CN104745511B - Composite bacteria agent for repairing heavy oil pollution coastline and preparation method and application - Google Patents
Composite bacteria agent for repairing heavy oil pollution coastline and preparation method and application Download PDFInfo
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- CN104745511B CN104745511B CN201510145572.3A CN201510145572A CN104745511B CN 104745511 B CN104745511 B CN 104745511B CN 201510145572 A CN201510145572 A CN 201510145572A CN 104745511 B CN104745511 B CN 104745511B
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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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- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D3/00—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances
- A62D3/02—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances by biological methods, i.e. processes using enzymes or microorganisms
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
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- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
- A62D2101/20—Organic substances
Abstract
The present invention relates to composite bacteria agent for repairing heavy oil pollution coastline and preparation method and application.The composite bacteria agent includes Sphingobacterium mizutae (Sphingobacterium mizutaii), Achromobacter xylosoxidans (Achromobacter xylosoxidans) and Pi Shi achromobacters (Achromobacter piechaudii), further, Sphingobacterium mizutae preserving number CGMCC No.8000；Achromobacter xylosoxidans deposit number CGMCC No.8022；Pi Shi achromobacter deposit number CGMCC No.8021.The composite bacteria agent salt resistant character of the present invention is good, and competitive advantage protrudes, and using the synergy of three kinds of bacterium, the heavy oil pollution to the especially refractory reason of coastline crude oil pollution has efficient repairing effect, is equally applicable for the reparation of oil spill salt affected soil.
Description
Technical field
The invention belongs to microbial technique and oil pollution coastline technical field of biological remediation, and in particular to a kind of energy is specially
Item degraded heavy oil pollutant is for the composite bacteria agent in reparation heavy oil pollution coastline and preparation method and application.
Background technology
Ocean is both the resource treasure-house that the mankind depend on for existence and development, is important transportation environment again.With oil demand
It is growing, Offshore Oil Industry, transport service develop rapidly, and offshore oil exploration, exploitation and transportation scale be not
Disconnected to expand, marine oil spill and contamination accident occur again and again, oil and products thereof have become be only second to blue-green algae, red tide it is second largest
Source of Marine Pollution.And when oil spill accident occurs in coastal waters, because tide and wave act on, the compromised general of shoreline environment institute
It is difficult to avoid that.China possesses 1.8 ten thousand kilometers of mainland coastline, 1.4 ten thousand kilometers of island coastline, is a mass of aquaculture along the line
Area, tourism landscape area, seashore industrial area and ecological preservation area, coastline oil spill will mean seashore ring once occurring
The decline of border quality, the destruction of the ecosystem, the loss of economic industry and the infringement of human health.And due to petroleum pollution
Physics slightly solubility, chemical stability, bio-toxicity and bioaccumulation effect, oil spill may influence coastline many decades extremely
Centuries, as long as it endangers weight, influence so that economic, efficient, environment-friendly oil pollution coastline Treatment process research and development
With important strategic importance.
It is biological prosthetic to refer to make the contaminant degradation in environment or innoxious contaminated using biology especially microorganism
The normal function of the ecosystem is able to the process recovered, have cost it is low, it is simple to operate, do not produce the notable excellent of secondary pollution
Point.In the early 1990s, bioremediation technology the U.S. Alaska prince William gulf oil spill improvement in by first into
Work(application, has started the beginning of oil spill coastline biological treating, is increasingly becoming what is studied and tackle key problems both at home and abroad in the past 20 years
Focus.
The biological prosthetic basis in oil spill coastline is metabolism of the microorganism to petroleum pollution, has been ground
Study carefully confirmation, many microorganisms can be using the alkane in oil, aromatic hydrocarbons, low molecule amount polycyclic aromatic hydrocarbon as single carbon source and energy in nature
Source is utilized, or the degraded of polycyclic aromatic hydrocarbon with high molecular weight is realized in a manner of Co metabolism.And after the generation of oil spill accident, seashore
Thread environment is often due to lack suitable microorganism, or stress and the shortage of nutritional condition because of petroleum pollution, causes micro- life
The quantity of thing is too low, slow-growing, active to be suppressed, self-purification capacity deficiency., on the one hand can be to sea using bioremediation technology
Efficient salt-tolerant oil degradation microbial inoculum is added in water front environment, to significantly improve flora and strain quantity, the microbial inoculum can be through indigenous bacterium
Kind acclimating is formed, and external source oil degradation bacteria or transgenosis bacterium can be also introduced on the premise of species security threat is not formed,
This method is also known as biological einforcement method；On the other hand, it is by adding required N, P nutrition or surfactant, improving bacterium
The growing environment of kind, improves the biological usability of its metabolic rate and pollutant, and this method is also known as biological promotion law.Biology
Repair and provide an effective governance way for oil spill environment, the effect day in remedying oil-polluted shoreline environment
Benefit is prominent.
