CN106635909B

CN106635909B - Crude oil degradation mixed bacteria, microbial inoculum and application thereof

Info

Publication number: CN106635909B
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: Changan University
Current assignee: Shaanxi Aomei Environmental Protection Technology Co.,Ltd.
Priority date: 2016-12-29
Filing date: 2016-12-29
Publication date: 2020-04-24
Anticipated expiration: 2036-12-29
Also published as: CN106635909A

Abstract

The invention specifically discloses a crude oil degradation mixed bacterium, which comprises at least two of marine crude oil degradation bacterium 6-10-1, Brevundimonas diminuta 1-8, Bacillus thuringiensis 3-1 or Pseudomonas flexi 3. Also discloses a microbial inoculum taking the marine crude oil degrading bacteria 6-10-1, Brevundimonas diminuta 1-8, Bacillus thuringiensis 3-1 or Pseudomonas flexi 3 as active ingredients and application thereof in degradation of crude oil polluted water and crude oil polluted soil. The crude oil degradation mixed bacteria and the crude oil degradation mixed bacteria microbial inoculum are easy for large-scale production, and have strong degradation effect on crude oil polluted water and crude oil polluted soil.

Description

Crude oil degradation mixed bacteria, microbial inoculum and application thereof

Technical Field

The invention relates to the technical field of bioengineering, and particularly relates to a crude oil degradation mixed bacterium, a microbial inoculum and application thereof.

Background

Alkanes, aromatic hydrocarbons, sulfur-containing compounds, sulfur-containing or nitrogen-containing heterocyclic compounds, and the like contained in petroleum are toxic and extremely harmful to humans and the environment. Crude oil is unprocessed petroleum, is a viscous black or dark brown liquid or solid formed by geological transition of natural organic substances, is usually mostly present underground or seabed, has pungent smell, and has a chain compound formed by combining carbon and hydrogen atoms as a main component, so that the crude oil has great harm to human living environments such as water bodies, soil, air and the like.

At present, the technologies for repairing petroleum-polluted water, soil and the like mainly comprise a physical repair technology, a chemical repair technology, a photocatalytic repair technology and a biological repair technology; bioremediation technology is a treatment method with policy, economic and technical attractiveness, is considered as the most potential technical method, and gradually develops into the most effective means for solving the problem of complex environmental pollution with good economic and environmental benefits.

Generally, one kind of microorganism can only degrade one or a few kinds of crude oil hydrocarbon, and different microorganisms have different degradation effects on the same pollution component, and multiple microorganisms can also have synergistic or competitive effects in the degradation process. However, the crude oil and its refined products contain very complicated crude oil types, and the crude oil components of crude oils from different sources are very different. Therefore, the microbial remediation technology has some limitations while being rapidly developed, a specific microorganism can only degrade a specific compound type, the propagation and metabolic activity of the microorganism are greatly influenced by environmental factors, and a large amount of death often occurs in the initial stage of adding the microorganism to soil.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide a crude oil degradation mixed bacterium with good crude oil degradation effect, a microbial inoculum and application thereof.

In order to achieve the purpose, the invention is realized by adopting the following technical scheme.

The mixed bacteria for degrading the crude oil are characterized by comprising at least two of marine crude oil degrading bacteria (Advenella kashmirensis)6-10-1, Brevundimonas diminuta (Brevundimonas diminuta)1-8, Bacillus thuringiensis (Bacillus thuringiensis)3-1 or Pseudomonas flexi (Pseudomonas genista) 3.

6-10-1 of the marine crude oil degrading bacteria (Advenella kashmirensis) is preserved in the China general microbiological culture Collection center of the institute of microbiology, China academy of sciences, No.3, North Chen West Lu 1 institute of North Chen, Tokyo, the city in 2016 within 2016, 09 and 12 days, and the preservation number is CGMCC No. 13003.

1-8 of Brevundimonas diminuta (Brevundimonas diminuta) is preserved in the general microbiological culture collection center of the China Committee for culture Collection of microorganisms of the institute of microbiology, No.3 of the institute of China academy of sciences, North Chen West Lu 1, the area facing the Sun, Beijing, in 2016 (09 months and 12 days), and the preservation number is CGMCC No. 13002.

The Bacillus thuringiensis (Bacillus thuringiensis)3-1 is preserved in the general microbiological culture collection center of the China institute of microbiology, national institute of sciences No.3, West Lu 1, North Chen, south China, Beijing, at 2016, 09 and 12 days, and the preservation number is CGMCC No. 13005.

The Pseudomonas flexi (Pseudomonas genic) 3 is preserved in the China general microbiological culture Collection center of the institute of microbiology, China institute of sciences, No.3, West Lu 1, Beijing, the south facing the Yangtze region at 2016, 12.09.10.2016, and the preservation number is CGMCC No. 13004.

