CN114075518A - Screening method of organic pollutant degrading bacteria - Google Patents
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
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Abstract
The invention provides a screening method of organic pollutant degrading bacteria, which comprises the following steps: s1, adding the soil sample polluted by organic pollutants into an inorganic salt liquid culture medium for culture and carrying out first enrichment; s2, coating the bacterial liquid after the first enrichment on a solid culture medium containing organic pollutants for culture and screening; and S3, selecting a single colony from the solid culture medium after culture and screening to perform liquid culture and secondary enrichment to obtain the degrading bacteria. The method avoids the complicated process that bacteria are completely separated by utilizing an LB (lysogeny broth) plate and are obtained through a large amount of re-screening in the traditional screening method, greatly shortens the screening period, improves the screening efficiency and is easy to find high-efficiency functional strains. And moreover, the problems that the organic solvent is easy to interfere, the contact between pollutants and strains is incomplete and the process is complicated in the double-layer flat plate separation method adopted in the prior art are solved.
Description
Technical Field
The invention relates to the technical field of microbial remediation of organic pollutants in the environment, and particularly relates to a screening method of organic pollutant degrading bacteria.
Background
Petroleum is one of the main energy pillars in the world today, and plays an indispensable role in the development of national economy. Leakage and discharge accidents are inevitable in the processes of oil exploitation, processing and transportation, wherein about 10 million tons of oil enter soil every year in China, the area of the polluted soil is about 500 million hectares, the structure and the function of the ecological system of the soil are damaged, and the health of human bodies is seriously harmed. The petroleum pollution problem of the main petrochemical and oil field exploitation areas (underground water and soil) in China is particularly prominent. The search for an economic and effective treatment technology without secondary pollution aiming at petroleum pollutants is a hot point which is very concerned by the environmental engineering community in recent years.
At present, the technology for restoring petroleum polluted environment mainly comprises the following steps: physical repair, chemical repair, biological repair, and the like. Compared with physical repair and chemical repair, the bioremediation method has the advantages of low cost, simplicity in operation, mild conditions, no secondary pollution and the like, and has a wide development prospect. Bioremediation refers to the biochemical metabolism of pollutants as carbon sources or energy sources by using microorganisms with special functions to realize the purpose of cell proliferation. The strain with high-efficiency degradation capability aiming at the target pollutant is the most key and important essential condition in the bioremediation technology, but the traditional separation and screening method has the defects of complex operation, low efficiency and easy omission of effective strains.
Chinese patent application CN 108300674A discloses a petroleum degrading bacterium and an obtaining method thereof and application thereof in degrading crude oil, wherein crude oil is used as a unique carbon source, the crude oil is diluted and coated by PBS solution after enrichment culture for 5-7 times, a single strain is separated, and then the single strain is inoculated into inorganic salt culture solution containing the crude oil, the degradation capability of the single strain is tested, so that the strain with high-efficiency degradation effect is obtained, the process is complicated, and the screening period is long. Chinese patent application CN 107058104 a discloses a method for culturing petroleum degrading bacteria, which is also flat coating, streaking, slant preservation, and finally testing the degradation performance of each strain. The Chinese patent application CN 106754538A discloses a separation and purification method of petroleum degradation bacteria oil 2-3 and application thereof, which is used for offshore oil spill degradation, and the method is also repeated streaking separation until strains which grow well and have single bacterial colonies are separated and screened. Chinese patent application CN 107418916 a discloses a method for screening polycyclic aromatic hydrocarbon high-efficiency degrading bacteria and the obtained high-efficiency degrading bacteria, wherein a double-layer plate is adopted for the configuration of the PAHs degrading bacteria screening plate, the PAHs mother liquor and the culture medium are mixed uniformly, and the subsequent operation is carried out after solidification. However, the following problems exist: acetone in the PAHs mother liquor needs to be completely volatilized before the culture medium is solidified, and is not easy to control; the distribution and thickness of PAHs in the upper flat plate have certain influence on the uptake of PAHs by strains, and effective strains are easy to miss; the manufacturing process is somewhat complicated. Chinese patent application CN 103224885A discloses a screening method of pollutant high-efficiency degrading bacteria based on micro-plate culture, in particular to a method for culturing pollutant high-efficiency degrading bacteria difficult to degrade by utilizing a 96-hole micro-plate, but the pollutant is required to be prepared into saturated matrix mother liquor, and the method is not suitable for crude oil and hydrophobic organic pollutants.
