CN110218689A - A kind of efficient offshore oil degradation composite bacteria agent and the preparation method and application thereof - Google Patents

A kind of efficient offshore oil degradation composite bacteria agent and the preparation method and application thereof Download PDF

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CN110218689A
CN110218689A CN201910614569.XA CN201910614569A CN110218689A CN 110218689 A CN110218689 A CN 110218689A CN 201910614569 A CN201910614569 A CN 201910614569A CN 110218689 A CN110218689 A CN 110218689A
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culture
composite bacteria
bacteria agent
degradation
offshore oil
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CN110218689B (en
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胡晓珂
陈卫卫
李俊德
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Yantai Institute of Coastal Zone Research of CAS
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Yantai Institute of Coastal Zone Research of CAS
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    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • 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
    • 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
    • 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
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/30—Organic compounds
    • C02F2101/32—Hydrocarbons, e.g. oil
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
    • C02F2103/08—Seawater, e.g. for desalination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A20/00—Water conservation; Efficient water supply; Efficient water use
    • Y02A20/20—Controlling water pollution; Waste water treatment
    • Y02A20/204—Keeping clear the surface of open water from oil spills

Abstract

The present invention relates to marine environment petroleum Treatment process fields, and in particular to a kind of preparation and application of the screening technique and composite bacteria agent of efficient offshore oil degradation bacteria.Composite bacteria agent contains the bacterial strain of false xanthomonas (Pseudoxanthomonas sp.S1-2), acinetobacter (Acinetobacter sp.HC8-3S) and enlightening thatch Bordetella (Dietzia sp.CN-3) mixing.Offshore oil degradation composite bacteria agent of the present invention is preferable to the degradation effect of petroleum in the environment of neutral meta-alkalescence, and is resistant to hypersaline environment, is particularly adapted to marine environment, expands its application range and application prospect in petroleum pollution in ocean improvement.

Description

A kind of efficient offshore oil degradation composite bacteria agent and the preparation method and application thereof
Technical field
The present invention relates to marine environment petroleum Treatment process fields, and in particular to a kind of efficient offshore oil drop Solve the preparation and application of the screening technique and composite bacteria agent of bacterium.
Background technique
Currently, being directed to petroleum pollution in ocean, the especially processing of Accidental Oil Spill accident, emergent management means include machine Tool sealing, oil spilling recycle and using chemical dispersants etc..Physical restoration is to utilize the different types of oil fence, oil-absorbing ship, with And natural or artificial synthesized adsorbent material etc. recycles petroleum.Detergent, solidifying oil is mainly added in chemical restoration into seawater Agent etc. fibrous gypsum oil.Although physical restoration and chemical restoration can quickly remove the petroleum largely leaked, physics is used Repairing method is difficult to completely remove the petroleum dissolved in Algorithms for Oil Slick and water；And with chemical remediations such as detergent, fuel thickeners Method actually adds chemical pollutant into ocean, causes secondary pollution.For seabed oil spilling or with water sports The oil pollution being deposited in bottom sediment, current physics and chemical restoration can not be administered effectively.Petroleum in situ The self-regeneration of degrading microorganism and exogenous oil degradation microorganism is artificially added, is current to solve marine sediment petroleum The major way of pollution.The hydro carbons overwhelming majority in petroleum can be decomposed by the microorganisms metabolism, finally be mineralized into CO2And H2O, Microorganism remediation becomes key technology.Microorganism can remove seawater surface oil film, decompose the petroleum hydrocarbon dissolved in seawater, right It is environmental-friendly, do not generate secondary pollution, have the advantages that physical restoration and chemical restoration it is incomparable, it is considered to be Solve the basic method of oil pollution.
Crude Oil-degrading Bacteria is the most thorough to its physiology, ecology and biotechnology research due to widely distributed.Research ratio More Crude Oil-degrading Bacteria includes acinetobacter, pseudomonas, alkane eating bacteria category, Nocardia, Micrococcus, enlightening Thatch Bordetella etc. more than 70 belong to.However, for complicated petroleum component, such as linear paraffin, branched paraffin, cycloalkane, aromatic hydrocarbon Deng the type of single enzyme producing microbe is less, concentration is lower, a small number of particular hydrocarbon classes that can only generally degrade or only degrades Thorough degradation to a certain stage, petroleum hydrocarbon generally requires multiple-microorganism synergistic effect, and compound bacteria or flora are to petroleum hydrocarbon Degradation effect is better than single culture.For example, Zhang Qiang in 2019 et al. discloses a kind of answering for oil-polluted soils reparation It closes bacterial preparation process and applies (publication number CN 109207413 A, application number 201811356554.X), pass through 4 plants of bacterium Compounding improves the degradation efficiency of petroleum, and single bacterial strain degradation rate is up to 39.3%, and composite bacteria agent has reached 49.8%. In addition, since indigenous microorganism possibly can not be metabolized petroleum or oil degradation bacteria in indigenous microorganism group composition completely Accounting is lower, and the biological reinforcing technology for adding Heterologous Microbial can be used to enhancing oil degradation.Therefore, using from getting dirty It contaminates the indigenous microorganism separated in the specific habitat (such as seawater or deposit) in place and carries out strain compounding, and then the original implemented Position biological reinforcing technology has apparent advantage, and the application potential for repairing petroleum pollution in ocean is big.
Summary of the invention
The present invention in view of the deficiency of the prior art, provides a kind of screening of efficient offshore oil degradation bacteria The preparation and application of method and composite bacteria agent.
To achieve the above object, the present invention uses technical solution:
A kind of efficient offshore oil degradation composite bacteria agent, composite bacteria agent contain false xanthomonas (Pseudoxanthomonas sp.S1-2), acinetobacter (Acinetobacter sp. HC8-3S) and enlightening thatch Bordetella The bacterial strain of (Dietzia sp.CN-3) mixing.
The composite bacteria agent is the mixed fermentation bacterium solution of the bacterial strain and culture presevation liquid is 0.75-1:1- by volume 1.5 ratio mixing；
Wherein, the culture presevation liquid presses the component of mass fraction: nonylphenol polyoxyethylene ether 4.6%-4.8%, and 12 Sodium alkyl benzene sulfonate 2.8%-3.0%, ethyl alcohol 2.2%-2.4%, citric acid 0.6%-0.8%, sodium sulphate 0.6%-0.8%, Potassium chloride 0.2%-0.4%, glycerol 0.2%-0.4%, acetic acid 0.1%-0.3%, xanthan gum 0.05%-0.07%, acetic acid second Ester 0.02%-0.04%, organic silicon defoamer 0.05%-0.07%, surplus are water.
