CN111394250A - Method for separating and purifying efficient petroleum hydrocarbon degrading bacteria from soil polluted by crude oil - Google Patents
Method for separating and purifying efficient petroleum hydrocarbon degrading bacteria from soil polluted by crude oil Download PDFInfo
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Abstract
The invention provides a method for separating and purifying efficient petroleum hydrocarbon degrading bacteria from crude oil contaminated soil, which comprises the steps of mixing and culturing an inorganic salt culture medium and a high-oil-content soil sample, carrying out acclimatization culture on a culture solution obtained after mixing and culturing by using a thin oil/inorganic salt culture medium, then carrying out acclimatization culture on a culture solution obtained after acclimatization culture by using a thick oil/inorganic salt culture medium, diluting the obtained culture solution, coating the diluted culture solution on a crude oil/inorganic salt solid plate culture medium and culturing; purifying to obtain purified strains with consistent forms, culturing the purified strains in a beef extract peptone culture medium, and inoculating the obtained bacterial liquid into a crude oil/inorganic salt liquid culture medium to perform a crude oil degradation experiment; and (3) measuring the crude oil degradation rate of the purified strain, and selecting the strain with higher crude oil degradation rate. The method can obtain the petroleum hydrocarbon degrading bacteria with the capability of degrading each component in the crude oil in a broad spectrum manner, and has wide applicability and higher use value.
Description
Technical Field
The invention relates to a method for separating and purifying efficient petroleum hydrocarbon degrading bacteria from crude oil polluted soil, belonging to the technical field of petroleum hydrocarbon biodegradation treatment.
Background
The petroleum as an important energy source is 'blood' of modern industry, but the petroleum brings rapidness and convenience to human beings and causes more and more serious environmental pollution. Petroleum is discharged to soil, underground water, ocean and the like in the processes of exploitation, transportation, storage and processing due to accidents or improper management, so that the environment is polluted, and the production and the life of human beings are directly harmed. The petroleum pollution treatment mainly comprises physical, chemical and microbial methods. The physical and chemical method has the defects of high energy consumption, high cost, serious secondary pollution and the like, and the microbial method enables the polluted environment to recover the natural function of the polluted environment by converting and removing petroleum pollutants in the new metabolic cycle of microorganisms, and has the advantages of low cost, small environmental influence and wide application range.
Due to the natural selection and elimination, different species of bacteria capable of degrading petroleum hydrocarbon generally exist in the environment polluted by petroleum hydrocarbon for a long time, and the bacteria are often the main force for bioremediation of the petroleum hydrocarbon pollutants in water or soil in the natural environment. Therefore, screening of high-efficiency petroleum hydrocarbon degrading bacteria is a research hotspot of the current microbial remediation technology of the petroleum-polluted soil.
At present, in the conventional method for enriching, separating and purifying degrading strains in the field, the carbon source used is usually crude oil with one component, and is relatively single. Such as: chinese patent CN 102021132 a discloses a process for acclimatizing oleophilic bacteria by gradually increasing the crude oil content of one component. However, the screened strain has poor tolerance and adaptability to heavy components in crude oil, and inhibits the strain from degrading thick oil with high content of the heavy components, so that the adaptability of the strain to the environment polluted by crude oil with various components is improved, and the degradation efficiency of the strain to crude oil with different components is promoted.
Disclosure of Invention
In order to solve the above disadvantages and shortcomings, it is an object of the present invention to provide a method for separating and purifying highly efficient petroleum hydrocarbon-degrading bacteria from soil contaminated with crude oil. The invention utilizes crude oil with gradually increased heavy components to carry out gradual enrichment domestication on petroleum hydrocarbon degrading bacteria, obtains the petroleum hydrocarbon degrading bacteria capable of degrading each component in the crude oil in a broad spectrum manner by separating and purifying the crude oil-containing soil, and finally realizes effective removal of the petroleum hydrocarbon in the soil.
The invention also aims to provide the pure bacterial strain of the high-efficiency petroleum hydrocarbon degrading bacteria obtained by the method for separating and purifying the high-efficiency petroleum hydrocarbon degrading bacteria from the soil polluted by the crude oil.
