CN106635909A

CN106635909A - Crude oil degradation mixed bacterium, microbial agent and application of microbial agent

Info

Publication number: CN106635909A
Application number: CN201611243167.6A
Authority: CN
Inventors: 李春荣; 张帆; 王文科; 申圆圆; 邓红章; 张慧慧; 姬雨; 王广华; 郭静茹
Original assignee: 长安大学
Current assignee: Shaanxi Aomei Environmental Protection Technology Co ltd
Priority date: 2016-12-29
Filing date: 2016-12-29
Publication date: 2017-05-10
Anticipated expiration: 2036-12-29
Also published as: CN106635909B

Abstract

The invention particularly discloses a crude oil degradation mixed bacterium which comprises at least two of a marine crude oil degradation bacterium 6-10-1, brevundimonas diminuta 1-8, bacillus thuringiensis 3-1 or pseudomonas geniculate 3. The invention further discloses a microbial agent which uses the marine crude oil degradation bacterium 6-10-1, the brevundimonas diminuta 1-8, the bacillus thuringiensis 3-1 or the pseudomonas geniculate 3 as an active ingredient, and application thereof to degradation of crude oil pollution water and crude oil pollution soil. The crude oil degradation mixed bacterium and the microbial agent thereof are easy for scale production, and have an excellent degradation effect on both the crude oil pollution water and the crude oil pollution soil.

Description

A kind of oil degradation Mixed Microbes, microbial inoculum and its application

Technical field

The present invention relates to technical field of bioengineering, and in particular to a kind of oil degradation Mixed Microbes, microbial inoculum and its application.

Background technology

The alkane for being included, aromatic hydrocarbon, sulfur-containing compound and sulfur-bearing or nitrogenous heterocyclic compound etc. in oil is With toxicity, to the very harmful of the mankind and environment.It is natural organic substance through geology and crude oil is crude oil A kind of sticky black for changing and being formed or dark-brown liquid or solid, are generally present in underground or sea mostly Bottom, and with penetrating odor, main component is the chain compound that hydrocarbon atom is combined to form, and it is to water body, soil, air Environment Deng human survival has greatly harm.

The recovery technique of water body, soil at present, to oil pollution etc. mainly has peripheral doses technology, chemical redemption skill Art, photocatalysis recovery technique and bioremediation technology；Bioremediation technology is one and has policy, economic and technical attraction Processing method, it is considered to be most potential technical method, and progressively develop into a kind of economic benefit and Environmental Effect Benefit is all good, solve the maximally effective means of complex environment pollution problem.

A kind of microorganism is only capable of degraded one kind or a few crude oil hydrocarbon for universal, and different microorganisms are to pollution of the same race The degradation effect of component is also different, and multiple-microorganism is in degradation process it is also possible that collaboration or Competition.But crude oil Product is refined oil with it, contained crude oil Jing species is extremely complex, and crude oil its crude oil Jing compositions in the not homologous place of production also have very Big difference.Therefore microorganism remediation technology also having some limitations property while developing rapidly, specific microorganism can only Degrade specific type of compounds, breeding and the metabolic activity of microorganism are affected larger by environmental factor, usually add to There is mortality in the starting stage of soil.

The content of the invention

For problems of the prior art, it is an object of the invention to provide the original that a kind of oil degradation works well Oil degraded Mixed Microbes, microbial inoculum and its application.

In order to achieve the above object, the present invention is employed the following technical solutions and is achieved.

(1) a kind of oil degradation Mixed Microbes, it is characterised in that including Crude oil from CNOOC degradation bacteria (Advenella Kashmirensis) 6-10-1, defect shortwave monad (Brevundimonas diminuta) 1-8, bacillus thuringiensis In (Bacillus thuringiensis) 3-1 or Pseudomonas geniculate (Pseudomonas geniculata) 3 at least two Kind.

Crude oil from CNOOC degradation bacteria (Advenella kashmirensis) 6-10-1 was in preservation on the 12nd in 09 month in 2016 Manage in the Chinese microorganism strain preservation of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica Committee's common micro-organisms center, preserving number is CGMCC No.13003.

Defect shortwave monad (Brevundimonas diminuta) 1-8 was preserved in north on 09 12nd, 2016 The Chinese microorganism strain preservation conservator of No. 3 Institute of Microorganism, Academia Sinica of institute of Jing Shi Chaoyang Districts North Star West Road 1 Meeting common micro-organisms center, preserving number is CGMCC No.13002.

Bacillus thuringiensis (Bacillus thuringiensis) 3-1 was preserved in north on 09 12nd, 2016 The Chinese microorganism strain preservation conservator of No. 3 Institute of Microorganism, Academia Sinica of institute of Jing Shi Chaoyang Districts North Star West Road 1 Meeting common micro-organisms center, preserving number is CGMCC No.13005.

The Pseudomonas geniculate (Pseudomonas geniculata) 3 was preserved in Beijing on 09 12nd, 2016 The China Committee for Culture Collection of Microorganisms of Chaoyang District North Star West Road No. 3 Institute of Microorganism, Academia Sinica of No. 1 institute is general Logical microorganism center, preserving number is CGMCC No.13004.

The 16S rDNA sequencing results of Crude oil from CNOOC degradation bacteria (Advenella kashmirensis) 6-10-1 are such as Shown in SEQ ID No.1.

The 16S rDNA sequencing result such as SEQ of defect shortwave monad (Brevundimonas diminuta) 1-8 Shown in ID No.2.

The 16S rDNA sequencing result such as SEQ of bacillus thuringiensis (Bacillus thuringiensis) 3-1 Shown in ID No.3.

The 16S rDNA sequencing results such as SEQ ID of the Pseudomonas geniculate (Pseudomonas geniculata) 3 Shown in No.4.

(2) a kind of oil degradation mixing bacteria agent, it is characterised in that its active component includes Crude oil from CNOOC degradation bacteria (Advenella kashmirensis) 6-10-1, defect shortwave monad (Brevundimonas diminuta) 1-8, Su Yun Golden bacillus (Bacillus thuringiensis) 3-1 or Pseudomonas geniculate (Pseudomonas geniculata) 3 In at least two.

Preferably, the oil degradation mixing bacteria agent is solid-state microbial inoculum.

(3) application of the oil degradation mixing bacteria agent in the degraded of crude oil pollution water body.

(4) application of the oil degradation mixing bacteria agent in oil contaminated soil degraded.