The volume ratio of 3 kinds of bacterial strains is 0.5-1:0.75-1:1-1.5 in mixed bacteria liquid in the mixed fermentation bacterium solution
The vacation xanthomonas Pseudoxanthomonas sp.S1-2, the entitled Chinese Typical Representative training of depositary institution Support object collection (CCTCC), address: wuchang, wuhan Luo Jia Shan, deposit number are CCTCC M 2019450, the deposit date is On June 12nd, 2019；
Acinetobacter Acinetobacter sp.HC8-3S, in the entitled China typical culture collection of depositary institution The heart (CCTCC), address: wuchang, wuhan Luo Jia Shan, deposit number are CCTCC M 2019449, and the deposit date is in June, 2019 12 days；
Enlightening thatch Bordetella (Dietzia sp.CN-3), the entitled China typical culture collection center of depositary institution (CCTCC), address: wuchang, wuhan Luo Jia Shan, deposit number are CCTCC M 2015537, and the deposit date is Septembers 15 in 2015 Day.
The mixed fermentation bacterium solution of the bacterial strain is collected by centrifugation precipitating and washs through BHB culture medium, and is resuspended to bacterium solution OD600 It is 1.0, is then mixed with culture presevation liquid.
A kind of preparation method of efficient offshore oil degradation composite bacteria agent, by false xanthomonas (Pseudoxanthomonas sp.S1-2), acinetobacter (Acinetobacter sp. HC8-3S) and enlightening thatch Bordetella The mixed fermentation bacterium solution of (Dietzia sp.CN-3) is collected by centrifugation precipitating and washs through BHB culture medium, and is resuspended to bacterium solution OD600It is 1.0, is then mixed with culture presevation liquid.
The mixed fermentation bacterium solution is by false xanthomonas (Pseudoxanthomonas sp. S1-2), not lever Pseudomonas (Acinetobacter sp.HC8-3S) and enlightening thatch Bordetella (Dietzia sp.CN-3) are inoculated in 2216E culture respectively Through level-one, secondary seed culture under being vibrated at 30-37 DEG C in base；The secondary seed solution of each bacterial strain is mixed in the ratio of 1:1:1 It closes in 2216E culture medium in 30-37 DEG C, the lower culture of oscillation to OD600For 2.0 to get arrive mixed fermentation bacterium solution.
A kind of application of efficient offshore oil degradation composite bacteria agent, the composite bacteria agent is in efficient degradation offshore oil In application.
The screening technique of degradation bacteria in a kind of efficient offshore oil degradation composite bacteria agent, by sample in containing 0.5% (m/v) at 30-37 DEG C, shaken cultivation 7-10 days in the BHB minimal medium of petroleum, primary dcreening operation is carried out；
Then by gained culture in petroliferous BHB minimal medium, at 30-37 DEG C, shaken cultivation 7-10 It, domestication culture repeatedly；Wherein, the content for taming initial petroleum in the culture medium of culture is 1%, and then oil content is successively It is 2%, 5%；Then enrichment culture object is obtained using petroleum as sole carbon source and the energy by gradient dilution method purifies and separates Bacterial strain.
The sample is the bottom sediment under oil spilling environment.
Beneficial effects of the present invention
Composite bacteria agent of the present invention is to filter out 3 plants of efficient offshore oil degradations for unique carbon source and the energy with petroleum Bacterial strain；Since 3 plants of bacterial strains are to the difference of petroleum component degradation property, it is mixed and in specified strain preservative fluid culture, acquisition Offshore oil degradation composite bacteria agent；Gained mix bacterium agent, can be further to the degradation capability of petroleum compared to single bacterial strain It significantly improves.In addition, offshore oil degradation composite bacteria agent of the present invention is in the environment of neutral meta-alkalescence to the degradation effect of petroleum Preferably, and it is resistant to hypersaline environment, is particularly adapted to marine environment, expand it in petroleum pollution in ocean improvement Application range and application prospect；Specifically:
(1) bacterial strain is truly realized using petroleum as sole carbon source and the energy, in oil degradation bacteria in composite bacteria agent of the present invention During screening, tame, isolating and purifying etc., used culture medium is all using petroleum as sole carbon source and the energy, in specific Under the conditions of the strain that screens can have better tolerance to petroleum, be conducive to improve it in practical oil polluted environment Survival ability, degradation efficiency etc..
(2) the efficient offshore oil degradation bacteria strains that present invention screening obtains, and the original having complementary advantages using degradation property It is then compounded, obtains offshore oil degradation composite bacteria agent；The present invention screens the 3 plants of bacterium obtained and drops to different petroleum the hydrocarbon components It is different to solve characteristic, respectively to C16-C20, C21-C30, C31-C37There is degradation effect outstanding.And there is collaboration after 3 plants of bacterium compoundings Facilitation can significantly improve the degradation efficiency of petroleum.
(3) present invention obtains efficient offshore oil degradation composite bacteria agent in the environment of neutral meta-alkalescence to the drop of petroleum Solution effect is preferable, repairs the effect of petroleum pollution in ocean to promote microbial bacterial agent, and be resistant to high salinity environment, It is particularly adapted to marine environment, expands application range and application prospect of the microbial inoculum in petroleum pollution in ocean improvement.
Detailed description of the invention
Fig. 1 is the gas chromatography-mass spectrum figure for not adding bacterium control group petroleum hydrocarbon degradation that the embodiment of the present invention mentions.
Fig. 2 is the gas-chromatography-for the Pseudoxanthomonas sp.S1-2 decomposing petroleum hydrocarbon that the embodiment of the present invention mentions Mass spectrogram.
Fig. 3 is the gas-chromatography-matter for the Acinetobacter sp.HC8-3S decomposing petroleum hydrocarbon that the embodiment of the present invention mentions Spectrogram.
Fig. 4 is the gas chromatography-mass spectrum figure for the Dietzia sp.CN-3 decomposing petroleum hydrocarbon that the embodiment of the present invention mentions.
Fig. 5 is the gas-chromatography-of offshore oil degradation composite bacteria agent (Mix) decomposing petroleum hydrocarbon that the embodiment of the present invention mentions Mass spectrogram.
Fig. 6 is Pseudoxanthomonas sp.S1-2, the Acinetobacter sp.HC8- that the embodiment of the present invention mentions The effect of 3S and Dietzia sp.CN-3 single bacterial strain and offshore oil degradation composite bacteria agent (Mix) decomposing petroleum hydrocarbon.