The invention also aims to provide application of the high-efficiency petroleum hydrocarbon degrading bacteria pure strain in remediation of soil polluted by crude oil.
In order to achieve the above objects, in one aspect, the present invention provides a method for separating and purifying highly efficient petroleum hydrocarbon-degrading bacteria from soil contaminated with crude oil, wherein the method comprises:
(1) mixing an inorganic salt culture medium with a high oil-content soil sample, and then culturing to obtain an enriched culture solution;
(2) mixing an inorganic salt culture medium with thin oil to obtain a mixed solution A, adding the enriched culture solution obtained in the step (1) into the mixed solution A, and culturing;
(3) mixing an inorganic salt culture medium with the thickened oil to obtain a mixed solution B, adding the enriched culture solution obtained in the step (2) into the mixed solution B, and then culturing;
(4) diluting the culture solution obtained in the step (3), coating the diluted culture solution on a crude oil/inorganic salt solid plate culture medium, and culturing; purifying to obtain purified strains with consistent forms;
(5) culturing the purified strain obtained in the step (4) in a beef extract peptone culture medium to obtain a bacterial liquid; then inoculating the bacterial liquid into a crude oil/inorganic salt liquid culture medium to carry out a crude oil degradation experiment; and then measuring the crude oil degradation rate of the purified strain, and selecting the strain with higher crude oil degradation rate as the pure strain of the high-efficiency petroleum hydrocarbon degrading strain.
According to a particular embodiment of the invention, in said method, said sample of high oil-bearing soil is a sample stored in a refrigerator at 4 ℃.
According to a particular embodiment of the invention, in the method, the oil content of the high oil-bearing soil sample is between 10% and 20%, wherein the oil content is calculated on the basis of the total weight of the air-dried soil.
According to a specific embodiment of the invention, in step (1) of the method, the ratio of the volume of the inorganic salt medium to the mass of the high oil-bearing soil sample is 9:1 to 10:1, and the units are m L and g respectively.
According to a specific embodiment of the present invention, in the method, the culturing is carried out at 25-30 ℃ and at 150rpm for 7-10 days in steps (1) - (4).
According to a specific embodiment of the present invention, in the method, based on the volume of the inorganic salt medium, the method comprises:
2.0 g/L g of ammonium nitrate, 1.5 g/L g of dipotassium phosphate, 3.0 g/L g of monopotassium phosphate, 0.1 g/L g of magnesium sulfate heptahydrate, 0.01 g/L g of anhydrous calcium chloride and 0.01 g/L g of disodium ethylene diamine tetraacetate.
According to a particular embodiment of the invention, in the method, the pH of the mineral salts medium is 7.5. + -. 0.1.
According to a specific embodiment of the invention, in the step (2) of the method, the concentration of the thin oil is 1-10 g/L based on the total volume of the inorganic salt culture medium and the enriched culture solution obtained in the step (1).
According to a particular embodiment of the invention, in step (2) of the process, the thin oil has a content of saturated hydrocarbons ranging from 65% to 75%, a content of aromatic hydrocarbons ranging from 15% to 25% and a content of gums and asphaltenes ranging from 5% to 15%, all based on 100% by weight of the thin oil.
According to a specific embodiment of the invention, in the step (2) of the method, the volume ratio of the enriched culture solution obtained in the step (1) to the mixed solution A is 0.1: 1.
According to a specific embodiment of the invention, the method further comprises the operation of performing cyclic acclimation culture on the enriched culture solution obtained in the step (2).
According to a specific embodiment of the present invention, in the step (2) of the method, the cyclic acclimation culture is a cyclic acclimation culture 2 to 3 times.
According to a specific embodiment of the invention, in the step (3) of the method, the concentration of the thick oil is 1-10 g/L based on the total volume of the inorganic salt culture medium and the enriched culture solution obtained in the step (2), and the dosage of the thick oil is the same as that of the thin oil in the step (2).
According to a specific embodiment of the present invention, in step (3), the heavy oil has a saturated hydrocarbon content of 30% to 40%, an aromatic hydrocarbon content of 15% to 25%, and a colloid and asphaltene content of 30% to 45%, based on the total weight of the heavy oil taken as 100%.