The sequencing result of 16S rDNA of the marine crude oil degrading bacterium (Advenella kashmirensis)6-10-1 is shown as SEQ ID No. 1.

The 16S rDNA sequencing result of the Brevundimonas diminuta (Brevundimonas diminuta)1-8 is shown as SEQID No. 2.

The 16S rDNA sequencing result of the Bacillus thuringiensis (Bacillus thuringiensis)3-1 is shown as SEQID No. 3.

The sequencing result of 16S rDNA of the Pseudomonas flexi (Pseudomonas genic) 3 is shown in SEQ ID No. 4.

(II) a crude oil degradation mixed bacteria agent, which is characterized in that the active ingredients comprise at least two of marine crude oil degradation bacteria (Advenella kashmirensis)6-10-1, Brevundimonas diminuta (Brevundimonas diminuta)1-8, Bacillus thuringiensis (Bacillus thuringiensis)3-1 or Pseudomonas flexuosa (Pseudomonas genista) 3.

Preferably, the crude oil degradation mixed bacterial agent is a solid bacterial agent.

And (III) the crude oil degradation mixed bacterial agent is applied to degradation of crude oil polluted water.

And (IV) the crude oil degradation mixed bacterial agent is applied to degradation of crude oil polluted soil.

Compared with the prior art, the invention has the beneficial effects that:

the crude oil degradation mixed bacteria and the crude oil degradation mixed bacteria microbial inoculum provided by the invention are easy for large-scale production, and have stronger degradation effects on crude oil polluted water and crude oil polluted soil; the microbial inoculum has strong environment adaptability, can grow smoothly in the presence of a large amount of organic matters, and takes crude oil as a carbon source, thereby achieving better degradation effect; the method has good application prospect in the realization of the remediation of the crude oil polluted wastewater and the crude oil polluted soil.

Drawings

The invention is described in further detail below with reference to the figures and specific embodiments.

FIG. 1 is a colony morphology of marine petroleum-degrading bacteria (Advenella kashmirensis)6-10-1 of the present invention on a beef extract peptone medium plate;

FIG. 2 is a microscopic morphological diagram of the marine petroleum-degrading bacterium (Advenella kashmirensis)6-10-1 of the present invention, at 1000-fold magnification.

FIG. 3 is a colony morphology of Brevundimonas diminuta (Brevundimonas diminuta)1-8 of the present invention on beef extract peptone medium plate;

FIG. 4 is a diagram showing the cell morphology under a microscope of Brevundimonas diminuta 1-8 of the present invention, wherein the magnification is 1600 times.

FIG. 5 is a colony morphology of Bacillus thuringiensis (Bacillus thuringiensis)3-1 of the present invention on a beef extract peptone medium plate;

FIG. 6 is a microscopic morphological diagram of Bacillus thuringiensis (Bacillus thuringiensis)3-1 of the present invention, at 4000-fold magnification.

FIG. 7 is a colony morphology of Pseudomonas flexi (Pseudomonas genic) 3 of the present invention on a beef extract peptone medium plate;

FIG. 8 is a microscopic cell morphology of Pseudomonas flexuosa (Pseudomonas genic) 3 of the present invention, at 1000-fold magnification;

FIG. 9 is a graph of wavelength vs. absorbance for crude oil I of example 3 of the present invention; in the figure, the abscissa is wavelength in nm and the ordinate is absorbance in 1;

FIG. 10 is a standard curve of crude oil No. I in example 3 of the present invention; in the figure, the abscissa is concentration in mg/L and the ordinate is absorbance in 1;

FIG. 11 is a standard curve of crude oil No. II of example 6 of the present invention; in the figure, the abscissa represents concentration in mg/L, and the ordinate represents absorbance in 1.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention.

EXAMPLE 1 isolation and purification of the Strain

(1) The source of the strain

The crude oil degradation mixed bacteria are respectively separated from the ground crude oil polluted soil of the oil collecting station of the oil production field with seven holes of the extended crude oil group. Collecting surface soil which is about 20cm away from the ground surface by adopting a plum blossom distribution method, and placing a sample into a refrigerator at 4 ℃ for aseptic preservation, wherein the physical and chemical properties of the sample are as follows: ammonium nitrogen 15.09ppm and nitrate nitrogen 0.72 ppm; nitrogen content 15.81 ppm; phosphorus content 82.05 ppm; the potassium content was 17.45 ppm; the organic content is 1.43 percent; the pH was 7.65.