In disclosed oil polluted environment biological renovation method, more for conventional soil, underground water oil spill, such as
CN 102453678A, CN 101603018A, CN 102382784A, CN 102021132A etc., and for the hydrology, geology, gas
Wait etc. increasingly complex, extreme coastline (or ocean) environment it is then relatively seldom.CN 102676424A disclose one plant from brill
Obtained Halophilic Bacillus Y-B is screened in well discarded slurry, petroleum industry high salt oily waste liquor is can be applied to and coastline is overflow
In oily sudden instability, however, single culture can only utilize certain or certain class component in oil, only multiple-microorganism
Synergy could realize that petroleum pollution each component is degraded to greatest extent.CN 102533615A disclose a kind of collaboration drop
Composite bacteria liquid of oil and preparation method thereof is solved, the composite bacteria liquid is made up of two plants of alkane eating bacterias and one plant of extra large bacillus, to alkane group
Divide and the degraded of aromatic component is more abundant, biological prosthetic, the involved bacterium in coastline or beach suitable for marine oil spill pollution
Kind is located away from China South Sea, SEDIMENT IN HUANGHAI SEA.CN 10171725A are with 4 kinds of bacterium such as bacillus pumilus, Venice acinetobacter calcoaceticus
The mixed fermentation liquid and fixation support of strain are that raw material is prepared into multiplexing microbial inoculum, can form competitive advantage in contaminated site, significantly
Increase the quantity of hydrocarbon degradation microorganism, improve the biodegradable efficiency of crude oil.CN 102491592A are into oil pollution beach
Apply combination of reclaimed grease, inorganic nutrients preparation and turned over, realize twice laid and the raising of repairing effect, this method belongs to raw
The technical standpoint that thing promotes.
The effect that above achievement administers oil pollution for bioremediation technology again gives sufficient affirmative, but due to
The domestic field is started late, and such technology there is no extensive practice in the oil spill environmental improvement of China coastline.Separately
Outside, as the aggravation of domestic and international petroleum resources anxiety situation, more heavy oil inferior are increasingly taken seriously, refining is mixed by domestic refinery
Amount increasingly increases, in addition to the main heavy oil base such as the Bohai Bay Oil in China, Liaohe Oil Field, Shengli Oil Field, a large amount of heavy oil warps
Increasingly increased from external import, heavy oil spill and the risk polluted by sea transport.Due to difficult for biological degradation component in heavy oil
Content is higher, and Biostatic is stronger, and biological prosthetic difficulty is bigger, researches and develops and efficiently repairs oil especially heavy oil pollution seashore
The special microbial inoculum of line is even more important.
The content of the invention
, can special drop it is an object of the present invention to provide a kind of special composite bacteria agent in remedying oil-polluted coastline
Solve crude oil particularly heavy oil pollutant.
Another object of the present invention is to provide the preparation method of the composite bacteria agent.
Another object of the present invention is to provide described composite bacteria agent repairing to restore oily pollution particularly heavy oil pollution sea
Application in water front or oil spill salt affected soil.
To achieve the above object, on the one hand, the invention provides a kind of compound bacteria for being used to repair heavy oil pollution coastline
Agent, the composite bacteria agent include Sphingobacterium mizutae (Sphingobacterium mizutaii), Achromobacter xylosoxidans
(Achromobacter xylosoxidans) and Pi Shi achromobacters (Achromobacter piechaudii).
According to specific embodiments of the present invention, present invention screening has obtained three plants of bacterium with efficient degradation oil function
Strain, XG-3, XG-14, XG-11 are respectively designated as in of the invention.Wherein：
Strain X G-3 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms on July 26th, 2013
Center (address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica), deposit number：CGMCC
No.8000；Classification And Nomenclature：Sphingobacterium mizutae Sphingobacterium mizutaii.The strain X G-3 of the present invention can be with
Heavy crude is survived for single carbon source, and its major physiological biochemical character is as follows：Sphingobacterium mizutae Sphingobacterium
Mizutaii XG-3 bacterium colony projection spherical in shape, it is translucent；Thalline is direct rod shape, no gemma, atrichia, and Gram's staining is cloudy
Property, G+C=54.6%；Oxidizing ferment and contact enzyme positive, nitrate reduction negative, it is impossible to hydrolysis starch, energy Oxidation of Alcohol production acid,
Energy glucose fermentation, maltose, sucrose etc..The strain good salt tolerance, it can be survived in 0~10% salinity range, salinity>
Its growth is slightly suppressed when 5%.Under simulated seawater salinity, Venezuela's weight in strain 10d to initial concentration for 1g/L
Oily degradation efficiency is up to more than 40%.
Strain X G-14 is preserved in China Committee for Culture Collection of Microorganisms's commonly micro- life on July 26th, 2013
Thing center (address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica), deposit number：
CGMCC No.8022；Classification And Nomenclature：Achromobacter xylosoxidans Achromobacter xylosoxidans.The bacterium of the present invention
Strain XG-14 can survive by single carbon source of heavy crude, and its major physiological biochemical character is as follows：Achromobacter xylosoxidans
Achromobacter xylosoxidans XG-14 bacterium colony moistening is glossy, and surface is smooth, neat in edge；Thalline is in quarter butt
Shape, there are 1-5 root peritricha, Gram-negative, G+C=54.0%；Oxidizing ferment, catalase, nitrate reduction are positive, and do not produce
Raw indoles, oxidation of ethanol production is sour, azymic glucose, not hydrolysis starch.The strain salt tolerance is good, when salinity reaches 7% still
Well-grown, it is slightly suppressed after reaching 10%.The strain is under simulated seawater salinity, the committee in 10d to initial concentration for 1g/L
Nei Ruila heavy oil degradation efficiency is up to more than 45%.
