CN115029275B - Chlorobenzene degrading bacterium and application thereof - Google Patents

Chlorobenzene degrading bacterium and application thereof Download PDF

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CN115029275B
CN115029275B CN202210719619.2A CN202210719619A CN115029275B CN 115029275 B CN115029275 B CN 115029275B CN 202210719619 A CN202210719619 A CN 202210719619A CN 115029275 B CN115029275 B CN 115029275B
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chlorobenzene
degrading
bacteria
acinetobacter
china
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CN115029275A (en
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王继华
黄金锐
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Harbin Normal University
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Harbin Normal University
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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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
    • B09C—RECLAMATION OF CONTAMINATED SOIL
    • B09C2101/00—In situ
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/30—Organic compounds
    • C02F2101/36—Organic compounds containing halogen
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
    • C02F2103/06—Contaminated groundwater or leachate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • 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
    • 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
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W10/00—Technologies for wastewater treatment
    • Y02W10/10—Biological treatment of water, waste water, or sewage

Abstract

A chlorobenzene degrading bacterium and application thereof relate to a microorganism. The chlorobenzene degrading strain can be applied to bioremediation treatment of chlorobenzene polluted sites, and solves the defect of poor pollutant removal effect of chlorobenzene biological treatment in the present stage. The chlorobenzene degrading bacteria are identified as being Acinetobacter oleaphilus (Acinetobacter oleivorans) WH-15 and are preserved in China center for type culture collection, and the preservation address is China university of Wuhan, china, with the preservation number of CCTCC NO: m2022059. The chlorobenzene bacteria are gram-negative aerobic bacteria, and can grow by taking chlorobenzene 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 chlorobenzene degrading bacteria has the function of degrading soil and underground water polluted by chlorobenzene, the degradation rate of chlorobenzene is up to 94.1%, and the secondary pollution to the environment is reduced. The invention is applied to the field of microbial degradation.