Therefore, a rapid and efficient screening method is urgently needed to be established for organic pollutants with complex components and strong hydrophobicity so as to construct a functional bacteria source library of the pollutants, thereby further promoting the development and application of biological treatment technology and reducing economic cost.
Disclosure of Invention
The invention mainly aims to provide a screening method of organic pollutant degrading bacteria, so as to shorten the screening period.
In order to achieve the above object, the present invention provides a screening method of organic pollutant degrading bacteria, comprising the following steps: s1, adding the soil sample polluted by organic pollutants into an inorganic salt liquid culture medium for culture and carrying out first enrichment; s2, coating the bacterial liquid after the first enrichment on a solid culture medium containing organic pollutants for culture and screening; and S3, selecting a single colony from the solid culture medium after culture and screening to perform liquid culture and secondary enrichment to obtain the degrading bacteria.
Further, between step S1 and step S2, the screening method further includes: a step of preparing a solid medium containing organic contaminants.
Further, the step of preparing a solid culture medium containing organic contaminants comprises: adding agar into an inorganic salt culture medium, and obtaining an agar culture medium after the agar is solidified; coating the organic pollutants in an agar culture medium to obtain a solid culture medium containing the organic pollutants; preferably, the organic contaminants are selected from crude oil or diesel; or the organic pollutant is polycyclic aromatic hydrocarbon organic matter and/or alkane organic matter.
Further, plating organic contaminants onto agar medium includes: diluting the organic pollutants by using a diluent to obtain diluted organic matters; the diluted organics were plated on agar medium.
Further, the diluent is petroleum ether, n-hexane, acetone or ethanol.
Further, in the solid culture medium, the mass-volume ratio of the organic pollutants to the bacterial liquid is 5: 1-3: 100; preferably, when the organic pollutant is crude oil or diesel oil, the mass-volume ratio of the organic pollutant to the bacterial liquid is 8: 1-10: 1; preferably, when the organic pollutant is polycyclic aromatic hydrocarbon, the mass-volume ratio of the organic pollutant to the bacterial liquid is 1: 100-3: 100; preferably, when the organic pollutants are alkanes, the mass-to-volume ratio of the organic pollutants to the bacterial liquid is 5: 1-8: 1.
Further, step S1 includes: s11, adding 0.8-1.5 g of soil sample polluted by organic pollutants into an inorganic salt liquid culture medium for shake culture for 2-4 days; s12, adding 0.4-0.7 g of organic pollutants into the inorganic salt liquid culture medium, and continuing culturing for 2-4 days; s13, carrying out continuous transfer enrichment culture according to the inoculation proportion of 5-15%; preferably, the inorganic salt liquid medium comprises: na (Na)2HPO4 0.5～0.7g/L，K2HPO4 0.1～0.3g/L，NaNO33-5 g/L, yeast powder 0.3-0.7 g/L, CaCl2 0.008～0.012g/L，FeSO4 0.008～0.012g/L，MgSO40.02-0.04 g/L, and a pH value of 7.0-7.5.
Further, in step S2, the bacterial liquid after the first enrichment is coated on a solid culture medium containing organic pollutants, and placed at 29-32 ℃ for inverted culture screening.
Further, step S3 includes the steps of: s31, selecting a single colony from the solid culture medium after culture screening; s32, placing a single colony in an LB liquid culture medium for secondary enrichment; s33, carrying out scribing purification on the bacteria liquid after the second enrichment to obtain degrading bacteria; preferably, the LB liquid medium includes: 4-6 g/L beef extract, 8-12 g/L peptone, 4-6 g/L NaCl, and 7.0-7.5 pH value.
Further, the organic pollutant is diesel oil, thick oil, light oil or heavy oil.
Further, the organic pollutant is a hydrophobic organic matter, preferably the hydrophobic organic matter comprises one or more of phenanthrene, pyrene, dibenzothiophene and carbazole.
