CN111705001A - Method for rapidly screening polycyclic aromatic hydrocarbon degrading bacteria - Google Patents
Method for rapidly screening polycyclic aromatic hydrocarbon degrading bacteria Download PDFInfo
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- CN111705001A CN111705001A CN202010712934.3A CN202010712934A CN111705001A CN 111705001 A CN111705001 A CN 111705001A CN 202010712934 A CN202010712934 A CN 202010712934A CN 111705001 A CN111705001 A CN 111705001A
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Abstract
The invention discloses a method for rapidly screening polycyclic aromatic hydrocarbon degrading bacteria, and belongs to the technical field of petroleum pollution remediation. The existing method for screening the bacteria is optimized in four aspects of shortening the experimental period of screening the microorganisms capable of degrading the polycyclic aromatic hydrocarbon, controlling the using amount of an organic solvent, using trace-level target pollutants, simplifying experimental operation and the like by using a Tecan infinite 200 enzyme-labeling instrument and a 96-well plate in combination with a 2,6-DCPIP reagent. Can realize the rapid screening of strains with polycyclic aromatic hydrocarbon degradation capability and provide strain resources as much as possible for the remediation of the soil polluted by petroleum.
Description
Technical Field
The invention relates to a method for rapidly screening polycyclic aromatic hydrocarbon degrading bacteria, and belongs to the technical field of petroleum pollution remediation.
Background
In 2014, the general survey of soil in China finds that the main pollutants of a typical plot and the soil around the plot are petroleum hydrocarbon and polycyclic aromatic hydrocarbon, and the percentage of the overproof points is higher than 20.3%. After a large amount of complex petroleum hydrocarbons enter soil, the structure and functions of the soil are irreversibly damaged, such as changing the granularity, porosity, permeability and water holding capacity of the soil, changing the fertility effect of the soil and inducing orientation change of microbial communities in the soil. Petroleum hydrocarbons are easy to adhere to plant root systems, so that the absorption of oxygen, moisture and nutrient elements by plants is influenced. PAHs have strong lipophilicity, can damage viscera and adipose tissues, have triple-cause toxicity, and have immunotoxic effect on adult fishes, wild small mammals and human beings.
At present, the methods for remedying the polluted soil include physical methods, chemical methods, biological methods and the like. The physical method has the advantages of simple equipment, easy operation and high efficiency. However, the process is essentially the transfer of contaminants, the nature of which has not changed. Chemical processing is a treatment technique that adds an exogenous agent to remove colloidal or dissolved contaminants. The chemical treatment method can effectively treat sudden high-toxicity pollution events, but the cost for treating the soil polluted by low-concentration petroleum hydrocarbon for a long time is high, and secondary pollution is easy to form. The biological method for restoring petroleum-polluted soil is to utilize the carbon source requirement of animal, plant and microbe to degrade petroleum hydrocarbon into volatile organic matter and carbon dioxide, water, hydrogen and other inorganic matter, so as to restore soil. The biological method is emphasized at home and abroad because of the advantages of simple operation, economy, strong adaptability, no secondary pollution and the like. Among them, the microbial remediation technology is the most widely used method.
In order to scientifically recognize the role of microorganisms in the environment of petroleum hydrocarbon-contaminated soil, it is necessary to optimize the existing method and further rapidly separate strains with efficient PAHs degradation effect from the petroleum hydrocarbon-contaminated soil.
At present, the method for screening petroleum degrading microorganisms is generally divided into the following steps: (1) enriching microorganisms in a liquid culture medium in a biological shaking table with proper temperature and rotation speed; (2) culturing with solid culture medium in constant temperature incubator at proper temperature, and separating and purifying strains with large morphological difference; (3) preparing an inorganic salt culture medium containing a target pollutant, wherein the target pollutant is a unique carbon source, putting the separated strain into the culture medium, culturing the strain in a biological shaking table at a proper temperature and rotation speed, and performing multiple times of subculture and domestication; (4) and extracting the residual target pollutants from the conical flask by a solvent extraction method, and further judging the degradation capability of the strain on petroleum hydrocarbons.