According to a specific embodiment of the present invention, in the step (3) of the method, the volume ratio of the enriched culture solution obtained in the step (2) to the mixed solution B is 0.1: 1.
According to a specific embodiment of the invention, the method further comprises the operation of performing cyclic acclimation culture on the culture solution obtained in the step (3).
According to a specific embodiment of the present invention, in the step (3) of the method, the cyclic acclimation culture is a cyclic acclimation culture 2 to 3 times.
According to a specific embodiment of the invention, in the step (4) of the method, the diluted culture solution obtained in the step (3) is spread on a crude oil/inorganic salt solid plate culture medium and cultured, and the method specifically comprises the following steps: diluting the culture solution obtained in step (3) with sterile water (such as 10-fold dilution method) to 10-4-10-6Then, 100. mu. L diluted culture medium (e.g., 10 of 100. mu. L each)-4、10-5、10-6Diluted culture solution) was spread on a crude oil/inorganic salt solid plate medium and cultured.
According to a specific embodiment of the present invention, in step (4) of the method, the purification step obtains a morphologically uniform purified strain, specifically comprising:
selecting dominant single colonies with different colors and forms, streaking and separating the single colonies for multiple times on a crude oil/inorganic salt solid plate culture medium to obtain purified strains with consistent forms through purification, and then storing the purified strains on a beef extract peptone solid culture medium for later use.
According to a specific embodiment of the present invention, in the step (4) of the method, the crude oil/inorganic salt solid plate medium is prepared by a preparation method comprising the following steps:
adding agar of 20 g/L (calculated by the total volume of the inorganic salt culture medium) into the inorganic salt culture medium to prepare an inorganic salt solid plate culture medium;
dissolving crude oil in n-hexane, preparing 10% (v/v) solution by taking the total volume of the obtained solution as a calculation reference, sucking 100 microliters of the solution, coating the solution on the surface of an inorganic salt solid plate culture medium, and obtaining the crude oil/inorganic salt solid plate culture medium after n-hexane is volatilized;
the inorganic salt medium comprises the following components in percentage by volume of the inorganic salt medium:
2.0 g/L g of ammonium nitrate, 1.5 g/L g of dipotassium phosphate, 3.0 g/L g of monopotassium phosphate, 0.1 g/L g of magnesium sulfate heptahydrate, 0.01 g/L g of anhydrous calcium chloride and 0.01 g/L g of disodium ethylene diamine tetraacetate.
According to a specific embodiment of the invention, in the step (5) of the method, the culture of the purified strain obtained in the step (4) in a beef extract peptone medium specifically comprises the following steps:
inoculating the purified strain into a beef extract peptone liquid culture medium of 100m L, and culturing for 2d at 30 ℃ and 150rpm to obtain a bacterial liquid for later use.
According to a specific embodiment of the invention, in the step (5), the bacterial liquid is inoculated into a crude oil/inorganic salt liquid culture medium to perform a crude oil degradation experiment, and the method comprises the following steps: inoculating the bacterial liquid into a crude oil/inorganic salt liquid culture medium according to the inoculation amount of 10 v% to perform a crude oil degradation experiment for 7d, and obtaining a strain with relatively high degradation rate.
According to a specific embodiment of the present invention, in the step (5) of the method, the concentration of crude oil in the crude oil/inorganic salt liquid medium is 1 to 10 g/L (calculated on the basis of the total volume of the crude oil/inorganic salt liquid medium).
According to a specific embodiment of the present invention, in step (5) of the method, the "crude oil" in the crude oil/inorganic salt liquid culture medium should be the same as the oil used in the degradation experiment, if the degradation object in the degradation experiment is thin oil, the culture medium used in step (5) is thin oil/inorganic salt liquid culture medium, and if the degradation object in the degradation experiment is thick oil, the culture medium used in step (5) is thick oil/inorganic salt liquid culture medium.
