CN110628671A - Separation method of cellulose and petroleum hydrocarbon degrading bacteria - Google Patents
Separation method of cellulose and petroleum hydrocarbon degrading bacteria Download PDFInfo
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- CN110628671A CN110628671A CN201910910605.7A CN201910910605A CN110628671A CN 110628671 A CN110628671 A CN 110628671A CN 201910910605 A CN201910910605 A CN 201910910605A CN 110628671 A CN110628671 A CN 110628671A
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- 229920002678 cellulose Polymers 0.000 title claims abstract description 26
- 239000001913 cellulose Substances 0.000 title claims abstract description 26
- 241000894006 Bacteria Species 0.000 title claims abstract description 25
- 230000000593 degrading effect Effects 0.000 title claims abstract description 15
- 239000003209 petroleum derivative Substances 0.000 title claims abstract description 14
- 238000000926 separation method Methods 0.000 title abstract description 7
- 235000010980 cellulose Nutrition 0.000 claims abstract description 25
- 238000000034 method Methods 0.000 claims abstract description 24
- 239000001768 carboxy methyl cellulose Substances 0.000 claims abstract description 23
- 239000010779 crude oil Substances 0.000 claims abstract description 21
- 229920002134 Carboxymethyl cellulose Polymers 0.000 claims abstract description 20
- 235000010948 carboxy methyl cellulose Nutrition 0.000 claims abstract description 20
- 239000008112 carboxymethyl-cellulose Substances 0.000 claims abstract description 20
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 16
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 16
- 239000002689 soil Substances 0.000 claims abstract description 15
- 229920000168 Microcrystalline cellulose Polymers 0.000 claims abstract description 13
- 235000019813 microcrystalline cellulose Nutrition 0.000 claims abstract description 13
- 239000008108 microcrystalline cellulose Substances 0.000 claims abstract description 13
- 229940016286 microcrystalline cellulose Drugs 0.000 claims abstract description 13
- 238000012216 screening Methods 0.000 claims abstract description 13
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims abstract description 9
- 238000012258 culturing Methods 0.000 claims abstract description 8
- 239000010902 straw Substances 0.000 claims abstract description 6
- 230000015556 catabolic process Effects 0.000 claims abstract description 5
- 238000006731 degradation reaction Methods 0.000 claims abstract description 5
- 241000380130 Ehrharta erecta Species 0.000 claims abstract description 3
- 239000002023 wood Substances 0.000 claims abstract description 3
- 239000001963 growth medium Substances 0.000 claims description 32
- 239000002609 medium Substances 0.000 claims description 14
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 8
- 239000008103 glucose Substances 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 239000003208 petroleum Substances 0.000 claims description 6
- 230000001954 sterilising effect Effects 0.000 claims description 6
- 239000000725 suspension Substances 0.000 claims description 6
- 230000003321 amplification Effects 0.000 claims description 5
- 239000007864 aqueous solution Substances 0.000 claims description 5
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 5
- 238000004659 sterilization and disinfection Methods 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 3
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 3
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 claims description 3
- 230000001580 bacterial effect Effects 0.000 claims description 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 3
- 239000008367 deionised water Substances 0.000 claims description 3
- 229910021641 deionized water Inorganic materials 0.000 claims description 3
- 238000012257 pre-denaturation Methods 0.000 claims description 3
- 239000011535 reaction buffer Substances 0.000 claims description 3
- 238000012163 sequencing technique Methods 0.000 claims description 3
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 claims description 3
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 claims description 3
- 230000002538 fungal effect Effects 0.000 claims description 2
- 238000000746 purification Methods 0.000 abstract description 4
- 230000000813 microbial effect Effects 0.000 abstract description 3
- 239000012154 double-distilled water Substances 0.000 description 3
- 244000025254 Cannabis sativa Species 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 1
- 241001136275 Sphingobacterium Species 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 239000002803 fossil fuel Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000002699 waste material Substances 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- 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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Abstract
The invention relates to the technical field of microbial separation, in particular to a separation method of cellulose and petroleum hydrocarbon degrading bacteria. A method for separating cellulose and petroleum hydrocarbon degrading bacteria, comprising the steps of: (1) enrichment culture: taking forest soil with perennial grass, wood, straw and fallen leaves, inoculating the forest soil on an enrichment medium by adopting a concentration gradient method, culturing until a single colony appears, transferring the forest soil to a flat plate with carboxymethyl cellulose as a unique carbon source for purification until the single colony appears; (2) re-screening: continuously passaging the single colonies on a flat plate with carboxymethyl cellulose as a unique carbon source; (3) directional screening: transferring the single colony to a flat plate taking microcrystalline cellulose powder as a unique carbon source until the single colony appears, and continuously carrying out passage; (4) crude oil degradation bacteria target screening: transferring the single colony to a crude oil flat plate by a flat plate marking method, and culturing until the single colony appears; (5) and (3) strain identification: amplifying 16SrDNA of a single colony, and comparing the 16SrDNA for identification, wherein the compared 16SrDNA is reserved for the bacteria.