(2) Separation and purification of bacterial strains

Adding 10g of crude oil contaminated soil sample into 100mL of enrichment liquid culture medium, and performing shake cultivation at 28 ℃ for 7d at 130 r/min; after the culture solution is turbid, 5mL of the culture solution is sucked and re-inoculated into a fresh enrichment medium, and the culture solution is continuously inoculated and enriched for 3 times in the same way as the culture conditions. Taking 1mL of the bacterial suspension, serially diluting with sterile water, respectively inoculating the bacterial suspension into beef extract peptone culture media, and culturing at a constant temperature of about 29 ℃ for 3 d; wherein, the enriched liquid culture medium comprises the following components: NaNO₃1.5g，(NH₄)SO₄1.5g，K₂HPO₄1g，MgSO₄·7H₂O 0.5g，KCl 0.5g，FeSO₄·7H₂O 0.01g，CaCl₂0.002g, 1000mL of distilled water, 1% of crude oil I (volume ratio), and pH 7.0. The beef extract peptone medium comprises the following components: 3.0g of beef extract, 10.0g of peptone, 5.0g of NaCl, 15.0g of nutrient agar and 1000mL of distilled water, wherein the pH value is 7.4-7.6. After the plate bacterial colonies are effectively separated, single bacterial colonies are respectively selected by an inoculating needle for plate streaking purification and separation according to the difference of the color, morphological characteristics and surface humidity of each bacterial colony. Inoculating each single strain on a slant, and refrigerating at 4 ℃ in a refrigerator for later use.

(3) Screening of crude oil degrading bacteria by oil dissolving ring method

Respectively inoculating the single strains on an oil-containing inorganic salt solid culture medium, culturing in an incubator at 37 ℃ for 10-15D, observing the existence of an oilphilic ring, and preliminarily determining the crude oil degradation capacity of the strains according to the ratio of the diameter (D) of the oilphilic ring to the diameter (D) of a bacterial colony; wherein the oily inorganic salt solid culture medium comprises the following components: 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 with 1mol/L sodium hydroxide or 1mol/L hydrogen chloride to the desired value. Inoculating the preliminarily screened crude oil degrading bacteria to a slant culture medium, and refrigerating for later use in a refrigerator at 4 ℃.

EXAMPLE 2 identification of the strains

(1) Morphological characteristics of the Strain

As shown in figure 1, after the marine petroleum degrading bacteria are cultured on a beef extract peptone medium at a constant temperature of 30 ℃ for 24h, the colony morphology is observed, and the strain 6-10-1 is round, complete in edge, smooth, opaque and milk-white on a flat plate of the beef extract peptone, and has a moist surface and no special odor. As shown in FIG. 2, the cells were in the form of short rods having a size of (0.41-1.02) μm.times. (0.41-1.53) μm and were nonfilamentous.

As shown in figure 3, after the brevundimonas diminuta is cultured on a beef extract peptone medium at a constant temperature of 30 ℃ for 24h, the colony morphology is observed, the bacterial strains 1-8 are light yellow on a beef extract peptone flat plate, the colony color is slightly deepened along with the prolonging of the culture time, the surface is smooth and moist, the bacterial colony is circular, the center is protruded, the edge is neat, and the bacterial colony is semitransparent. As shown in FIG. 4, the bacterial cells were in the form of short rods or rods, and the cells were aligned individually and had a size of 0.5. mu. m.times. (0.1-0.3) μm.

As shown in figure 5, after culturing the bacillus thuringiensis on a beef extract peptone medium at a constant temperature of 30 ℃ for 24h, the colony morphology is observed, and the strain 3-1 is round, jagged on the edge, convex in the middle, creamy yellow, smooth and moist in surface on a flat plate of the beef extract peptone. As shown in FIG. 6, the cells were short rods with sizes of (1.2-1.5) μm.times. (3-5) μm.

As shown in FIG. 7, the colony morphology of the Pseudomonas flexuosa is observed after the Pseudomonas flexuosa is cultured on a beef extract peptone medium at a constant temperature of 30 ℃ for 24 hours, and the strain 3 is light yellow initially, darker with the increase of the age of the strain and yellow finally. The colony is characterized by being a faint yellow colony with a needle point size, being regular and round, having a smooth and moist surface and a neat edge, and being not easy to stain after gram staining. As shown in FIG. 8, the bacterial cells were rod-shaped, short rod-shaped, and gram-negative bacteria having a size of (1-1.5). mu.m.times.0.5. mu.m.

(2) Biochemical characteristics

According to Bergey's Manual of bacteria identification and Manual of identification of common bacteria systems, the physiological and biochemical identification of marine oil-degrading bacteria, Brevundimonas diminuta, Bacillus thuringiensis and Pseudomonas flexi is carried out, and the identification results are shown in tables 1-4.

