CN104894016A - Halomonas axialensis and application thereof in degrading of decabromodiphenyl ether - Google Patents

Halomonas axialensis and application thereof in degrading of decabromodiphenyl ether Download PDF

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CN104894016A
CN104894016A CN201510266347.5A CN201510266347A CN104894016A CN 104894016 A CN104894016 A CN 104894016A CN 201510266347 A CN201510266347 A CN 201510266347A CN 104894016 A CN104894016 A CN 104894016A
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halomonas
axialensis
swir
decabromodiphenyl oxide
degrading
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CN104894016B (en
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邵宗泽
王万鹏
蔡庆涛
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CHINA OCEAN MINERAL RESOURCES R&D ASSOCIATION
Third Institute of Oceanography SOA
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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
    • 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
    • A—HUMAN NECESSITIES
    • A62—LIFE-SAVING; FIRE-FIGHTING
    • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
    • A62D3/00—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances
    • A62D3/02—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances by biological methods, i.e. processes using enzymes or microorganisms
    • 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
    • A—HUMAN NECESSITIES
    • A62—LIFE-SAVING; FIRE-FIGHTING
    • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
    • A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
    • A62D2101/20—Organic substances
    • A62D2101/28—Organic substances containing oxygen, sulfur, selenium or tellurium, i.e. chalcogen

Abstract

The invention provides halomonas axialensis and application of the halomonas axialensis in degrading of decabromodiphenyl ether, and relates to the field of microbe application. The halomonas axialensis SWIR-CL 71 has a collection number of CCTCC No.M2015211 and can be applied to the degrading of decabromodiphenyl ether. After the halomonas axialensis SWIR-CL 71 is cultured for 8 days in a degrading culture medium using decabromodiphenyl ether as the unique carbon source under the conditions that the pH is 7.4, the temperature is 28 DEG C, and the rotating frequency of a shake bed is 160rpm, the degrading rate of the halomonas axialensis for decabromodiphenyl ether is up to 87%.

