CN110656079A - Preparation method of naphthalene restoration and degradation microbial inoculum in petroleum hydrocarbon polluted soil - Google Patents
Preparation method of naphthalene restoration and degradation microbial inoculum in petroleum hydrocarbon polluted soil Download PDFInfo
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- CN110656079A CN110656079A CN201911062265.3A CN201911062265A CN110656079A CN 110656079 A CN110656079 A CN 110656079A CN 201911062265 A CN201911062265 A CN 201911062265A CN 110656079 A CN110656079 A CN 110656079A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/36—Adaptation or attenuation of cells
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
Abstract
The invention discloses a preparation method of a naphthalene restoration and degradation microbial inoculum in petroleum hydrocarbon polluted soil, which comprises the steps of weighing a certain amount of petroleum hydrocarbon polluted soil, culturing in a culture medium to obtain a culture bacterial solution; inoculating the culture bacterial liquid into an inorganic salt culture medium, carrying out constant-temperature shaking culture, carrying out acclimatization culture by utilizing gradient pressure screening, diluting according to gradient after culture, adding physiological saline, coating a flat plate, culturing in a constant-temperature incubator, selecting complete bacterial colonies to be cultured in an enrichment culture medium, and obtaining enrichment culture bacterial liquid; inoculating the enrichment culture bacterial liquid into a culture medium, carrying out constant-temperature oscillation culture to logarithmic phase, adding glycerol, mixing, and freezing and storing to obtain the naphthalene restoration and degradation bacterial agent. The screening and separating method of the naphthalene restoration degradation strain is relatively simple, has no secondary pollution, has high degradation rate on naphthalene in the petroleum hydrocarbon polluted soil, realizes high-efficiency restoration on the naphthalene in the petroleum hydrocarbon polluted soil through a biological strengthening process, and has wide engineering application prospect.
Description
Technical Field
The invention relates to a preparation method of a naphthalene restoration and degradation microbial inoculum in petroleum hydrocarbon polluted soil, belonging to the technical field of microbial restoration of polluted soil.
Background
Naphthalene is a fused ring aromatic hydrocarbon with a 2-ring structure, has the characteristics of low toxicity and high efficiency compared with other substances of Polycyclic Aromatic Hydrocarbons (PAHs), and is widely used for producing phthalic anhydride, dye intermediates, rubber auxiliaries, pesticides and the like. Studies have reported that naphthalene exposure can lead to deleterious effects of hemolytic anemia, liver and nervous system injury, cataracts, and retinal hemorrhage. In 10 months in 2017, the international cancer research institution of the world health organization has listed naphthalene in a list of 2B carcinogens, and China also listed in the first list of priority control chemicals in 12 months in 2017. Naphthalene is widely present in ecological environment, such as surface water, underground water, soil, sediment and the like, and high-concentration naphthalene is detected in water bodies of seven large watersheds in China. Research shows that naphthalene has extremely high toxicity to aquatic organisms and may have long-term adverse effects on the water environment.
In recent years, the problem of soil pollution in China is increasingly prominent, the soil environment quality is directly related to the safety of agricultural products, the overproof rate of pesticide residues in main agricultural products is as high as 16% -20%, the soil pollution becomes one of important obstacles for limiting international trade and social and economic sustainable development of agricultural products in China, and the polluted soil is repaired and treated urgently. Naphthalene is a common organic pollutant in soil, and particularly has the characteristics of strong hydrophobicity, high treatment difficulty and the like in places such as petroleum refining, waste incineration, coking plants and the like. At present, the treatment methods for PAHs (polycyclic aromatic hydrocarbons) such as naphthalene in polluted soil mainly comprise chemical leaching, advanced oxidation, electric restoration and the like, but the defects of damaging soil aggregates, generating secondary pollution and the like generally exist. In contrast, the microbial treatment technology is considered to be an environment-friendly technology, has the advantages of rapidness, safety, low cost and the like, and is also a main process for converting and removing organic pollutants in environmental soil.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: a method for treating naphthalene in petroleum hydrocarbon polluted soil by microorganisms is provided.