Fig. 7 is Pseudoxanthomonas sp.S1-2, the Acinetobacter sp.HC8- that the embodiment of the present invention mentions 3S and Dietzia sp.CN-3 single bacterial strain and the different petroleum the hydrocarbon component (C of offshore oil degradation composite bacteria agent (Mix) degradation16- C20, C21-C30, C31-C37) effect.
Fig. 8 be Pseudoxanthomonas sp. S1-2 under the condition of different pH that the embodiment of the present invention mentions, Acinetobacter sp.HC8-3S and Dietzia sp.CN-3 single bacterial strain and offshore oil degradation composite bacteria agent (Mix) The effect of decomposing petroleum hydrocarbon.
Fig. 9 be Pseudoxanthomonas sp.S1-2 under the conditions of the different NaCl concentrations that the embodiment of the present invention mentions, Acinetobacter sp.HC8-3S and Dietzia sp.CN-3 single bacterial strain and offshore oil degradation composite bacteria agent (Mix) The effect of decomposing petroleum hydrocarbon.
Specific embodiment
A specific embodiment of the invention is described further below in conjunction with example, it is noted that retouch in this place The specific embodiment stated is simply to illustrate that with the present invention is explained, it is not limited to the present invention.
The present invention is using the bottom sediment of Peng Lai 19-3 oil spilling platform as object, with petroleum for unique carbon source and the energy, Filter out 3 plants of efficient offshore oil degradation bacteria strains.According to their differences to petroleum component degradation property, and using independently The culture presevation liquid of research and development is prepared for offshore oil degradation composite bacteria agent.Compared to single bacterial strain, drop of the microbial inoculum to petroleum Solution ability significantly improves.In addition, the offshore oil is degraded, composite bacteria agent imitates the degradation of petroleum in the environment of neutral meta-alkalescence Fruit is preferable, and is resistant to hypersaline environment, is particularly adapted to marine environment, expands it in petroleum pollution in ocean improvement Application range and application prospect.
Embodiment 1
The screening technique of offshore oil degradation bacteria, screening and culturing medium used in the method and degradation culture medium are all With petroleum for unique carbon source and the energy.Specific steps are as follows:
1) offshore oil degradation bacteria strain source
(1) sample of offshore oil degradation bacteria is derived from the bottom sediment of Peng Lai 19-3 oil spilling platform.
(2) petroleum is derived from the viscous crude of Dongying city Shengli Oil Field, viscosity 4896mPas.
2) screening of offshore oil degradation bacteria
(1) primary dcreening operation of offshore oil degradation bacteria
The above-mentioned sediment sample of 10g is weighed to be put into through high-temperature sterilization (121 DEG C, 20min) treated 100mL BHB In minimal medium, wherein the petroleum of the addition above-mentioned record of 0.5g is as unique carbon source and the energy.30 DEG C, 180rpm shakes It is cultivated 7 days in bed.
(2) domestication of offshore oil degradation bacteria
Bacterium solution 5mL after switching primary dcreening operation is in fresh 100mL BHB minimal medium, wherein the addition above-mentioned note of 1g The petroleum of load is as unique carbon source and the energy, 30 DEG C, cultivates 7 days in 180rpm shaking table；Culture solution 5mL is taken to repeat the above behaviour Make, and improve the petroleum concentration in minimal medium, is followed successively by 2g, 5g, is then trained in 30 DEG C, 180rpm shaking table respectively It supports 7 days and carries out domestication culture.
(3) offshore oil degradation bacteria isolates and purifies
A. gradient dilution method separation of bacterial is used.It is sterile that 9mL is added in bacterium solution 1mL after taking above-mentioned domestication enrichment culture In 0.9% (m/v) physiological saline, it is successively diluted to 10-2、10-3、10-4Gradient dilution liquid, each three groups of gradient work parallel.
B. the 100 μ L of bacterium solution of three kinds of different gradients is taken to be coated on BHA culture medium flat plate (containing 0.5% petroleum) respectively.30 DEG C constant temperature obtains the single bacterium colony of a variety of bacterium by screening respectively, but passes through preliminary verifying, and the degradation effect of three kinds of bacterium is most It is good, carry out subsequent experimental.It is sequenced by 16S rRNA, identifies strain classification.
C. the single colonie for selecting different shape, by the single colonie formed after above-mentioned culture a few hours respectively in BHA culture medium Plate carries out scribing line separation on (containing 0.5% petroleum).A-b is repeated above operation, until forming the single purifying bacterium colony of form. Strain idenfication is carried out using 16s rRNA sequencing approach.
Can be using petroleum as the single bacterium colony of sole carbon source and the energy by above-mentioned acquisition, respectively false xanthomonas (Pseudoxanthomonas sp.S1-2), acinetobacter (Acinetobacter sp.HC8-3S) and enlightening thatch Bordetella (Dietzia sp.CN-3)。
It obtains the single bacterium colony of a variety of bacterium respectively by screening, but passes through preliminary verifying, the degradation effect of three kinds of bacterium Most preferably, subsequent experimental is carried out.It is sequenced by 16S rRNA, identifies strain classification.
Above-mentioned oil degradation bacterial strain, bacterial strain are Pseudoxanthomonas sp.S1-2, entitled China, depositary institution Type Tissue Collection (CCTCC), deposit number are CCTCC M 2019450.The deposit date is on June 12nd, 2019.
Above-mentioned oil degradation bacterial strain, bacterial strain are Acinetobacter sp.HC8-3S, the entitled Chinese allusion quotation of depositary institution Type culture collection (CCTCC), deposit number are CCTCC M 2019449, and the deposit date is on June 12nd, 2019.
Above-mentioned oil degradation bacterial strain, bacterial strain are Dietzia sp.CN-3, the entitled Chinese Typical Representative culture of depositary institution Collection (CCTCC), deposit number are CCTCC M 2015537.The deposit date is on September 15th, 2015.
Wherein, the morphological feature of three plants of bacterium is as follows:
Pseudoxanthomonas sp.S1-2 is Gram-negative bacteria, and bacterium colony is in faint yellow, round, surface wettability, Edge comparison rule shows that shape is rod-short under scanning electron microscope.
Acinetobacter sp.HC8-3S is Gram-negative bacteria, and bacterium colony is creamy white, round, and surface is smooth wet Moisten, regular edges, shows that shape is spherical under scanning electron microscope.
Dietzia sp.CN-3 is gram-positive bacteria, and bacterium colony is in orange, circle, and surface is smooth wet, regular edges, Show that shape is rod-short under scanning electron microscope.
D. colony inoculation after purification is cultivated a few hours in 30 DEG C of constant incubators in inclined-plane Storaged media, is saved It is stand-by in 4 DEG C of refrigerators.