Description

Axial seamount Halomonas and the application in degrading decabromodiphenyl ether thereof
Technical field
The present invention relates to microbe application field, specifically relate to axial seamount Halomonas and the application in degrading decabromodiphenyl ether thereof.
Background technology
Poly Brominated Diphenyl Ethers (Polybrominated diphenyl ethers, PBDEs) is the aromatics of a series of bromine atom.According to the number of bromine atoms on phenyl ring and the difference of position, Poly Brominated Diphenyl Ethers always has 209 kinds of isomerss, and its maximum purposes is added in matrix material as fire retardant to improve fire performance.At present, PBDEs has been widely used in the commercial prods such as electronic and electrical equipment, automatic control equipment, material of construction and textiles.Commercial main use 3 kinds of brominated flame-retardants: pentabromo-Biphenyl Ether (penta-BDE), octa-BDE (octa-BDE) and decabromodiphenyl oxide (deca-BDE).Pentabromo-Biphenyl Ether (penta-BDE) and octa-BDE octa-BDE (octa-BDE) are lastingly and be present in environment, living organism widely even in blood of human body and milk, potential harm is formed to ecotope and HUMAN HEALTH, has stopped the use to the two in the certain areas of Europe and the U.S..
Decabromodiphenyl oxide (deca-BDE) has the advantages such as flame retardant properties is good, manufacturing process is ripe, Heat stability is good, moderate cost and hypotoxicity due to it, remains the based flame retardant that market demand amount is larger.Decabromodiphenyl oxide (deca-BDE), as a kind of additive flame retardant, is be combined on product for physically, is therefore easily discharged in environment in production, use, disposal process.Decabromodiphenyl oxide (deca-BDE) detected at air, soil, water body and settling and biosphere, distribution range extend over the entire globe at present, even also detect in the polar bear body far away from Arctic region.And its concentration in the environment in the past few decades in increase rapidly.
Decabromodiphenyl oxide (deca-BDE) is similar with persistent organism, has the features such as difficult degradation, fat-solubility, biological magnification, by food web transfer, makes the biology being in high trophic level be subject to serious murder by poisoning, is finally detrimental to health.Have Results of Animal to show decabromodiphenyl oxide (deca-BDE) in recent years can Endocrine system and process of immune regulation have an impact: newborn mice is exposed to decabromodiphenyl oxide (deca-BDE) can affect its Adulthood independent behaviour and cholinomimetic system sensitivity, in abiotic environment and in part biological body, debromination can be there is and generate low bromobiphenyl ether (Tetra-NonaBDEs) in decabromodiphenyl oxide (deca-BDE), or change into hydroxyl PBDEs and methoxyl group PBDEs, more high toxicity is larger for low bromobiphenyl ether bioavailability, a series of chronic disease due to hormonal imbalance and liver dysfunction can be caused.Decabromodiphenyl oxide (deca-BDE) is classified as potential carcinogen matter by EPA (EPA), and its potential healthy effect caused can not be ignored.
Less about the research of decabromodiphenyl oxide aerobic degradation at present.
Summary of the invention
The object of the present invention is to provide axial seamount Halomonas (Halomonas axialensis) SWIR-CL71.
Another object of the present invention is to provide the application of axial seamount Halomonas (Halomonas axialensis) SWIR-CL71 of axle in degrading decabromodiphenyl ether.
Axial seamount Halomonas (Halomonas axialensis) SWIR-CL71, be preserved in China typical culture collection center on April 8th, 2015, address: Wuhan, China Wuhan University, postcode: 430072, preservation center deposit number is CCTCC NO.M 2015211.
Axial seamount Halomonas (Halomonas axialensis) SWIR-CL71 is by the ocean surface deposit (E70.04 ° being collected in south west Indian Ocean, S25.32 °) middle separation screening, belong to Halomonas section (Halomonadaceae); The sole carbon source that the enrichment isolation of this bacterial strain adopts isoconcentration to add pollutent to grow as it, at artificial seawater substratum MMC (NaCl 24g/L, NH 4nO 31g/L, KCl 0.7g/L, KH 2pO 42.0g/L, Na 2hPO 43.0g/L, pH 7.4, MgSO 4decabromodiphenyl oxide-dichloromethane solution and trace element (CaCl 2mg/L, FeCl is added 7g/L) 36H 2o 50mg/L, CuSO 40.5mg/L, MnCl 2.H 2o 0.5mg/L, ZnSO 4.7H 2o 10mg/L), decabromodiphenyl oxide concentration is 100mg/L, pH7.4.This bacterium colony is circular, and regular edges is neat, and oyster white is smooth, moistening, protruding; Thalline is microbot shape, Gram-negative.
Axial seamount Halomonas (Halomonas axialensis) SWIR-CL71 can apply in degrading decabromodiphenyl ether.
The method of degrading decabromodiphenyl ether can be: it is in the liquid nutrient medium of sole carbon source that axial seamount Halomonas (Halomonas axialensis) SWIR-CL71 is inoculated in decabromodiphenyl oxide, is placed in shaking table enrichment culture under lucifuge condition.
Decabromodiphenyl oxide degraded substratum DM (Degradation Medium) is artificial seawater substratum MMC (NaCl 24g/L, NH 4nO 31g/L, KCl 0.7g/L, KH 2pO 42.0g/L, Na 2hPO 43.0g/L, pH 7.4, MgSO 4decabromodiphenyl oxide-dichloromethane solution and trace element (CaCl 2mg/L, FeCl is added 7g/L) 36H 2o 50mg/L, CuSO 40.5mg/L, MnCl 2.H 2o 0.5mg/L, ZnSO 4.7H 2o 10mg/L), decabromodiphenyl oxide concentration is 50mg/L, pH7.4.
The condition of described degraded is pH7.4, and temperature is 28 DEG C, and the slewing rate of shaking table is 160rpm.
In described decabromodiphenyl oxide degraded substratum, decabromodiphenyl oxide is sole carbon source.
Culture measures through GC-MSD, can analysis axis to seamount Halomonas (Halomonas axialensis) SWIR-CL71 to the degradation effect of decabromodiphenyl oxide, when taking decabromodiphenyl oxide as sole carbon source, axial seamount Halomonas (Halomonas axialensis) SWIR-CL71 has certain Degradation to decabromodiphenyl oxide.
Beneficial effect of the present invention is as follows:
Axial seamount Halomonas (Halomonas axialensis) SWIR-CL71 of the present invention is at pH7.4,28 DEG C, the rotational frequency of shaking table is 160rpm, cultivate 8 days in the degraded substratum taking decabromodiphenyl oxide as sole carbon source, the degradation rate of this bacterium to decabromodiphenyl oxide reaches 87%.