On the other hand, the invention also provides a pure bacterial strain of the high-efficiency petroleum hydrocarbon degrading bacteria obtained by the method for separating and purifying the high-efficiency petroleum hydrocarbon degrading bacteria from the soil polluted by the crude oil.
In another aspect, the invention also provides application of the high-efficiency petroleum hydrocarbon degrading bacteria pure bacterial strain in remediation of soil polluted by crude oil.
The method for separating and purifying the high-efficiency petroleum hydrocarbon degrading bacteria from the soil polluted by the crude oil comprises the steps of firstly adding a high-oil-content soil sample into a conical flask added with an inorganic salt culture medium to enrich the petroleum hydrocarbon degrading bacteria, then inoculating the obtained enriched culture solution into crude oil/inorganic salt culture media with different components to continue enrichment and domestication, and finally obtaining pure strains capable of efficiently degrading the petroleum hydrocarbon through separation and purification.
The pure petroleum hydrocarbon degrading strain screened by the invention is derived from the soil polluted by crude oil, so that the pure petroleum hydrocarbon degrading strain has strong adaptability to the soil, can quickly become dominant bacteria after entering the soil, occupies a certain ecological position, effectively promotes the degradation of petroleum hydrocarbons, optimizes a soil microbial system, improves the physical properties of the soil, enhances the enzymatic activity of the soil, and repairs hardened and degenerated soil.
In the method for separating and purifying the high-efficiency petroleum hydrocarbon degrading bacteria from the soil polluted by the crude oil, the crude oil with different components is used as the only carbon source in the screening process, so the obtained petroleum hydrocarbon degrading bacteria strain can adapt to the soil environment polluted by the crude oil with different components, the properties of the crude oil can be changed in the growth and propagation processes of the petroleum hydrocarbon degrading bacteria strain, the components of the crude oil can be changed, and the effects of emulsifying and dispersing the crude oil and efficiently degrading petroleum pollutants in the soil are achieved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to these drawings without inventive labor.
FIG. 1 is a schematic diagram showing the degradation rate of the strain A, the strain B and the strain C in thin oil and thick oil respectively in application example 1 and application example 2 of the present invention;
FIG. 2 is a graph showing the relationship between the degradation rate of the strain A, the degradation rate of the strain B and the degradation rate of the strain C on the thick oil in the soil and the time in application example 3 of the present invention.
Detailed Description
In order to clearly understand the technical features, objects and advantages of the present invention, the following detailed description of the technical solutions of the present invention will be made with reference to the following specific examples, which should not be construed as limiting the implementable scope of the present invention.
Example 1
The embodiment provides a method for separating and purifying high-efficiency petroleum hydrocarbon degrading bacteria from soil polluted by crude oil, wherein the method comprises the following steps:
carrying out on-site sampling in a certain oil field, setting sampling points in blocks with serious petroleum hydrocarbon pollution, taking back a sample of high-oil-content soil (with the oil content of 10-12 wt%) and storing the sample in a refrigerator at 4 ℃ for later use;
1. 10g of crude oil contaminated soil (high oil content soil) is added into an erlenmeyer flask added with 90m L inorganic salt culture medium to carry out enrichment culture of the petroleum hydrocarbon degrading bacteria, wherein the culture is carried out for 7 days in a shaking culture box with the temperature of 30 ℃ and the rotating speed of 150 rpm.
The inorganic salt culture medium comprises 2.0 g/L of ammonium nitrate, 1.5 g/L of dipotassium phosphate, 3.0 g/L of monopotassium phosphate, 0.1 g/L of magnesium sulfate heptahydrate, 0.01 g/L of anhydrous calcium chloride and 0.01 g/L of disodium ethylene diamine tetraacetate, and the pH value of the culture medium is 7.5 +/-0.1;
2. 0.1g of a thin oil (the concentration of the thin oil is 1 g/L; the content of saturated hydrocarbon in the thin oil is 71.32%, the content of aromatic components is 20.87%, the content of colloid is 6.47%, and the content of asphaltene is 3.01%) and a 90m L inorganic salt medium are added into an Erlenmeyer flask, the culture solution 10m L enriched in the step 1 is added into the Erlenmeyer flask, the Erlenmeyer flask is placed in a shaking incubator at the temperature of 30 ℃ and the rotation speed of 150rpm for culture for 7 days, and then the culture is circularly acclimatized and cultured for 3 times according to the method.