By applying the technical scheme of the invention, the bacterial liquid is coated on the solid separation culture medium containing the petroleum substances and other hydrophobic organic matters for culture and screening by only adopting a single-layer solid inorganic salt culture medium. The method avoids the complicated process that bacteria are completely separated by utilizing an LB (lysogeny broth) plate and are obtained through a large amount of re-screening in the traditional screening method, greatly shortens the screening period, improves the screening efficiency and is easy to find high-efficiency functional strains. And moreover, the problems that the organic solvent is easy to interfere, the contact between pollutants and strains is incomplete and the process is complicated in the double-layer flat plate separation method adopted in the prior art are solved. And the organic pollutants in the method can provide carbon sources for microorganisms, so that the microorganisms capable of directly utilizing the carbon sources can grow, the screening efficiency of screening degradation strains is improved, the method is suitable for different oil products (light crude oil, thick oil, heavy crude oil, diesel oil and the like, the degradation effect of the oil product concentration can be represented by the total petroleum hydrocarbon or oil content) or hydrophobic organic matters (phenanthrene, pyrene, dibenzothiophene, carbazole and the like, the degradation effect can be represented by a specific compound composition) with different structures, and the method is suitable for further popularization.
Detailed Description
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail with reference to examples.
As mentioned in the background, the remediation of petroleum-contaminated environments is increasingly tending towards the adoption of bioremediation techniques. The existing screening method of the degrading bacteria aiming at the petroleum pollutants has the defects of complex operation, long screening period and the like. In order to shorten the screening period, in an exemplary embodiment of the present application, there is provided a screening method of an organic pollutant-degrading bacterium, the screening method including the steps of: s1, adding the soil sample polluted by organic pollutants into an inorganic salt liquid culture medium for culture and carrying out first enrichment; s2, coating the bacterial liquid after the first enrichment on a solid culture medium containing organic pollutants for culture and screening; s3, selecting a single colony from the solid culture medium after culture and screening for liquid culture and secondary enrichment; and S4, carrying out streak purification on the bacterial liquid after the second enrichment to obtain the degrading bacteria.
According to the screening method of the degrading bacteria, the bacterial liquid is coated on a solid separation culture medium containing petroleum substances and other hydrophobic organic matters for culture and screening only by adopting a single-layer solid inorganic salt culture medium. The method avoids the complicated process that bacteria are completely separated by utilizing an LB (lysogeny broth) plate and are obtained through a large amount of re-screening in the traditional screening method, greatly shortens the screening period, improves the screening efficiency and is easy to find high-efficiency functional strains. And moreover, the problems that the organic solvent is easy to interfere, the contact between pollutants and strains is incomplete and the process is complicated in the double-layer flat plate separation method adopted in the prior art are solved. And the organic pollutants in the method can provide carbon sources for microorganisms, so that the microorganisms capable of directly utilizing the carbon sources can grow, the screening efficiency of screening degradation strains is improved, the method is suitable for different oil products (light crude oil, thick oil, heavy crude oil, diesel oil and the like, the degradation effect of the oil product concentration can be represented by the total petroleum hydrocarbon or oil content) or hydrophobic organic matters (phenanthrene, pyrene, dibenzothiophene, carbazole and the like, the degradation effect can be represented by a specific compound composition) with different structures, and the method is suitable for further popularization.
Between step S1 and step S2, the screening method further includes: a step of preparing a solid medium containing organic contaminants. In a preferred embodiment, the step of preparing a solid culture medium containing organic contaminants comprises: adding agar into an inorganic salt culture medium, and obtaining an agar culture medium after the agar is solidified; and coating the organic pollutants in an agar culture medium to obtain a solid culture medium containing the organic pollutants.
Usually, most of organic pollutants are hydrophobic, so although the organic pollutants are added in the previous enrichment culture process for culture and primary screening, the screening conditions are relatively mild, and the main purpose of enriching strains is still achieved. However, in the preparation of a solid medium, organic contaminants cannot be uniformly mixed with a liquid inorganic salt medium, and thus it is difficult to obtain a medium in which organic contaminants are uniformly distributed even if agar is added to solidify it. Therefore, the organic pollutants are uniformly coated on the surface of the solid culture medium after the agar is solidified, so that bacteria and the organic pollutants are uniformly distributed on the surface of the solid culture medium when bacteria liquid is subsequently coated on the solid culture medium containing the organic pollutants, and the strains with resistance to the organic pollutants can be screened by fully utilizing the screening conditions of the organic pollutants.
