrient solution, that is, obtain containing degraded oil
The mixture of 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 carbon source is added without, flora can only be filtered out and can utilize petroleum hydrocarbon by the use of petroleum hydrocarbon as carbon source, and then cultivation
As the flora of carbon source, while the decomposing petroleum hydrocarbon mixed bacterial stronger in order to obtain adaptability；In addition, the present invention is used simultaneously
Oil field mining liquid and oily sludge are sieved respectively as sample according to the different attribute of flora in oil field mining liquid and oily sludge
Gas production is selected more than 0.4 μm of ol/d and 1.15 μm of sample of ol/d, and then is mixed, obtaining being capable of decomposing petroleum hydrocarbon mixing
The mixture of flora；Finally, the present invention also contemplates optimal oil field collection liquid nutrient solution and optimal oily sludge nutrient solution simultaneously
Mixed proportion, so that it is guaranteed that the mixed bacterial for finally giving has outstanding degradation capability.
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, after gathering 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 at -5 DEG C after sampling
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：The oil field mining liquid or oily sludge of 30-40g are taken, and 60mL the first inorganic salts enriched mediums fill
Enter 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 first inorganic salts enriched medium for using are：KH2PO45.0g,
K2HPO45.0g, NH4Cl 5.0g, NaCl 1.0g, MgCl22.0g, CaCl20.1g, dusty yeast 0.5g, Cys salt
Hydrochlorate 0.5g, iron ammonium sulfate 0.5g and resazurin 1.0mg, add water and are settled to 1L.
The need for the first 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 that vitamin is water-soluble in the first inorganic salts enriched medium
Liquid 5mL, the composition and concentration of vitamin are in 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 unit are also included in the first inorganic salts enriched medium
Composition and concentration in the plain aqueous solution are：NTA 1.5g/L, cobalt chloride 0.1g/L, anhydrous Manganese chloride 0.1g/L, chlorine
Change ferrous 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 first inorganic salts enriched medium is：Resazurin, the vitamin aqueous solution will be removed in culture medium
With the other components mixing outside trace element water-soluble liquid, heating is constantly passed through nitrogen while boiling, until culture medium is by powder
Color becomes colorless, and stops heating, after after system cooling, adds the vitamin aqueous solution and trace element water-soluble liquid, is added after sterilizing
Final mass than the nine water vulcanized sodium for 0.03%, final mass than the sodium acid carbonate for 0.02%, and resazurin, protect by sealing
Deposit.
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
Before sludge carries out Anaerobic culturel, carried out just to the first inorganic salts enriched medium of 10 times of weight is added in the oily sludge
Level culture 6-8 hours, primary culture takes supernatant and proceeds Anaerobic culturel after terminating.
In the present invention, the methane production for generating is measured using chromatograph, wherein the temperature of FID 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 the programming rate of 5 DEG C/min after keeping 15min
200 DEG C, and 5min is kept, gas is collected from sample top using gas collector during sampling, then taken with sealing injection device again
Sample and sample introduction, sampling volume are 0.3mL.
In step S4, the supernatant 15ml by the mixed sample of different proportion is taken, and add the enrichment training of the second inorganic salts
Base 60mL is supported, is cultivated 260 days.
The composition and content of the second inorganic salts enriched medium be：KH2PO45.0g, K2HPO45.0g, NH4Cl
5.0g, NaCl 1.0g, MgCl22.0g, CaCl20.1g, dusty yeast 0.5g, L-cysteine hydrochloride 0.5g, ferrous sulfate
Ammonium 0.5g and resazurin 1.0mg, adds water and is settled to 1L.
Also include vitamin aqueous solution 5mL in the second inorganic salts enriched medium, tieed up in the vitamin aqueous solution
Raw plain composition and concentration are：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 trace element water-soluble liquid 5mL, the trace element water-soluble liquid in the second inorganic salts enriched medium
In composition and concentration be：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.
The compound method of the second inorganic salts enriched medium is：Resazurin, the vitamin aqueous solution will be removed in culture medium
With the other components mixing outside trace element water-soluble liquid, heating is constantly passed through nitrogen while boiling, until culture medium is by powder
Color becomes colorless, and stops heating, after after system cooling, adds the vitamin aqueous solution and trace element water-soluble liquid, is added after sterilizing
Final mass than the nine water vulcanized sodium for 0.03%, final mass than the sodium acid carbonate for 0.02%, and resazurin, protect by sealing
Deposit.
In order to verify the gas of final generation for the flora in oil field collection liquid or oily sludge is produced, it is preferred that described
Screening technique also includes verifying cultivation results using sterilizing group：Trained according to the anaerobic condition using the sample after sterilizing
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 to produce 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.
The mixture containing decomposing petroleum hydrocarbon mixed bacterial is obtained, flora therein is separated, you can obtain anaerobism
The flora of decomposing petroleum hydrocarbon methane phase.