Compared with prior art, beneficial effects of the present invention are：

The oil degradation Mixed Microbes and oil degradation mixing bacteria agent that the present invention is provided are easy to large-scale production, dirty to crude oil Dye water body and oil contaminated soil are respectively provided with stronger degradation effect；The microbial inoculum strong environmental adaptability, can be in large amount of organic Smooth growth under conditions of presence, with crude oil as carbon source, so as to reach preferable degradation effect；Realize crude oil pollution waste water and Good application prospect is presented in the reparation of oil contaminated soil.

Description of the drawings

Below in conjunction with the accompanying drawings the present invention is described in further details with specific embodiment.

Fig. 1 is offshore oil degradation bacteria (Advenella kashmirensis) 6-10-1 of the present invention in beef extract albumen Colonial morphology figure on peptone culture medium flat plate；

Fig. 2 is that 6-10-1 is under the microscope for offshore oil degradation bacteria (Advenella kashmirensis) of the invention Thalli morphology figure, in figure, multiplication factor be 1000 times.

Fig. 3 is defect shortwave monad (Brevundimonas diminuta) 1-8 of the present invention in beef extract-peptone Colonial morphology figure on culture medium flat plate；

Fig. 4 is that 1-8 is under the microscope for defect shortwave monad (Brevundimonas diminuta) of the invention Thalli morphology figure, in figure, multiplication factor is 1600 times.

Fig. 5 is bacillus thuringiensis (Bacillus thuringiensis) 3-1 of the present invention in beef extract-peptone Colonial morphology figure on culture medium flat plate；

Fig. 6 is that 3-1 is under the microscope for bacillus thuringiensis (Bacillus thuringiensis) of the invention Thalli morphology figure, in figure, multiplication factor is 4000 times.

Fig. 7 is Pseudomonas geniculate (the Pseudomonas geniculata) 3 of the present invention in beef extract-peptone culture Colonial morphology figure on base flat board；

Fig. 8 is the thalline under the microscope of Pseudomonas geniculate (Pseudomonas geniculata) 3 of the present invention Aspect graph, in figure, multiplication factor is 1000 times；

Fig. 9 is the wavelength-absorbance curve figure of I crude oil in the embodiment of the present invention 3；In figure, abscissa is wavelength, unit For nm, ordinate is absorbance, and unit is 1；

Figure 10 is the canonical plotting of I crude oil in the embodiment of the present invention 3；In figure, abscissa is concentration, and unit is mg/ L, ordinate is absorbance, and unit is 1；

Figure 11 is the canonical plotting of II crude oil in the embodiment of the present invention 6；In figure, abscissa is concentration, and unit is mg/ L, ordinate is absorbance, and unit is 1.

Specific embodiment

Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will It will be appreciated that the following example is merely to illustrate the present invention, and it is not construed as limiting the scope of the present invention.

Embodiment 1 bacterial strain is isolated and purified

(1) bacterium source

The oil degradation Mixed Microbes of the present invention are individually separated former from the inner hole oil recovery field oil gathering station landing of crude oil group seven is extended Soil contaminated by crude oil.Method collection is layouted away from earth's surface 20cm or so topsoil using plum blossom, sample is put into into the aseptic guarantor of 4 DEG C of refrigerators Hide, its physicochemical property is：Ammonium nitrogen 15.09ppm, nitrate nitrogen 0.72ppm；Nitrogen content 15.81ppm；Phosphorus content 82.05ppm；Potassium Content 17.45ppm；The content of organic matter 1.43%；PH is 7.65.

(2) bacterial strain is isolated and purified

10g oil contaminated soils sample is added in 100mL enriched liquid culture mediums, 28 DEG C, 130r/min shaking table cultures 7d；After nutrient solution muddiness, draw 5mL nutrient solutions and transfer again in fresh enriched medium, it is identical with above-mentioned condition of culture Continuous switching enrichment culture 3 times.The above-mentioned bacteria suspensions of 1mL are taken, after being serially diluted with sterilized water, beef extract-peptone is inoculated in respectively In culture medium, 29 DEG C or so incubated 3d；Wherein, the composition of enriched liquid culture medium is：NaNO₃1.5g, (NH₄)SO₄ 1.5g, K₂HPO₄1g, MgSO₄·7H₂O 0.5g, KCl 0.5g, FeSO₄·7H₂O 0.01g, CaCl₂0.002g, distilled water 1000mL, crude oil No. I 1% (volume ratio), pH 7.0.The composition of beef-protein medium is：Beef extract 3.0g, peptone 10.0g, NaCl 5.0g, nutrient agar 15.0g, distilled water 1000mL, pH 7.4-7.6.After flat-plate bacterial colony is efficiently separated, root According to the difference of each colony colour, morphological feature and surface moisture, carry out plate streaking with transfer needle picking individual colonies respectively and purify Separate.Each single strain is inoculated in into inclined-plane, 4 DEG C of refrigerator cold-storages are standby.

(3) molten scraper ring method screens oil degradation bacterium

Above-mentioned single bacterium bacterial classification is inoculated in respectively on oil-containing inorganic salts solid medium, is placed in 37 DEG C of incubators and is cultivated 10 ～15d, observation whether there is thermophilic scraper ring, and primarily determines that the crude oil of bacterial strain according to the ratio of thermophilic scraper ring diameter (D) and colony diameter (d) Degradation capability；Wherein, the composition of oil-containing inorganic salts solid medium is：NaNO₃1.5g, (NH₄)₂SO₄1.5g, K₂HPO₄1g, MgSO₄·7H₂O 0.5g, KCl 0.5g, FeSO₄·7H₂O 0.01g, CaCl₂0.002g, distilled water 1000mL, crude oil 1.5mL, pH 7.0, pH value is adjusted to required value with the NaOH or 1mol/L hydrogen chloride of 1mol/L.By the crude oil of preliminary screening Degradation bacteria is inoculated in slant medium, and 4 DEG C of refrigerator cold-storages are standby.

The identification of the bacterial strain of embodiment 2

(1) morphological feature of bacterial strain

As shown in figure 1, by the offshore oil degradation bacteria after 30 DEG C of incubated 24h on beef-protein medium Observation colonial morphology, bacterial strain 6-10-1 is rounded on the flat board of beef extract-peptone, edge is complete, smooth, opaque, milk is white Color, surface wettability, without special odor.As shown in Fig. 2 thalline is in rod-short, size is (0.41-1.02) μ m (0.41- 1.53) μm, atrichia.