Description
Technical Field
The invention relates to the technical field of microbial separation, in particular to a separation method of cellulose and petroleum hydrocarbon degrading bacteria.
Background
Cellulose is a renewable resource which is distributed most widely and in the largest quantity in nature, and with the increasing exhaustion of fossil fuels and the prominent problem of environmental pollution, the development of natural cellulose draws the wide attention of scientists. How to convert natural cellulose into energy which can be used by people becomes a new topic which troubles people. Many microorganisms have the ability to degrade petroleum and are widely distributed in nature, drawing a wide range of attention from scientists. At present, the natural cellulose is mostly treated by burning or naturally rotting, so that the environment is seriously polluted and great waste is caused. Meanwhile, with the acceleration of the industrialization process, the petroleum pollution also becomes a great problem which puzzles people, and the microbial degradation of petroleum also becomes a hotspot of scientific research. Therefore, the bacterial strain which can degrade both cellulose and petroleum is found to have profound significance for environmental protection.
Disclosure of Invention
In order to solve the above problems, it is an object of the present invention to provide a method for separating cellulose and petroleum hydrocarbon-degrading bacteria, which can degrade both cellulose and petroleum hydrocarbon.
In order to achieve the above object, the present invention provides a method for separating cellulose and petroleum hydrocarbon degrading bacteria, comprising the steps of:
(1) enrichment culture: dissolving forest soil with perennial grass, wood, straw and fallen leaves in a mixed aqueous solution of glucose and carboxymethyl cellulose to prepare a soil suspension, inoculating the soil suspension on an enrichment culture medium by adopting a concentration gradient method, culturing until a single colony appears, transferring the single colony to a culture medium plate with the carboxymethyl cellulose as a unique carbon source by adopting a plate scribing method, and purifying until the single colony appears;
(2) re-screening: continuously transferring and passaging the single colony obtained in the step (1) on a culture medium plate with carboxymethyl cellulose as a unique carbon source for 3-5 times until the colony grows stably;
(3) directional screening: transferring the single colony obtained in the step (2) to a culture medium plate taking microcrystalline cellulose powder as a unique carbon source until the single colony appears, and continuously carrying out passage for 3-5 times until the colony grows stably;
(4) crude oil degradation bacteria target screening: transferring the single colony obtained in the step (3) to a crude oil culture medium plate by a plate marking method, culturing until the single colony appears, carrying out passage 5 generations on the single colony growing on the crude oil culture medium plate, and storing the stably inherited strain to-80 ℃ for later use;
(5) and (3) strain identification: amplifying the 16SrDNA of the single colony obtained in the step (4), and comparing the 16SrDNA with EZbiocloud to identify the 16SrDNA, wherein the 16SrDNA is reserved for the bacterial genus and discarded for other fungal genera after comparison.