The procedure of coating the organic contaminants onto the agar medium is slightly different depending on the type of the particular contaminant. In a preferred embodiment, the step of plating the organic contaminants onto the agar medium comprises: diluting the organic pollutants by using a diluent to obtain diluted organic matters; the diluted organics were plated on agar medium.
The petroleum products with strong fluidity, such as diesel oil, light crude oil (light oil) and the like, can be directly and uniformly coated in a solid separation culture medium. For oil products with poor fluidity and high viscosity such as heavy oil or heavy oil, the oil products can be diluted by petroleum ether (60-90 ℃) and then coated evenly. From the chemical structure, for other hydrophobic organic matters such as polycyclic aromatic hydrocarbon, stock solution can be prepared by acetone and the like, and then the coating operation is carried out. After the acetone is completely volatilized, the coating needs to be performed.
The above-mentioned solvents can be used as diluents for organic pollutants, whether petroleum ether (60-90 ℃) or acetone. In addition to these two, the diluent may also be n-hexane or ethanol. Experiments prove that the diluent comprises petroleum ether and acetone, and normal hexane is similar to the petroleum ether and can play a certain role in diluting oil products with poor fluidity; ethanol, like acetone, is more volatile and can be used as a diluent.
In the screening method, in order to improve the screening efficiency, shorten the screening period and improve the effectiveness of screening the strains, the organic pollutants coated in the solid culture medium and the coated bacterial liquid can be reasonably proportioned according to different pollutant types. In a preferred embodiment, the mass-to-volume ratio of the organic pollutants to the bacterial liquid is 5: 1-3: 100.
When the organic pollutant is crude oil, the dosage ratio of the organic pollutant and the crude oil is preferably controlled within the range of 8: 1-10: 1; when the organic pollutant is polycyclic aromatic hydrocarbon, the dosage ratio of the polycyclic aromatic hydrocarbon to the organic pollutant is preferably controlled within the range of 1: 100-3: 100; when the organic pollutants are alkanes, the dosage ratio of the organic pollutants and the alkanes is preferably controlled within the range of 5: 1-8: 1.
The step S1 is a conventional operation, and the culture enrichment can be performed according to a known method. In a preferred embodiment of the present application, step S1 includes: s11, adding 0.8-1.5 g of soil sample polluted by organic pollutants into an inorganic salt liquid culture medium, and performing shaking table shaking culture for 2-4 days; s12, heating 0.4-0.7 g of organic pollutants in the inorganic salt liquid culture medium, and continuing culturing for 2-4 days; s13, carrying out continuous transfer enrichment culture according to the inoculation proportion of 5-15%; preferably, the inorganic salt liquid medium comprises: na (Na)2HPO4 0.5～0.7g/L，K2HPO4 0.1～0.3g/L，NaNO33-5 g/L, yeast powder 0.3-0.7 g/L, CaCl2 0.008～0.012g/L，FeSO4 0.008～0.012g/L，MgSO4 0.02About 0.04g/L and a pH value of 7.0 to 7.5.
The soil sample polluted by organic pollutants is placed in an inorganic salt liquid culture medium for culture, and enrichment culture is expanded through multiple times of transfer, so that soil strains enough for subsequent screening are obtained, saturated screening is facilitated, and strains are prevented from being omitted.
In the step S2, when the solid medium obtained after the agar is solidified is cultured, the culture dish is usually placed in an incubator, and inverted culture is performed with a suitable temperature set. In a preferred embodiment, the bacterial liquid after the first enrichment is coated in a solid culture medium containing organic pollutants, and is placed at 29-32 ℃ for inverted culture screening. Preferably, the culture is carried out at 30 ℃ or 31 ℃. The specific culture time may be 24-48 hours, or may be shortened or delayed by a few hours, for example, 15 hours, 16 hours, 17 hours, 18 hours, 20 hours, 22 hours, or 50 hours, 54 hours. However, if the colonies grow aseptically, usually for a long time, under the condition that other culture conditions are normal, it is not a strain that can degrade the organic contaminants in a high probability. The colonies that can grow in a short time may be strains with stronger degradation activity.