As shown in figure 3, by the defect shortwave monad after 30 DEG C of incubated 24h on beef-protein medium Observation colonial morphology, bacterial strain 1-8 is in light yellow on the flat board of beef extract-peptone, with the prolongation of incubation time, bacterium colony face Color is slightly deepened, and surface is smooth, moistening, and bacterium colony is rounded, and central protrusion, neat in edge is presented translucent.As shown in figure 4, In quarter butt, bar-shaped, cell is in single arrangement to somatic cells, and size is 0.5 μ m (0.1-0.3) μm.

As shown in figure 5, by the bacillus thuringiensis after 30 DEG C of incubated 24h on beef-protein medium Observation colonial morphology, bacterial strain 3-1 is rounded on the flat board of beef extract-peptone, and edge is serrated, intermediate projections, yellow fraction Color, surface is smooth, moistening.As shown in fig. 6, thalline is rod-short, size is (1.2-1.5) μ m (3-5) μm.

As shown in fig. 7, the Pseudomonas geniculate is seen after 30 DEG C of incubated 24h on beef-protein medium Colonial morphology is examined, originally bacterial strain 3 is in faint yellow, and with the increase of cell age, color is more and more deeper, finally in yellow.The bacterium colony is special The faint yellow bacterium colony for needle point size is levied, in regular circle shapes, the smooth moistening in surface, neat in edge after Gram's staining, is difficult Color.As shown in figure 8, somatic cells are in quarter butt, bar-shaped, size is 0.5 μm of (1-1.5) μ m, is Gram-negative bacteria.

(2) biochemical characteristic

According to《Primary Jie Shi Bacteria Identifications handbook》With《Common bacteria system identification handbook》To offshore oil degradation bacteria, defect Shortwave monad, bacillus thuringiensis and Pseudomonas geniculate carry out Physiology and biochemistry identification, and qualification result is as shown in table 1-4.

The Physiology and biochemistry qualification result of the offshore oil degradation bacteria 6-10-1 bacterial strain of table 1

Index	Characteristic
		Gram's staining	—
Glucose	+
		Wood sugar	+
Fructose	+
		Acetate	+
Lactose	+
		Maltose	—
Sucrose	—
		Oxidizing ferment	+
Catalase	+
		Urase	+
Nitrate reducibility	—

Note："-" represents that reaction is feminine gender；"+" represents that reaction is the positive.

The Physiology and biochemistry qualification result of the defect shortwave monad 1-8 bacterial strains of table 2

The Physiology and biochemistry qualification result of the bacillus thuringiensis 3-1 bacterial strains of table 3

Note："-" represents that reaction is feminine gender；"+" represents that reaction is the positive

The bacterial strain Physiology and biochemistry qualification result of the Pseudomonas geniculate of table 4 (Pseudomonas geniculata) 3

(3) the 16S rDNA identifications of bacterial strain

The genomic DNA of Crude oil from CNOOC degradation bacteria is extracted respectively with DNA extraction kit, then carries out 16S rDNA genes Clone, sequencing, its 16S rDNA sequencing result is as shown in SEQ ID No.1；The genomic DNA of defect shortwave monad is extracted, Then 16S rDNA gene clonings, sequencing are carried out, its 16S rDNA sequencing result is as shown in SEQ ID No.2；Extract Su Yunjin The genomic DNA of bacillus, then carries out 16S rDNA gene clonings, sequencing, its 16S rDNA sequencing result such as SEQ ID Shown in No.3；The genomic DNA of Pseudomonas geniculate is extracted, 16S rDNA gene clonings, sequencing, its 16S rDNA is then carried out Sequencing result is as shown in SEQ ID No.4.

Degraded of the oil degradation Mixed Microbes bacteria suspension of embodiment 3 to crude oil pollution water body

Aseptically, picking isolates and purifies the Crude oil from CNOOC degradation bacteria (Advenella kashmirensis) of acquisition 6-10-1, defect shortwave monad (Brevundimonas diminuta) 1-8, bacillus thuringiensis (Bacillus Thuringiensis) 3-1 and Pseudomonas geniculate (Pseudomonas geniculata) 3 are inoculated in respectively the beef of 100mL In cream peptone fluid nutrient medium, shaking table Amplification Culture culture 48h；Wherein, the composition of beef extract-peptone fluid nutrient medium is： Beef extract 3.0g, peptone 10.0g, NaCl 5.0g, nutrient agar 15.0g, distilled water 1000mL, pH 7.4-7.6.PH value is used The NaOH or 1mol/L hydrogen chloride of 1mol/L is adjusted to required value.Medium sterilization condition is：Temperature is 121～126 DEG C, when Between be 25min.

(1) preparation of oil degradation Mixed Microbes bacteria suspension

1) Crude oil from CNOOC degradation bacteria (Advenella kashmirensis) 6-10-1 bacterium solutions 8mL of Amplification Culture are taken, is lacked Sunken shortwave monad (Brevundimonas diminuta) 1-8 bacterium solutions 3mL, bacillus thuringiensis (Bacillus Thuringiensis) 3-1 bacterium solutions 1mL are homogenously mixed together, and obtain 1# oil degradation plastc rings；

2) Crude oil from CNOOC degradation bacteria (Advenella kashmirensis) 6-10-1 bacterium solutions 4mL of Amplification Culture, curved are taken 3 bacterium solutions 1mL of bent pseudomonad (Pseudomonas geniculata), bacillus thuringiensis (Bacillus Thuringiensis) 3-1 bacterium solutions 1mL are homogenously mixed together, and obtain 2# oil degradation plastc rings.

3) defect shortwave monad (Brevundimonas diminuta) 1-8 bacterium solutions 4mL, the bending for taking Amplification Culture is false Monad (Pseudomonas geniculata) 3 bacterium solutions 1mL, bacillus thuringiensis (Bacillus thuringiensis) 3-1 bacterium solutions 1mL are homogenously mixed together, and obtain 3# oil degradation plastc rings.

4) Crude oil from CNOOC degradation bacteria (Advenella kashmirensis) 6-10-1 bacterium solutions 6mL and the Soviet Union of Amplification Culture are taken Cloud gold bacillus (Bacillus thuringiensis) 3-1 bacterium solutions 2mL are homogenously mixed together, and obtain the mixing of 4# oil degradations Bacteria suspension.