TABLE 1 physiological and biochemical identification results of marine petroleum-degrading bacteria 6-10-1

Index (I)	Characteristics of
		Gram stain	—
Glucose	+
		Xylose	+
Fructose	+
		Acetate salt	+
Lactose	+
		Maltose	—
Sucrose	—
		Oxidase enzyme	+
Catalase enzyme	+
		Urease	+
Nitrate reducibility	—

Note: "-" indicates that the reaction was negative; "+" indicates a positive reaction.

TABLE 2 physiological and biochemical identification results of Brevundimonas diminuta 1-8 strains

TABLE 3 physiological and biochemical identification results of Bacillus thuringiensis 3-1 strains

Note: "-" indicates that the reaction was negative; "+" indicates a positive reaction

TABLE 4 physiological and biochemical identification results of the strain of Pseudomonas flexi (Pseudomonas genic) 3

(3) 16S rDNA identification of strains

Respectively extracting the genome DNA of the marine crude oil degrading bacteria by using a DNA extraction kit, and then cloning and sequencing 16S rDNA genes, wherein the 16S rDNA sequencing result is shown as SEQ ID No. 1; extracting genome DNA of the Brevundimonas diminuta, and then carrying out 16S rDNA gene cloning and sequencing, wherein the 16S rDNA sequencing result is shown as SEQ ID No. 2; extracting the genome DNA of the bacillus thuringiensis, and then carrying out 16S rDNA gene cloning and sequencing, wherein the 16S rDNA sequencing result is shown as SEQ ID No. 3; extracting genome DNA of Pseudomonas flexi, cloning and sequencing 16S rDNA gene, wherein the 16S rDNA sequencing result is shown in SEQ ID No. 4.

Example 3 degradation of crude oil-degrading mixed bacterial suspension to crude oil-contaminated water

Under the aseptic condition, selecting, separating and purifying obtained marine crude oil degrading bacteria (Advenella kashmirensis)6-10-1, Brevundimonas diminuta (Brevundimonas diminuta)1-8, Bacillus thuringiensis (Bacillus thuringiensis)3-1 and Pseudomonas flexi (Pseudomonas genic) 3 to be respectively inoculated in 100mL beef extract peptone liquid culture medium, and carrying out shake enlarged culture for 48 h; wherein, the components of the beef extract peptone liquid medium are as follows: 3.0g of beef extract, 10.0g of peptone, 5.0g of NaCl, 15.0g of nutrient agar and 1000mL of distilled water, wherein the pH value is 7.4-7.6. The pH is adjusted to the desired value with 1mol/L sodium hydroxide or 1mol/L hydrogen chloride. The culture medium sterilization conditions were: the temperature is 121-126 ℃, and the time is 25 min.

(1) Preparation of crude oil degradation mixed bacteria suspension

1) Uniformly mixing 8mL of ocean crude oil degrading bacteria (Advenella kashmirensis)6-10-1 bacteria liquid, 3mL of Brevundimonas diminuta (Brevundimonas diminuta)1-8 bacteria liquid and 1mL of Bacillus thuringiensis (Bacillus thuringiensis)3-1 bacteria liquid which are subjected to amplification culture together to obtain a 1# crude oil degrading mixed bacteria suspension;

2) uniformly mixing 4mL of marine crude oil degrading bacteria (Advenella kashmirensis)6-10-1 bacteria liquid, 1mL of Pseudomonas flexuralis (Pseudomonas genic) 3 bacteria liquid and 1mL of Bacillus thuringiensis (Bacillus thuringiensis)3-1 bacteria liquid which are subjected to amplification culture together to obtain a 2# crude oil degrading mixed bacteria suspension.

3) Uniformly mixing 4mL of a strain solution of Brevundimonas diminuta (Brevundimonas diminuta)1-8, 1mL of a strain solution of Pseudomonas flexi (Pseudomonas genic) 3 and 1mL of a strain solution of Bacillus thuringiensis (Bacillus thuringiensis)3-1, which are subjected to amplification culture, to obtain a suspension of the 3# crude oil degradation mixed strain.

4) 6mL of the marine crude oil degrading bacteria (Advenella kashmirensis)6-10-1 bacteria liquid subjected to amplification culture and 2mL of the Bacillus thuringiensis (Bacillus thuringiensis)3-1 bacteria liquid are uniformly mixed together to obtain the No.4 crude oil degrading mixed bacteria suspension.

5) Uniformly mixing 3mL of the marine crude oil degrading bacteria (Advenella kashmirensis)6-10-1 bacteria liquid subjected to amplification culture and 3mL of the Bacillus thuringiensis (Bacillus thuringiensis)3-1 bacteria liquid to obtain a 5# crude oil degrading mixed bacteria suspension.

6) Uniformly mixing 3mL of the marine crude oil degrading bacteria (Advenella kashmirensis)6-10-1 bacteria liquid subjected to amplification culture and 3mL of the Pseudomonas flexuralis (Pseudomonas genic) 3 bacteria liquid to obtain a No. 6 crude oil degrading mixed bacteria suspension.