Strain X G-11 is preserved in China Committee for Culture Collection of Microorganisms's commonly micro- life on July 26th, 2013
Thing center (address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica), deposit number：
CGMCC No.8021；Classification And Nomenclature：Pi Shi achromobacter Achromobacter piechaudii.The strain X G-11 of the present invention
It can be survived by single carbon source of heavy crude, its major physiological biochemical character is as follows：Pi Shi achromobacters Achromobacter
Piechaudii XG-11 bacterium colonies are smaller, circular, colourless to arrive canescence, and slightly raised, surface is smooth and edge is complete；Thalline is ball
It is shaft-like, there is motility, Gram-negative, G+C=54.3%, oxidizing ferment, contact enzyme positive, nitrate reduction is positive,
Edwardsiella hoshinae, hydrolysis starch, energy glucose fermentation, xylose production be not sour.The strain salt tolerance is preferable, in 0~7% salinity model
Enclose it is interior can survive, salinity be 7% when it is slightly suppressed.The strain is 1g/L to initial concentration in 10d under simulated seawater salinity
Venezuela's heavy oil degradation efficiency up to more than 50%.
It is thus preferable in composite bacteria agent of the invention, the Sphingobacterium mizutae is that preserving number is CGMCC
No.8000 Sphingobacterium mizutae.Achromobacter xylosoxidans (Achromobacter in composite bacteria agent
Xylosoxidans), Pi Shi achromobacters (Achromobacter piechaudii) can have oil in the prior art
XG-14, XG-11 that the bacterial strain (being preferably sibling species bacterial strain) of degradation function or present invention screening obtain.
Preferably, in composite bacteria agent of the invention, the Achromobacter xylosoxidans are that deposit number is CGMCC
No.8022 Achromobacter xylosoxidans.Sphingobacterium mizutae (Sphingobacterium in composite bacteria agent
Mizutaii), Pi Shi achromobacters (Achromobacter piechaudii) can have oil degradation in the prior art
XG-3, XG-11 that the bacterial strain (being preferably sibling species bacterial strain) of function or present invention screening obtain.
Preferably, in composite bacteria agent of the invention, the Pi Shi achromobacters are that deposit number is CGMCC No.8021
Pi Shi achromobacters.Sphingobacterium mizutae (Sphingobacterium mizutaii), xylose oxidation nothing in composite bacteria agent
Color bacillus (Achromobacter xylosoxidans), can be that the bacterial strain with oil degradation function is (excellent in the prior art
Elect sibling species bacterial strain as) or present invention screening obtained XG-3, XG-14.
The composite bacteria agent of the present invention, mainly using the synergy of three kinds of Salt And Alkali Tolerance heavy oil degrading bacterias, remove heavy oil
Pollution, so as to provide a kind of efficient, economic, environment-friendly oil pollution coastline biological renovation method.
According to specific embodiments of the present invention, in composite bacteria agent of the invention, the Sphingobacterium mizutae, xylose oxygen
It is 1~10 to change the ratio between viable count of achromobacter and Pi Shi achromobacters：1~10：1~10.
According to specific embodiments of the present invention, composite bacteria agent of the invention can be solid-state bacteria preparation, or liquid bacterium
Preparation.Directly the thalline of three kinds of strains or zymotic fluid containing the strain can directly be mixed, compounding obtains institute
The composite bacteria agent stated.When composite bacteria agent is liquid bacteria preparation, the cell concentration of each strain is controllable to 10 in microbial inoculum8~
109CFU/mL.For convenience of preservation or transport, it can further concentrate or even dry.
On the other hand, the invention provides the preparation method of described composite bacteria agent, this method to include：
Sphingobacterium mizutae, Achromobacter xylosoxidans, Pi Shi achromobacters are inoculated in the liquid LB of sterilizing respectively
In culture medium, in 25 DEG C~30 DEG C lucifuge condition Shaking cultures, to late log phase is grown, respectively as seed liquor；
Above-mentioned seed liquor is inoculated with the nutrition fermentation medium of sterilizing respectively with volume ratio 2%~5%, 25 DEG C~30
DEG C culture to each strain cell density be 108~109CFU/mL, obtain zymotic fluid；Preferably, the nutrition fermentation medium bag
Include beef extract, peptone and ASM compositions；
Above-mentioned zymotic fluid is compounded, or further concentration or dry as needed, composite bacteria agent is made.
In the specific embodiment of the present invention, special composite bacteria agent is repaired in the oil pollution coastline that is used for of the invention
It is by Sphingobacterium mizutae Sphingobacterium mizutaii XG-3, Achromobacter xylosoxidans
Achromobacter xylosoxidans XG-14, Pi Shi achromobacter Achromobacter piechaudii XG-11 tri-
Kind strain fermentating liquid is according to 1：0.8~1.2：(0.8~1.2) is uniformly mixed, and the cell concentration of each strain is 10 in microbial inoculum7
~108CFU/mL。
In the more specific embodiment of the present invention, the method for preparing the composite bacteria agent of the invention includes：
(1) prepared by actication of culture and seed liquor：Preservation of bacteria strain Sphingobacterium mizutae of the present invention, xylose are aoxidized
Achromobacter, Pi Shi achromobacters are inoculated in the LB liquid medium of sterilizing respectively, in 25 DEG C~30 DEG C, 140rpm, lucifuge
Condition Shaking culture, activation is to growth late log phase, respectively as seed liquor.Preferably, above-mentioned every liter of LB culture mediums contain 10.0g
Tryptone, 5.0g yeast extracts, 5.0g NaCl, pH 7.4.