On the basis of above scheme, the BHB culture medium are as follows:
BHB (Bushnell-Haas Broth) culture medium: magnesium sulfate 0.2g, calcium chloride 0.02g, potassium dihydrogen phosphate 1g, Dipotassium hydrogen phosphate 1g, ammonium nitrate 1g, frerrous chloride 0.05g, sodium chloride 25g, distilled water are settled to 1000mL, and adjusting pH is 7.0 ±0.2.121 DEG C, high pressure steam sterilization 20min.
The BHA culture medium are as follows:
BHA (Bushnell-Haas Agar) culture medium: magnesium sulfate 0.2g, calcium chloride 0.02g, potassium dihydrogen phosphate 1g, phosphorus Sour hydrogen dipotassium 1g, ammonium nitrate 1g, frerrous chloride 0.05g, sodium chloride 25g, agar 15g, distilled water are settled to 1000mL, adjust PH is 7.0 ± 0.2.121 DEG C, high pressure steam sterilization 20min.
The 16s rRNA sequence information of above-mentioned three kinds of degradation bacterias
1) Dietzia sp.CN-3 (the entitled China typical culture collection center of depositary institution, deposit number CCTCC M 2015537)
16s rRNA sequence (1399bp)
GCTTACCATGCAAGTCGAACGGTAAGGCCCCTTCGGGGGTACACGAGTGGCGAACGGGTG AGTAACA CGTGGGTAATCTGCCCTGCACTTCGGGATAAGCCTGGGAAACTGGGTCTAATAC CGGATATGAACTCCTGCCGCA TGGTGGGGGTTGGAAAGTTTTTCGGTGCAGGATGAGCCCG CGGCCTATCAGCTTGTTGGTGGGGTAATGGCCTAC CAAGGCGACGACGGGTAGCCGGCCTG AGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACG GGAGGCAGCAG TGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGGGGGATGACGG TCT TCGGATTGTAAACCCCTTTCAGTAGGGACGAAGCGCAAGTGACGGTACCTGCAGAAGA AGCACCGGCTAACTACG TGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTGTCCGG AATTACTGGGCGTAAAGAGCTCGTAGGCGGTT TGTCACGTCGTCTGTGAAATCCCTCGGCT TAACCGGGGGCGTGCAGGCGATACGGGCAGACTTGAGTACTACAGG GGAGACTGGAATTC CTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTC TGGGTAGTAACTGACGCTGAGGAGCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTG GTAGTCCATGCCGTA AACGGTGGGCGCTAGGTGTGGGGTCCTTCCACGGACTCCGTGCCGT AGCTAACGCATTAAGCGCCCCGCCTGGGG AGTACGGCCGCAAGGCTAAAACTCAAAGGAA TTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCG ATGCAACGCGAAGAAC CTTACCTAGGCTTGACATATACAGGACGACGGCAGAGATGTCGTTTCCCTTGTGGCTTG TA TACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAAC GAGCGCAACCC CTGTCTCATGTTGCCAGCACGTAATGGTGGGGACTCGTGAGAGACTGCCG GGGTCAACTCGGAGGAAGGTGGGGA TGACGTCAAATCATCATGCCCCTTATGTCTAGGGCT TCACACATGCTACAATGGCTAGTACAGAGGGCTGCGATA CCGCGAGGTGGAGCGAATCCC TTAAAGCTGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGG AGTCGCT AGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCG TCACGT CATGAAAGTCGGTAACACCCGAAGCCGGTGGCCTAACCCTTGTGGAGGGAGCCG TCGAAGG
2) Pseudoxanthomonas sp.S1-2 (protect by the entitled China typical culture collection center of depositary institution Hiding number is CCTCC M 2019450)
16s rRNA sequence (1412bp)
AAGTCGAACGGCAGCACAGGAGAGCTTGCTCTCTGGGTGGCGAGTGGCGGACGGGTGAGG AATACAT CGGAATCTACCTTTTCGTGGGGGATAACGTAGGGAAACTTACGCTAATACCGCA TACGACCTACGGGTGAAAGTG GGGGACCGCAAGGCCTCACGCGATTAGATGAGCCGATGT CCGATTAGCTAGTTGGCGGGGTAATGGCCCACCAAG GCGACGATCGGTAGCTGGTCTGAG AGGATGATCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAG GCAGCAGTG GGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATACCGCGTGGGTGAAGAAGGCC TTCGG GTTGTAAAGCCCTTTTGTTGGGAAAGAAATCCTGTCGATTAATACTCGGTGGGGAT GACGGTACCCAAAGAATAA GCACCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAG GGTGCAAGCGTTACTCGGAATTACTGGGCGTAAA GCGTGCGTAGGTGGTGGTTTAAGTCTG CTGTGAAAGCCCTGGGCTCAACCTGGGAATTGCAGTGGATACTGGGTC ACTAGAGTGTGGT AGAGGGATGCGGAATTTCTGGTGTAGCAGTGAAATGCGTAGAGATCAGAAGGAACATCCG T GGCGAAGGCGGCATCCTGGGCCAACACTGACACTGAGGCACGAAAGCGTGGGGAGCAAA CAGGATTAGATACCCT GGTAGTCCACGCCCTAAACGATGCGAACTGGATGTTGGGTGCAAC TTGGCACCCAGTATCGAAGCTAACGCGTTA AGTTCGCCGCCTGGGGAGTACGGTCGCAAGA CTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAG TATGTGGTTTAATTCG ATGCAACGCGAAGAACCTTACCTGGTCTTGACATCCACGGAACTTTCCAGAGATGGATT GG TGCCTTCGGGAACCGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGT TGGGTTAAGTC CCGCAACGAGCGCAACCCTTGTCCTTAGTTGCCAGCACGTAATGGTGGGA ACTCTAAGGAGACCGCCGGTGACAA ACCGGAGGAAGGTGGGGATGACGTCAAGTCATCAT GGCCCTTACGACCAGGGCTACACACGTACTACAATGGTGG GGACAGAGGGCTGCAAACCC GCGAGGGTGAGCCAATCCCAGAAACCCTATCTCAGTCCGGATTGGAGTCTGCAAC TCGACT CCATGAAGTCGGAATCGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGG GCCTTGT ACACACCGCCCGTCACACCATGGGAGTTTGTTGCACCAGAAGCAGGTAGCTTAA CCTTCGGGAGGGCGCTGCCA
3) Acinetobacter sp.HC8-3S (the entitled China typical culture collection center of depositary institution, preservation Number be CCTCC M 2019449)
16s rRNA sequence (1426bp)