However, the above method still has the following problems: (1) separating strains which are possible to degrade petroleum hydrocarbon from a polluted environment, determining that the strains need longer time for being the petroleum hydrocarbon degrading bacteria after repeated screening for multiple passages, and easily causing the change of microbial properties and the loss of advantageous properties after repeated subculture; (2) in order to extract target pollutants remained in the conical flask as much as possible in experiments, a large amount of organic solvent is often used, the cost is wasted, unnecessary pollution is generated, and the organic solvent is generally volatile and toxic and harmful to the physical health of research personnel; (3) the gravimetric method is inaccurate in measuring the residual target pollutants, and the analytical method of a precision instrument (such as CG-MS, LC-MS and the like) has higher requirements on the instrument precision, the selection of a chromatographic column, the preparation of a standard curve and the operation of researchers; (4) certain amount of petroleum hydrocarbons such as PAHs or crude oil are required to be used as target pollutants.
Disclosure of Invention
Aiming at the defects or improvement requirements of the existing method, the invention provides a method for combining a Tecan infinite 200 enzyme-labeling instrument, a 96-pore plate and a 2,6-DCPIP reagent, aiming at rapidly separating strains with high-efficiency degradation effect on PAHs by using trace experimental medicines, providing as much strain resources as possible for petroleum hydrocarbon soil remediation, providing support for preparing soil petroleum pollution in-situ remediation microbial inoculum in the later stage and reducing the problem of unnecessary pollution in the experimental process.
The method comprises the following specific steps:
1. experimental reagent
The experimental reagent comprises naphthalene, anthracene, phenanthrene, pyrene, fluoranthene, n-hexane and 2, 6-dichlorophenol indophenol (2, 6-DCPIP).
2. Preparation of culture medium
Bushnell-Haas (BH) Medium: KH (Perkin Elmer)2PO4：1.0g/L，K2HPO4：1.0g/L，NH4NO3：1.0g/L，MgSO4·7H2O：0.2g/L，FeCl3：0.05g/L，CaCl2·2H2O: 0.02g/L, pH: sterilizing at 121 deg.C for 30min at 6.8-7.2.
Luria-Bertani (LB) Medium: tryptone: 10g/L, yeast extract: 5g/L, NaCl: 10g/L, pH value: sterilizing at 121 deg.C for 30min at 7.0-7.4.
3. Separation and enhancement of dominant bacteria
(1) And (3) naturally drying the soil sample, grinding the soil sample by using a sterilization mortar, and sieving the ground soil sample by using a nylon sieve with the diameter of 2mm to remove foreign matters such as stones, branches and the like.
(2) 1g of soil sample is uniformly weighed, 1% of crude oil is added, the mixture is put into a conical flask with 100mL of LB liquid culture medium, and the conical flask is placed in a temperature-controlled shaking table at 36 ℃ and shaken at 120rmp for enrichment culture for 2-3 d.
(3) And 3d, taking 1mL of culture solution, coating the culture solution on an LB solid culture medium plate by using a dilution coating plate method, placing the plate in a constant-temperature incubator at 36 ℃, and carrying out inverted culture for 3-7 d.
(4) Observing colony color and morphology, selecting single colony strain with large difference, inoculating the strain on LB solid culture medium plate by plate streaking separation method, and placing in a constant temperature incubator at 36 deg.C for inverted culture.
(5) The dominant bacteria were cultured in a BH liquid medium with 4.0mg/mL PAHs solution (naphthalene: anthracene: phenanthrene: pyrene: fluoranthene: 2:1: 2:2) as a sole carbon source for enrichment. 1mL of PAHs solution is uniformly distributed at the bottom of the conical flask, and the solvent is volatilized in a fume hood. 100mL of BH broth was added to the flask, sealed with a silica gel plug, and sterilized at 121 ℃ for 20 min.
(6) Inoculating the separated strain with inoculating loop, placing in 36 deg.C temperature-controlled shaking table, shaking at 120rmp, placing in temperature-controlled shaking table, and performing intensive culture for 2-3 d.
(7) Inoculating the strain to slant solid culture medium, and storing in refrigerator at 4 deg.C. It is activated in liquid culture medium before application.