5) Crude oil from CNOOC degradation bacteria (Advenella kashmirensis) 6-10-1 bacterium solutions 3mL and the Soviet Union of Amplification Culture are taken Cloud gold bacillus (Bacillus thuringiensis) 3-1 bacterium solutions 3mL are homogenously mixed together, and obtain the mixing of 5# oil degradations Bacteria suspension.

6) Crude oil from CNOOC degradation bacteria (Advenella kashmirensis) 6-10-1 bacterium solutions 3mL of Amplification Culture and curved are taken 3 bacterium solutions 3mL of bent pseudomonad (Pseudomonas geniculata) are homogenously mixed together, and obtain 6# oil degradations Mixed Microbes and hang Liquid.

(2) degraded of the oil degradation Mixed Microbes bacteria suspension to crude oil pollution water body

Test method：

(1) measure of I crude oil maximum absorption wavelength

Oil and products thereof has certain Absorption Characteristics in ultraviolet region, the aromatic compound with phenyl ring in petroleum component Object wave length is generally 250～260nm, and carries the compound wavelength of conjugated double bond typically between 215～230nm, there is research Show, when being determined using ultraviolet spectrometry light method, 255nm or so be crude oil and the range of choice of mink cell focus, 225nm or so with for The range of choice of the oil product of light oil and oil plant.Under 100mg/L concentration, according to the wavelength and absorbance data that obtain, paint System supplies the wavelength-absorbance curve of examination I crude oil as shown in figure 9, wherein, and I crude oil picks up from the inner hole of prolongation Petroleum Group seven and adopts Oily field oil gathering station, density is 0.65g/mL.

(2) drafting of I crude oil calibration curve

Take 0 respectively, 2,2.5,5,6,7mg I crude oil in 50mL colorimetric cylinders, use petroleum ether constant volume, concentration point now Not Wei 0,40,50,100,120,140mg/L, under 258nm wavelength, with petroleum ether as reference solution, the suction of bioassay standard concentration Luminosity.Abscissa is concentration (mg/L), and ordinate is absorbance.Draw the mark of concentration (the mg/L)-absorbance (A) of I crude oil Directrix curve is as shown in Figure 10.

(3) measure of oil degradation rate

1# oil degradation plastc rings, 2# oil degradation plastc rings, the 3# oil degradation Mixed Microbes of 6mL are taken respectively Suspension, 4# oil degradation plastc rings, 5# oil degradation plastc rings, 6# oil degradation plastc rings, and connect respectively Plant in I crude oil 1mL/100mL inorganic salt liquid culture mediums, at 30 DEG C, 7d is cultivated in 120rpm thermostatic control oscillator vibrations, train Nutrient solution uses 5:It is 2～3 that 1 (V/V) sulfuric acid solution adjusts pH value, methyl alcohol 2-4mL is added, with the crude oil ether of 15mL in 60～90 DEG C of conditions Lower extraction three times, extract proceeds to 50mL volumetric flasks and constant volume Jing after anhydrous sodium sulfate drying.

By petroleum ether extraction liquid Jing after being serially diluted, with petroleum ether as reference, by its phase of determined by ultraviolet spectrophotometry The absorbance answered, according to the calibration curve of concentration (the mg/L)-absorbance of I crude oil, determines petroleum ether extract respective sample Concentration, according to concentration calculate degradation efficiency.

While degradation rate of the calculating oil degradation Mixed Microbes to I crude oil, determined respectively respectively using identical method The oil degradation rate of single bacterium.

The computational methods of oil degradation rate refer to below equation：

η (%)=(1-Ct/Co) × 100%

Wherein：η-oil degradation rate；Crude oil concentration in the presence of Ct- bacterial strains；Co- compares crude oil concentration.

When above-mentioned crude oil Mixed Microbes are carried out to the degradation effect of crude oil pollution water body, while former with the ocean of equivalent respectively Oily degradation bacteria (Advenella kashmirensis) 6-10-1 and bacillus thuringiensis (Bacillus Thuringiensis) 3-1 degrades to crude oil pollution water body, and its degradation results is contrasted.

Result of the test：

The degradation rate of 1#, 2#, 3#, 4#, 5#, 6# crude oil Mixed Microbes bacteria suspension, such as table 5 are calculated according to above-mentioned computing formula It is shown.

All degradation rate of the crude oil Mixed Microbes bacteria suspension of table 5 to crude oil pollution water body

Numbering	Degradation rate/%
		1#	74.12
2#	78.92
		3#	65.22
4#	72.35
		5#	65.28
6#	74.01

As shown in Table 5, all degradation rate highest of the 2# crude oil Mixed Microbes to crude oil I polluted-waters, liquid amount 100mL, In total inoculum concentration 6mL, the liquid inorganic salt culture medium of crude oil concentration 1%, in 7d oil degradation rate up to 78.92%, than single Bacterial classification 6-10-1,3,3-1 it is all high in identical conditions next week degradation rate, show there is certain synergy between these three bacterium, 1# crude oil Mixed Microbes also show identical result.And 3# crude oil Mixed Microbes under identical conditions to crude oil I polluted-waters All degradation rates are all low compared with single culture, illustrate that the synergy between 1-8,3 and 3-1 is not good, 6# crude oil Mixed Microbes and 4# crude oil Mixed Microbes also show identical result, and degradation effect is good.

The investment of embodiment 4 prepares oil degradation Mixed Microbes solid fungicide

Sodium alginate (Sodium alginate, SA) is that one kind has future and extensive types of natural gel, and quilt It is known as low price and there is nontoxicity to microorganism.Addition carrier material can strengthen mechanical strength, the tolerance of SA gels And provide adsorption site for microorganism.With sodium alginate as embedded material, CaCl₂Solution is crosslinking agent, and alpha-lactose is nutrition Thing adsorbent.

Preparation method is followed the steps below：

1. the alpha-lactose of 4g sodium alginates, 1.5g biomass carbons, 0.5g is mixed, and adds 100mL distilled water slowly to stir Mix, note activated carbon stirs outward during stirring, stirring amplitude is unsuitable excessive, prevents a large amount of bubbles from entering sodium alginate molten Liquid, stirs autoclaving 20min under the conditions of the solution is placed in into 121 DEG C after terminating.