(2) fermented and cultured of strain：Above-mentioned seed liquor is inoculated with volume ratio 2%~5% respectively sterilized into 20L
In " beef extract/peptone-ASM " fermentation medium, 28 DEG C, pH 7.4, throughput 1.8L~2.2L/ of fermentation processes temperature
Min, mixing speed 150rpm, fermentation time 48h.At the end of fermented and cultured, the cell density of each strain is 108~109CFU/
mL.Preferably, above-mentioned " beef extract/peptone-ASM " every liter of beef extract containing 5g of fermentation medium, 10g peptones, the micro members of 2mL
Plain liquid (every liter of COCl containing 0.1g2·6H2O, 0.425g Mn02·4H2O, 0.05g ZnCl2, 0.01g NiCl2·6H2O,
0.015g CuS04·5H2O, 0.01g Na2MoO4·2H2O, 0.01g Na2SeO4·2H2O) and certain ASM compositions (including
24g NaCl, 0.7g KCl, 7.0g MgSO4·7H2O, 1.0g NH4NO3And 2.0g KH2PO4)。
(3) zymotic fluid compounding and concentration：The fermentation culture of 3 plants of strains is uniformly mixed and produces special composite bacteria agent.Separately
Outside, can by microbial inoculum, low-temperature centrifugation concentrates suitable multiple, such as 5 times of concentration under 4 DEG C, 8000rpm, or be further dried and be made
Solid-state bacteria preparation, so that preservation is transported.Each fermented liquid concentrates (or being further dried) after can mixing, or concentrate respectively (or enter
One step dry) after remix compounding.
The present invention composite bacteria agent can at 4 DEG C Cord blood more than 10 months.
On the other hand, present invention also offers described Sphingobacterium mizutae Sphingobacterium mizutaii
XG-3, Achromobacter xylosoxidans Achromobacter xylosoxidans XG-14, Pi Shi achromobacters
It is special that Achromobacter piechaudii XG-11 independent microbial inoculum (zymotic fluid) or composite bacteria agent repairing restores oily pollution
It is not the application in heavy oil pollution coastline or oil spill salt affected soil.It is uniformly to spray described microbial inoculum during concrete application
Spill in seashore (being sprayed during seawater low tide one) or salt affected soil surface.
Beneficial effects of the present invention：Special microbial inoculum salt resistant character provided by the invention is good, and competitive advantage protrudes, particularly profit
With the synergy of three kinds of single bacteriums, there is the heavy oil pollution for the especially refractory reason of coastline crude oil pollution efficient repair to imitate
Fruit, be equally applicable in the reparation of oil spill salt affected soil, nutritional agents, surfactant facilitation under, 40d pairs
The clearance of 5g/kg heavy oil pollution is respectively up to 60% and more than 75%.
Brief description of the drawings
Fig. 1 is Sphingobacterium mizutae Sphingobacterium mizutaii XG-3, Achromobacter xylosoxidans
Achromobacter xylosoxidans XG-14, Pi Shi achromobacters Achromobacter piechaudii XG-11 and
Degradation effect comparison diagram of its mixed bacterial to Venezuela's heavy oil.
Fig. 2 is Sphingobacterium mizutae Sphingobacterium mizutaii XG-3 sequencing sequence figure.
Fig. 3 is Achromobacter xylosoxidans Achromobacter xylosoxidans XG-14 sequencing sequence figure.
Fig. 4 is Pi Shi achromobacter Achromobacter piechaudii XG-11 sequencing sequence figure.
Fig. 5 is indoor oil spill coastline analogue means figure.
Fig. 6 is repairing effect of the special composite bacteria agent to simulation oil pollution coastline.
Microorganism for proprietary program preserves：
(1) Sphingobacterium mizutae Sphingobacterium mizutaii XG-3 of the invention
Preservation date：On July 26th, 2013；
Depositary institution：China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC)；
Depositary institution address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica
Deposit number：CGMCC No.8000；
Classification And Nomenclature：Sphingobacterium mizutae (Sphingobacterium mizutaii).
(2) Achromobacter xylosoxidans Achromobacter xylosoxidans XG-14 of the invention
Preservation date：On July 26th, 2013；
Depositary institution：China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC)；
Depositary institution address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica
Deposit number：CGMCC No.8022；
Classification And Nomenclature：Achromobacter xylosoxidans (Achromobacter xylosoxidans).
(3) Pi Shi achromobacter Achromobacter piechaudii XG-11 of the invention
Preservation date：On July 26th, 2013；
Depositary institution：China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC)；
Depositary institution address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica
Deposit number：CGMCC No.8021；
Classification And Nomenclature：Pi Shi achromobacters (Achromobacter piechaudii).
Embodiment
The implementation of technical solution of the present invention and possessed beneficial effect are described in detail below by way of specific embodiment, but not
Can regard as to the present invention can practical range any restriction.It is operation dated in detail according to institute in following each embodiments
The routine techniques in category field is carried out.In each embodiment, affiliated simulated seawater salinity is by using artificial seawater culture medium
(Artificial Seawater Medium, ASM) is realized, every liter of NaCl containing 24g, 0.7g KCl, 7.0g MgSO4·7H2O,
1.0g NH4NO3And 2.0g KH2PO4, pH 7.5.
The enrichment and screening of the oil degradation strain of embodiment 1
Strain separating of the present invention was once seriously overflow in 2010 in Talien New Port petroleum pipeline explosion accident
The Huanghai coast line of oily pollution.