(2) Degradation of crude oil polluted water body by crude oil degradation mixed bacteria suspension

The test method comprises the following steps:

(1) measurement of maximum absorption wavelength of crude oil No. I

The petroleum and the products thereof have certain absorption characteristics in an ultraviolet region, the wavelength of aromatic compounds with benzene rings in petroleum components is generally 250-260 nm, and the wavelength of compounds with conjugated double bonds is generally 215-230 nm, and researches show that when the petroleum and the products thereof are measured by an ultraviolet spectrophotometry, about 255nm is the selection range of crude oil and heavy oil, and about 225nm is the selection range of light oil and oil products of oil refineries. Under the concentration of 100mg/L, according to the obtained wavelength and absorbance data, a wavelength-absorbance curve of the crude oil I to be tested is drawn as shown in FIG. 9, wherein the crude oil I is collected from an extended oil group seven-mile oil field oil collecting station, and the density is 0.65 g/mL.

(2) Drawing of No. I crude oil standard curve

Taking crude oil I of 0, 2, 2.5, 5, 6 and 7mg respectively, putting the crude oil in a 50mL colorimetric cylinder, determining the volume by using petroleum ether, wherein the concentration at the moment is 0, 40, 50, 100, 120 and 140mg/L respectively, and measuring the absorbance of the standard concentration by using the petroleum ether as a reference solution at the wavelength of 258 nm. The abscissa represents concentration (mg/L) and the ordinate represents absorbance. A standard curve plotting the concentration (mg/L) and absorbance (A) of crude oil No. I is shown in FIG. 10.

(3) Determination of crude oil degradation Rate

Respectively taking 6mL of 1# crude oil degradation mixed bacterial suspension, 2# crude oil degradation mixed bacterial suspension, 3# crude oil degradation mixed bacterial suspension, 4# crude oil degradation mixed bacterial suspension, 5# crude oil degradation mixed bacterial suspension and 6# crude oil degradation mixed bacterial suspension, respectively inoculating the suspensions in 1mL/100mL inorganic salt liquid culture medium of No. I crude oil, culturing for 7d in a 30 ℃, 120rpm constant-temperature water bath oscillator, adjusting the pH value of the culture solution to be 2-3 by using 5:1(V/V) sulfuric acid solution, adding 2-4mL of methanol, extracting for three times by using 15mL of crude oil ether at the temperature of 60-90 ℃, drying the extract by using anhydrous sodium sulfate, transferring the extract into a 50mL volumetric flask and fixing the volume.

After the petroleum ether extract is serially diluted, the corresponding absorbance of the petroleum ether extract is measured by an ultraviolet spectrophotometry method by taking the petroleum ether as a reference, the concentration of a corresponding sample of the petroleum ether extract is determined according to a standard curve of the concentration (mg/L) -absorbance of No. I crude oil, and the degradation efficiency is calculated according to the concentration.

And (3) calculating the degradation rate of the crude oil degradation mixed bacteria to the crude oil I, and simultaneously respectively measuring the crude oil degradation rate of each single bacterium by adopting the same method.

The method for calculating the degradation rate of crude oil refers to the following formula:

η(％)＝(1-Ct/Co)×100％

wherein η -crude oil degradation rate, Ct-crude oil concentration in the presence of strain, and Co-control crude oil concentration.

When the degradation effect of the crude oil mixed bacteria on the crude oil polluted water body is carried out, the crude oil polluted water body is degraded by the same amount of marine crude oil degrading bacteria (Advenella kashmirensis)6-10-1 and bacillus thuringiensis (Bacillus thuringiensis)3-1 respectively, and the degradation results are compared.

And (3) test results:

the degradation rates of the 1#, 2#, 3#, 4#, 5#, and 6# crude oil mixed bacteria suspensions were calculated according to the above calculation formulas, as shown in table 5.

TABLE 5 degradation rate of crude oil mixed bacteria suspension on crude oil polluted water body

Numbering	Percent of degradation/%)
		1#	74.12
2#	78.92
		3#	65.22
4#	72.35
		5#	65.28
6#	74.01

As can be seen from Table 5, the peripheral degradation rate of the No.2 crude oil mixed bacteria to the No. I crude oil polluted water body is the highest, the degradation rate of the crude oil in 7d in a liquid inorganic salt culture medium with the liquid loading amount of 100mL, the total inoculation amount of 6mL and the crude oil concentration of 1% can reach 78.92%, and the peripheral degradation rate is higher than that of the single bacteria 6-10-1, 3 and 3-1 under the same condition, which indicates that certain synergistic effect exists among the three bacteria, and the No.1 crude oil mixed bacteria also show the same result. The peripheral degradation rate of the 3# crude oil mixed bacteria to the crude oil I polluted water body is lower than that of a single bacterial strain under the same condition, which shows that the synergistic effect among 1-8, 3 and 3-1 is poor, and the 6# crude oil mixed bacteria and the 4# crude oil mixed bacteria also show the same result and have good degradation effect.