2. after subject to sterilization, 30 DEG C or so are cooled to, are separately added into the mixed according to the degraded of the Crude Oil of embodiment 3 of 20mL 1#, 2#, 4#, 6# crude oil Mixed Microbes bacteria suspension for activating 36h obtained by bacterium bacteria suspension preparation method is closed, is mixed respectively Uniformly, 1#, 2#, 4#, 6# mixed liquor is respectively obtained.

3. with 10mL disposable syringes 1#, 2#, 4#, 6# mixed liquor is clamp-oned into the CaCl that mass percent is 4% respectively₂ In solution, 24h is crosslinked, forms 1#, 2#, 4#, 6# and fix microballoon, 1#, 2#, 4#, 6# oil degradation that as prepared by investment is mixed Close bacterium solid fungicide.

4. filter, then with aseptic water washing three times, in being immersed in physiological saline, 4 DEG C save backup.

The absorption method of embodiment 5 prepares oil degradation Mixed Microbes solid fungicide

With biological material and its correspondence charcoal as destination carrier, oil degradation Mixed Microbes are fixed by absorption method.

Fixing means is followed the steps below：

1. 1.5g biomass carbons are separately added in 6 conical flasks equipped with 100mL beef-protein mediums, and point Not Jia Ru 20mL according to the Crude Oil of embodiment 3 degrade Mixed Microbes bacteria suspension preparation method obtained by the 1# for activating 36h, 2#, 4#, 6# crude oil Mixed Microbes bacteria suspension, mixing.

2. it is in the shaking table of 120r/min, to fix 18 hours each conical flask to be placed in into rotating speed under the conditions of 30 DEG C.

3. then 10min is centrifuged under the conditions of 3000rpm, removes supernatant, lower sediment is cleaned into 2 with sterilized water water It is secondary.

4. it is placed in culture dish and is dried in 30 DEG C of baking ovens, 1#, 2#, 3#, 4#, 5# crude oil that as prepared by absorption method Degraded Mixed Microbes solid fungicide, in being immersed in physiological saline, 4 DEG C save backup.

Degraded of the oil degradation Mixed Microbes solid fungicide of embodiment 6 to crude oil pollution water body

1#, 2#, 4#, 6# oil degradation Mixed Microbes solid fungicide prepared by embodiment 4 and embodiment 5 is respectively used to into drop Solution crude oil pollution water body, it is specific as follows：

Take respectively 5g investments preparation 1#, 2#, 4#, 6# oil degradation Mixed Microbes solid fungicide and 5g absorption methods prepare 1#, 2#, 4#, 6# oil degradation Mixed Microbes solid fungicide, is respectively connected to 1mL II crude oil/100mL crude oil minimal mediums In 30 DEG C, after the incubated 7d of 120r/min, determine its removal effect to crude oil；Wherein, II crude oil picks up from Changqing oilfields, Density is 0.87g/mL, and the measure of the maximum absorption wavelength of II crude oil and the drafting of canonical plotting are with the I in embodiment 3 Number crude oil, the calibration curve of II crude oil is as shown in figure 11.

At the same time, it is respectively adopted and prepares Crude oil from CNOOC drop with embodiment 4 and the identical investment of embodiment 5 and absorption method Solution bacterium (Advenella kashmirensis) 6-10-1 and defect shortwave monad (Brevundimonas diminuta) 1-8 Solid fungicide, and using Crude oil from CNOOC degradation bacteria (Advenella kashmirensis) 6-10-1 and defect shortwave monad The solid fungicide of (Brevundimonas diminuta) 1-8 is degraded to crude oil pollution water body, and contrast its with 1#, 2#, The degradation effect of 3#, 4#, 5# oil degradation Mixed Microbes solid fungicide is contrasted, and degradation effect is shown in Table 6.

In addition, taking the 1# oil degradation plastc rings of 1.2mL and 4.2mL, 2# oil degradation plastc rings, 4# respectively Oil degradation plastc ring, 6# oil degradation plastc rings, and II crude oil 1mL/100mL inorganic salt liquids are inoculated in respectively In body culture medium, cultural method is contrasted its degradation effect with the degradation effect of solid pharmaceutical preparation with embodiment 3, is as a result seen Table 6.

The degradation rate of the crude oil Mixed Microbes solid fungicide of table 6 and crude oil Mixed Microbes bacteria suspension to II crude oil pollution water bodys

From the result of the test of table 6, under identical conditions, the solid fungicide of crude oil Mixed Microbes and its bacteria suspension, crude oil The solid fungicide and its bacteria suspension of degraded single bacterium 6-10-1,1-8 is respectively provided with the effect that degraded is repaired to crude oil pollution water body, and Degradation effect of 1#, 2#, 3#, 4#, 5# crude oil Mixed Microbes solid fungicide prepared by investment and absorption method to crude oil pollution water body More preferably.This is because immobilized microorganism technology is that free cell is positioned in the region of restriction so as to which holding activity simultaneously can The technology recycled, and with microbe density height, long action time, anti-poor environment ability be strong, microorganism be lost in it is few etc. Advantage, so unit immobilized microbial inoculum is than liquid bacterial agent degradation effect more preferably.

In addition, the degradation effect of the solid fungicide of contrast two methods preparation understands, under identical conditions, prepared by absorption method The degradation effect of solid fungicide is better than degradation effect of the solid fungicide of investment preparation to II crude oil pollution water bodys.This be because The microbial biomass adsorbed on biomass carbon in the solid fungicide prepared for two methods differs caused, micro organism quantity More, its solid fungicide is better to the degradation effect of crude oil pollution water body.

Degraded of the oil degradation Mixed Microbes solid fungicide of embodiment 7 to oil contaminated soil

1#, 2#, 3#, 4#, 5# oil degradation Mixed Microbes solid fungicide prepared by embodiment 4 and embodiment 5 is used respectively It is specific as follows in degrading crude oil contaminated soil：

Crude oil pollution soil sample：The soil sample that 1mm sieves dry was weighed, was carried out by crude oil pollution degree and the quality for weighing soil sample Weigh and stirred evenly in addition soil sample, crude oil pollution degree is 10g/kg, according to C：N：P is 100：10：1 ratio admixes (NH₄)₂SO₄With KH₂PO₄As N sources and P sources.Particularly, the nutriment of microorganism also includes C, but oil pollution result in the big of C sources Amount increases, it is sufficient to ensure its supply, it is not necessary to additionally add.The physicochemical property of soil sample is：Ammonium nitrogen 5.75ppm, nitrate nitrogen 4.70ppm；Nitrogen content 10.45ppm；Phosphorus content 16.41ppm；Potassium content 76.45ppm；The content of organic matter 2.35%；PH is 7.27。

Test method：

Take respectively 5g investments preparation 2# and 4# oil degradation Mixed Microbes solid fungicides and 5g absorption methods prepare 2# and 4# oil degradation Mixed Microbes solid fungicides, are respectively connected to 100gII crude oil pollution concentration of the moisture control in 12%-15% For in 1% soil, 120r/min, determine its removal effect to II crude oil after 30 DEG C of incubated 7d, 14d, 21d, obtain Go out its degradation rate.