The step of selecting a single colony for liquid culture enrichment in step S3 is also a conventional step, and may be performed according to conventional procedures. In a preferred embodiment of the present application, the step S3 includes the steps of: s31, selecting a single colony from the solid culture medium after culture screening; s32, placing a single colony in an LB liquid culture medium for secondary enrichment; s33, carrying out scribing purification on the bacteria liquid after the second enrichment to obtain degrading bacteria; preferably, the LB liquid medium includes: 4-6 g/L beef extract, 8-12 g/L peptone, 4-6 g/L NaCl, and 7.0-7.5 pH.
Different single colonies are respectively selected for enrichment culture, so that enrichment products of a plurality of different strains are obtained. And the purpose of scribing and purifying the bacteria liquid after the second enrichment is to further improve the purity of the strain, so that the bacteria liquid can be conveniently prepared into a bacteria source liquid for subsequent degradation performance investigation.
According to the screening method, the organic pollutants are classified from the petroleum product types, and can be diesel oil, thick oil, light oil or heavy oil. From the perspective of organic structure components, the organic pollutants are hydrophobic organic matters, and preferably the hydrophobic organic matters comprise one or more of phenanthrene, pyrene, dibenzothiophene and carbazole. The screening method is high in efficiency and wide in application range. Not only heavy oil, other diesel oil, viscous oil and other oil products contain polycyclic aromatic hydrocarbon, and the screening method aims at screening strains with degradation effect on total petroleum hydrocarbon of oil products with different properties. The method can also improve the screening effect on a specific oil degradation bacterium.
The advantageous effects of the present application will be further described with reference to specific examples. It should be noted that the degradation rate is calculated according to the following formula in the following experiment:
η＝(C0-C1)/C0。
wherein, C0Denotes blank control experiment (without addition of bacteria), C1The degradation experiments (spiking) are shown. A value greater than 0 is considered to be a degradation efficiency.
Example 1
Screening crude oil degrading bacteria:
(1) four soil samples with different pollution degrees around the Shengli oil field are taken, and three soil samples are parallel to each other. Adding 1g of soil sample into 100mL of inorganic salt culture solution, culturing for three days at 30 ℃ of a shaking table and 160rpm, then continuously adding 0.5g of crude oil, continuously culturing for three days, and then switching to a fresh petroleum inorganic salt culture medium for acclimatization for one week.
The inorganic salt culture medium comprises the following components in percentage by weight: na (Na)2HPO4 0.6g/L，K2HPO4 0.2g/L，NaNO34g/L, 0.5g/L of yeast powder and CaCl2 0.01g/L，FeSO4 0.01g/L，MgSO40.03g/L, and the pH is adjusted to 7.2. Sterilizing at 121 deg.C for 20min with CaCl2And MgSO4Separately sterilized, FeSO4And (5) filtering and sterilizing.
(2) Preparing inorganic salt solid culture medium, sterilizing, pouring into a plate culture dish, and solidifying the plate. Taking petroleum ether (boiling range 60-90 ℃) solution of crude oil, and slowly and uniformly coating the petroleum ether solution on a flat plate by using a coating device (the concentration ratio of petroleum ether is not determined, and the prepared oil product is generally required to have flowability due to different oil product sources, viscosities and properties, and can be uniformly coated on the surface of the flat plate). Wherein, the mass volume ratio of the crude oil to the bacteria liquid is 8: 1.
(3) sterile water is used for preparing culture solution with the concentration gradient of 10-3To 10-6Adding 100 mu L of bacterial liquid into the solidified plate culture dish, dipping the coater into absolute ethyl alcohol, igniting the absolute ethyl alcohol on an alcohol lamp for sterilization, cooling the coater, coating the absolute ethyl alcohol uniformly clockwise or anticlockwise until the surface of the plate is astringent. Placing into an incubator, and performing inversion culture at constant temperature of 30 ℃ for 24-48 h.
(4) And (3) observing the plate regularly, after bacterial colonies grow out, selecting bacterial colonies with different forms, inoculating the bacterial colonies to an LB culture medium for enrichment, and preparing a bacterial source liquid after streaking and purifying.
(5) Inoculating 10% of bacteria source liquid into a crude oil degradation culture medium, and culturing for 7d to determine the degradation rate. Wherein the crude oil degradation culture medium is an inorganic salt culture medium, and crude oil is added into the inorganic salt culture medium, and the concentration of the crude oil in the inorganic salt culture medium is 5 g/L.