Specific lock out operation is carried out as follows：Coastline bank is added into the sterilized wide spectrum nutrient solutions of 100mL
Beach sample 2g and a small amount of liquid microelement, the shaking table enrichment culture 5d under the conditions of 25 DEG C~30 DEG C, 140rpm, lucifuge；Continuous two
After secondary enrichment processing, take 1mL pregnant solutions transfer the 1g/L of heavy oil containing Venezuela ASM culture mediums in, add a small amount of micro member
Plain liquid, 25 DEG C~30 DEG C, 140rpm, cultivate 7d under the conditions of lucifuge, then by nutrient solution identical, fresh containing Venezuela's weight
Continuous switching is cultivated 3 times in oily ASM culture mediums.The nutrient solution after the 3rd switching is taken according to 10,102、103、104、105And 106Times
Gradient dilution is carried out, and takes 103、104、105Times dilution is respectively coated on LB culture plates, and training is inverted in 28 DEG C of incubators
Support 5d~7d；According to the colonial morphology of different microorganisms on flat board, distinguish picking with oese and come out and rule to fresh LB
Flat board, continue 28 DEG C be inverted culture, repeat line 2~3 times, produce can be using Venezuela's heavy oil as single carbon source single bacterium.Institute
Obtain strain and be separately added into sterilized 30% glycerite, the preservation under -70 DEG C of ultralow temperature.
Every liter of K containing 0.3g in above-mentioned wide spectrum nutrient solution2HPO4, 0.05g MgSO4·7H2O, 0.5g glucose, 0.3g boron
Acid, 0.5g soluble starches, 0.5g casamino acids, 0.5g peptones, 0.5g beef extracts；pH 7.5.Oil-containing ASM culture mediums
In every liter of heavy oil of Venezuela containing 1g, 24g NaCl, 0.7g KCl, 7.0g MgSO4·7H2O, 1.0g NH4NO3And 2.0g
KH2PO4, pH 7.4.Every liter of tryptone containing 10.0g of LB culture mediums, 5.0g yeast extracts, 5.0g NaCl, pH 7.5, Gu
Body flat board adds 17g agar.Above culture medium is before use through moist heat sterilization 30min under 120 DEG C, 0.1MPa.Above-mentioned micro member
Plain every liter of every liter of COCl containing 0.1g containing liquid microelement of liquid2·6H2O, 0.425g MnO2·4H2O, 0.05g ZnCl2, 0.01g
NiCl2·6H2O, 0.015g CuS04·5H2O, 0.01g Na2MoO4·2H2O, 0.01g Na2SeO4·2H2O。
The further investigation of oil degradation performance is carried out for all strains of single carbon source to be able to Venezuela's heavy oil.
Concrete operation step is as follows：Alkene crude oil 1g (1%, m/v) mixes in addition Venezuela in the ASM culture mediums to be sterilized to 100mL, is inoculated with
Each single bacterium suspensions of 5mL, not to be inoculated with the experimental group of bacteria suspension as control, 30 DEG C, 150rpm, determine after lucifuge culture 10d it is residual
Remaining TPHs, each processing set 3 repetitions.After 10d degrades, remaining TPHs amount is respectively 0.563g in ASM culture mediums,
0.515g, 0.413g, 0.872g, 0.798g, 0.831g, 0.902g, 0.858g etc..Wherein, three optimal plant weight oil degradation bacterias
To 1% heavy oil clearance more than 40% (43.7%, 48.5%, 58.7%), the degradation efficiency of remaining strain is 20%
Left and right.Final screening obtains three plants of bacterium of oil degradation efficiency high, and XG-3, XG-14, XG-11 are respectively designated as in the present invention.
XG-3, XG-14, XG-11 physio-biochemical characteristics and strain idenfication：
With the observation of LB solid mediums the present invention relates to the colonial morphology of strain, with violet staining, light microscope oil
The cellular morphology of each thalline of sem observation, and according to《The outstanding Bacteria Identification handbook of uncle》Method in (Science Press, 1984), to this
The physiological and biochemical property of three kinds of single bacteriums is analyzed involved by invention.The STb gene of three kinds of strains is extracted, carries out 16SrRNA analyses.
(1) XG-3 bacterium colony projection spherical in shape, it is translucent；Thalline is direct rod shape, no gemma, atrichia, Gram's staining
Feminine gender, G+C=54.6%；Oxidizing ferment and contact enzyme positive, nitrate reduction negative, it is impossible to hydrolysis starch, can Oxidation of Alcohol production
Acid, energy glucose fermentation, maltose, sucrose etc..16SrRNA sequencing sequences are referring to SEQ ID No.1 and Fig. 2.XG-3 and trough
Sphingobacterium has highest homology (being more than 98%), is accredited as Sphingobacterium mizutae Sphingobacterium
mizutaii。
The Sphingobacterium mizutae Sphingobacterium mizutaii XG-3 good salt tolerances of the present invention, 0~
It can be survived in 10% salinity range, salinity>Its growth is slightly suppressed when 5%.Under simulated seawater salinity, strain 10d
It is interior to Venezuela's heavy oil degradation efficiency that initial concentration is 1g/L up to more than 40%.Strain X G-3 is July 26 in 2013
Day is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (address：BeiChen West Road, Chaoyang District, BeiJing City 1
Number institute 3, Institute of Microorganism, Academia Sinica), deposit number：CGMCC No.8000；Classification And Nomenclature：Trough sphingol bar
Bacterium Sphingobacterium mizutaii.