Soil petrochina hydrocarbon content assay method, is carried out according to the following steps：

1. weigh 5g and air-dry soil sample, in being put into 50mL plastic centrifuge tubes.

2. 10mL carbon tetrachloride (analysis is pure), ultrasonic extraction 1h (temperature is less than 30 DEG C), leaching liquor are added with pipette During 25mL volumetric flasks are filled into Jing after anhydrous sodium sulfate drying, continuation 2 × 10mL carbon tetrachloride hot dipping two times, each 20min, Filtrate is incorporated to after drying volumetric flask, uses carbon tetrachloride constant volume.

3., in 288nm wavelength, with carbon tetrachloride as the absorbance of reference determination sample, reference standard is bent for spectrophotometer The anti-concentration for pushing away crude oil of line.

When 2# and 6# oil degradation Mixed Microbes solid fungicides are carried out to the test of the degradation effect of original contaminated soil, together When using the solid fungicide of defect shortwave monad (Brevundimonas diminuta) 1-8 as control sample, by test knot It is as shown in table 7 to the degradation results of oil contaminated soil that fruit obtains oil degradation Mixed Microbes solid fungicide.

Degradation rate of the oil degradation Mixed Microbes solid fungicide of table 7 to oil contaminated soil

From the result of the test of above-mentioned table 7,2# and 4# crude oil Mixed Microbes solid fungicides prepared by investment and absorption method The effect that degraded is repaired, and prolongation over time are also respectively provided with to oil contaminated soil, degradation efficiency is better, itself and degraded are imitated The degradation effect contrast of the solid fungicide of really good defect shortwave monad (Brevundimonas diminuta) 1-8, this The crude oil Mixed Microbes solid fungicide of invention to the degradation effect of oil contaminated soil more preferably.

Crude oil from CNOOC degradation bacteria 6-10-1, defect shortwave monad 1- are investigated using the test method of above-described embodiment 3-7 8th, the Mixed Microbes of other combinations of bacillus thuringiensis 3-1 or Pseudomonas geniculate 3 are to crude oil pollution water body and crude oil pollution The degraded repair of soil, as a result finds, it is respectively provided with degraded repair.

Although with a general description of the specific embodiments the present invention is described in detail in this specification, But on the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art. Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to claimed model Enclose.

SEQUENCE LISTING

SEQ ID No.1

<110>Chang An University

<120>A kind of oil degradation Mixed Microbes, microbial inoculum and its application