GCGCGCGCTACCAGCAGTCGAGCGGGGAAGGTAGCTTGCTACTGGACCTAGCGGCGGACG GGTGAGT AATGCTTAGGAATCTGCCTATTAGTGGGGGACAACATTCCGAAAGGAATGCTA ATACCGCATACGTCCTACGGGA GAAAGCAGGGGACCTTCGGGCCTTGCGCTAATAGATGA GCCTAAGTCGGATTAGCTAGTTGGTGGGGTAAAGGCC TACCAAGGCGACGATCTGTAGCG GGTCTGAGAGGATGATCCGCCACACTGGGACTGAGACACGGCCCAGACTCCT ACGGGAGG CAGCAGTGGGGAATATTGGACAATGGGGGGAACCCTGATCCAGCCATGCCGCGTGTGTGA AGAAGG CCTTATGGTTGTAAAGCACTTTAAGCGAGGAGGAGGCTACTAGTATTAATACTAC TGGATAGTGGACGTTACTCG CAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTA ATACAGAGGGTGCGAGCGTTAATCGGATTTACTG GGCGTAAAGCGTGCGTAGGCGGCCAT TTAAGTCAAATGTGAAATCCCCGAGCTTAACTTGGGAATTGCATTCGAT ACTGGATGGCTA GAGTATGGGAGAGGATGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAG GA ATACCGATGGCGAAGGCAGCCATCTGGCCTAATACTGACGCTGAGGTACGAAAGCATG GGGAGCAAACAGGATTA GATACCCTGGTAGTCCATGCCGTAAACGATGTCTACTAGCCGTT GGGGCCTTTGAGGCTTTAGTGGCGCAGCTAA CGCGATAAGTAGACCGCCTGGGGAGTACG GTCGCAAGACTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGC GGTGGAGCATGTGG TTTAATTCGATGCAACGCGAAGAACCTTACCTGGCCTTGACATACTAGAAACTTTCCAGAG ATGGATTGGTGCCTTCGGGAATCTAGATACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTC GTGAGATGTTGGGT TAAGTCCCGCAACGAGCGCAACCCTTTTCCTTACTTGCCAGCATTTCG GATGGGAACTTTAAGGATACTGCCAGT GACAAACTGGAGGAAGGCGGGGACGACGTCAAG TCATCATGGCCCTTACGGCCAGGGCTACACACGTGCTACAAT GGTCGGTACAAAGGGTTGC TACCTAGCGATAGGATGCTAATCTCAAAAAGCCGATCGTAGTCCGGATTGGAGTCT GCAAC TCGACTCCATGAAGTCGGAATCGCTAGTAATCGCGGATCAGAATGCCGCGGTGAATACGTT CCCGGGCC TTGTACACACCGCCCGTCACACCATGGGAGTTTGTTGCACCAGAAGTAGGTAG TCTAACCGCAAGGAGACGCTAC CCGGCCTGCTAC
Embodiment 2
The preparation method of efficient offshore oil degradation composite bacteria agent:
(1) first order seed culture
Respectively to above-mentioned three kinds of petroleum hydrocarbon degradation bacterium Pseudoxanthomonas sp.S1-2, Acinetobacter Sp.HC8-3S and Dietzia sp.CN-3 carries out activation culture.Concrete operations are as follows: aseptic condition takes in test tube slant culture medium Oil degradation bacteria is in 10mL 2216E culture medium, 30 DEG C, cultivates in 180rpm shaking table, as primary seed solution.
(2) secondary seed culture
5mL Pseudoxanthomonas sp.S1-2, Acinetobacter sp.HC8- are taken under aseptic condition respectively 3S and Dietzia sp.CN-3 primary seed solution is linked into 100 mL 2216E culture mediums, inoculum concentration 5%, 30 DEG C, It is cultivated in 180rpm shaking table, as secondary seed solution.
(3) mixed fermentation
Adjusted respectively under aseptic condition Pseudoxanthomonas sp.S1-2, Acinetobacter sp.HC8-3S and The OD of Dietzia sp.CN-3 secondary seed solution600=1.0, and 10mL is taken respectively, total 30mL secondary seed solution is linked into In 600mL 2216E culture medium, 30 DEG C, mixed fermentation is carried out in 180rpm shaking table to OD600It is 2.0, mixed fermentation bacterium is made Liquid.
(4) the efficiently preparation of offshore oil degradation composite bacteria agent
Above-mentioned mixed fermentation liquid is centrifuged (8000rpm, 10min), gained is precipitated and is washed with BHB culture medium, repeats 3 It is secondary, and resuspended bacterium solution is carried out to OD with BHB culture medium600It is 1.0, takes appropriate mixed fermentation liquid and culture presevation liquid with 1:1's Volume ratio mixing obtains the offshore oil degradation composite bacteria agent.
The culture presevation liquid includes the component of following mass fraction: nonylphenol polyoxyethylene ether 4.6%, dodecyl Benzene sulfonic acid sodium salt 2.8%, ethyl alcohol 2.2%, citric acid 0.6%, sodium sulphate 0.6%, potassium chloride 0.2%, glycerol 0.2%, acetic acid 0.1%, xanthan gum 0.05%, ethyl acetate 0.02%, organic silicon defoamer 0.05%, water 88.58%.
The 2216E culture medium are as follows:
2216E culture medium: peptone 5g, yeast powder 1g, ironic citrate 0.1g, sodium chloride 19.45g, magnesium chloride 5.98g, Sodium sulphate 3.24g, calcium chloride 1.8g, potassium chloride 0.55g, sodium carbonate 0.16g, potassium bromide 0.08g, strontium chloride 0.034g, boric acid 0.022g, sodium metasilicate 0.004g, sodium fluoride 0.0024g, sodium nitrate 0.0016g, disodium hydrogen phosphate 0.008g determine distilled water Hold to 1000mL, adjusting pH is 7.6 ± 0.2.
Embodiment 3
The efficient degradation of offshore oil degradation bacteria strains and composite bacteria agent to petroleum
By above-mentioned three kinds of offshore oil degradation bacterias Pseudoxanthomonas sp.S1-2, Acinetobacter Sp.HC8-3S and Dietzia sp.CN-3 and offshore oil degradation composite bacteria agent are living in 2216E culture medium respectively Change culture, then washs 3 times and resuspended bacterium solution to OD using BHB culture medium600It is 1.0, contains according to 10% inoculum concentration access In the 100mL BHB culture medium of petroleum concentration 0.5% (w/v)；The petroleum concentration 0.5% (w/v) of bacterium is not added in setting simultaneously 100 mL BHB culture mediums are control group, and every group three parallel；Then in 30 DEG C, 180rpm shaking table culture 7 days, culture solution is used In the measurement of oil degradation effect.
