CN115058362B - Strain for degrading carbon tetrachloride and application thereof - Google Patents
Strain for degrading carbon tetrachloride and application thereof Download PDFInfo
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- CN115058362B CN115058362B CN202210723656.0A CN202210723656A CN115058362B CN 115058362 B CN115058362 B CN 115058362B CN 202210723656 A CN202210723656 A CN 202210723656A CN 115058362 B CN115058362 B CN 115058362B
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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
- C12N1/205—Bacterial isolates
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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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- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K17/00—Soil-conditioning materials or soil-stabilising materials
- C09K17/14—Soil-conditioning materials or soil-stabilising materials containing organic compounds only
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C2101/00—In situ
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/20—Air quality improvement or preservation, e.g. vehicle emission control or emission reduction by using catalytic converters
Abstract
A strain for degrading carbon tetrachloride and application thereof relate to a microorganism. The invention provides a strain for degrading carbon tetrachloride and application thereof, which are applied to bioremediation treatment of a carbon tetrachloride polluted site and solve the defect of poor pollutant removal effect of carbon tetrachloride biological treatment at the present stage. The bacteria are identified to belong to Acinetobacter (Acinetobacter) and are preserved in China center for type culture Collection, and the preservation address is CCTCC NO: M2022058 of university of Wuhan in Wuhan, china. The carbon tetrachloride degrading bacteria are gram negative aerobic bacteria, and can grow by taking carbon tetrachloride as a unique carbon source; growing in an environment with the temperature of 20-40 ℃ and the pH value of 5.0-9.0; the carbon tetrachloride degrading bacteria has the function of degrading the soil polluted by carbon tetrachloride, the degradation rate of the carbon tetrachloride is up to 90.91%, and the secondary pollution to the environment is reduced. The invention is applied to the field of microbial degradation.
Description
Technical Field
The invention relates to the field of microbial degradation, in particular to a strain for degrading carbon tetrachloride and application thereof.
Background
Carbon tetrachloride (CCl) 4 Carbon Tetrachloride) is a common organic solvent of chlorinated hydrocarbon, is colorless transparent liquid with special smell, is extremely volatile and is a common extinguishing agent, and is also an excellent solvent for paint, resin, rubber and the like. It is used both in laboratory and industry as a solvent and extractant. Carbon tetrachloride is quite stable and difficult to decompose. In surface water, the concentration is generally low because the surface water volatilizes and disappears in the atmosphere; in groundwater and underground soil, however, since the volatilization and diffusion are small, the biological decomposition is weak, and thus, it is difficult to self-clean the soil once it is contaminated with the substance. Carbon tetrachloride not only can affect the normal growth of plants, but also can affect the health of human bodies through a food chain. The highest limit of carbon tetrachloride in drinking water is 2 mug/L specified in sanitary Standard for Drinking Water (GB 5749-2006). Thus, study of biological degradation of carbon tetrachlorideThe degradation can be quickened, and the method has important significance for protecting the environmental health and reducing the human safety risk.
Soil contaminated with carbon tetrachloride is currently relatively difficult to treat. The research on carbon tetrachloride degrading microbes is less common at home and abroad, aiming at the research, high-efficiency degrading bacteria of carbon tetrachloride are screened, degradation characteristics of the high-efficiency degrading bacteria are known, degradation paths of the high-efficiency degrading bacteria are detected, microbial agent resources can be provided for environmental treatment technologies such as in-situ bioremediation of carbon tetrachloride contaminated soil, and the like, so that a foundation is laid for reducing and eliminating the problem of carbon tetrachloride contaminated soil.
Disclosure of Invention
The invention aims to provide a strain for degrading carbon tetrachloride and application thereof, which can be applied to in-situ bioremediation of carbon tetrachloride-polluted soil and provide assistance for reducing and eliminating the problem of carbon tetrachloride-polluted soil.
The carbon tetrachloride degrading bacteria WY-C10 of the invention are identified to belong to Acinetobacter (Acinetobacter sp.) WY-C10, and are preserved in China center for type culture Collection, with a preservation address: university of martial arts, chinese, date of preservation: 2022.1.11, accession number: cctccc No. M2022058.
The carbon tetrachloride degrading bacteria WY-C10 are used for degrading carbon tetrachloride in polluted soil.
Further, the concentration of the carbon tetrachloride is 150-750 mug/L.
Further, the concentration of the carbon tetrachloride is 200-500 mug/L.
The invention relates to a preparation of carbon tetrachloride degrading bacteria WY-C10.