<130> 2016

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 1402

<212> DNA

<213>Offshore oil degradation bacteria（Advenella kashmirensis）6-10-1

<400> 1

GCAGCGGGAA AGTAGCTTGC TACTTTTGCC GGCGAGTGGC GAACGGGTGA GTAATGTATC 60

GGAACGTGCC CAGTAGCGGG GGATAACTAC GCGAAAGCGT GGCTAATACC GCATACGCCC 120

TACGGGGGAA AGGGGGGGAT CTTAGGACCT CTCACTATTG GAGCGGCCGA TATCGGATTA 180

GCTAGTTGGT GGGGTAAAGG CCTACCAAGG CGACGATCCG TAGCTGGTTT GAGAGGACGA 240

CCAGCCACAC TGGGACTGAG ACACGGCCCA GACTCCTACG GGAGGCAGCA GTGGGGAATT 300

TTGGACAATG GGGGAAACCC TGATCCAGCC ATCCCGCGTG TGCGATGAAG GCCTTCGGGT 360

TGTAAAGCAC TTTTGTCAGG GAAGAAAAGG TTTCGGATAA TACCCGGAAC TGATGACGGT 420

ACCTGAAGAA TAAGCACCGG CTAACTACGT GCCAGCAGCC GCGGTAATAC GTAGGGTGCA 480

AGCGTTAATC GGAATTACTG GGCGTAAAGC GTGCGCAGGC GGTTCGGAAA GAAAGATGTG 540

AAATCCCAGG GCTCAACCTT GGAACTGCAT TTTTAACTAC CGAACTAGAG TATGTCAGAG 600

GGGGGTGGAA TTCCACGTGT AGCAGTGAAA TGCGTAGATA TGTGGAGGAA CACCGATGGC 660

GAAGGCAGCC CCCTGGGATA ATACTGACGC TCATGCACGA AAGCGTGGGG AGCAAACAGG 720

ATTAGATACC CTGGTAGTCC ACGCCCTAAA CGATGTCAAC TAGCTGTTGG GCCCTTCGGG 780

GCTTAGTAGC GCAGCTAACG CGTGAAGTTG ACCGCCTGGG GAGTACGGTC GCAAGATTAA 840

AACTCAAAGG AATTGACGGG GACCCGCACA AGCGGTGGAT GATGTGGATT AATTCGATGC 900

AACGCGAAAA ACCTTACCTA CCCTTGACAT GTCTGGAATC CTGAAGAGAT TTAGGAGTGC 960

TCGCAAGAGA ACCGGAACAC AGGTGCTGCA TGGCTGTCGT CAGCTCGTGT CGTGAGATGT 1020

TGGGTTAAGT CCCGCAACGA GCGCAACCCT TGTCATTAGT TGCTACATTT AGTTGAGCAC 1080

TCTAATGAGA CTGCCGGTGA CAAACCGGAG GAAGGTGGGG ATGACGTCAA GTCCTCATGG 1140

CCCTTATGGG TAGGGCTTCA CACGTCATAC AATGGTCGGG ACAGAGGGTT GCCAAACCGC 1200

AAGGTGGAGC TAATCTCATA AACCCGATCG TAGTCCGGAT TGCAGGCTGC AACTCGCCTG 1260

CATGAAGTCG GAATCGCTAG TAATCGCGGA TCAGCATGTC GCGGTGAATA CGTTCCCGGG 1320

TCTTGTACAC ACCGCCCGTC ACACCATGGG AGTGGGTTTT ACCAGAAGTA GTTAGCCTAA 1380

CCGCAAGGGG GGCGATACCA CG 1402

SEQ ID No.2

<210> 2

<211> 1206

<212> DNA

<213>Defect shortwave monad（Brevundimonas diminuta）1-8

<400> 2

GGTGAGTAAC ACGTGGGAAC GTGCCTTTAG GTTCGGAATA GCTCCTGGAA ACGGGTGGTA 60

ATGCCGAATG TGCCCTTCGG GGGAAAGATT TATCGCCTTT AGAGCGGCCC GCGTCTGATT 120

AGCTAGTTGG TGAGGTAACG GCTCACCAAG GCGACGATCA GTAGCTGGTC TGAGAGGATG 180

ACCAGCCACA CTGGGACTGA GACACGGCCC AGACTCCTAC GGGAGGCAGC AGTGGGGAAT 240

CTTGCGCAAT GGGCGAAAGC CTGACGCAGC CATGCCGCGT GAATGATGAA GGTCTTAGGA 300

TTGTAAAATT CTTTCACCGG GGACGATAAT GACGGTACCC GGAGAAGAAG CCCCGGCTAA 360

CTTCGTGCCA GCAGCCGCGG TAATACGAAG GGGGCTAGCG TTGCTCGGAA TTACTGGGCG 420

TAAAGGGCGC GTAGGCGGAT CGTTAAGTCA GAGGTGAAAT CCCAGGGCTC AACCCTGGAA 480

CTGCCTTTGA TACTGGCGAT CTTGAGTATG AGAGAGGTAT GTGGAACTCC GAGTGTAGAG 540

GTGAAATTCG TAGATATTCG GAAGAACACC AGTGGCGAAG GCGACATACT GGCTCATTAC 600

TGACGCTGAG GCGCGAAAGC GTGGGGAGCA AACAGGATTA GATACCCTGG TAGTCCACGC 660

CGTAAACGAT GATTGCTAGT TGTCGGGCTG CATGCAGTTC GGTGACGCAG CTAACGCATT 720

AAGCAATCCG CCTGGGGAGT ACGGTCGCAA GATTAAAACT CAAAGGAATT GACGGGGGCC 780

CGCACAAGCG GTGGAGCATG TGGTTTAATT CGAAGCAACG CGCAGAACCT TACCACCTTT 840

TGACATGCCT GGACCGCCAC GGAGACGTGG CTTTCCCTTC GGGGACTAGG ACACAGGTGC 900

TGCATGGCTG TCGTCAGCTC GTGTCGTGAG ATGTTGGGTT AAGTCCCGCA ACGAGCGCAA 960

CCCTCGCCAT TAGTTGCCAT CATTTAGTTG GGAACTCTAA TGGGACTGCC GGTGCTAAGC 1020

CGGAGGAAGG TGGGGACGAC GTCAAGTCCT CATGGCCCTT ACAGGGTGGG CTACACACGT 1080

GCTACAATGG CAACTACAGA GGGTTAATCC TTAAAAGTTG TCTCAGTTCG GATTGTCCTC 1140

TGCAACTCGA GGGCATGAAG TGGGAATCGC TAGTAATCGC GGATCCGCAT GCCGCGGTGA 1200

ATACGT

SEQ ID No.3

<210> 3

<211> 1404

<212> DNA

<213>Bacillus thuringiensis（Bacillus thuringiensis）3-1

<400> 3

TAAGAGCTTG CTCTTATGAA GTTAGCGGCG GACGGGTGAG TAACACGTGG GTAACCTGCC 60

CATAAGACTG GGATAACTCC GGGAAACCGG GGCTAATACC GGATAACATT TTGAACTGCA 120

TGGTTCGAAA TTGAAAGGCG GCTTCGGCTG TCACTTATGG ATGGACCCGC GTCGCATTAG 180

CTAGTTGGTG AGGTAACGGC TCACCAAGGC AACGATGCGT AGCCGACCTG AGAGGGTGAT 240

CGGCCACACT GGGACTGAGA CACGGCCCAG ACTCCTACGG GAGGCAGCAG TAGGGAATCT 300

TCCGCAATGG ACGAAAGTCT GACGGAGCAA CGCCGCGTGA GTGATGAAGG CTTTCGGGTC 360

GTAAAACTCT GTTGTTAGGG AAGAACAAGT GCTAGTTGAA TAAGCTGGCA CCTTGACGGT 420

ACCTAACCAG AAAGCCACGG CTAACTACGT GCCAGCAGCC GCGGTAATAC GTAGGTGGCA 480

AGCGTTATCC GGAATTATTG GGCGTAAAGC GCGCGCAGGT GGTTTCTTAA GTCTGATGTG 540

AAAGCCCACG GCTCAACCGT GGAGGGTCAT TGGAAACTGG GAGACTTGAG TGCAGAAGAG 600

GAAAGTGGAA TTCCATGTGT AGCGGTGAAA TGCGTAGAGA TATGGAGGAA CACCAGTGGC 660

GAAGGCGACT TTCTGGTCTG TAACTGACAC TGAGGCGCGA AAGCGTGGGG AGCAAACAGG 720

ATTAGATACC CTGGTAGTCC ACGCCGTAAA CGATGAGTGC TAAGTGTTAG AGGGTTTCCG 780

CCCTTTAGTG CTGAAGTTAA CGCATTAAGC ACTCCGCCTG GGGAGTACGG CCGCAAGGCT 840

GAAACTCAAA GGAATTGACG GGGGCCCGCA CAAGCGGTGG AGCATGTGGT TTAATTCGAA 900

GCAACGCGAA GAACCTTACC AGGTCTTGAC ATCCTCTGAA AACCCTAGAG ATAGGGCTTC 960