And (4) conclusion: after 15 days of screening, 27 bacterial colonies with different bacterial strain forms are grown, and after streaking and purifying, 7d degradation experiments are carried out, 17 bacterial strains in the bacterial colonies have degradation efficiency, and the effective rate of the screening method is 62.96%.
Example 2
Screening diesel oil degrading bacteria:
the domestication and separation method of the diesel oil degrading bacteria is similar to the screening method of the crude oil degrading bacteria, and the only difference is that in the preparation of the inorganic salt solid culture medium, 100 mu L of diesel oil is uniformly smeared on a flat plate by using an applicator (wherein, the mass-volume ratio of the diesel oil to the bacterial liquid is 8: 1).
And (4) conclusion: after 15 days of screening, 34 colonies with different strain forms are grown, and 7d degradation experiments are carried out after streak purification, wherein 28 colonies have degradation efficiency, and the effective rate of the screening method is 82.35%.
Example 3
Screening polycyclic aromatic hydrocarbon degrading bacteria:
(1) selecting phenanthrene, pyrene, carbazole and dibenzothiophene, preparing acetone stock solution (10g/L), filtering and sterilizing by using a 0.22 mu m filter membrane, adding 400 mu L of acetone stock solution into 100mL of inorganic salt culture medium which is sterilized at high temperature, carrying out shaking culture overnight in a shaking table at 30 ℃ and 160rpm, adding 1g of petroleum-polluted soil sample after acetone is completely volatilized, and carrying out shaking culture in the shaking table for 3 d.
(2) The culture broth 10% was transferred to fresh medium for further culture 7 d.
(3) Preparing an inorganic salt solid separation culture medium. 100 mu L of acetone solution of polycyclic aromatic hydrocarbon is coated on the inorganic salt solid culture medium until the acetone is completely volatilized. Get 10-4、10-5、10-6And 10-7The bacterial liquid with four dilutions is diluted and coated (wherein, the mass volume ratio of the polycyclic aromatic hydrocarbon to the bacterial liquid is 1:100 (namely 0.01g:1mL), and cultured in an incubator for 24-48h at 30 ℃.
And (4) conclusion: after 12 days of screening, 17 colonies (phenanthrene) with different bacterial strain forms are grown, 10 bacterial strains with degradation function are grown, and the effective rate of the screening method is 58.82%. 12 colonies (pyrene) with different strain forms are grown in total, 8 strains with degradation function are grown, and the effective rate of the screening method is 66.67%. Total 11 bacterial colonies (carbazole) with different bacterial strain forms are grown, 6 bacterial strains with degradation function are obtained, and the effective rate of the screening method is 54.54%. 13 colonies (dibenzothiophene) with different strain forms grow out, 5 strains with degradation effect are obtained, and the effective rate of the screening method is 38.46%.
Example 4
Example 4 differs from example 1 only in that: in the solid medium, the mass-to-volume ratio of the crude oil to the bacterial liquid was 8:1(g: mL).
And (4) conclusion: after 15 days of screening, 27 bacterial colonies with different bacterial strain forms are grown, and after streaking and purifying, 7d degradation experiments are carried out, 17 bacterial strains in the bacterial colonies have degradation efficiency, and the effective rate of the screening method is 62.96%.
Example 5
Example 5 differs from example 1 only in that: in the solid culture medium, the mass-volume ratio of the crude oil to the bacterial liquid is 10: 1.
And (4) conclusion: after 15 days of screening, 24 bacterial colonies with different bacterial strain forms are grown, and after streaking and purifying, 7d degradation experiments are carried out, and 13 bacterial strains in the bacterial colonies have degradation efficiency, wherein the effective rate of the screening method is 54.17%.
Example 6
Screening of n-hexadecane degrading bacteria:
(1) four soil samples with different pollution degrees around the Shengli oil field are taken, and three soil samples are parallel to each other. Adding 1g of soil sample into 100mL of inorganic salt culture solution, culturing for three days at 30 ℃ of a shaking table and 160rpm, then continuously adding 1mL of n-hexadecane, continuously culturing for three days, and then switching to a fresh n-hexadecane inorganic salt culture medium for acclimatization for one week.
The inorganic salt culture medium comprises the following components in percentage by weight: na (Na)2HPO4 0.6g/L，K2HPO4 0.2g/L，NaNO34g/L, 0.5g/L of yeast powder and CaCl2 0.01g/L，FeSO4 0.01g/L，MgSO40.03g/L, and the pH is adjusted to 7.2. Sterilizing at 121 deg.C for 20min with CaCl2And MgSO4Separately sterilized, FeSO4And (5) filtering and sterilizing.