After culture 7 days, culture solution is poured into separatory funnel, extracts petroleum in three times with 100mL n-hexane.By Oil sample is settled to 25mL, and separates petroleum the hydrocarbon component using column chromatography by filter, concentration.Key step includes: to weigh Silica gel, aluminium oxide and anhydrous sodium sulfate carry out dress column, loading, n-hexane elution, methylene chloride and n-hexane mixed solution and wash De-, methanol elution, saturated hydrocarbons, aromatic hydrocarbon and the colloid in petroleum component are successively separated.
The degradation effect of petroleum hydrocarbon is measured using gas chromatography-mass spectrometry (GC-MS), degradation map is such as Shown in Fig. 1-5.By 7 days degradation experiments, compared with control group (Fig. 1), Pseudoxanthomonas sp.S1-2, The experiment of Acinetobacter sp.HC8-3S and Dietzia sp. CN-3 single bacterial strain and offshore oil degradation composite bacteria agent In group, apparent variation is had occurred in the peak of petroleum hydrocarbon each component, and different strains have the alkane degradation effect of different chain length aobvious The difference of work.
Total petroleum hydrocarbon degrades situation as shown in fig. 6, Pseudoxanthomonas sp.S1-2, Acinetobacter Sp.HC8-3S and Dietzia sp.CN-3 single bacterial strain and offshore oil degradation composite bacteria agent divide the degradation rate of total petroleum hydrocarbon It Wei 81.8%, 85.6%, 88.9% and 94.1%.In addition, by different chain length petroleum hydrocarbon C16-C20, C21-C30, C31- C37Degradation situation analyzed (see Fig. 7), find Pseudoxanthomonas sp.S1-2 to C16-C20Degradation effect Most preferably, reached 98.5%, and to overlength alkane C31-C37Degradation effect it is weaker, only 45.7%； Acinetobacter sp.HC8-3S is to C16-C20Degradation effect is 90.5%, to overlength chain C31-C37Degradation effect it is preferable, Reach 66.5%；Dietzia sp.CN-3 bacterial strain is to C16-C20And C21-C30Degradation effect preferably (> 94%), to C31- C37Alkane degradation effect is slightly weak；The offshore oil degradation obtained is mixed by three plants of degradation bacteria mixed fermentations and with culture presevation liquid Composite bacteria agent is to C16-C20And C21-C30The degradation effect of petroleum hydrocarbon is obviously improved, and total petroleum hydrocarbon degradation rate reaches 94.1%.In short, using three plants of offshore oil degradation bacterias to the difference of different chain length petroleum hydrocarbon degradation characteristic, resulting ocean Oil degradation composite bacteria agent has reached mutual supplement with each other's advantages, realizes the efficient degradation to petroleum hydrocarbon.
Embodiment 4
Degradation under the conditions of different pH and NaCl concentration to petroleum:
(1) influence of the pH to oil degradation effect
The pH for adjusting the 100mL BHB culture medium containing petroleum concentration 0.5% (w/v) is respectively 5,6,7,8,9, and respectively Access the Pseudoxanthomonas sp.S1-2, Acinetobacter sp.HC8-3S of 5% (v/v) preactivated culture With Dietzia sp.CN-3 single bacterial strain and offshore oil degradation composite bacteria agent.0.5% (the w/ of petroleum concentration for not adding bacterium is set V) BHB culture medium is control group, and every group three parallel.After being placed in 30 DEG C, cultivating 7 days in the shaking table of 180rpm, GC-MS is used Different strains are measured to the degradation effect of petroleum hydrocarbon (referring to Fig. 8).
As seen from Figure 8: when pH is 5, the degradation efficiency of each single bacterial strain and composite bacteria agent is minimum (< 50%)； When pH is 6, the degradation efficiency of Acinetobacter sp.HC8-3S, Dietzia sp. CN-3 and composite bacteria agent increase (> 68%)；Under neutrality and alkaline condition that pH is 7,8,9, Pseudoxanthomonas sp.S1-2, Acinetobacter The degradation efficiency of sp.HC8-3S and Dietzia sp.CN-3 single bacterial strain and composite bacteria agent significantly improves, 83% with On；And the degradation efficiency of composite bacteria agent reaches peak 92.3% when pH is 8.As it can be seen that three plants of bacterium are in neutral meta-alkalescence Environment in degradation effect it is preferable, and through mutual supplement with each other's advantages, composite bacteria agent to alkalinity tolerance improve it is more obvious. Marine environment is weakly alkaline environment, and the alkaline-resisting characteristic of composite bacteria agent expands it and applies model in petroleum pollution in ocean improvement It encloses and application prospect.
(2) influence of the NaCl concentration to oil degradation effect
The NaCl concentration for adjusting the 100mL BHB culture medium containing petroleum concentration 0.5% (w/v) is respectively 10g/L, 20g/ L, 40g/L, 60g/L, 80g/L, and be respectively connected to Pseudoxanthomonas sp.S1-2 that 5% (v/v) cultivated in advance, Acinetobacter sp.HC8-3S and Dietzia sp.CN-3 single bacterial strain and offshore oil degradation composite bacteria agent.Setting Not plus the BHB culture medium of the petroleum concentration 0.5% (w/v) of bacterium is control group, and every group three parallel.It is placed in 30 DEG C, 180rpm Shaking table in cultivate 7 days after, with GC-MS measurement different strains to the degradation effect of petroleum hydrocarbon (referring to Fig. 9).
As seen from Figure 9: under the conditions of different NaCl concentrations, Pseudoxanthomonas sp. S1-2 is in 40g/L With highest degradation efficiency, with the raising of concentration, degrading activity is substantially reduced；Acinetobacter sp.HC8-3S and Two plants of bacterium of Dietzia sp.CN-3 are preferable to the tolerance of NaCl concentration, and within the scope of 10-80g/L, degradation rate is both greater than 66%, especially CN-3, under the conditions of the high salinity of 80g/L, degradation efficiency still maintains 82.5%.Composite bacteria agent pair The tolerance of NaCl concentration significantly improves, and degradation rate is also able to maintain 85% or more under the conditions of high salinity, reaches in 40g/L Highest 92.8%.In short, composite bacteria agent improves the degradation efficiency under the conditions of high salinity, salt tolerance is strong, is particularly suitable for ocean ring Border and other high salinity environments.