In order to solve the technical problem, the invention provides a preparation method of a naphthalene restoration and degradation microbial inoculum in petroleum hydrocarbon polluted soil, which is characterized by comprising the following steps:
step 1): culturing petroleum hydrocarbon polluted soil in an LB culture medium to activate indigenous microorganisms to obtain a culture bacterial liquid;
step 2): inoculating the activated culture bacterial liquid into an inorganic salt culture medium, placing the inorganic salt culture medium in a constant-temperature shaking table for shaking culture, performing acclimatization culture by using a gradient pressure screening method, diluting according to gradient after culture, adding physiological saline, coating a flat plate, and placing the flat plate in a constant-temperature incubator for culture; after the culture is finished, selecting a colony which is uniform in size and complete to perform enrichment culture in a solid culture medium;
step 3): and inoculating the enriched and cultured bacterial liquid into an LB culture medium, placing the LB culture medium in a constant-temperature shaking table for shaking culture until the logarithmic phase is reached, adding glycerol, mixing, and freezing and storing to obtain the naphthalene restoration and degradation microbial inoculum.
Preferably, the ratio of the petroleum hydrocarbon contaminated soil to the LB medium in the step 1) is 1g (8-12) L, the concentration of the LB medium is 20-30 g/L, and the culture time is 24 h.
Preferably, the inoculum size of the activated culture solution in the step 2) is 5 wt%; the temperature of the constant temperature shaking table is 37 ℃, the oscillation rate is 100-200 r/min, and the oscillation time is 4-7 d; the temperature of the constant temperature incubator is 37 ℃, and the incubation time is 2-5 days.
Preferably, theThe inorganic salt culture medium in the step 2) contains 1g/L Na2HPO4、0.75g/L KH2PO4、0.5g/L NH4Cl、0.1g/L MgSO4·7H2O、0.01g/L CaCl2、0.5g/L NaNO3、0.03g/L FeSO4·7H2O and 0.005L/L microelement solution with the pH value of 7.0-7.2; wherein the microelement solution contains 5g/L CuCl2·2H2O、1g/L ZnCl2、0.5g/L CoCl2·6H2O and 0.06g/L H3BO3。
Preferably, the naphthalene concentration in the step 2) is 5-50 mg/L through gradient pressure screening, and the volume ratio of the culture solution to the normal saline in the gradient dilution is 10-6～10-1。
Preferably, the solid culture medium in the step 2) is an inorganic salt culture medium containing agar, wherein the mass percentage of the agar is 1.5-5%.
Preferably, the volume ratio of the glycerol to the culture solution in the step 2) is 1 (0.6-0.8).
Preferably, the inoculation amount of the bacterial liquid in the step 3) is 5 wt%.
Compared with the prior art, the invention has the following beneficial effects:
(1) the method obtains the naphthalene restoration degradation indigenous strains by screening and separating from the petroleum hydrocarbon polluted soil after activation, domestication, enrichment and purification, identifies the strains as Paenibacillus subtilis through 16S rDNA gene sequencing, and can perform good growth by taking naphthalene as a unique carbon source and energy. The screening and separating method of the degrading strain is relatively simple, has no secondary pollution, and has high degradation rate on the naphthalene in the petroleum hydrocarbon polluted soil.
(2) The invention activates indigenous microorganisms of the polluted soil by utilizing nutrition enhancement, obtains naphthalene restoration degradation strains by screening and separating, realizes high-efficiency restoration of naphthalene in the petroleum hydrocarbon polluted soil by a biological enhancement process, and has wide engineering application prospect.
Drawings
FIG. 1 is a scanning electron microscope image of a naphthalene repair degradation strain;
FIG. 2 is a construction diagram of naphthalene repair degradation strain phylogeny.
Detailed Description
In order to make the invention more comprehensible, preferred embodiments are described in detail below with reference to the accompanying drawings.
Examples
A method for preparing a naphthalene restoration and degradation microbial inoculum in petroleum hydrocarbon polluted soil comprises the following specific steps:
step 1: weighing 5g of petroleum hydrocarbon polluted soil, and culturing the petroleum hydrocarbon polluted soil in an LB culture medium with the concentration of 25g/L for 24h to activate indigenous microorganisms to obtain a culture solution;
step 2: inoculating the activated culture bacterial liquid into an inorganic salt culture medium (the formula is that the culture bacterial liquid contains 1g/L Na) according to the inoculation amount of 5wt percent2HPO4、0.75g/L KH2PO4、0.5g/L NH4Cl、0.1g/L MgSO4·7H2O、0.01g/L CaCl2、0.5g/L NaNO3、0.03g/L FeSO4·7H2O and 0.005L/L microelement solution with the pH value of 7.0-7.2; wherein the microelement solution contains 5g/L CuCl2·2H2O、1g/L ZnCl2、0.5g/L CoCl2·6H2O and 0.06g/L H3BO3) Placing the mixture in a constant-temperature shaking table at 37 ℃ for shake culture at the oscillation speed of 100r/min, performing acclimatization culture by using a gradient pressure screening method, wherein the naphthalene adding concentrations are respectively 5mg/L, 10mg/L, 20mg/L, 30mg/L, 40mg/L and 50mg/L, and the acclimatization culture is performed for 5 days under each concentration condition, and after the culture, the volume ratio of a culture solution to normal saline is 10-6～10-1Diluting, coating a flat plate, and culturing in a constant-temperature incubator at 37 ℃ for 5 d; after the culture is finished, selecting a colony with uniform and complete size to perform enrichment culture in a solid culture medium, wherein the mass percentage of agar in the solid culture medium is 1.5%;
and step 3: inoculating the enriched and cultured bacterial liquid into an LB culture medium according to the inoculation amount of 5 wt%, placing the LB culture medium in a constant-temperature shaking table at 37 ℃ for shaking culture until the logarithmic phase is reached, wherein the shaking rate is 100r/min, then adding glycerol, wherein the volume ratio of the glycerol to the cultured bacterial liquid is 1:0.6, mixing, and freezing and storing to obtain the naphthalene restoration degradation bacterial agent.