Description

Chlorobenzene degrading bacterium and application thereof
Technical Field
The invention relates to the field of microorganisms, in particular to chlorobenzene degrading bacteria and application thereof.
Background
Chlorobenzene is an organic matter with a wide pollution surface and high toxicity and bitter almond taste, exists in various industrial waste water, such as waste water discharged by industries of textile, pharmacy, steel and the like, is one of important risk pollutants for sudden water quality pollution of raw water in water supply factories, and forms a potential threat to urban water supply safety. The chlorobenzene has very stable property, so the chlorobenzene has serious environmental hazard, is easy to accumulate in human bodies, has inhibition effect on nervous systems, anesthetizes muscles and forms serious threat to the health of human bodies. Chlorobenzene has strong toxic action on growth, development and reproduction of aquatic organisms. Chlorobenzene compounds are a relatively harmful class of organic pollutants, and therefore control and treatment of such pollutants is particularly important. In conclusion, the research on degradation biodegradation of chlorobenzene degrading bacteria has important significance for environmental remediation.
According to investigation, along with the rapid development of industrial production, the quantity of chlorobenzene-containing industrial wastewater discharged into an environmental system is obviously increased, chlorobenzene degrading bacteria are screened and the degrading capability is explored, a certain reference is provided for improving the repairing potential of the degrading bacteria in the natural environment and for breeding pollutant degrading bacteria in other pollution sites, and theoretical basis is provided for environmental treatment technologies such as bioremediation of chlorobenzene-polluted groundwater.
Disclosure of Invention
The invention aims to provide chlorobenzene degrading bacteria and application thereof, which can be applied to in-situ bioremediation of chlorobenzene-polluted soil and underground water and provide assistance for reducing and eliminating the problem of chlorobenzene pollution to the environment.
The chlorobenzene degrading bacteria of the invention are identified to belong to Acinetobacter oleaginous (Acinetobacter oleivorans) WH-15, and are preserved in China center for type culture collection, and the preservation addresses are: the preservation time of the university of Wuhan in Wuhan, china: 2022.01.11, accession number is: cctccc NO: m2022059.
The chlorobenzene degrading bacteria WH-15 are gram-negative aerobic bacteria, and can grow by taking chlorobenzene 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 optimal growth pH value of the chlorobenzene degrading bacteria WH-15 is 7.0, and the optimal growth temperature is 35 ℃. Chlorobenzene degrading bacteria WH-15 were grown on beef extract-peptone medium at 28deg.C for 3 days, and the colonies were round, translucent in beige color, smooth and moist in surface, regular in edge, free of halo, and central microprotrusions (shown in FIG. 1).
Conventional physiological and biochemical identification is carried out on chlorobenzene degrading bacteria WH-15 according to the Bojie's bacteriology identification manual, and the experimental result is as follows: the method has the advantages of positive contact enzyme reaction, negative oxidase reaction, positive citric acid utilization, negative starch hydrolysis, negative methyl red test reaction, negative nitrate reduction reaction and negative Fu Pu test reaction.
The chlorobenzene degrading bacterium WH-15 is subjected to 16S rDNA sequencing and homology comparison to obtain the most similar species Acinetobacter (Acinetobacter oleivorans) with the homology of 99.89%. The chlorobenzene degrading bacteria WH-15 is determined to be the Acinetobacter oleaphilium of the Acinetobacter genus by combining with the morphological characteristics, growth conditions and physiological and biochemical identification results, and is named as the chlorobenzene degrading bacteria WH-15.
The chlorobenzene degrading bacterium WH-15 has the function of degrading soil polluted by chlorobenzene, the degradation rate of chlorobenzene 24h of 40mg/L is 94.1%, and the degradation rate of chlorobenzene 24h of 320mg/L is 38.3%. And simultaneously, the secondary pollution to the environment is reduced. The chlorobenzene degrading bacterium WH-15 has important significance for reducing and eliminating the underground water and soil polluted by chlorobenzene.
The chlorobenzene degrading bacteria WH-15 belongs to Acinetobacter (Acinetobacter oleivorans) and is preserved in China center for type culture collection, and the preservation address is the preservation number of the university of Wuhan in Wuhan, china, CCTCC NO: m2022059.
Drawings
FIG. 1 is a streaked photograph of a colony of chlorobenzene-degrading bacterium WH-15;
FIG. 2 shows phylogenetic tree constructed by chlorobenzene degrading bacteria WH-15 and similar strain gene sequences.
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 chlorobenzene degrading bacteria WH-15 is obtained by primary screening and secondary screening of groundwater polluted by chlorobenzene in a pollution site in the city of Jiang Susheng Changzhou.
Chlorobenzene has certain toxic action on thalli, and the concentration gradient domestication method of the pollutant can enable strains adapting to the concentration of the pollutant to survive and gradually adapt to the environment along with the domestication process to grow and reproduce.
The screening process is as follows: (1) 10mL of polluted underground water is taken and filled into a culture medium containing 200mL of chlorobenzene inorganic salt (chlorobenzene concentration is 40 mg/L), and the culture medium 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 is sucked up according to 10 percentInoculum size was added to a newly prepared strain selection medium with chlorobenzene as the sole carbon source for subculture while increasing chlorobenzene to 80mg/L. (3) 1mL of bacterial liquid is sucked from the cultured culture solution, and the bacterial liquid is diluted to 10 according to a gradient dilution method -1 ～10 -6 The gradient liquid was applied to a yeast extract solid medium in an amount of 100. Mu.L, and the yeast extract solid medium was uniformly applied with a sterilization applicator, and cultured 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 were transferred to a chlorobenzene-containing inorganic salt medium (chlorobenzene concentration 40 mg/L), and cultured for 5d to verify whether they had degradation ability. And (3) placing the chlorobenzolized strain in a refrigerator at the temperature of minus 20 ℃ for preservation by adopting a glycerol preservation method.
The re-screening is to screen out the strain with better degradation efficiency based on the condition of 35 ℃ on the basis of whether the strain has higher chlorobenzene degradation rate.
In this embodiment, the chlorobenzene 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 chlorobenzene solution.
The routine physiological and biochemical identification is carried out on the chlorobenzene degrading bacteria WH-15 obtained by screening according to the Berger's bacteriology identification handbook. 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 a PCR product, entrusting a Federation standard technical service company to carry out 16S rDNA sequencing, carrying out homology comparison on a sequencing result and a registered 16S rDNA sequence in Genbank by using BLAST software, finding that the most similar species of chlorobenzene degrading bacteria WH-15 obtained by screening in the embodiment is Acinetobacter (Acinetobacter oleivorans), the homology reaches 99.89%, and determining that the chlorobenzene degrading bacteria WH-15 is Acinetobacter oleaphilus of the Acinetobacter by combining with morphological characteristics, growth conditions and physiological biochemical identification results of the bacteria, and naming the chlorobenzene degrading bacteria WH-15 as the chlorobenzene degrading bacteria WH-15. Based on the 16S rDNA sequencing results, homologous sequences were found in the GenBank database using Blast tool and related software such as MEGA 5.0 provided by NCBI, and phylogenetic tree was established (as shown in FIG. 2).
Example 2
Chlorobenzene degradation experiments were performed on the chlorobenzene degrading bacteria WH-15 obtained by screening in example 1:
(1) Construction of degradation System
And (3) preparing an inorganic salt liquid culture medium with chlorobenzene as the only carbon source, so that the concentration of chlorobenzene is 40mg/L and 320mg/L, inoculating the strain to be tested into a conical flask filled with the inorganic salt culture medium under the aseptic condition, and fully shaking uniformly. The reaction system is placed in a constant temperature shaking incubator at 35 ℃ and 150r/min, and the chlorobenzene content in the system is measured after 24 hours.
(2) Detection and analysis of chlorobenzene
The chlorobenzene degradation capacity is measured by adopting the following instrument: gas chromatography: 7890A-5975C gas chromatograph-mass spectrometer (GC-MS), agilent technologies Inc. of America. The chromatographic conditions are as follows: the column was HP-5MS (30.0mX1250 μm,0.25 μm); the initial temperature of the chromatographic column is kept at 40 ℃ for 5min, the temperature is increased to 120 ℃ at the speed of 5 ℃/min, and the temperature is increased to 280 ℃ at the speed of 10 ℃/min and kept for 10min; the temperature of the gasification chamber is 250 ℃; the temperature of the transmission line is 280 ℃; a carrier gas He; the carrier gas flow rate is 1.0mL/min; no split flow is produced.
The chlorobenzene degradation rate of the chlorobenzene degradation bacterium WH-15 of the embodiment reaches 94.1 percent when the chlorobenzene is cultured for 24 hours at the concentration of 40 mg/L. The concentration of the chlorobenzene is 80mg/L, and the degradation rate of the chlorobenzene reaches 78.3 percent when the culture is carried out for 24 hours. The concentration of the chlorobenzene is 160mg/L, and the degradation rate of the chlorobenzene reaches 45.2 percent when the culture is carried out for 24 hours. The concentration of the chlorobenzene is 320mg/L, and the degradation rate of the chlorobenzene reaches 38.3 percent when the culture is carried out for 24 hours. Experiments prove that the chlorobenzene degrading bacteria WH-15 has the function of treating chlorobenzene polluted soil, has the best treatment effect on 40mg/L chlorobenzene, and still has ideal treatment effect on 320mg/L chlorobenzene with high concentration. The chlorobenzene degrading bacterium WH-15 provided by the invention can treat chlorobenzene-polluted soil without adding other chemical agents, can provide microbial agent resources for in-situ bioremediation of chlorobenzene-polluted groundwater, and has a wide application prospect.
In the physiological and biochemical experiments of microorganisms, different chemical reagents are generally used to detect the metabolic effects and metabolites of bacteria on different substances, and are used for identifying bacteria. The main experiments comprise amylase, contact enzyme, oxidase, nitrate, citrate, methyl red, V-P experiment, indole experiment and the like, and the results of the experiments are shown in table 1, wherein the results of the experiments are shown in the microbiological experiments and the "Berger's identification manual" for carrying out physiological and biochemical identification experiments on WH-15 bacteria, and the results of the analyses are shown in the "Berger's identification manual". Wherein "+" indicates a positive reaction and "-" indicates a negative reaction.
TABLE 1 physiological and biochemical identification experiment results of chlorobenzene degrading bacteria