4. Preparation of bacterial suspension
20mL of the enhanced culture seed was placed in a 50mL sterilized centrifuge tube in a clean bench. Centrifuging for 10min with a high speed centrifuge (4000 rpm), discarding supernatant, suspending the precipitate with sterilized normal saline (50 mL), centrifuging again, washing for 3 times, and measuring the concentration of the bacterial suspension with ultraviolet spectrophotometer with normal saline as reference.
5.2,6-DCPIP color reaction polycyclic aromatic hydrocarbons degradation test
(1) Firstly, using 8-channel pipettor or 12-channel pipettor to take 5 microliter of target pollutant in a sterile 96-well plate, and placing on a super clean bench for 5-10min to volatilize the solvent. 160. mu.L of BH medium, 15. mu.L of bacterial suspension (step 4) and 20. mu.L of 2,6-DCPIP bio-chromogenic reagent (37.5. mu.g/mL) were continuously transferred into the above 96-well plate to simulate a contaminated microenvironment.
(2) Putting the 96-well plate into a sterilized plastic sealing box, and laying two layers of gauze which are fully soaked with water at the bottom of the box to ensure that the water surface is lower than the opening of the 96-well plate. Placing the mixture in a constant temperature incubator for cultivation.
(3) OD was measured at 0h, 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, 24h, 36h, 48h, 60h and 72h using a Tecan infinite 200 microplate reader600. And the color change of the 96-well plate was observed at the corresponding time.
6. Drawing a growth curve chart of strains
And drawing a growth curve of the strain by taking the time as an abscissa and taking a light absorption value at 600nm as an ordinate, wherein the corresponding slope represents the growth trend of the strain in the time period.
Drawings
FIG. 1 is a technical flow chart of the present invention.
FIG. 2 is a graph showing the growth of the bacterial species. (wherein A is a growth curve chart of the halomonas in a naphthalene environment, B is a growth curve chart of the halomonas in an anthracene environment, C is a growth curve chart of the halomonas in a phenanthrene environment, and D is a growth curve chart of the halomonas in a PAHs environment.)
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more clearly understood, the present embodiment is exemplified by Halomonas (Halomonas anticriensis sp.) and the present invention is further described in detail below with reference to the embodiments and the accompanying drawings.
Example 1:
target pollutants, strains and the like are added into a 96-well plate to simulate a polluted microenvironment, naphthalene is used as a single carbon source and energy substance, blue-to-white of the 2,6-DCPIP reagent is found to occur in about 8 hours, and the strains have metabolic activity in the microenvironment.
As can be seen from FIG. 2A, the strain is tolerant to naphthalene, when naphthalene is taken as a single carbon source and energy substance, the strain can rapidly grow within 2h-4h, and can grow for the second time within 6h-10h, and probably in a naphthalene environment, the strain mobilizes naphthalene-related degradation genes, and basically shows a descending trend after 24h, and possibly nutrient substances are consumed to be depleted.
Example 2:
target pollutants, strains and the like are added into a 96-well plate to simulate a polluted microenvironment, anthracene is used as a single carbon source and energy substance, blue-to-white of the 2,6-DCPIP reagent is found to occur in about 8 hours, and the strains have metabolic activity in the microenvironment.
As can be seen from FIG. 2B, the strain has tolerance to anthracene, and when anthracene is used as a single carbon source and energy substance, the strain can rapidly grow within 2h-4h, and can grow for the second time within 8h-10h, the strain has naphthalene-related gene degradation, and basically has a descending trend after 24h, and the nutrient substances are probably consumed.
Example 3:
target pollutants, strains and the like are added into a 96-well plate to simulate a polluted microenvironment, phenanthrene is used as a single carbon source and energy substance, blue-to-white of the 2,6-DCPIP reagent is found within about 10 hours, and the strains have metabolic activity in the microenvironment.
As can be seen from FIG. 2C, the strain is tolerant to phenanthrene, when phenanthrene is used as a single carbon source and energy source substance, the strain can rapidly grow within 0h-4h, and rapidly grow for the second time within 8h-10h, the strain is probably a dominant degradation strain of phenanthrene, and basically shows a descending trend after 24h, and possibly nutrients are consumed.