Beneficial effects of the present invention are：The screening technique using oil field mining liquid and oily sludge as sample, to wherein
Flora carry out Anaerobic culturel, in the case where carbon source is added without, flora by the use of petroleum hydrocarbon as carbon source, and then can only cultivate sieve
Selecting can be by the use of petroleum hydrocarbon as the flora of carbon source, while the decomposing petroleum hydrocarbon mixed bacterial stronger in order to obtain adaptability；
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 gas production more than 0.4 μm of ol/d and 1.15 μm of sample 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 optimal oil field collection liquid nutrient solution simultaneously
With the mixed proportion of optimal oily sludge nutrient solution, so that it is guaranteed that the mixed bacterial for finally giving has outstanding degradation capability.
Specific 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, as sample；
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 the oily sludge of 5.8-11.3% by the oil content of oil pollution, and anaerobism is preserved at -5 DEG C after sampling,
Numbering is TH1-TH6 respectively；Wherein, the physicochemical property of YH1-YH6 is as shown in table 1.
Table 1：The physicochemical property of YH1-YH6
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 the oil field mining liquid of 30-40g or contain
Greasy dirt, and 60mL the first inorganic salts enriched mediums are filled into 120mL aseptic bottles, and enrichment culture is until blake bottle at 30 DEG C
The middle methane production that can detect stabilization, culture terminates.
The composition and content of the first inorganic salts enriched medium for using be：KH2PO45.0g, K2HPO45.0g,
NH4Cl 5.0g, NaCl 1.0g, MgCl22.0g, CaCl20.1g, dusty yeast 0.5g, L-cysteine hydrochloride 0.5g, sulphur
Sour ferrous ammonium 0.5g and resazurin 1.0mg, adds water and is settled to 1L.
Also include vitamin aqueous solution 5mL in the first inorganic salts enriched medium, tieed up in the vitamin aqueous solution
Raw plain composition and concentration are：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 trace element water-soluble liquid 5mL, the trace element water-soluble liquid in the first inorganic salts enriched medium
In composition and concentration be：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, added
The vitamin aqueous solution and trace element water-soluble liquid, add final mass than nine water vulcanized sodium, the final matter for 0.03% 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, primary culture is carried out to the first inorganic salts enriched medium of 10 times of weight is added in the oily sludge
6-8 hours, primary culture took supernatant and proceeds Anaerobic culturel after terminating.
S3, cultivation results are determined：After culture 400 days, the methane production of measurement oil field mining liquid and oily sludge；
In the present invention, the methane production for generating is measured using chromatograph, wherein the temperature of FID 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 the programming rate of 5 DEG C/min after keeping 15min
200 DEG C, and 5min is kept, gas is collected from sample top using gas collector during sampling, then taken with sealing injection device again
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℃；150 DEG C of injector of gasification；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 with sealing injection device again, sampling volume：
0.3mL, various concentrations are diluted to by calibrating gas high pure nitrogen, are 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 |
S4：Strain mixes：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
1.15 μm of samples of ol/d, 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, by optimal oil field collection liquid nutrient solution and most
Excellent oily sludge nutrient solution is mixed by 1: 1,1: 2,1: 3,1: 5 and 2: 1,3: 1,5: 1 of volume ratio respectively, used as sample point
Not according to anaerobic condition culture, methane production is measured, filter out methane yield maximum nutrient solution, that is, obtain containing degraded oil
The mixture of hydrocarbon mixed bacterial.
Can be learnt from table 2, YH5 and TH2 meet the requirements, so YH5 and TH2 is trained respectively as optimal oil field collection liquid
Nutrient solution and optimal oily sludge nutrient solution, are respectively 1: 1,1: 2,1: 3,1: 5 and 2: 1,3: 1,5: 1 with volume ratio by YH5 and TH2
Mixed, as sample respectively according to anaerobic condition culture, measured methane production.
The supernatant 15ml by the mixed sample of different proportion is taken, and adds the second inorganic salts enriched medium 60mL,
Culture 260 days.
The composition and content of the second inorganic salts enriched medium be：KH2PO45.0g, K2HPO45.0g, NH4Cl
5.0g, NaCl 1.0g, MgCl22.0g, CaCl20.1g, dusty yeast 0.5g, L-cysteine hydrochloride 0.5g, ferrous sulfate
Ammonium 0.5g and resazurin 1.0mg, adds water and is settled to 1L.
Also include vitamin aqueous solution 5mL in the second inorganic salts enriched medium, tieed up in the vitamin aqueous solution
Raw plain composition and concentration are：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 trace element water-soluble liquid 5mL, the trace element water-soluble liquid in the second inorganic salts enriched medium
In composition and concentration be：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.