(2) XG-14 bacterium colony moistening is glossy, and surface is smooth, neat in edge；Thalline is in rod-short, there is 1-5 root peritricha,
Gram-negative, G+C=54.0%；Oxidizing ferment, catalase, nitrate reduction are positive, edwardsiella hoshinae, oxidation of ethanol
Production is sour, azymic glucose, not hydrolysis starch.16SrRNA sequencing sequences are referring to SEQ ID No.2 and Fig. 3.XG-14 and xylose
Xylosoxidan has highest homology (being more than 98%), is accredited as Achromobacter xylosoxidans Achromobacter
xylosoxidans。
The Achromobacter xylosoxidans Achromobacter xylosoxidans XG-14 salt tolerances of the present invention are good, salinity
Still well-grown when reaching 7%, it is slightly suppressed after reaching 10%.The strain is under simulated seawater salinity, to initial dense in 10d
Venezuela's heavy oil degradation efficiency for 1g/L is spent up to more than 45%.Strain X G-14 is on July 26th, 2013 is preserved in
State's Microbiological Culture Collection administration committee common micro-organisms center (address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, in
Institute of microbiology of the academy of sciences of state), deposit number：CGMCC No.8022；Classification And Nomenclature：Achromobacter xylosoxidans
Achromobacter xylosoxidans。
(3) Pi Shi achromobacters Achromobacter piechaudii XG-11 bacterium colonies are smaller, circular, colourless to greyish white
Color, slightly raised, surface is smooth and edge is complete；Thalline is club shape, has motility, Gram-negative, G+C=
54.3%, oxidizing ferment, contact enzyme positive, nitrate reduction is positive, edwardsiella hoshinae, not hydrolysis starch, can glucose fermentation, wood
Sugar production acid.16SrRNA sequencing sequences are referring to SEQ ID No.3 and Fig. 4.XG-11 and Pi Shi achromobacters have highest homologous
Property (be more than 98%), be accredited as Pi Shi achromobacter Achromobacter piechaudii.
The Pi Shi achromobacter Achromobacter piechaudii XG-11 salt tolerances of the present invention are preferable, 0~7%
Salinity range in can survive, salinity be 7% when it is slightly suppressed.The strain is under simulated seawater salinity, to initial dense in 10d
Venezuela's heavy oil degradation efficiency for 1g/L is spent up to more than 50%.Strain X G-11 is on July 26th, 2013 is preserved in
State's Microbiological Culture Collection administration committee common micro-organisms center (address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, in
Institute of microbiology of the academy of sciences of state), deposit number：CGMCC No.8021；Classification And Nomenclature：Pi Shi achromobacters
Achromobacter piechaudii。
The degradation property of the single bacterium of embodiment 2 and mixed bacterial to Venezuela's heavy oil
Using Venezuela's heavy oil as pollutant, the stone of above single culture and mixed bacteria is investigated under simulated seawater salinity
Oily degradation property.Carry out as follows：(1) the liquid LB of the strain 200uL addition sterilizings of low-temperature preservation is pipetted with liquid-transfering gun
In culture medium, the vibration activation 48h under the conditions of 25 DEG C~30 DEG C, 140rpm, switching fresh LB is repeated 1 time.Now, take
5000rpm centrifuges 10min to turbid solution in growth logarithmic phase at room temperature, and collection thalline is simultaneously clear with sterile phosphate buffer
Wash, after so repeating at least 3 times, specified strain density (OD is diluted to phosphate buffer600), it is prepared into single culture and hangs
Liquid.Every liter of K containing 21.759g in above-mentioned phosphate buffer2HPO4·H2O, 33.48g Na2HPO4·12H2O, 8.79g
KH2PO4, 5.09g NH4Cl, pH 7.4.(2) each single bacterium suspension is inoculated in 100mL simulation oil pollution ASM culture mediums,
Venezuela's heavy oil content is 1g/L, and bacterial suspension inoculation amount is 5% (volume ratio), 25 DEG C~30 DEG C, 140rpm, lucifuge condition
Lower shaken cultivation 10d, determine single bacterium oil degradation efficiency；It is separately compound bacteria suspension by three kinds of single bacterium suspension mixed in equal amounts, carries out
Identical is investigated.Wherein, each processing sets 3 repetitions, and right as feminine gender instead of bacteria suspension to add equivalent sterile distilled water
According to.(3) degrade after 10d, the content of zymotic fluid residual crude oil is carried out after carbon tetrachloride extraction, constant volume using Infrared Oil Determination Instrument
Measure, degradation rate (%)=(1- residual crude oils amount/original crude oil amount) × 100.
Sphingobacterium mizutae Sphingobacterium mizutaii XG-3 of the present invention, xylose oxidation are colourless
Bacillus Achromobacter xylosoxidans XG-14, Pi Shi achromobacter Achromobacter piechaudii XG-
11 and its mixed bacterial Fig. 1 is shown in the degradation efficiency of Venezuela's heavy oil.Wherein, under simulated seawater salinity, by 10d drop
Solution, Sphingobacterium mizutae Sphingobacterium mizutaii XG-3 are reachable to the degradation efficiency of Venezuela's heavy oil
41.4%, Achromobacter xylosoxidans Achromobacter xylosoxidans XG-14 are up to the colourless bars of 47.5%, Pi Shi
Bacterium Achromobacter piechaudii XG-11 degradation efficiency highest, reach 51.6%, three synergistic bacterium effects processing
Better, clearance is up to 58.2%.