TCCTTCGGGA GCAGAGTGAC AGGTGGTGCA TGGTTGTCGT CAGCTCGTGT CGTGAGATGT 1020

TGGGTTAAGT CCCGCAACGA GCGCAACCCT TGATCTTAGT TGCCATCATT AAGTTGGGCA 1080

CTCTAAGGTG ACTGCCGGTG ACAAACCGGA GGAAGGTGGG GATGACGTCA AATCATCATG 1140

CCCCTTATGA CCTGGGCTAC ACACGTGCTA CAATGGACGG TACAAAGAGC TGCAAGACCG 1200

CGAGGTGGAG CTAATCTCAT AAAACCGTTC TCAGTTCGGA TTGTAGGCTG CAACTCGCCT 1260

ACATGAAGCT GGAATCGCTA GTAATCGCGG ATCAGCATGC CGCGGTGAAT ACGTTCCCGG 1320

GCCTTGTACA CACCGCCCGT CACACCACGA GAGTTTGTAA CACCCGAAGT CGGTGGGGTA 1380

ACCTTTTTGG AGCCAGCCGC CTAA 1404

SEQ ID No.4

<210> 4

<211> 1319

<212> DNA

<213>Pseudomonas geniculate（Pseudomonas geniculata）3

<400> 4

AGCACAGAGG AGCTTGCTCC TTGGGTGGCG AGTGGCGGAC GGGTGAGGAA TACATCGGAA 60

TCTACTCTGT CGTGGGGGAT AACGTAGGGA AACTTACGCT AATACCGCAT ACGACCTACG 120

GGTGAAAGCA GGGGACCTTC GGGCCTTGCG CGATTGAATG AGCCGATGTC GGATTAGCTA 180

GTTGGCGGGG TAAAGGCCCA CCAAGGCGAC GATCCGTAGC TGGTCTGAGA GGATGATCAG 240

CCACACTGGA ACTGAGACAC GGTCCAGACT CCTACGGGAG GCAGCAGTGG GGAATATTGG 300

ACAATGGGCG CAAGCCTGAT CCAGCCATAC CGCGTGGGTG AAGAAGGCCT TCGGGTTGTA 360

AAGCCCTTTT GTTGGGAAAG AAATCCAGCT GGCTAATACC CGGTTGGGAT GACGGTACCC 420

AAAGAATAAG CACCGGCTAA CTTCGTGCCA GCAGCCGCGG TAATACGAAG GGTGCAAGCG 480

TTACTCGGAA TTACTGGGCG TAAAGCGTGC GTAGGTGGTC GTTTAAGTCC GTTGTGAAAG 540

CCCTGGGCTC AACCTGGGAA CTGCAGTGGA TACTGGGCGA CTAGAGTGTG GTAGAGGGTA 600

GCGGAATTCC TGGTGTAGCA GTGAAATGCG TAGAGATCAG GAGGAACATC CATGGCGAAG 660

GCAGCTACCT GGACCAACAC TGACACTGAG GCACGAAAGC GTGGGGAGCA AACAGGATTA 720

GATACCCTGG TAGTCCACGC CCTAAACGAT GCGAACTGGA TGTTGGGTGC AATTTGGCAC 780

GCAGTATCGA AGCTAACGCG TTAAGTTCGC CGCCTGGGGA GTACGGTCGC AAGACTGAAA 840

CTCAAAGGAA TTGACGGGGG CCCGCACAAG CGGTGGAGTA TGTGGTTTAA TTCGATGCAA 900

CGCGAAGAAC CTTACCTGGC CTTGACATGT CGAGAACTTT CCAGAGATGG ATGGGTGCCT 960

TCGGGAACTC GAACACAGGT GCTGCATGGC TGTCGTCAGC TCGTGTCGTG AGATGTTGGG 1020

TTAAGTCCCG CAACGAGCGC AACCCTTGTC CTTAGTTGCC AGCACGTAAT GGTGGGAACT 1080

CTAAGGAGAC CGCCGGTGAC AAACCGGAGG AAGGTGGGGA TGACGTCAAG TCATCATGGC 1140

CCTTACGGCC AGGGCTACAC ACGTACTACA ATGGTAGGGA CAGAGGGCTG CAAGCCGGCG 1200

ACGGTAAGCC AATCCCAGAA ACCCTATCTC AGTCCGGATT GGAGTCTGCA ACTCGACTCC 1260

ATGAAGTCGG AATCGCTAGT AATCGCAGAT CAGCATTGCT GCGGTGAATA CGTTCCCGG 1319

Claims

1. a kind of oil degradation Mixed Microbes, it is characterised in that including Crude oil from CNOOC degradation bacteria (Advenella Kashmirensis) 6-10-1, defect shortwave monad (Brevundimonas diminuta) 1-8, bacillus thuringiensis In (Bacillus thuringiensis) 3-1 or Pseudomonas geniculate (Pseudomonas geniculata) 3 at least two Kind.

2. oil degradation Mixed Microbes according to claim 1, it is characterised in that the Crude oil from CNOOC degradation bacteria (Advenella kashmirensis) 6-10-1 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms The heart, preserving number is CGMCC No.13003；Defect shortwave monad (Brevundimonas diminuta) 1-8 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is CGMCC No.13002；The Su Yun is golden Bacillus (Bacillus thuringiensis) 3-1 is preserved in the common micro- life of China Committee for Culture Collection of Microorganisms Thing center, preserving number is CGMCC No.13005；The Pseudomonas geniculate (Pseudomonas geniculata) 3 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is CGMCC No.13004.

3. oil degradation Mixed Microbes according to claim 1, it is characterised in that the Crude oil from CNOOC degradation bacteria The 16S rDNA sequencing results of (Advenella kashmirensis) 6-10-1 are as shown in SEQ ID No.1；The defect is short The 16S rDNA sequencing results of ripple monad (Brevundimonas diminuta) 1-8 are as shown in SEQ ID No.2；The Soviet Union The 16S rDNA sequencing results of cloud gold bacillus (Bacillus thuringiensis) 3-1 are as shown in SEQ ID No.3；Institute The 16S rDNA sequencing results of Pseudomonas geniculate (Pseudomonas geniculata) 3 are stated as shown in SEQ ID No.4.

4. a kind of oil degradation mixing bacteria agent, it is characterised in that its active component includes Crude oil from CNOOC degradation bacteria (Advenella kashmirensis) 6-10-1, defect shortwave monad (Brevundimonas diminuta) 1-8, Su Yun Golden bacillus (Bacillus thuringiensis) 3-1 or Pseudomonas geniculate (Pseudomonas geniculata) 3 In at least two.

5. oil degradation mixing bacteria agent according to claim 4, it is characterised in that the oil degradation mixing bacteria agent For solid-state microbial inoculum.

6. application of the oil degradation mixing bacteria agent described in claim 4 or 5 in the degraded of crude oil pollution water body.

7. application of the oil degradation mixing bacteria agent described in claim 4 or 5 in oil contaminated soil degraded.