The inorganic salt culture medium comprises 2.0 g/L of ammonium nitrate, 1.5 g/L of dipotassium phosphate, 3.0 g/L of monopotassium phosphate, 0.1 g/L of magnesium sulfate heptahydrate, 0.01 g/L of anhydrous calcium chloride and 0.01 g/L of disodium ethylene diamine tetraacetate, and the pH value of the culture medium is 7.5 +/-0.1;
3. 0.1g of thick oil (the thick oil concentration is 1 g/L; the saturated hydrocarbon content in the thick oil is 36.67%, the aromatic content is 23.18%, the colloid content is 26.32%, and the asphaltene content is 14.39%) and 90m L inorganic salt medium are added into an erlenmeyer flask, 10m L of the culture solution enriched in the step 2 is added into the erlenmeyer flask, the erlenmeyer flask is placed in a shaking incubator at the temperature of 30 ℃ and the rotation speed of 150rpm for culture for 7 days, and then the cyclic acclimation culture is carried out for 3 times according to the method.
The inorganic salt culture medium comprises 2.0 g/L of ammonium nitrate, 1.5 g/L of dipotassium phosphate, 3.0 g/L of monopotassium phosphate, 0.1 g/L of magnesium sulfate heptahydrate, 0.01 g/L of anhydrous calcium chloride and 0.01 g/L of disodium ethylene diamine tetraacetate, and the pH value of the culture medium is 7.5 +/-0.1;
4. diluting the culture solution obtained by enrichment culture in step 3 by 10-fold dilution method, and respectively taking 10-4、10-5、10-6The preparation method comprises the following specific steps of adding agar with the concentration of 20 g/L into the inorganic salt culture medium to prepare an inorganic salt solid plate culture medium, dissolving crude oil into normal hexane to prepare a 10% (v/v) solution, sucking 100 mu L of the solution, coating the solution on the surface of the inorganic salt solid plate culture medium, preparing the crude oil/inorganic salt solid plate culture medium after the normal hexane volatilizes, culturing the culture medium in a constant-temperature incubator at 30 ℃ for 96 hours, selecting single bacterial colonies with different colors and shapes, carrying out scribing separation on the crude oil/inorganic salt solid plate culture medium for multiple times to purify to obtain purified bacterial strains with consistent shapes, storing the purified bacterial strains on a beef extract peptone solid culture medium, culturing to obtain A bacterial liquid, and keeping the A bacterial liquid for later use.
Application example 1
In the present application, a thin oil degradation test (conventional operation in the art) was performed using the pure strain of the efficient petroleum hydrocarbon-degrading bacteria (denoted as strain A) obtained in example 1, and 10 v% of the bacterial solution A was inoculated into a thin oil/inorganic salt liquid medium (the thin oil content was 5 g/L; the saturated hydrocarbon content in the thin oil was 71.32 wt%, the aromatic content was 20.87 wt%, the colloidal content was 6.47 wt%, and the asphaltene content was 3.01 wt%), and the thin oil/inorganic salt liquid medium used was composed of 2.0 g/L ammonium nitrate, 1.5 g/L dipotassium hydrogen phosphate, 3.0 g/L potassium dihydrogen phosphate, 0.1 g/L magnesium sulfate heptahydrate, 0.01 g/L calcium chloride, 0.01 g/L disodium ethylenediaminetetraacetate, and the pH value was 7.5. + -. 0.1), and the degradation rate of the strain A was determined to be the highest after the oil degradation test was performed using the pure strain A obtained in example 1, and the test result was determined to be the highest degradation rate of the thin oil degradation test in example 1.
After 2 rounds of domestication (3 times per round of domestication) of the petroleum hydrocarbon degrading bacteria in the step 1 of the example 1 are sequentially domesticated by using only the thin oil (1 g/L) as a carbon source, a pure petroleum hydrocarbon degrading strain (marked as a B strain) obtained by separation and purification is used as a comparison and is used in the thin oil degradation test (the conventional operation in the field) as above, and the experimental result shows that the degradation rate of the B strain to the thin oil is only 56%.