Embodiment 3 is related to the salt resistant character of strain
Being influenceed by seawer tide and evaporation, the salinity of oil spill shoreline environment is higher, and the change of complexity is presented,
It is to influence microorganism growth metabolism and emulative key factor.The present embodiment is carried out as follows：The strain concentration such as take
Sphingobacterium mizutae Sphingobacterium mizutaii XG-3, Achromobacter xylosoxidans Achromobacter
Xylosoxidans XG-14, Pi Shi achromobacter Achromobacter piechaudii XG-11 suspensions by volume 5%
Ratio be inoculated in NaCl mass concentrations gradient be 0,1%, 3%, 5%, 7%, 10% LB nutrient solutions in, 28 DEG C,
After lower culture 24h is vibrated under the conditions of 140rpm, lucifuge, the OD of each mixed liquor is determined600, according to each dominant bacteria of evaluation
The Saline alkali tolerance energy of kind.If the OD of bacterium solution600>0.25, then it is assumed that strain well-grown in the saline environment, its salt tolerance mark
For " ++ ", if OD600>0.15, it is believed that strain can grow in the saline environment, and its salt tolerance is designated as "+", if OD600<
0.05, then it is assumed that the strain can not grow, and refer to table 1.Sphingobacterium mizutae, Achromobacter xylosoxidans, skin as seen from table
Family name's achromobacter bacterium has preferable salt resistant character, has well adapting to property and competition in the shoreline environment of high salinity
Property.
Table 1XG-3, XG-14, XG-11 salt resistant character
The preparation method of the remedying oil-polluted coastline special project composite bacteria agent of embodiment 4
Remedying oil-polluted coastline special project composite bacteria agent is by Sphingobacterium mizutae Sphingobacterium
Mizutaii XG-3, Achromobacter xylosoxidans Achromobacter xylosoxidans XG-14, Pi Shi achromobacters
Achromobacter piechaudii XG-11 zymotic fluid is uniformly mixed, and the fermentation of each strain can use routine
Fermentation process is carried out, and following public method is only one of which, is not intended to limit the present invention.Implementation steps are：
(1) prepared by actication of culture and seed liquor.Preservation of bacteria strain Sphingobacterium mizutae of the present invention, xylose are aoxidized
Achromobacter, Pi Shi achromobacters are inoculated in the LB liquid medium of sterilizing respectively, in 25 DEG C~30 DEG C, 140rpm, lucifuge
Condition Shaking culture, activation prepare seed liquor to growth late log phase.
(2) fermented and cultured of strain.Above-mentioned seed liquor is respectively connected to the sterilized hairs of 20L with 2%~5% inoculum concentration
In ferment culture medium, 25 DEG C, pH 7.4, throughput 1.8L~2.2L/min, mixing speed 150rpm of fermentation processes temperature, hair
Ferment time 48h.Above-mentioned fermentation medium uses beef extract/peptone-ASM culture mediums, every liter of beef extract containing 5g, 10g peptones,
2mL liquid microelements and ASM medium components (24g NaCl, 0.7g KCl, 7.0g MgSO4·7H2O, 1.0g NH4NO3And
2.0g KH2PO4).At the end of fermented and cultured, the cell density of each strain is 108~109CFU/mL。
(3) compound and concentrate.By the fermentation culture of 3 plants of strains according to 1：1：1 volume ratio, which uniformly mixes, produces special project
Composite bacteria agent.In addition, can by microbial inoculum under 4 DEG C, 8000rpm low-temperature centrifugation concentrate 5 times, so as to preservation transport, the shelf-life 10
Month.
Shaking flask repairing effect of the 5 special composite bacteria agent of embodiment to oil pollution seashore deposit
The specific embodiment of the present embodiment is：
(1) preparation of oil pollution seashore deposit.The pollution-free seashore deposit of uniform particle sizes is chosen, it is a per 50g
Dispensed with 150mL conical flasks, 121 DEG C, 0.1MPa, 30min moist heat sterilization；Venezuela's heavy oil is fully molten with dichloromethane
Solution, add in above-mentioned deposit and stir in the ratio of 5g heavy oil/kg deposits, 38 DEG C volatilize solvent and quiet in fume hood
Put standby after 24h reaches stable.
(2) special composite bacteria agent reparation is added.It is 10 by cell density8~109CFU/mL three kinds of single bacterium zymotic fluids are uniform
Mixing, structure oil pollution coastline are repaired special composite bacteria agent, added in the ratio of 50mL microbial inoculums/kg deposits, together
Shi Tianjia NaCl solids adjustment salinity is 3%, and moisturizing presses C to moisture content 30%：N：P=100：20：3 add nutritional agents, i.e.,
(NH4)2SO44.01g/kg、KH2PO40.57g/kg, after stirring 25 DEG C~30 DEG C, 140rpm, lucifuge condition Shaking culture
12d.The processing for replacing microbial inoculum using equivalent sterilized water sets 3 repetitions as negative control, each processing.
(3) after Degrading experiment terminates, carbon tetrachloride solvent is added into deposit, is extracted using soxhlet extraction methods remaining
Oil, the content of deposit Crude Oil is determined after constant volume with Infrared Oil Determination Instrument, calculate oil removal effect.
As a result show, oil pollution coastline of the invention repairs special composite bacteria agent and Venezuela's heavy oil pollution is repaiied
Effect is answered up to 60.8%.
Simulation repairing effect of the 6 special composite bacteria agent of embodiment to oil spill coastline
The biological prosthetic test platform in oil spill coastline is built in laboratory, simulates oil spill seashore and its tide ring
Border, repaired using the special composite bacteria agent of the present invention.
The oil spill seashore analogue means is as shown in Figure 5.By aerator cylinder 1, seawater storage tank 2, peristaltic pump 3,
The biological prosthetic part of reactor 4 four composition, wherein biological prosthetic reactor is cylindrical configuration, material is polymethyl methacrylate
(lucite), its middle column body internal diameter 190mm, interior high 280mm, top is provided with blow vent, oil pollution is placed in reactor
Intertidalite 5.Intake-outlet aperture 10mm, it is connected through pvc pipe with peristaltic pump, for simulated seawater tide environment.