Example 4 preparation of crude oil degrading mixed bacteria solid microbial inoculum by embedding method

Sodium Alginate (SA) is a promising and wide-ranging type of natural gel and is well recognized as being inexpensive and non-toxic to microorganisms. The addition of carrier materials enhances mechanical strength, tolerance of the SA gel, and provides adsorption sites for microorganisms. Sodium alginate as embedding material, CaCl₂The solution is cross-linking agent, and α -lactose is nutrient adsorbent.

The preparation method comprises the following steps:

① mixing 4g sodium alginate, 1.5g biomass carbon, 0.5g α -lactose, adding 100mL distilled water, stirring slowly while stirring, stirring activated carbon uniformly, and keeping stirring amplitude not too large to prevent a large amount of bubbles from entering sodium alginate solution, and autoclaving the solution at 121 deg.C for 20min after stirring.

② after sterilization, cooling to about 30 ℃, respectively adding 20mL of the 1#, 2#, 4#, 6# crude oil mixed bacterial suspension which is activated for 36h and obtained by the preparation method of the crude oil degradation mixed bacterial suspension in the embodiment 3, and respectively mixing uniformly to obtain 1#, 2#, 4# and 6# mixed liquid.

③ the 1#, 2#, 4#, 6# mixed solution is squeezed into CaCl with mass percent of 4% by using 10mL disposable syringes respectively₂In the solution, crosslinking is carried out for 24 hours to form 1#, 2#, 4# and 6# fixed microspheres, namely 1#, 2#, 4# and 6# crude oil degradation mixed bacteria solid microbial inoculum prepared by an embedding method.

④ filtering, washing with sterile water three times, soaking in physiological saline, and storing at 4 deg.C.

Example 5 preparation of crude oil degradation Mixed bacteria solid microbial inoculum by adsorption method

The biomass material and the corresponding biochar are used as target carriers, and crude oil degradation mixed bacteria are fixed by an adsorption method.

The fixing method comprises the following steps:

① adding 1.5g biomass carbon into 6 conical flasks filled with 100mL beef extract peptone medium, respectively, adding 20mL 1#, 2#, 4#, and 6# crude oil mixed bacterial suspension activated for 36h obtained by the preparation method of crude oil degradation mixed bacterial suspension in example 3, respectively, and mixing.

② Each Erlenmeyer flask was placed in a shaker at 30 ℃ and a rotation speed of 120r/min and fixed for 18 hours.

③ it is then centrifuged at 3000rpm for 10min, the supernatant removed and the lower pellet washed 2 times with sterile water.

④ and oven drying in 30 deg.C oven in culture dish to obtain solid bacterial preparation of No.1, No.2, No.3, No.4, and No. 5 crude oil degradation mixed bacteria prepared by adsorption method, soaking in physiological saline, and storing at 4 deg.C.

Example 6 degradation of crude oil-degrading mixed bacteria solid microbial inoculum to crude oil-contaminated water

The 1#, 2#, 4#, and 6# crude oil degradation mixed bacteria solid microbial inoculum prepared in the embodiment 4 and the embodiment 5 are respectively used for degrading crude oil polluted water bodies, and the concrete steps are as follows:

respectively taking 5g of crude oil degradation mixed bacteria solid microbial inoculum of No.1, No.2, No.4 and No. 6 prepared by an embedding method and 5g of crude oil degradation mixed bacteria solid microbial inoculum of No.1, No.2, No.4 and No. 6 prepared by an adsorption method, respectively inoculating the crude oil degradation mixed bacteria solid microbial inoculum into a No. II crude oil/100 mL crude oil inorganic salt culture medium, culturing at constant temperature of 30 ℃ and 120r/min for 7 days, and then measuring the removal effect of the crude oil degradation mixed microbial inoculum; wherein, the crude oil II is collected from the Changqing oil field, the density is 0.87g/mL, the maximum absorption wavelength of the crude oil II is measured and the standard curve graph is drawn in the same way as the crude oil I in the example 3, and the standard curve of the crude oil II is shown in figure 11.

Meanwhile, solid microbial inoculum of marine crude oil degrading bacteria (Advenella kashmirensis)6-10-1 and Brevundimonas diminuta (Brevundimonas diminuta)1-8 are prepared by the same embedding method and adsorption method as in examples 4 and 5 respectively, crude oil polluted water is degraded by using the solid microbial inoculum of the marine crude oil degrading bacteria (Advenella kashmirensis)6-10-1 and Brevundimonas diminuta (Brevundimonas diminuta)1-8, and the degradation effect is compared with that of mixed bacteria solid microbial inoculum of crude oil degrading bacteria No.1, 2, 3, 4 and 5, and the degradation effect is shown in Table 6.