Through physiological and biochemical identification, 16S rDNA gene sequencing analysis and BLAST database comparison, the naphthalene repair and degradation strain is rod-shaped (shown in figure 1), and is identified as Paenibacillus naphalinus naphthalones (shown in figure 2). Adding the prepared naphthalene restoration and degradation microbial inoculum according to the inoculation amount of 5 wt% into an inorganic salt culture medium containing naphthalene with the concentration of 50mg/L, and placing the mixture in a constant-temperature shaking table at 37 ℃ for shaking culture for 5 days, wherein the shaking speed is 100 r/min. The naphthalene degradation removal rate is 51.9 percent through measurement.
Example 2
A method for preparing a naphthalene restoration and degradation microbial inoculum in petroleum hydrocarbon polluted soil comprises the following specific steps:
step 1: weighing 3g of petroleum hydrocarbon polluted soil, and culturing the petroleum hydrocarbon polluted soil in an LB culture medium with the concentration of 25g/L for 24h to activate indigenous microorganisms to obtain a culture solution;
step 2: inoculating the activated culture bacterial liquid into an inorganic salt culture medium (the formula is that the culture bacterial liquid contains 1g/L Na) according to the inoculation amount of 5wt percent2HPO4、0.75g/L KH2PO4、0.5g/L NH4Cl、0.1g/L MgSO4·7H2O、0.01g/L CaCl2、0.5g/L NaNO3、0.03g/L FeSO4·7H2O and 0.005L/L microelement solution with the pH value of 7.0-7.2; wherein the microelement solution contains 5g/L CuCl2·2H2O、1g/L ZnCl2、0.5g/L CoCl2·6H2O and 0.06g/L H3BO3) Placing the mixture in a constant-temperature shaking table at 37 ℃ for shake culture at the oscillation speed of 200r/min, performing acclimatization culture by using a gradient pressure screening method, wherein the naphthalene adding concentrations are respectively 5mg/L, 10mg/L, 20mg/L, 30mg/L, 40mg/L and 50mg/L, and the acclimatization culture is performed for 5 days under each concentration condition, and after the culture, the volume ratio of a culture solution to normal saline is 10-6～10-1Diluting, coating a flat plate, and culturing in a constant-temperature incubator at 37 ℃ for 5 d; after the culture is finished, selecting a colony with uniform and complete size to perform enrichment culture in a solid culture medium, wherein the mass percentage of agar in the solid culture medium is 5%;
and step 3: inoculating the enriched and cultured bacterial liquid into an LB culture medium according to the inoculation amount of 5 wt%, placing the LB culture medium in a constant-temperature shaking table at 37 ℃ for shaking culture until the logarithmic phase is reached, wherein the shaking rate is 200r/min, then adding glycerol, wherein the volume ratio of the glycerol to the cultured bacterial liquid is 1:0.8, mixing, and freezing and storing to obtain the naphthalene restoration degradation bacterial agent.
Through physiological and biochemical identification, 16S rDNA gene sequencing analysis and BLAST database comparison, the naphthalene repair and degradation strain is rod-shaped (shown in figure 1), and is identified as Paenibacillus naphalinus naphthalones (shown in figure 2). Adding the prepared naphthalene restoration and degradation microbial inoculum into an inorganic salt culture medium containing naphthalene with the concentration of 50mg/L according to the inoculation amount of 5 wt%, and placing the mixture in a constant-temperature shaking table at 37 ℃ for shaking culture for 5 days at the shaking speed of 200 r/min. The naphthalene degradation removal rate is 83.6 percent through measurement.