The carbon tetrachloride degrading bacteria WY-C10 are gram-negative aerobic bacteria, and can grow by taking carbon tetrachloride as a unique carbon source; growing in an environment with the temperature of 20-40 ℃ and the pH value of 3.0-11.0; the optimal growth pH value of the carbon tetrachloride degrading bacteria WY-C10 is 7.0, and the optimal growth temperature is 35 ℃. The colony form of the carbon tetrachloride degrading bacteria WY-C10 on the solid culture medium is grey white, round and semitransparent, which shows that the bacteria are smooth, moist, free of wrinkles, neat in edge and easy to pick up.
The conventional physiological and biochemical identification of the carbon tetrachloride degrading bacteria WY-C10 is carried out according to the Bojie's bacteriology identification manual, and the experimental result is as follows: the method has the advantages of negative contact enzyme reaction, negative oxidase reaction, positive citric acid utilization, negative starch hydrolysis, negative methyl red test reaction, negative nitrate reduction reaction, negative Fu Pu test reaction and positive indole production test.
The homology of the carbon tetrachloride degrading bacteria WY-C10 is 99.79% after 16S rDNA sequencing and homology comparison, and the most similar species is Acinetobacter (Acinetobacter FZW.22). The method combines the morphological characteristics, growth conditions and physiological and biochemical identification results of bacteria to determine that the carbon tetrachloride degrading bacteria WY-C10 is a new strain of Acinetobacter genus and named as carbon tetrachloride degrading bacteria WY-C10.
The carbon tetrachloride degrading bacteria WY-C10 has the function of degrading carbon tetrachloride, the degradation rate of 450 mug/L of carbon tetrachloride for 3 days is 90.91%, and the secondary pollution to the environment is reduced. The carbon tetrachloride degrading bacteria WY-C10 provided by the invention are helpful for reducing and eliminating the problem of carbon tetrachloride pollution to soil, and have higher application value.
The carbon tetrachloride degrading bacteria WY-C10 belong to Acinetobacter (Acinetobacter FZW.22) and are preserved in China center for type culture Collection, and the preservation address is CCTCC NO: M2022058 at university of Wuhan in Wuhan, china.
Drawings
FIG. 1 is a streak photograph of a colony of carbon tetrachloride degrading bacteria WY-C10;
FIG. 2 is a phylogenetic tree constructed by the gene sequences of carbon tetrachloride degrading bacteria WY-C10 and similar strains.
Detailed Description
For the purposes of clarity, technical solutions and advantages of embodiments of the present invention, the spirit of the present disclosure will be described in detail below, and any person skilled in the art, after having appreciated the embodiments of the present disclosure, may make changes and modifications to the techniques taught by the present disclosure without departing from the spirit and scope of the present disclosure.
The exemplary embodiments of the present invention and the descriptions thereof are intended to illustrate the present invention, but not to limit the present invention.
Example 1
The carbon tetrachloride degrading bacteria WY-C10 are obtained by preliminary screening and then re-screening of the soil polluted by carbon tetrachloride in a polluted site in the city of Jiang Susheng Changzhou.
Carbon tetrachloride has certain toxic action on thalli, and the pollutant concentration gradient domestication method can enable strains adapting to the pollutant concentration to survive and gradually adapt to the environment along with the domestication process to grow and reproduce.
The screening process is as follows: (1) 10g of polluted soil is taken and filled into a culture medium containing 200mL of inorganic salt of carbon tetrachloride (the carbon tetrachloride concentration is 150 mu g/L), a proper amount of glass beads are added, and the mixture is placed into a shaking table at 35 ℃ and at 150r/min for constant temperature shaking culture. (2) After 7 days of culture, the upper layer bacterial suspension was aspirated, and added to a newly configured strain selection medium with carbon tetrachloride as the sole carbon source in an inoculum size of 10% for subculture while increasing carbon tetrachloride to 300. Mu.g/L. After that, the mass concentration of the carbon tetrachloride is gradually increased according to 300 mug/L, 450 mug/L, 600 mug/L and 750 mug/L, and the carbon tetrachloride is domesticated and cultured for 5 generations. (3) And (3) sucking 1mL of bacterial liquid from the 5-generation culture liquid, diluting the bacterial liquid into liquid with a gradient of 10 < -1 > -10 < -6 > according to a gradient dilution method, coating 100 mu L of liquid on a yeast extract solid culture medium, uniformly coating by using a sterilization coating rod, and culturing in a constant-temperature incubator at 37 ℃. (4) After independent colonies are grown, morphological characteristics of each colony are observed for screening, the screened colonies are picked up by using an inoculating loop, and streaked and separated on a beef extract solid medium. Repeated streak purification is carried out until single bacterial colony is screened to obtain pure bacteria. The obtained pure bacteria are transferred into a carbon tetrachloride-containing inorganic salt culture medium (carbon tetrachloride concentration is 150 mug/L) and cultured for 5 days to verify whether the pure bacteria have degradation capability. And (3) placing the carbon tetrachloride degradation strain in a refrigerator at the temperature of minus 20 ℃ for preservation by adopting a glycerol preservation method for standby.