In addition, 1.2mL and 4.2mL of the mixed bacterial suspension for degrading crude oil 1# respectively, the mixed bacterial suspension for degrading crude oil 2# respectively, the mixed bacterial suspension for degrading crude oil 4# respectively and the mixed bacterial suspension for degrading crude oil 6# respectively are taken and inoculated into the inorganic salt liquid culture medium of crude oil II 1mL/100mL respectively, the culture method is the same as that of example 3, the degradation effect is compared with that of a solid preparation, and the result is shown in table 6.

TABLE 6 degradation rate of crude oil mixed bacteria solid microbial inoculum and crude oil mixed bacteria suspension on crude oil polluted water body II

From the test results in table 6, it can be seen that, under the same condition, the solid microbial inoculum of the crude oil mixed bacteria and the bacterial suspension thereof, the solid microbial inoculum of the crude oil degradation single bacteria 6-10-1 and 1-8 and the bacterial suspension thereof all have the effect of degradation and remediation on the crude oil polluted water body, and the solid microbial inoculum of the crude oil mixed bacteria 1#, 2#, 3#, 4# and 5# prepared by the embedding method and the adsorption method has better degradation effect on the crude oil polluted water body. This is because the microorganism immobilization technology is a technology that locates free cells in a limited area, so that the free cells can maintain activity and be recycled, and has the advantages of high microorganism density, long action time, strong adverse environment resistance, less microorganism loss and the like, so that the degradation effect of the unit immobilized microbial inoculum is better than that of the liquid microbial inoculum.

In addition, the degradation effect of the solid microbial inoculum prepared by the two methods is compared, and under the same condition, the degradation effect of the solid microbial inoculum prepared by the adsorption method is better than that of the solid microbial inoculum prepared by the embedding method on the water body polluted by the crude oil II. The reason is that the solid microbial inoculum prepared by the two methods has different microbial amounts adsorbed on biomass charcoal, and the solid microbial inoculum has better degradation effect on crude oil polluted water bodies when the microbial amount is more.

Example 7 degradation of crude oil-contaminated soil by Mixed bacteria solid microbial inoculum for crude oil degradation

The 1#, 2#, 3#, 4#, and 5# crude oil degradation mixed bacteria solid microbial inoculum prepared in example 4 and example 5 are respectively used for degrading crude oil polluted soil, and the concrete steps are as follows:

crude oil contaminated soil sample: weigh the soil sample that 1mm sieve dried, weigh according to crude oil pollution degree and the quality of weighing soil sample and add in the soil sample and stir evenly, crude oil pollution degree is 10g/kg, according to C: n: p is 100: 10: 1 part of (NH)₄)₂SO₄And KH₂PO₄As an N source and a P source. Specifically, the nutrients of the microorganisms also include C, but petroleum pollution causes a large increase in the source of C, sufficient to ensure its supply, without the need for additional dosing. The physicochemical properties of the soil sample are as follows: 5.75ppm of ammonium nitrogen and 4.70ppm of nitrate nitrogen; nitrogen content 10.45 ppm; phosphorus content 16.41 ppm; potassium content 76.45 ppm; the organic content is 2.35%; the pH was 7.27.

The test method comprises the following steps:

respectively taking 5g of the mixed bacteria solid microbial inoculum for degrading the crude oil No.2 and No.4 prepared by the embedding method and 5g of the mixed bacteria solid microbial inoculum for degrading the crude oil No.2 and No.4 prepared by the adsorption method, respectively inoculating the mixed bacteria solid microbial inoculum into soil with the water content of 12-15 percent and the pollution concentration of the crude oil No. 100g being 1 percent, culturing at constant temperature of 120r/min and 30 ℃ for 7d, 14d and 21d, and then determining the removal effect of the mixed bacteria solid microbial inoculum on the crude oil No. II to obtain the degradation rate of the mixed bacteria solid microbial inoculum.

The method for measuring the content of petroleum hydrocarbon in soil comprises the following steps:

① A5 g sample of air-dried soil was weighed into a 50mL plastic centrifuge tube.

② adding 10mL carbon tetrachloride (analytically pure) with a pipette, ultrasonic extracting for 1h (temperature not more than 30 ℃), drying the extract with anhydrous sodium sulfate, filtering into a 25mL volumetric flask, hot-dipping with 2 × 10mL carbon tetrachloride twice for 20min each time, drying the filtrate, adding into the volumetric flask, and diluting to desired volume with carbon tetrachloride.