Example 4:
target pollutants, strains and the like are added into a 96-well plate to simulate a polluted microenvironment, PAHs is used as a single carbon source and energy substance, and the fact that the color of the 2,6-DCPIP reagent is lightened after about 24 hours is found, and the strains have metabolic activity in the microenvironment.
As can be seen from FIG. 2D, the strains have tolerance to PAHs, when PAHs are used as single carbon source and energy source substances, the PAHs can rapidly grow within 0h-2h, and the PAHs can grow for the second time within 20h-22h, the tolerance of the strains to mixed PAHs is obviously inferior to the tolerance to single naphthalene, anthracene and phenanthrene, the strains basically decline after 22h, and the nutrients are probably consumed until the total.
Claims (4)
1. A method for rapidly screening polycyclic aromatic hydrocarbon degrading bacteria is characterized by comprising the following specific steps:
(1) enriching and culturing a soil sample polluted by petroleum hydrocarbon in a liquid culture medium, and adding 1% of crude oil to obtain a strain with the petroleum hydrocarbon degradation capability;
(2) preparing a solid culture medium, and separating and purifying culturable strains by using a dilution coating flat plate method and a flat plate marking method;
(3) respectively adding naphthalene, anthracene, phenanthrene and mixed polycyclic aromatic hydrocarbon into an inorganic salt culture medium as a single carbon source, and screening the separated strains;
(4) the polycyclic aromatic hydrocarbon degrading bacteria are rapidly screened by utilizing the 2,6-DCPIP decoloration principle.
2. The application of the method for rapidly screening the polycyclic aromatic hydrocarbon degrading bacteria as claimed in claim 1, wherein the method is applied to screening the microorganisms having the capability of degrading the aromatic hydrocarbons and the microorganisms having the capability of degrading pollutants such as petroleum crude oil, petrochemical products and the like.
3. The application of the method for rapidly screening the polycyclic aromatic hydrocarbon degrading bacteria as claimed in claim 2, wherein the method is applied to screening the microorganisms with the capability of degrading the vegetable oil and the biodiesel.
4. The use of the method of claim 2 for rapid screening of polycyclic aromatic hydrocarbon-degrading bacteria in petroleum-contaminated water.
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| CN202010712934.3A CN111705001A (en) | 2020-07-23 | 2020-07-23 | Method for rapidly screening polycyclic aromatic hydrocarbon degrading bacteria |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101245366A (en) * | 2008-03-07 | 2008-08-20 | 中国科学院沈阳应用生态研究所 | Improved solid double-layer flat plate method for quick high-flux sifting motion of polycyclic aromatic hydrocarbon degradation bacterium |
| US20140144074A1 (en) * | 2012-11-26 | 2014-05-29 | National Taiwan Ocean University | Process of rapid isolating monostroma latissimum filamentous bodies for mass-scale breeding |
| CN109609428A (en) * | 2018-12-24 | 2019-04-12 | 北京高能时代环境技术股份有限公司 | The screening technique of polycyclic aromatic hydrocarbon-degrading bacteria |
| CN113941419A (en) * | 2021-09-22 | 2022-01-18 | 中山市环行电器科技有限公司 | Groove cutter and garbage disposal device |
-
2020
- 2020-07-23 CN CN202010712934.3A patent/CN111705001A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101245366A (en) * | 2008-03-07 | 2008-08-20 | 中国科学院沈阳应用生态研究所 | Improved solid double-layer flat plate method for quick high-flux sifting motion of polycyclic aromatic hydrocarbon degradation bacterium |
| US20140144074A1 (en) * | 2012-11-26 | 2014-05-29 | National Taiwan Ocean University | Process of rapid isolating monostroma latissimum filamentous bodies for mass-scale breeding |
| CN109609428A (en) * | 2018-12-24 | 2019-04-12 | 北京高能时代环境技术股份有限公司 | The screening technique of polycyclic aromatic hydrocarbon-degrading bacteria |
| CN113941419A (en) * | 2021-09-22 | 2022-01-18 | 中山市环行电器科技有限公司 | Groove cutter and garbage disposal device |
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Application publication date: 20200925 |