Sequence table
<110>Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences
<120>a kind of efficient offshore oil degradation composite bacteria agent and the preparation method and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1399
<212> DNA
<213>gram-positive bacteria (Dietzia sp. CN-3)
<400> 1
gcttaccatg caagtcgaac ggtaaggccc cttcgggggt acacgagtgg cgaacgggtg 60
agtaacacgt gggtaatctg ccctgcactt cgggataagc ctgggaaact gggtctaata 120
ccggatatga actcctgccg catggtgggg gttggaaagt ttttcggtgc aggatgagcc 180
cgcggcctat cagcttgttg gtggggtaat ggcctaccaa ggcgacgacg ggtagccggc 240
ctgagagggt gatcggccac actgggactg agacacggcc cagactccta cgggaggcag 300
cagtggggaa tattgcacaa tgggcgaaag cctgatgcag cgacgccgcg tgggggatga 360
cggtcttcgg attgtaaacc cctttcagta gggacgaagc gcaagtgacg gtacctgcag 420
aagaagcacc ggctaactac gtgccagcag ccgcggtaat acgtagggtg cgagcgttgt 480
ccggaattac tgggcgtaaa gagctcgtag gcggtttgtc acgtcgtctg tgaaatccct 540
cggcttaacc gggggcgtgc aggcgatacg ggcagacttg agtactacag gggagactgg 600
aattcctggt gtagcggtga aatgcgcaga tatcaggagg aacaccggtg gcgaaggcgg 660
gtctctgggt agtaactgac gctgaggagc gaaagcatgg gtagcgaaca ggattagata 720
ccctggtagt ccatgccgta aacggtgggc gctaggtgtg gggtccttcc acggactccg 780
tgccgtagct aacgcattaa gcgccccgcc tggggagtac ggccgcaagg ctaaaactca 840
aaggaattga cgggggcccg cacaagcggc ggagcatgtg gattaattcg atgcaacgcg 900
aagaacctta cctaggcttg acatatacag gacgacggca gagatgtcgt ttcccttgtg 960
gcttgtatac aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt 1020
cccgcaacga gcgcaacccc tgtctcatgt tgccagcacg taatggtggg gactcgtgag 1080
agactgccgg ggtcaactcg gaggaaggtg gggatgacgt caaatcatca tgccccttat 1140
gtctagggct tcacacatgc tacaatggct agtacagagg gctgcgatac cgcgaggtgg 1200
agcgaatccc ttaaagctgg tctcagttcg gattggggtc tgcaactcga ccccatgaag 1260
tcggagtcgc tagtaatcgc agatcagcaa cgctgcggtg aatacgttcc cgggccttgt 1320
acacaccgcc cgtcacgtca tgaaagtcgg taacacccga agccggtggc ctaacccttg 1380
tggagggagc cgtcgaagg 1399
<210> 2
<211> 1412
<212> DNA
<213>Gram-negative bacteria (Pseudoxanthomonas sp. S1-2)
<400> 2
aagtcgaacg gcagcacagg agagcttgct ctctgggtgg cgagtggcgg acgggtgagg 60
aatacatcgg aatctacctt ttcgtggggg ataacgtagg gaaacttacg ctaataccgc 120
atacgaccta cgggtgaaag tgggggaccg caaggcctca cgcgattaga tgagccgatg 180
tccgattagc tagttggcgg ggtaatggcc caccaaggcg acgatcggta gctggtctga 240
gaggatgatc agccacactg gaactgagac acggtccaga ctcctacggg aggcagcagt 300
ggggaatatt ggacaatggg cgcaagcctg atccagccat accgcgtggg tgaagaaggc 360
cttcgggttg taaagccctt ttgttgggaa agaaatcctg tcgattaata ctcggtgggg 420
atgacggtac ccaaagaata agcaccggct aacttcgtgc cagcagccgc ggtaatacga 480
agggtgcaag cgttactcgg aattactggg cgtaaagcgt gcgtaggtgg tggtttaagt 540
ctgctgtgaa agccctgggc tcaacctggg aattgcagtg gatactgggt cactagagtg 600
tggtagaggg atgcggaatt tctggtgtag cagtgaaatg cgtagagatc agaaggaaca 660
tccgtggcga aggcggcatc ctgggccaac actgacactg aggcacgaaa gcgtggggag 720
caaacaggat tagataccct ggtagtccac gccctaaacg atgcgaactg gatgttgggt 780
gcaacttggc acccagtatc gaagctaacg cgttaagttc gccgcctggg gagtacggtc 840
gcaagactga aactcaaagg aattgacggg ggcccgcaca agcggtggag tatgtggttt 900
aattcgatgc aacgcgaaga accttacctg gtcttgacat ccacggaact ttccagagat 960
ggattggtgc cttcgggaac cgtgagacag gtgctgcatg gctgtcgtca gctcgtgtcg 1020
tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg tccttagttg ccagcacgta 1080
atggtgggaa ctctaaggag accgccggtg acaaaccgga ggaaggtggg gatgacgtca 1140
agtcatcatg gcccttacga ccagggctac acacgtacta caatggtggg gacagagggc 1200
tgcaaacccg cgagggtgag ccaatcccag aaaccctatc tcagtccgga ttggagtctg 1260
caactcgact ccatgaagtc ggaatcgcta gtaatcgcag atcagcattg ctgcggtgaa 1320
tacgttcccg ggccttgtac acaccgcccg tcacaccatg ggagtttgtt gcaccagaag 1380
caggtagctt aaccttcggg agggcgctgc ca 1412
<210> 3
<211> 1426
<212> DNA
<213>Gram-negative bacteria (Acinetobacter sp. HC8-3S)
<400> 3
gcgcgcgcta ccagcagtcg agcggggaag gtagcttgct actggaccta gcggcggacg 60
ggtgagtaat gcttaggaat ctgcctatta gtgggggaca acattccgaa aggaatgcta 120
ataccgcata cgtcctacgg gagaaagcag gggaccttcg ggccttgcgc taatagatga 180
gcctaagtcg gattagctag ttggtggggt aaaggcctac caaggcgacg atctgtagcg 240
ggtctgagag gatgatccgc cacactggga ctgagacacg gcccagactc ctacgggagg 300
cagcagtggg gaatattgga caatgggggg aaccctgatc cagccatgcc gcgtgtgtga 360
agaaggcctt atggttgtaa agcactttaa gcgaggagga ggctactagt attaatacta 420
ctggatagtg gacgttactc gcagaataag caccggctaa ctctgtgcca gcagccgcgg 480
taatacagag ggtgcgagcg ttaatcggat ttactgggcg taaagcgtgc gtaggcggcc 540
atttaagtca aatgtgaaat ccccgagctt aacttgggaa ttgcattcga tactggatgg 600
ctagagtatg ggagaggatg gtagaattcc aggtgtagcg gtgaaatgcg tagagatctg 660
gaggaatacc gatggcgaag gcagccatct ggcctaatac tgacgctgag gtacgaaagc 720
atggggagca aacaggatta gataccctgg tagtccatgc cgtaaacgat gtctactagc 780
cgttggggcc tttgaggctt tagtggcgca gctaacgcga taagtagacc gcctggggag 840
tacggtcgca agactaaaac tcaaatgaat tgacgggggc ccgcacaagc ggtggagcat 900
gtggtttaat tcgatgcaac gcgaagaacc ttacctggcc ttgacatact agaaactttc 960
cagagatgga ttggtgcctt cgggaatcta gatacaggtg ctgcatggct gtcgtcagct 1020
cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccttttcc ttacttgcca 1080
gcatttcgga tgggaacttt aaggatactg ccagtgacaa actggaggaa ggcggggacg 1140
acgtcaagtc atcatggccc ttacggccag ggctacacac gtgctacaat ggtcggtaca 1200
aagggttgct acctagcgat aggatgctaa tctcaaaaag ccgatcgtag tccggattgg 1260
agtctgcaac tcgactccat gaagtcggaa tcgctagtaa tcgcggatca gaatgccgcg 1320
gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca ccatgggagt ttgttgcacc 1380
agaagtaggt agtctaaccg caaggagacg ctacccggcc tgctac 1426

Claims (9)

  1. The composite bacteria agent 1. a kind of efficient offshore oil is degraded, it is characterised in that: composite bacteria agent contains false xanthomonas (Pseudoxanthomonas sp.S1-2), acinetobacter (Acinetobacter sp.HC8-3S) and enlightening thatch Bordetella The bacterial strain of (Dietzia sp.CN-3) mixing.