Compared with the existing microorganism separation technology, firstly, the invention takes the forest soil with the grass, the straw and the fallen leaves all the year round as the material, and the forest soil with the grass, the straw and the fallen leaves all the year round is rich in more bacteria capable of degrading cellulose and is easier to separate to obtain the bacteria capable of degrading cellulose; secondly, sequentially screening the bacteria for degrading the cellulose by adopting a culture medium plate taking carboxymethyl cellulose as a unique carbon source and a culture medium plate taking microcrystalline cellulose powder as a unique carbon source, so that the bacteria for degrading the cellulose can be accurately obtained; finally, a crude oil culture medium plate is adopted to screen bacteria which can degrade cellulose and petroleum hydrocarbon.
Preferably, the enrichment medium has a formula of 0.5g KNO3，0.5gKH2PO4， 0.4gMgSO4.7H2O，0.01gFeSO4.7H2O, 0.5g glucose, pH 7.0-7.2.
Preferably, the formula of the medium with carboxymethyl cellulose as the only carbon source is 0.5g KNO3，0.5gKH2PO4，0.4gMgSO4.7H2O，0.01gFeSO4.7H2O, 3g of sodium carboxymethyl cellulose, and the pH value is 7.0-7.2.
Preferably, the formula of the culture medium with the microcrystalline cellulose powder as the only carbon source is 0.5g KNO3，0.5gKH2PO4，0.4gMgSO4.7H2O，0.01gFeSO4.7H2O, 3g of microcrystalline cellulose powder, and the pH value is 7.0-7.2.
Preferably, the crude oil culture medium has a formula of 1.0gNH4NO3，2.0gK2HPO4， 1.0gKH2PO4，0.05g0.05gMgSO4.7H2O，0.01gCaCl2And adding 0.5-1.0 g/L of crude oil into the sterilized culture medium after sterilization for standby.
Preferably, the step (5) of amplifying the 16SrDNA comprises amplifyingThe primer is 16S universal primer: 27F: AGAGTTTGATCCTGGCTCAG and 1492R AGAGTTTGATCCTGGCTCAG; the amplification reaction system is as follows: 10 XPCR reaction buffer 5. mu.l, 25mmol/L MgSO4Mu.l of 2. mu.l, 5. mu.l of 10mmol dNTP, 1. mu.l of each 10nmol/L primer, a colony serving as a DNA template, 1. mu.l of KoD-Plus DNA polymerase and a volume of 50. mu.l of double-distilled deionized water. The reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; 30 seconds at 95 ℃, 30 seconds at 50 ℃ and 2.00min at 68 ℃ for 30 cycles in total; extending for 10min at 68 ℃; keeping at 15 deg.C for 10 min. Sequencing of the amplified sequence was performed by an ABI3100 sequencer.
Drawings
FIG. 1 is a graph of the growth of bacteria on crude oil medium obtained in the examples;
FIG. 2 is a graph of the growth of bacteria on microcrystalline cellulose medium obtained in the example;
FIG. 3 is a graph of the growth of bacteria on carboxymethyl cellulose medium obtained in the examples.
Detailed Description
For a better understanding of the present invention, the following detailed description of the invention will be given with reference to specific examples.
A method for separating cellulose and petroleum hydrocarbon degrading bacteria, comprising the steps of:
preparation of a culture medium:
enrichment medium (0.5 gKNO)3，0.5gKH2PO4，0.4gMgSO4.7H2O， 0.01gFeSO4.7H2O, 0.5g of glucose, pH7.0-7.2, constant volume of 1L double distilled water, and sterilization at 115 ℃;
a mixed aqueous solution of glucose and carboxymethyl cellulose (mixed aqueous solution of 0.2% glucose and 0.2 carboxymethyl cellulose);
carboxymethyl cellulose Medium (0.5 gKNO)3，0.5gKH2PO4，0.4gMgSO4.7H2O， 0.01gFeSO4.7H2O, 3g of sodium carboxymethylcellulose, pH7.0-7.2, constant volume of 1L double distilled water, and sterilization at 121 ℃ for 20 min);
microcrystalline cellulose Medium (0.5 gKNO)3，0.5gKH2PO4，0.4gMgSO4.7H2O， 0.01gFeSO4.7H2O, 3g of microcrystalline cellulose powder, pH7.0-7.2, constant volume of 1L double distilled water, and sterilizing at 121 ℃ for 20 min);
crude oil medium (1.0 gNH)4NO3，2.0gK2HPO4，1.0gKH2PO4， 0.05g0.05gMgSO4.7H2O，0.01gCaCl2And adding 0.5-1.0 g/L of crude oil into the sterilized culture medium for later use after sterilizing the culture medium until the volume of distilled water is 1000 mL).