After sequentially acclimating the petroleum hydrocarbon degrading bacteria in the step 1 of the example 1 for 2 rounds (acclimatization is performed for 3 times per round) by only using the thickened oil (1 g/L) as a carbon source, separating and purifying the obtained pure petroleum hydrocarbon degrading strain (marked as C strain) as a comparison, and using the pure petroleum hydrocarbon degrading strain in the same thin oil degradation test (conventional operation in the field), the experimental result shows that the degradation rate of the C strain to the thin oil is only 64%.
In the application example 1, a schematic diagram of comparison of the degradation rates of the strains A, B and C to the thin oil is shown in FIG. 1; as can be seen from FIG. 1, the degradation rate of the strain A on the thin oil is respectively improved by 29 percent and 13 percent compared with that of the strain B and the strain C.
Application example 2
In the application, a thick oil degradation test (conventional operation in the field) is carried out by using the high-efficiency petroleum hydrocarbon degrading bacteria pure strain (marked as strain A) obtained in example 1, 10 v% of the strain A is inoculated into a thick oil/inorganic salt culture medium (the content of the thick oil is 5 g/L; the content of saturated hydrocarbon in the thick oil is 36.67%, the content of aromatic components is 23.18%, the content of colloid is 26.32%, and the content of asphaltene is 14.39%), the thick oil/inorganic salt culture medium comprises 2.0 g/L of ammonium nitrate, 1.5 g/L of dipotassium hydrogen phosphate, 3.0 g/L of potassium dihydrogen phosphate, 0.1 g/L of magnesium sulfate heptahydrate, 0.01 g/L of anhydrous calcium chloride, 0.01 g/L of disodium ethylene diamine tetraacetate, and the pH value is 7.5 +/-0.1), the thick oil degradation test is carried out by culturing the thick oil degradation test for 7 days at 30 ℃ and 150rpm, and the degradation condition of the thick oil degradation test is determined after the test is finished.
The experimental results show that: the A bacterial strain obtained in the embodiment 1 of the invention can effectively degrade thick oil, and the degradation rate is 42%.
After 2 rounds of domestication (3 times per round of domestication) of the petroleum hydrocarbon degrading bacteria in the step 1 of the example 1 are sequentially domesticated by only using thin oil (1 g/L) as a carbon source, a pure petroleum hydrocarbon degrading strain (marked as a B strain) obtained by separation and purification is used as a comparison and is used for a degradation test of thick oil (conventional operation in the field), and the experimental result shows that the degradation rate of the B strain to the thick oil is only 31%.
After sequentially acclimating the petroleum hydrocarbon degrading bacteria in the step 1 of the example 1 for 2 rounds (acclimatization is performed for 3 times per round) by only using the thickened oil (1 g/L) as a carbon source, separating and purifying the obtained petroleum hydrocarbon degrading pure strain (marked as C strain) as a comparison, and using the petroleum hydrocarbon degrading pure strain in a thickened oil degradation test (routine operation in the field), wherein the experimental result shows that the degradation rate of the C strain to the thickened oil is 33%.
In application example 2, a schematic diagram of comparison of the degradation rates of the strain a, the strain B and the strain C to thick oil is shown in fig. 1; as can be seen from FIG. 1, the degradation rate of the strain A on the thick oil is improved by 35% and 27% respectively compared with that of the strain B and the strain C.
Application example 3
In the application example, the degradation test of thick oil in soil (routine operation in the field) is carried out by adopting the pure strain of the efficient petroleum hydrocarbon degrading bacteria (marked as strain A) obtained in the example 1, the content of the thick oil in the soil is 5 percent (the mass of the thick oil in each gram of air-dried soil), the adding amount of the bacterial liquid A is 10m L of the bacterial liquid A added into each 100g of soil, in addition, urea and dipotassium hydrogen phosphate are added as nutrient sources, and after the mixture is fully stirred and uniformly mixed, the microbial degradation test is carried out at the temperature of 30 ℃.