  2. The composite bacteria agent 2. efficient offshore oil according to claim 1 is degraded, it is characterised in that: the composite bacteria agent is institute The mixed fermentation bacterium solution and culture presevation liquid for stating bacterial strain are the ratio mixing of 0.75-1:1-1.5 by volume；
    Wherein, the culture presevation liquid presses the component of mass fraction: nonylphenol polyoxyethylene ether 4.6%-4.8%, dodecyl Benzene sulfonic acid sodium salt 2.8%-3.0%, ethyl alcohol 2.2%-2.4%, citric acid 0.6%-0.8%, sodium sulphate 0.6%-0.8%, chlorination Potassium 0.2%-0.4%, glycerol 0.2%-0.4%, acetic acid 0.1%-0.3%, xanthan gum 0.05%-0.07%, ethyl acetate 0.02%-0.04%, organic silicon defoamer 0.05%-0.07%, surplus are water.
    The volume ratio of 3 kinds of bacterial strains is 0.5-1:0.75-1:1-1.5 in mixed bacteria liquid in the mixed fermentation bacterium solution.
  3. The composite bacteria agent 3. efficient offshore oil according to claim 1 is degraded, it is characterised in that:
    The vacation xanthomonas Pseudoxanthomonas sp.S1-2, the entitled Chinese Typical Representative culture of depositary institution Collection (CCTCC), address: wuchang, wuhan Luo Jia Shan, deposit number are CCTCC M 2019450, and the deposit date is 2019 On June 12, in；
    Acinetobacter Acinetobacter sp.HC8-3S, the entitled China typical culture collection center of depositary institution (CCTCC), address: wuchang, wuhan Luo Jia Shan, deposit number are CCTCC M 2019449, and the deposit date is June 12 in 2019 Day；
    Enlightening thatch Bordetella (Dietzia sp.CN-3), the entitled China typical culture collection center of depositary institution (CCTCC), Address: wuchang, wuhan Luo Jia Shan, deposit number are CCTCC M 2015537, and the deposit date is on September 15th, 2015.
  4. 4. by efficient offshore oil degradation composite bacteria agent described in claim 1-3 any one, it is characterised in that: the bacterium The mixed fermentation bacterium solution of strain is collected by centrifugation precipitating and washs through BHB culture medium, and is resuspended to bacterium solution OD600Be 1.0, then with bacterium Kind preservative fluid mixing.
  5. 5. a kind of preparation method of efficient offshore oil degradation composite bacteria agent described in claim 1, it is characterised in that: will be false Xanthomonas (Pseudoxanthomonas sp.S1-2), acinetobacter (Acinetobacter sp.HC8-3S) and The mixed fermentation bacterium solution of enlightening thatch Bordetella (Dietzia sp.CN-3) is collected by centrifugation precipitating and washs through BHB culture medium, and through being resuspended To bacterium solution OD600It is 1.0, is then mixed with culture presevation liquid.
  6. 6. the preparation method of efficient offshore oil degradation composite bacteria agent as described in claim 5, it is characterised in that: described mixed Closing zymocyte liquid is by false xanthomonas (Pseudoxanthomonas sp.S1-2), acinetobacter (Acinetobacter sp.HC8-3S) and enlightening thatch Bordetella (Dietzia sp.CN-3) are inoculated in respectively in 2216E culture medium Through level-one, secondary seed culture under being vibrated at 30-37 DEG C；The secondary seed solution of each bacterial strain is mixed again in the ratio of 1:1:1 In 30-37 DEG C, the lower culture of oscillation to OD in 2216E culture medium600For 2.0 to get arrive mixed fermentation bacterium solution.
  7. 7. a kind of application of efficient offshore oil degradation composite bacteria agent described in claim 1, it is characterised in that: described compound Application of the microbial inoculum in efficient degradation offshore oil.
  8. 8. the screening technique of degradation bacteria, feature exist in efficient offshore oil degradation composite bacteria agent described in a kind of claim 1 In: by sample at 30-37 DEG C, shaken cultivation 7-10 days in the BHB minimal medium containing 0.5% (m/v) petroleum, carry out just Sieve；
    Then by gained culture in petroliferous BHB minimal medium, at 30-37 DEG C, shaken cultivation 7-10 days, repeatedly Domestication culture；Wherein, the content for taming initial petroleum in the culture medium of culture is 1%, and then oil content is followed successively by 2%, 5%；Then enrichment culture object is obtained using petroleum as the bacterial strain of sole carbon source and the energy by gradient dilution method purifies and separates.
  9. 9. by the screening technique of degradation bacteria in efficient offshore oil degradation composite bacteria agent described in claim 8, it is characterised in that: The sample is the bottom sediment under oil spilling environment.
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