(1) Enrichment culture: packaging forest soil with accumulated plants, straws and fallen leaves all the year round in northeast China with sterile bags, taking back, and storing at-20 ℃ for later use; dissolving 1g of forest soil in a glucose carboxymethyl cellulose mixed aqueous solution to prepare a soil suspension, standing and incubating for 3 hours at 37 ℃, inoculating the soil suspension on an enrichment medium flat plate by adopting a concentration gradient method, culturing for 6 days in a thermostat at 28 ℃ until a single colony appears, and transferring the single colony to the carboxymethyl cellulose medium flat plate by adopting a flat plate scribing method for purification until the single colony appears;
(2) re-screening: transferring the single colony on the carboxymethyl cellulose culture medium plate to another carboxymethyl cellulose culture medium plate by adopting a plate marking method for purification until the single colony appears;
(3) directional screening: transferring the re-screened single colony to a microcrystalline cellulose culture medium flat plate by adopting a flat plate marking method for further purification to obtain a single colony;
(4) crude oil degradation bacteria target screening: and (4) transferring the single colony obtained in the step (3) to a crude oil culture medium plate by a plate marking method, culturing until the single colony appears, carrying out passage 5 generations on the single colony growing on the crude oil culture medium plate, and storing the stably inherited strain to-80 ℃ for later use.
(5) And (3) strain identification: amplifying the 16SrDNA of the single colony obtained in the step (4), wherein the amplification primer is a 16S universal primer: 27F: AGAGTTTGATCCTGGCTCAG and 1492R AGAGTTTGATCCTGGCTCAG; the amplification reaction system is as follows: 10 XPCR reaction buffer 5. mu.l, 25mmol/L MgSO4Mu.l of 2. mu.l, 5. mu.l of 10mmol dNTP, 1. mu.l of each 10nmol/L primer, a colony serving as a DNA template, 1. mu.l of KoD-Plus DNA polymerase and a volume of 50. mu.l of double-distilled deionized water. The reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min;30 seconds at 95 ℃, 30 seconds at 50 ℃ and 2.00min at 68 ℃ for 30 cycles in total; extending for 10min at 68 ℃; keeping at 15 deg.C for 10 min. Sequencing of the amplified sequence was performed by an ABI3100 sequencer. The sequence of the 16SrDNA obtained by amplification is shown as SEQ ID NO.1, and is identified by comparison of BLAST and EZbiochoud, the comparison result is that the Sphingobacterium bacteria are reserved for standby, and other species are discarded or stored for standby according to the needs.