The re-screening is based on whether the strain has higher carbon tetrachloride degradation rate or not under the condition of 35 ℃ further on the basis of the primary screening, and the strain with better degradation efficiency is screened out through the re-screening.
In this embodiment, the carbon tetrachloride selection medium is composed of 3.5g Na per liter 2 HPO 4 ·2H 2 O，1g K 2 HPO 4 ，0.5g (NH 4 ) 2 SO 4 ，0.1g MgCl 2 ·6H 2 O，0.05g Ca(NO 3 ) 2 ·4H 2 O, pH value is 7.0-7.2, sterilizing at 121 deg.c for 20min, cooling and adding sterilized carbon tetrachloride stock solution.
And carrying out conventional physiological and biochemical identification on the carbon tetrachloride degrading bacteria WY-C10 obtained by screening according to the Berger's bacteriology identification manual. Extracting bacterial DNA, carrying out PCR amplification on the obtained bacterial genome DNA by using a bacterial 16S rDNA universal primer, carrying out rubber tapping to recover PCR products, entrusting a Federation standard technical service company to carry out 16S rDNA sequencing, carrying out homology comparison on the sequencing result and a registered 16S rDNA sequence in Genbank by using BLAST software, finding that the most similar species of carbon tetrachloride degrading bacteria WY-C10 obtained by screening in the embodiment is Acinetobacter FZW.22, determining that the homology is 99.65 percent, combining with the morphological characteristics, growth conditions and physiological and biochemical identification results of bacteria, and determining that carbon tetrachloride degrading bacteria WY-C10 is a new strain of rhodococcus and is named as carbon tetrachloride degrading bacteria WY-C10; the sequences were submitted to the GenBank database, accession number MT150631. Based on the 16S rDNA sequencing results, homologous sequences were found in the GenBank database using Blast tool and related software such as MEGA5.0 provided by NCBI, and phylogenetic tree was established (as shown in FIG. 2).
Example 2
Functional verification is carried out on the carbon tetrachloride degrading bacteria WY-C10 obtained in the embodiment 1:
adding carbon tetrachloride into sterilized inorganic salt culture solution to make carbon tetrachloride concentration in culture medium 450 μg/L, adding prepared experimental bacterial solution with inoculation amount of 3%, culturing at 35deg.C, 150r/min and pH7.0 for 3d, and quantitatively detecting degradation condition of carbon tetrachloride by gas chromatograph. And the corresponding degradation rate is calculated. The degradation rate of WY-C10 to 450 mug/L of carbon tetrachloride for 3 days is 90.91%, and the strain has strong practical application potential. Experiments prove that the carbon tetrachloride degrading bacteria WY-C10 have the function of treating carbon tetrachloride polluted soil, do not need to add other chemical preparations, can provide microbial agent resources for in-situ bioremediation of carbon tetrachloride polluted soil, and have wide application prospects.
The chromatographic conditions for measuring the carbon tetrachloride are OV-17 quartz capillary chromatographic column, gasification chamber 200 ℃, detection chamber 230 ℃, column temperature 60 ℃, carrier gas: nitrogen, sample injection amount 10 μl, column flow rate: 1.2mL/min, tail blowing: 50mL/min.
The morphological structure observation results of the screened carbon tetrachloride degrading bacteria are shown in table 1. The physiological and biochemical identification experiments were performed on WY-C10, and the results were analyzed according to the "Berger's bacteria identification Manual", and the experimental results are shown in Table 2. Wherein "+" indicates a positive reaction and "-" indicates a negative reaction.
Table 1 morphological observations of WY-C10
Table 2 WY-C10 physiological and biochemical identification experiment results
Claims (5)
1. A strain for degrading carbon tetrachloride, which is characterized in that the strain is Acinetobacter (Acinetobacter sp.) WY-C10 and is preserved in China center for type culture Collection, and the preservation address is: university of martial arts, chinese, date of preservation: 2022, 1 month 11 days, deposit number: cctccc No. M2022058.
2. Use of a strain of degrading carbon tetrachloride according to claim 1 for degrading carbon tetrachloride in contaminated soil.
3. The use of a strain for degrading carbon tetrachloride according to claim 2, wherein the concentration of carbon tetrachloride is 150-750 μg/L.
4. The use of a strain for degrading carbon tetrachloride according to claim 2 or 3, wherein the concentration of carbon tetrachloride is 200-500 μg/L.
5. A formulation of the carbon tetrachloride degrading strain of claim 1.
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| CN107904188B (en) * | 2017-11-07 | 2020-07-10 | 广东工业大学 | Achromobacter with monomethylamine degradation capacity and application thereof |
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