Claims (8)
1. A preparation method of a naphthalene restoration and degradation microbial inoculum in petroleum hydrocarbon polluted soil is characterized by comprising the following steps:
step 1): culturing petroleum hydrocarbon polluted soil in an LB culture medium to activate indigenous microorganisms to obtain a culture bacterial liquid;
step 2): inoculating the activated culture bacterial liquid into an inorganic salt culture medium, placing the inorganic salt culture medium in a constant-temperature shaking table for shaking culture, performing acclimatization culture by using a gradient pressure screening method, diluting according to gradient after culture, adding physiological saline, coating a flat plate, and placing the flat plate in a constant-temperature incubator for culture; after the culture is finished, selecting a colony which is uniform in size and complete to perform enrichment culture in a solid culture medium;
step 3): and inoculating the enriched and cultured bacterial liquid into an LB culture medium, placing the LB culture medium in a constant-temperature shaking table for shaking culture until the logarithmic phase is reached, adding glycerol, mixing, and freezing and storing to obtain the naphthalene restoration and degradation microbial inoculum.
2. The preparation method of the naphthalene restoration and degradation microbial inoculum in the petroleum hydrocarbon polluted soil as claimed in claim 1, wherein the ratio of the petroleum hydrocarbon polluted soil in the step 1) to the LB culture medium is 1g (8-12) L, the concentration of the LB culture medium is 20-30 g/L, and the culture time is 24 h.
3. The method for preparing the naphthalene restoration and degradation microbial inoculum in the petroleum hydrocarbon polluted soil as claimed in claim 1, wherein the inoculum size of the activated culture bacterial liquid in the step 2) is 5 wt%; the temperature of the constant temperature shaking table is 37 ℃, the oscillation rate is 100-200 r/min, and the oscillation time is 4-7 d; the temperature of the constant temperature incubator is 37 ℃, and the incubation time is 2-5 days.
4. The method for preparing the naphthalene restoration and degradation microbial inoculum in the petroleum hydrocarbon polluted soil as recited in claim 1, wherein the inorganic salt culture medium in the step 2) contains 1g/L Na2HPO4、0.75g/L KH2PO4、0.5g/L NH4Cl、0.1g/L MgSO4·7H2O、0.01g/L CaCl2、0.5g/L NaNO3、0.03g/L FeSO4·7H2O and 0.005L/L microelement solution with the pH value of 7.0-7.2; wherein the microelement solution contains 5g/L CuCl2·2H2O、1g/L ZnCl2、0.5g/L CoCl2·6H2O and 0.06g/L H3BO3。
5. The method for preparing the naphthalene restoration and degradation microbial inoculum in the petroleum hydrocarbon polluted soil as claimed in claim 1, wherein the naphthalene concentration in the step 2) is 5-50 mg/L through gradient pressure screening, and the volume ratio of the culture bacterial liquid to the normal saline in the gradient dilution is 10-6～10-1。
6. The method for preparing the naphthalene restoration and degradation microbial inoculum in the petroleum hydrocarbon polluted soil as recited in claim 1, wherein the solid culture medium in the step 2) is an inorganic salt culture medium containing agar, wherein the mass percent of the agar is 1.5% -5%.
7. The preparation method of the naphthalene restoration and degradation microbial inoculum in the petroleum hydrocarbon polluted soil according to claim 1, wherein the volume ratio of the glycerol to the culture bacterial liquid in the step 2) is 1 (0.6-0.8).
8. The method for preparing the naphthalene restoration and degradation microbial inoculum in the petroleum hydrocarbon polluted soil as recited in claim 1, wherein the inoculation amount of the bacterial liquid in the step 3) is 5 wt%.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113522961A (en) * | 2021-06-07 | 2021-10-22 | 广东石油化工学院 | Device and method for restoring soil polluted by petroleum hydrocarbon organic matters |
| CN114029340A (en) * | 2021-11-09 | 2022-02-11 | 中国科学院南京土壤研究所 | Application of biological PRB of biological carbon coupled microorganism in restoration of polycyclic aromatic hydrocarbon polluted site |
| CN115415299A (en) * | 2022-08-29 | 2022-12-02 | 东华大学 | Method for repairing pyrene contaminated soil through low-temperature heat treatment-pyrene degradation microbial inoculum combination |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113522961A (en) * | 2021-06-07 | 2021-10-22 | 广东石油化工学院 | Device and method for restoring soil polluted by petroleum hydrocarbon organic matters |
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