(2) Preparing inorganic salt solid culture medium, sterilizing, pouring into a plate culture dish, and solidifying the plate. N-hexadecane was sterilized with a 0.22 μm microporous membrane and then slowly and uniformly applied to a plate using an applicator. Wherein the mass-volume ratio of the n-hexadecane to the bacterial liquid is 5: 1.
(3) Sterile water is used for preparing culture solution with the concentration gradient of 10-3To 10-6Adding 100 mu L of bacterial liquid into the solidified plate culture dish, dipping the coater into absolute ethyl alcohol, igniting the absolute ethyl alcohol on an alcohol lamp for sterilization, cooling the coater, coating the absolute ethyl alcohol uniformly clockwise or anticlockwise until the surface of the plate is astringent. Placing into an incubator, and performing inversion culture at constant temperature of 30 ℃ for 24-48 h.
(4) And (3) observing the plate regularly, after bacterial colonies grow out, selecting bacterial colonies with different forms, inoculating the bacterial colonies to an LB culture medium for enrichment, and preparing a bacterial source liquid after streaking and purifying.
(5) Inoculating 10% of bacteria source liquid into a crude oil degradation culture medium, and culturing for 7d to determine the degradation rate. Wherein the n-hexadecane degradation culture medium is an inorganic salt culture medium, and n-hexadecane is added into the inorganic salt culture medium, and the concentration of the n-hexadecane in the inorganic salt culture medium is 7.75g/L (10 mL/L).
And (4) conclusion: after screening for 15 days, 38 bacterial colonies with different bacterial strain forms are grown, streaking and purifying are carried out, and then 7d degradation experiments are carried out, 26 bacterial strains in the bacterial colonies have degradation efficiency, and the effective rate of the screening method is 68.42%.
Example 7
Example 7 differs from example 1 only in that: in the solid culture medium, the mass-volume ratio of the n-hexadecane to the bacterial liquid is 8: 1.
And (4) conclusion: after screening for 15 days, 35 colonies with different strain forms are grown, and after streaking and purifying, 7d degradation experiments are carried out, 22 of the colonies are found to have degradation efficiency, and the effective rate of the screening method is 62.86%.
Comparative example 1
Chinese patent CN 108300674A discloses a petroleum degrading bacterium and an obtaining method thereof and application thereof in degrading crude oil, in particular to an enrichment, separation and purification method of Mycobacterium sp.CSC-6. Namely: firstly, adding an oil sludge sample into an inorganic salt culture medium, culturing for 7-10d on a shaking table at 30 ℃, then continuously transferring and enriching for 5-7 times, continuously diluting for 6 times, coating a flat plate with culture solutions with different dilution gradients, culturing for 3-7d, respectively selecting bacteria with different quantities, different colors and colony forms, respectively streaking, purifying and culturing to obtain single bacteria. Finally, the mixture is subjected to shaking culture on a shaking table at the temperature of 30 ℃ for 3 to 7 days, and degradation efficiency is measured. The screening time is about 41 days to 84 days, and the bacterial strain CSC-6 with high-efficiency and stable degradation effect on crude oil is obtained.
From the above description, it can be seen that the above-described embodiments of the present invention achieve the following technical effects: the invention provides an improved method for quickly and efficiently screening aerobic degradation bacteria of petroleum and hydrophobic organic matters, which only needs to adopt a single-layer solid inorganic salt culture medium to directly and uniformly coat petroleum substances and other hydrophobic organic matters on a solid separation culture medium, then a proper amount of bacterial liquid is absorbed and coated on the surface of the culture medium until the culture medium becomes astringent, and finally, the constant-temperature culture is carried out until bacterial strains grow out. The method greatly shortens the process that bacteria are completely separated by utilizing an LB flat plate in the traditional screening method and are subjected to a large amount of re-screening to obtain effective degrading bacteria, improves the screening efficiency and is easy to find high-efficiency functional strains; solves the problems of easy interference of organic solvent, incomplete contact of pollutants and strains and complicated process in a double-layer flat plate separation method. The method is suitable for different oil products (light crude oil, thick oil, heavy crude oil, diesel oil and the like) and hydrophobic organic matters (phenanthrene, pyrene, dibenzothiophene, carbazole and the like), and is suitable for further popularization.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (11)
1. A screening method of organic pollutant degrading bacteria is characterized by comprising the following steps:
s1, adding the soil sample polluted by organic pollutants into an inorganic salt liquid culture medium for culture and carrying out first enrichment;
s2, coating the bacterial liquid after the first enrichment on a solid culture medium containing the organic pollutants for culture and screening;
and S3, selecting a single colony from the solid culture medium after culture and screening to perform liquid culture and secondary enrichment to obtain the degrading bacteria.