③ the spectrophotometer measures the absorbance of the sample at 288nm wavelength with carbon tetrachloride as reference, and the crude oil concentration is deduced against the standard curve.

When the test of the degradation effect of the mixed bacteria solid microbial inoculum for degrading crude oil of # 2 and # 6 on the original contaminated soil is carried out, the solid microbial inoculum of Brevundimonas diminuta (Brevundimonas diminuta)1-8 is used as a control sample, and the degradation result of the mixed bacteria solid microbial inoculum for degrading crude oil on the crude oil contaminated soil obtained from the test result is shown in Table 7.

TABLE 7 degradation rate of crude oil degradation mixed bacteria solid microbial inoculum to crude oil contaminated soil

From the test results in table 7, it can be seen that the mixed crude oil bacterium solid microbial inoculum prepared by the embedding method and the adsorption method has the effect of degrading and repairing the crude oil contaminated soil, the degradation efficiency is better along with the time extension, and compared with the degradation effect of the solid microbial inoculum of Brevundimonas diminuta (Brevundimonas diminuta)1-8, which has a good degradation effect, the mixed crude oil bacterium solid microbial inoculum of the present invention has a better degradation effect on the crude oil contaminated soil.

The test methods of the above examples 3 to 7 were used to examine the degradation and remediation effects of the mixed bacteria of marine crude oil degrading bacteria 6-10-1, Brevundimonas diminuta 1-8, Bacillus thuringiensis 3-1 or Pseudomonas flexi 3 on the crude oil contaminated water and crude oil contaminated soil, and as a result, it was found that all of them had the degradation and remediation effects.

Although the present invention has been described in detail in this specification with reference to specific embodiments and illustrative embodiments, it will be apparent to those skilled in the art that modifications and improvements can be made thereto based on the present invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

SEQUENCE LISTING

SEQ ID No.1

<110> Changan university

<120> crude oil degradation mixed bacteria, microbial inoculum and application thereof

<130>2016

<160>4

<170>PatentIn version 3.5

<210>1

<211>1402

<212>DNA

<213> Marine Petroleum-degrading bacterium (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> Brevundimonas diminuta (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 flexi (Pseudomonas genicula) 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 crude oil degradation mixed bacteria is characterized by comprising at least two of marine crude oil degradation bacteria Advenella kashmirensis6-10-1, Brevundimonas diminuta (Brevundimonas diminuta)1-8, Bacillus thuringiensis (Bacillus thuringiensis)3-1 or Pseudomonas flexi (Pseudomonas genia) 3; the marine crude oil degrading bacteria Advenella kashmirensis6-10-1, Brevundimonas diminuta 1-8, Bacillus thuringiensis (Bacillus thuringiensis)3-1 and Pseudomonas flexus (Pseudomonas genista) 3 are respectively separated from the ground crude oil polluted soil of the oil gathering station of the seven-cavity oil production field of the extended crude oil group;

the marine crude oil degrading bacteria Advenella kashmirensis6-10-1 are preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 13003; the sequencing result of 16S rDNA of the marine crude oil degrading bacterium Advenella kashmirensis6-10-1 is shown as SEQ ID No. 1;

the Brevundimonas diminuta (Brevundimonas diminuta)1-8 is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 13002; the sequencing result of 16S rDNA of the Brevundimonas diminuta (Brevundimonas diminuta)1-8 is shown as SEQ ID No. 2;

the Bacillus thuringiensis (Bacillus thuringiensis)3-1 is preserved in the China general microbiological culture Collection center of the culture Collection of microorganisms with the preservation number of CGMCC No. 13005; the 16S rDNA sequencing result of the bacillus thuringiensis (Bacillus thuringiensis)3-1 is shown as SEQ ID No. 3;

the Pseudomonas flexuosa (Pseudomonas genic) 3 is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 13004; the sequencing result of the 16S rDNA of the Pseudomonas flexi (Pseudomonas genic) 3 is shown in SEQ ID No. 4.

2. A mixed crude oil-degrading bacterial agent characterized in that its active ingredients comprise at least two of the marine crude oil-degrading bacteria Advenella kashmirensis6-10-1, Brevundimonas diminuta (Brevundimonas diminuta)1-8, Bacillus thuringiensis (Bacillus thuringiensis)3-1 or Pseudomonas flexi (Pseudomonas geniculata)3 according to claim 1.

3. The crude oil degradation mixed bacterial agent of claim 2, wherein the crude oil degradation mixed bacterial agent is a solid bacterial agent.

4. The use of the mixed crude oil degrading bacterial agent as claimed in claim 2 or 3 in the degradation of water polluted by crude oil.

5. The use of the crude oil degradation mixed bacterial agent of claim 2 or 3 in the degradation of soil polluted by crude oil.