Sequentially domesticating the petroleum hydrocarbon degrading bacteria in the step 1 of the example 1 by using only thin oil (1 g/L) as a carbon source for 2 rounds (domestication for 3 times per round) and then separating and purifying the obtained petroleum hydrocarbon degrading pure strain (marked as B strain), and sequentially domesticating the petroleum hydrocarbon degrading bacteria in the step 1 of the example 1 by using only thick oil (1 g/L) as a carbon source for 2 rounds (domestication for 3 times per round) and then separating and purifying the obtained petroleum hydrocarbon degrading pure strain (marked as C strain) as a comparison to carry out a degradation test of the thick oil in the soil, wherein the test parameters are the same as those of the test of degrading the thick oil in the soil by using the A strain.
After the test is finished, the degradation rates of the three strains to the thick oil in the soil at different times are respectively measured, and a graph of a relation between the degradation rates of the three strains to the thick oil in the soil and the time is shown in fig. 2, and as can be seen from fig. 2, the strain A obtained in the embodiment 1 of the invention can effectively degrade the crude oil in the soil, the degradation rate of the crude oil in the soil is 37.4% after 56 days of degradation, and is respectively improved by 44.9% and 33.1% compared with the degradation rates of the strain B (25.8%) and the strain C (28.1%) under the same conditions.
In addition, after the strain A provided by the embodiment 1 of the invention is adopted to degrade the thick oil in the soil, the proportion of aromatic components, colloid and asphaltene in the thick oil is obviously reduced compared with the strain B and the strain C, which shows that the strain A has better tolerance and degradation effect on heavy components in the thick oil in the process of repairing the soil by using the strain A.
In conclusion, the method for separating and purifying the high-efficiency petroleum hydrocarbon degrading bacteria from the soil polluted by the crude oil provided by the invention is a method for gradually enriching and domesticating the petroleum hydrocarbon degrading bacteria by utilizing the crude oil with gradually increased heavy components, compared with a single-component crude oil domesticating method, the method provided by the invention obtains the petroleum hydrocarbon degrading bacteria capable of improving the degrading capability of the petroleum hydrocarbon degrading bacteria on the crude oil with different components by separating and purifying the crude oil-containing soil, and the degrading rate of the screened petroleum hydrocarbon degrading bacteria on the crude oil with higher heavy component content is obviously increased.
The above description is only exemplary of the invention and should not be taken as limiting the scope of the invention, so that the invention is intended to cover all modifications and equivalents of the embodiments described herein. In addition, the technical features and the technical inventions of the present invention, the technical features and the technical inventions, and the technical inventions can be freely combined and used.
Claims (23)
1. A method for separating and purifying high-efficiency petroleum hydrocarbon degrading bacteria from soil polluted by crude oil, which is characterized by comprising the following steps:
(1) mixing an inorganic salt culture medium with a high oil-content soil sample, and then culturing to obtain an enriched culture solution;
(2) mixing an inorganic salt culture medium with thin oil to obtain a mixed solution A, adding the enriched culture solution obtained in the step (1) into the mixed solution A, and culturing;
(3) mixing an inorganic salt culture medium with the thickened oil to obtain a mixed solution B, adding the enriched culture solution obtained in the step (2) into the mixed solution B, and then culturing;
(4) diluting the culture solution obtained in the step (3), coating the diluted culture solution on a crude oil/inorganic salt solid plate culture medium, and culturing; purifying to obtain purified strains with consistent forms;
(5) culturing the purified strain obtained in the step (4) in a beef extract peptone culture medium to obtain a bacterial liquid; then inoculating the bacterial liquid into a crude oil/inorganic salt liquid culture medium to carry out a crude oil degradation experiment; and then measuring the crude oil degradation rate of the purified strain, and selecting the strain with higher crude oil degradation rate as the pure strain of the high-efficiency petroleum hydrocarbon degrading strain.