Claims (5)

1. The chlorobenzene degrading bacterium is characterized in that the bacterium is Acinetobacter oleaginous (Acinetobacter oleivorans) WH-15 and is preserved in China center for type culture collection, and the preservation address is: the preservation time of the university of Wuhan in Wuhan, china: 2022.01.11, accession number is: cctccc NO: m2022059.
2. The use of a strain of chlorobenzene degrading bacteria according to claim 1, characterized in that it is used for degrading chlorobenzene with a concentration of 40-320 mg/L.
3. The use of a strain of chlorobenzene degrading bacteria according to claim 2, characterized in that the chlorobenzene concentration is 40-250 mg/L.
4. The use of a strain of chlorobenzene degrading bacteria according to claim 2 or 3, characterized in that the chlorobenzene concentration is 40-100 mg/L.
5. A formulation of a chlorobenzene degrading bacterium according to claim 1.
CN202210719619.2A 2022-06-23 2022-06-23 Chlorobenzene degrading bacterium and application thereof Active CN115029275B (en)

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KR101636852B1 (en) * 2014-10-07 2016-07-06 주식회사 오이코스 Method And Agent For Soil Purification By Petroleum-Decomposing Microbe
CN108220211B (en) * 2018-04-02 2019-12-31 中国科学院微生物研究所 Acinetobacter oleophilic NMB17 and application thereof in plant disease control

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