The compound method of the second inorganic salts enriched medium is：Resazurin, the vitamin aqueous solution will be removed in culture medium
With the other components mixing outside trace element water-soluble liquid, heating is constantly passed through nitrogen while boiling, until culture medium is by powder
Color becomes colorless, and stops heating, after after system cooling, adds the vitamin aqueous solution and trace element water-soluble liquid, is added after sterilizing
Final mass than the nine water vulcanized sodium for 0.03%, final mass than the sodium acid carbonate for 0.02%, and resazurin, protect by sealing
Deposit.
Take and be shown in Table 3 by the methane production of the mixed sample of different proportion
Table 3：The methane production of the mixed sample of different proportion
From upper table, it can be seen that when YH5 and TH2 volume ratio be 1: 1 when, the generation efficiency highest of methane.
In order to verify the gas of final generation for the flora in oil field collection liquid or oily sludge is produced, the screening technique
Also include verifying cultivation results using 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 final methane for producing are 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.
The above, specific embodiment only of the invention, but protection scope of the present invention is not limited thereto, and it is any
Those familiar with the art the invention discloses technical scope in, change or replacement can be readily occurred in, should all contain
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 (9)
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, as sample；
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 of measurement oil field mining liquid and oily sludge；
S4：Strain mixes：Gas production is more than 1.15 more than gas production in 0.4 μm of ol/d and oily sludge in screening oil field collection liquid
The sample of μm ol/d, takes nutrient solution respectively as optimal oil field collection liquid nutrient solution and optimal oily sludge nutrient solution, and will most
Excellent oil field collection liquid nutrient solution and optimal oily sludge nutrient solution are mixed, and by optimal oil field collection liquid nutrient solution and optimal are contained
Greasy dirt nutrient solution is mixed by 1: 1,1: 2,1: 3,1: 5 and 2: 1,3: 1,5: 1 of volume ratio respectively, is pressed respectively as sample
According to anaerobic condition culture, methane production is measured, filter out methane yield maximum nutrient solution, that is, obtain mixed containing decomposing petroleum hydrocarbon
Close the mixture of flora.
2. screening technique according to claim 1, it is characterised in that in step S4, take by the mixed sample of different proportion
The supernatant 15ml of product, and the second inorganic salts enriched medium 60mL is added, cultivate 260 days.
3. screening technique according to claim 2, it is characterised in that the composition of the second inorganic salts enriched medium and
Content is：KH2PO45.0g, K2HPO45.0g, NH4Cl5.0g, NaCl1.0g, 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.
4. screening technique according to claim 3, it is characterised in that also include in the second inorganic salts enriched medium
Vitamin aqueous solution 5mL, the composition and concentration of vitamin are in the vitamin aqueous solution：Biotin 2.0mg/L, vitamin
B9 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.
5. screening technique according to claim 4, it is characterised in that also include in the second inorganic salts enriched medium
Trace element water-soluble liquid 5mL, composition and concentration in the trace element water-soluble liquid are：NTA 1.5g/L, chlorination
Cobalt 0.1g/L, anhydrous Manganese chloride 0.1g/L, frerrous chloride 0.1g/L, CoCL2 6H2O 0.18g/L, zinc chloride 0.1g/L, five water
Copper sulphate 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 chlorides
0.025g/L。
6. screening technique according to claim 5, it is characterised in that the preparation side of the second inorganic salts enriched medium
Method is：Other components in culture medium in addition to resazurin, the vitamin aqueous solution and trace element water-soluble liquid are mixed, heating is boiled
Nitrogen constantly is passed through while boiling, until culture medium is become colorless by pink colour, stops heating, after after system cooling, add dimension life
The plain aqueous solution and trace element water-soluble liquid, add final mass than the nine water vulcanized sodium for 0.03%, final mass ratio after sterilizing
It is 0.02% sodium acid carbonate, and resazurin, sealing preserve.
7. screening technique according to claim 1, it is characterised in that surveyed to the methane production for generating using chromatograph
Fixed, wherein the temperature of FID is 200 DEG C, and the temperature of the injector that gasifies is 150 DEG C；The initial temperature of chromatographic column is 35 DEG C, keeps 15min
200 DEG C are warming up to the programming rate of 5 DEG C/min afterwards, and keep 5min, received from sample top using gas collector during sampling
Collection gas, is then sampled and sample introduction with sealing injection device again, and sampling volume is 0.3mL.
8. screening technique according to claim 1, it is characterised in that also including being tested cultivation results using sterilizing group
Card：Using the sample after sterilizing according to the anaerobic condition culture, if the nothing after anaerobic condition culture of the sample after final sterilization
Methane is produced, then prove that methane gas is produced by the flora in sample.
9. according to any described screening techniques of claim 1-8, it is characterised in that obtain containing decomposing petroleum hydrocarbon mixed bacterial
Mixture, flora therein is separated, you can obtain the flora of anaerobic degradation petroleum hydrocarbon methane phase.
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