2. The screening method of claim 1, wherein between the step S1 and the step S2, the screening method further comprises: a step of preparing a solid medium containing the organic contaminant.
3. The screening method according to claim 2, wherein the step of preparing a solid medium containing the organic contaminant comprises:
adding agar into an inorganic salt culture medium, and obtaining an agar culture medium after the agar is solidified;
and coating the organic pollutants in the agar culture medium to obtain a solid culture medium containing the organic pollutants.
4. The screening method of claim 3, wherein plating the organic contaminants onto the agar medium comprises:
diluting the organic pollutants by using a diluent to obtain diluted organic matters;
spreading the diluted organic matter in the agar culture medium.
5. The screening method according to claim 4, wherein the diluent is petroleum ether, n-hexane, acetone or ethanol.
6. The screening method according to any one of claims 1 to 5, wherein the mass-to-volume ratio of the organic pollutants to the bacterial liquid in the solid medium is 5: 1-3: 100;
preferably, when the organic pollutant is crude oil or diesel oil, the mass-volume ratio of the organic pollutant to the bacterial liquid is 8: 1-10: 1;
preferably, when the organic pollutant is polycyclic aromatic hydrocarbon, the mass-to-volume ratio of the organic pollutant to the bacterial liquid is 1: 100-3: 100;
preferably, when the organic pollutants are alkanes, the mass-to-volume ratio of the organic pollutants to the bacterial liquid is 5: 1-8: 1.
7. The screening method according to claim 6, wherein the step S1 includes:
s11, adding 0.8-1.5 g of the soil sample polluted by the organic pollutants into the inorganic salt liquid culture medium for shake culture for 2-4 days;
s12, adding 0.4-0.7 g of the organic pollutants into the inorganic salt liquid culture medium, and continuing to culture for 2-4 days;
s13, carrying out continuous transfer enrichment culture according to the inoculation proportion of 5-15%;
preferably, the inorganic salt liquid medium includes: na (Na)2HPO4 0.5～0.7g/L，K2HPO4 0.1～0.3g/L，NaNO33-5 g/L, yeast powder 0.3-0.7 g/L, CaCl2 0.008～0.012g/L，FeSO4 0.008～0.012g/L，MgSO40.02-0.04 g/L, and a pH value of 7.0-7.5.
8. The screening method according to claim 6, wherein in step S2, the bacterial liquid after the first enrichment is coated on a solid culture medium containing the organic pollutants, and is placed at 29-32 ℃ for inverted culture screening.
9. The screening method according to claim 6, wherein the step S3 includes the steps of:
s31, selecting a single colony from the solid culture medium after culture screening;
s32, placing the single colony in an LB liquid culture medium for the second enrichment;
s33, carrying out streak purification on the secondarily enriched bacterial liquid to obtain the degrading bacteria;
preferably, the LB liquid medium includes: 4-6 g/L beef extract, 8-12 g/L peptone, 4-6 g/L NaCl, and the pH value is 7.0-7.5.
10. The screening method according to claim 6, wherein the organic contaminant is diesel oil, heavy oil, light oil, or heavy oil.
11. The screening method according to claim 6, wherein the organic contaminant is a hydrophobic organic substance, preferably the hydrophobic organic substance comprises one or more of phenanthrene, pyrene, dibenzothiophene and carbazole.
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| CN116396898A (en) * | 2023-03-10 | 2023-07-07 | 江苏诚冉环境修复工程有限公司 | 1, 2-trichloroethane degrading bacterium and application thereof |
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| CN108949634A (en) * | 2018-08-08 | 2018-12-07 | 东南大学 | The oil degradation bacteria and its separation method of a kind of degradable heavy crude and application |
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