The obtained bacteria capable of degrading cellulose and petroleum hydrocarbon are respectively inoculated to a crude oil culture medium, a microcrystalline cellulose culture medium and a carboxymethyl cellulose culture medium for culture, and the results are shown in fig. 1, fig. 2 and fig. 3, which show that the bacteria capable of degrading cellulose and petroleum hydrocarbon obtained in the embodiment can be proved to degrade petroleum hydrocarbon and cellulose by growing on the culture medium with crude oil and cellulose as the only carbon sources.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (6)
1. A method for separating cellulose and petroleum hydrocarbon degrading bacteria comprises the following steps:
(1) enrichment culture: dissolving forest soil with perennial grass, wood, straw and fallen leaves in a mixed aqueous solution of glucose and carboxymethyl cellulose to prepare a soil suspension, inoculating the soil suspension on an enrichment culture medium by adopting a concentration gradient method, culturing until a single colony appears, transferring the single colony to a culture medium plate with the carboxymethyl cellulose as a unique carbon source by adopting a plate scribing method, and purifying until the single colony appears;
(2) re-screening: continuously transferring and passaging the single colony obtained in the step (1) on a culture medium plate with carboxymethyl cellulose as a unique carbon source for 3-5 times until the colony grows stably;
(3) directional screening: transferring the single colony obtained in the step (2) to a culture medium plate taking microcrystalline cellulose powder as a unique carbon source until the single colony appears, and continuously carrying out passage for 3-5 times until the colony grows stably;
(4) crude oil degradation bacteria target screening: transferring the single colony obtained in the step (3) to a crude oil culture medium plate by a plate marking method, culturing until the single colony appears, carrying out passage 5 generations on the single colony growing on the crude oil culture medium plate, and storing the stably inherited strain to-80 ℃ for later use;
(5) and (3) strain identification: amplifying the 16SrDNA of the single colony obtained in the step (4), and comparing the 16SrDNA with EZbiocloud to identify the 16SrDNA, wherein the 16SrDNA is reserved for the bacterial genus and discarded for other fungal genera after comparison.
2. The method of claim 1, wherein the enrichment medium is formulated to have a concentration of 0.5g KNO3，0.5gKH2PO4，0.4gMgSO4.7H2O，0.01gFeSO4.7H2O, 0.5g glucose, pH 7.0-7.2.
3. The method of claim 1, wherein the medium with carboxymethyl cellulose as a sole carbon source has a formula of 0.5g KNO3，0.5gKH2PO4，0.4gMgSO4.7H2O，0.01gFeSO4.7H2O, 3g of sodium carboxymethyl cellulose, and the pH value is 7.0-7.2.
4. The method for separating cellulose and petroleum hydrocarbon-degrading bacteria according to claim 1, wherein the formula of the medium with microcrystalline cellulose powder as the sole carbon source is 0.5g KNO3，0.5gKH2PO4，0.4gMgSO4.7H2O，0.01gFeSO4.7H2O, 3g of microcrystalline cellulose powder, and the pH value is 7.0-7.2.
5. The method of claim 1 for isolating cellulose and petroleum hydrocarbon degrading bacteriaCharacterized in that the formula of the crude oil culture medium is 1.0gNH4NO3，2.0gK2HPO4，1.0gKH2PO4，0.05g0.05gMgSO4.7H2O，0.01gCaCl2And adding 0.5-1.0 g/L of crude oil into the sterilized culture medium after sterilization for standby.
6. The method of claim 1, wherein the step of amplifying the 16SrDNA in step (5) comprises amplifying the 16S universal primer: 27F: AGAGTTTGATCCTGGCTCAG and 1492R AGAGTTTGATCCTGGCTCAG; the amplification reaction system is as follows: 10 XPCR reaction buffer 5. mu.l, 25mmol/LMgSO4Mu.l of 2. mu.l, 5. mu.l of 10mmol dNTP, 1. mu.l of each 10nmol/L primer, a colony serving as a DNA template, 1. mu.l of KoD-Plus DNA polymerase and a volume of 50. mu.l of double-distilled deionized water. The reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; 30 seconds at 95 ℃, 30 seconds at 50 ℃ and 2.00min at 68 ℃ for 30 cycles in total; extending for 10min at 68 ℃; keeping at 15 deg.C for 10 min. Sequencing of the amplified sequence was performed by an ABI3100 sequencer.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104830708A (en) * | 2015-02-02 | 2015-08-12 | 天津科技大学 | Crude oil degrading bacteria strain and application thereof |
| CN104877930A (en) * | 2015-04-24 | 2015-09-02 | 南开大学 | Separating and screening method for saline-alkaline-resistant bacteria degrading petroleum hydrocarbon |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104830708A (en) * | 2015-02-02 | 2015-08-12 | 天津科技大学 | Crude oil degrading bacteria strain and application thereof |
| CN104877930A (en) * | 2015-04-24 | 2015-09-02 | 南开大学 | Separating and screening method for saline-alkaline-resistant bacteria degrading petroleum hydrocarbon |
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