2. The method of claim 1, wherein the high oil soil sample is a sample stored in a 4 ℃ refrigerator.
3. The method of claim 1, wherein the oil content of the high oil content soil sample is between 10% and 20%.
4. The method according to claim 1, wherein in the step (1), the ratio of the volume of the inorganic salt culture medium to the mass of the high oil-bearing soil sample is 9:1-10:1, and the units are m L and g respectively.
5. The method according to claim 1, wherein the culturing in steps (1) to (4) is carried out at 25 to 30 ℃ and at a rotation speed of 120 and 150rpm for 7 to 10 days.
6. The method according to claim 1, comprising, based on the volume of the inorganic salt medium:
2.0 g/L g of ammonium nitrate, 1.5 g/L g of dipotassium phosphate, 3.0 g/L g of monopotassium phosphate, 0.1 g/L g of magnesium sulfate heptahydrate, 0.01 g/L g of anhydrous calcium chloride and 0.01 g/L g of disodium ethylene diamine tetraacetate.
7. The method of claim 1 or 6, wherein the pH of the mineral salts medium is 7.5 ± 0.1.
8. The method according to claim 1, wherein in the step (2), the concentration of the thin oil is 1-10 g/L based on the total volume of the inorganic salt culture medium and the enriched culture solution obtained in the step (1).
9. The method as claimed in claim 1 or 8, wherein in the step (2), the content of saturated hydrocarbon is 65-75%, the content of aromatic hydrocarbon is 15-25%, and the content of colloid and asphaltene is 5-15%, based on 100% of the total weight of the thin oil.
10. The method according to claim 1, wherein in step (2), the volume ratio of the enriched culture solution obtained in step (1) to the mixed solution A is 0.1: 1.
11. The method according to claim 1, further comprising performing a cyclic acclimatization culture on the enriched culture solution obtained in step (2).
12. The method according to claim 11, wherein the cyclic acclimation culture is a cyclic acclimation culture 2-3 times.
13. The method according to claim 1, wherein in the step (3), the concentration of the thick oil is 1-10 g/L based on the total volume of the inorganic salt culture medium and the enriched culture solution obtained in the step (2), and the amount of the thick oil is the same as that of the thin oil in the step (2).
14. The method as claimed in claim 1 or 13, wherein in the step (3), the saturated hydrocarbon content is 30-40%, the aromatic hydrocarbon content is 15-25%, and the colloid and asphaltene content is 30-45% based on 100% of the total weight of the thick oil.
15. The method according to claim 1, wherein in step (3), the volume ratio of the enriched culture solution obtained in step (2) to the mixed solution B is 0.1: 1.
16. The method according to claim 1, further comprising performing a cyclic acclimatization culture on the culture solution obtained in step (3).
17. The method of claim 16, wherein the cyclic acclimatization culture is a cyclic acclimatization culture 2-3 times.
18. The method according to claim 1, wherein in the step (4), the diluted culture solution obtained in the step (3) is spread on a crude oil/inorganic salt solid plate culture medium and cultured, and the method specifically comprises the following steps: diluting the culture solution obtained in the step (3) to 10 ℃ with sterile water-4-10-6The culture broth diluted to 100. mu. L was applied to a crude oil/inorganic salt solid plate medium and cultured.
19. The method according to claim 1, wherein in the step (5), the culturing is carried out at 30 ℃ and 150rpm for 2 d.
20. The method of claim 1, wherein in the step (5), the bacterial liquid is inoculated into a crude oil/inorganic salt liquid culture medium to perform a crude oil degradation experiment, and the method comprises the following steps: the bacterial liquid is inoculated into a crude oil/inorganic salt liquid culture medium according to the inoculation amount of 10 v% so as to carry out a crude oil degradation experiment of 7 d.
21. The method as claimed in claim 1 or 20, wherein in the step (5), the concentration of the crude oil in the crude oil/inorganic salt liquid culture medium is 1-10 g/L.
22. The pure strain of efficient petroleum hydrocarbon-degrading bacteria obtained by the method for separating and purifying efficient petroleum hydrocarbon-degrading bacteria from soil contaminated by crude oil according to any one of claims 1 to 21.
23. The use of the pure strain of efficient petroleum hydrocarbon degrading bacteria of claim 22 in remediation of soil contaminated with crude oil.
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