Following preparation is carried out before on-test：Free of contamination seashore deposit cross 2mm sieve, 121 DEG C, 0.1MPa,
The processing of 30min moist heat sterilizations, takes deposit 4kg to be put into device, opens peristaltic pump and introduces artificial seawater and run 6 tides
Phase, seashore deposit is set to reach balance and stability；Take 20g Venezuela heavy oil to be mixed with a small amount of artificial seawater, stir energetically, be
System operation is spilt in seawater surface to uniformly being poured during big tide water level, continues to run with complete 4 damp phases, make crude oil with ebb tide seawater by
It is progressive enter deposit space between, and reach stable, hereafter can add special composite bacteria agent and carry out simulation reparative experiment.Above-mentioned people
Work seawater is using the Mocledon formulas shown in table 2, seawater salinity 3% or so；Tide law simulation Dalian coastline regular half
Diurnal tide, duration of rise 6h7min, ebb last 6h18min, and each damp phase is approximately 12h.
Table 2Mocledon artificial seawater formulas
By three kinds of single bacterium zymotic fluids etc. that cell density is 108~109CFU/mL than being mixed into special composite bacteria agent, add
Amount is set to 100mL, test run 40 days, takes the sediment sample of 10cm~15cm below top layer, is taken out using carbon tetrachloride Soxhlet
- remaining the oil content of Infrared Oil Determination Instrument method measure is put forward, calculates the repairing effect of microbial inoculum.Not add the processing of microbial inoculum as negative right
According to, while separately set two groups of experiments as a comparison, one group on the basis of microbial inoculum is added by (NH4)2SO44.01g/kg、
KH2PO40.57g/kg extra-nutrition agent, one group adds rhanolipid as biosurfactant on the basis of microbial inoculum, nutritional agents
80mg/kg, improve the biological usability of heavy oil pollutant, improve repairing effect.
As shown in fig. 6, in figure, " Bac " represents to add microbial inoculum (Bacteria) above-mentioned result of implementation；" BN " expression " microbial inoculum+
Nutritional agents " (Bacteria+Nutrient)；" BNR " expression " microbial inoculum+nutritional agents+rhamnolipid " (Bacteria+Nutrient+
Rhamnolipid), " NC " represents negative control (Negative Control).It can be seen that special composite bacteria agent is only added to initial
Concentration is that the clearance of 5g/kg Venezuela heavy oil is 61.7%；Add special composite bacteria agent and be aided with nutritional agents, to auspicious in committee
The clearance for drawing heavy oil is 64.9%；Special composite bacteria agent, nutritional agents and biosurfactant are added, is removed up to highest
Rate 76.3%.
Made it can be seen that special composite bacteria agent is repaired in oil pollution coastline of the present invention using the collaboration of three kinds of single bacteriums
With being polluted for coastline crude oil pollution especially heavy oil has efficient repairing effect, in addition, the salt tolerance of microbial inoculum is strong, also may be used
In reparation applied to oil spill salt affected soil.
Claims (7)
1. a kind of composite bacteria agent for being used to repair heavy oil pollution coastline, the composite bacteria agent is by Sphingobacterium mizutae
(Sphingobacterium mizutaii), Achromobacter xylosoxidans (Achromobacter xylosoxidans) and
Pi Shi achromobacters (Achromobacter piechaudii) form；
In the composite bacteria agent, the viable count of the Sphingobacterium mizutae, Achromobacter xylosoxidans and Pi Shi achromobacters it
Than for 1~10：1~10：1~10；
Wherein, the Sphingobacterium mizutae is the Sphingobacterium mizutae that preserving number is CGMCC No.8000；
The Achromobacter xylosoxidans are the Achromobacter xylosoxidans that deposit number is CGMCC No.8022；
The Pi Shi achromobacters are the Pi Shi achromobacters that deposit number is CGMCC No.8021.
2. composite bacteria agent according to claim 1, the composite bacteria agent is solid-state or liquid bacteria preparation.
3. composite bacteria agent according to claim 2, wherein, the cell concentration of each strain is 10 in the liquid bacteria preparation7~
108CFU/mL。
4. the preparation method of the composite bacteria agent described in any one of claims 1 to 3, this method include：
The liquid LB that Sphingobacterium mizutae, Achromobacter xylosoxidans, Pi Shi achromobacters are inoculated in sterilizing respectively is cultivated
In base, in 25 DEG C~30 DEG C lucifuge condition Shaking cultures, to late log phase is grown, respectively as seed liquor；
Above-mentioned seed liquor is inoculated with the nutrition fermentation medium of sterilizing respectively with volume ratio 2%~5%, 25 DEG C~30 DEG C trainings
It is 10 to support to the cell density of each strain8~109CFU/mL, obtain zymotic fluid；
Above-mentioned zymotic fluid is compounded, or further concentration or dry as needed, composite bacteria agent is made；
Wherein, the nutrition fermentation medium includes beef extract, peptone and ASM compositions；Wherein described ASM compositions are every liter of training
Supporting base includes 24g NaCl, 0.7g KCl, 7.0g MgSO4·7H2O, 1.0g NH4NO3And 2.0g KH2PO4。
5. the composite bacteria agent described in any one of claims 1 to 3 is in remedying oil-polluted coastline or oil spill salt affected soil
In application.
6. application according to claim 5, wherein, the oil pollution is Venezuela's heavy oil pollution.
7. application according to claim 5, wherein, it is that described composite bacteria agent is uniformly sprayed on seashore or salt affected soil
Surface.
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