The present invention, screens for enriched sample with great oceanic basin mud, be separated efficient decabromodiphenyl oxide aeration bacteria, and is applied to the research of decabromodiphenyl oxide degraded.The present invention has important scientific meaning and using value for the degradation pathway widening BDE-209.
Accompanying drawing explanation
The phylogenetic tree of Fig. 1 constructed by the 16S rRNA sequence based on SWIR-CL71 bacterial strain.
Fig. 2 is BDE-209 toluene-octane-iso mixing solutions (v/v=1: 9) standardized solution total ions chromatogram.
Fig. 3 is BDE-209 characteristic spectrum (m/z).
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.
Embodiment one
One, the separation andpreconcentration of axial seamount Halomonas (Halomonas axialensis) SWIR-CL71
1, the enrichment of axial seamount Halomonas (Halomonas axialensis) SWIR-CL71, abstraction and purification
Substratum:
Artificial seawater substratum (MMC): containing NaCl 24g, NH in often liter of substratum 4nO 31g, KCl 0.7g, KH 2pO 42.0g, Na 2hPO 43.0g, pH 7.4, MgSO 47g (adding after sterilizing).
Trace element salts solution: containing CaCl 2mg, FeCl in often liter of micro-salts solution 36H 2o 50mg, CuSO 40.5mg, MnCl 2.H 2o 0.5mg, ZnSO 4.7H 2o 10mg.
Raw bacterio-agar 2216 substratum in sea and Hai Sheng bacterial context soup 2216 substratum are bought in Bi Di medicine equipment (Shanghai) Co., Ltd..
Decabromodiphenyl oxide mother liquor: organic solvent is methylene dichloride, decabromodiphenyl oxide concentration is 1g/L.
Axial seamount Halomonas (Halomonas axialensis) SWIR-CL71 of the present invention is through enrichment, separation, purifying and obtaining from the surface deposit of south west Indian Ocean.
Example enrichment and single strain separation purification method: get 3 ~ 5g sediment sample and make an addition in the Erlenmeyer flask of the 250mL containing 100mL substratum (artificial seawater substratum+micro-salts solution), add decabromodiphenyl oxide as the sole carbon source required for strain growth, concentration is 100mg/L.Erlenmeyer flask masking foil is done lucifuge process, is placed in 28 DEG C, 160rpm shaking table concussion cultivation.Point 4 stages are cultivated in concussion, the time in per stage is 4w, the difference in 4 stages is in the enrichment medium of first stage, add 5mL sea raw bacterial context soup 2216 substratum, for thalli growth provides abundant nutritive substance, and second and third, four stages did not add this substratum.Get 1mL enrichment culture liquid on last stage as next stage bacterial classification source be inoculated in new substratum.After fourth stage enrichment culture terminates, get the enrichment culture liquid of 1mL fourth stage, with the aseptic ultrapure water property entered 10 times of gradient dilutions, then get 10 -5~ 10 -7diluent is doubly coated on raw bacterio-agar 2216 plate culture medium in sea.Flat board after coating is placed in 28 DEG C of constant incubator lucifuges and cultivates 5 days, chooses single bacterium colony of different shape from flat board, and purifying that sea raw bacterio-agar 2216 decabromodiphenyl oxide plate culture medium is rule, collects part thalline for extracting DNA, simultaneously preservation of bacteria strain.
The acquisition of axial seamount Halomonas (Halomonas axialensis) SWIR-CL71: 100mL substratum (artificial seawater substratum+micro-salts solution), using decabromodiphenyl oxide (50mg/L) substratum that is sole carbon source as experimental group, do not add the substratum of decabromodiphenyl oxide as a control group, experimental group and control group set up three repeating groups.Single strain after access purifying, makes the OD600 value of substratum in 0.23 ~ 0.25 scope.Postvaccinal experimental group and control group are placed in 28 DEG C, the concussion of the shaking table of 160rpm cultivates (substratum does lucifuge process).After cultivating 5d, get enrichment culture liquid, under OD600 condition, record light absorption value.And by the decabromodiphenyl oxide degradation bacteria strains that visual inspection finally obtains, in experiment, be numbered SWIR-CL71.
2, the qualification of bacterial strain
(1) morphological specificity of thalline and bacterium colony:
Adopt conventional Bacterial Physiological biochemical identification method and electron microscope observation, the cell of axial seamount Halomonas (Halomonas axialensis) SWIR-CL71 is Gram-negative, 2216E solid plate substratum cultivates 48h, colonial morphology is circular, neat in edge, oyster white, smooth, moistening, protruding, colony diameter is 1 ~ 1.5mm.
(2) hereditary feature of thalline:
By to the 16S rRNA sequencing of axial seamount Halomonas (Halomonas axialensis) SWIR-CL71 with carry out homology analysis on Ez-biocloud website, the homology of the 16S rRNA sequence of this sequence and type strain Halomonas axialensis Althf1 (T) AF212206 reaches 99.79%.According to Strain Designation rule, bacterial strain SWIR-CL71 is named as axial seamount Halomonas (Halomonas axialensis) SWIR-CL71.Based on SWIR-CL71 bacterial strain 16S rRNA sequence constructed by phylogenetic tree (as Fig. 1).The 16S rRNA sequence of axial seamount Halomonas (Halomonas axialensis) SWIR-CL71 is as follows:
Comprehensively above-mentioned physio-biochemical characteristics, 16S rRNA gene order comparison result, the axial seamount Halomonas in the present invention belongs to red Bacillaceae, axial seamount Halomonas (Halomonas axialensis) SWIR-CL71 of called after.
Embodiment two
Axial seamount Halomonas (Halomonas axialensis) SWIR-CL71 is to the degradation analysis of decabromodiphenyl oxide:
Artificial seawater substratum (MMC): containing NaCl 24g, NH in often liter of substratum 4nO 31g, KCl 0.7g, KH 2pO 42.0g, Na 2hPO 43.0g, pH 7.4, MgSO 47g (adding after sterilizing).
Trace element salts solution: containing CaCl 2mg, FeCl in often liter of micro-salts solution 36H 2o 50mg, CuSO 40.5mg, MnCl 2.H 2o 0.5mg, ZnSO 4.7H 2o 10mg.
Enrichment medium EM (Enrichment Medium): sea raw bacterial context soup 2216 substratum.
Decabromodiphenyl oxide degraded substratum: 100mL MMC substratum+trace element.
Axial seamount Halomonas (Halomonas axialensis) SWIR-CL71 multiplication culture: under sterile conditions, axial seamount Halomonas inoculation is cultivated in the sterilizing Erlenmeyer flask of the 1L of (2216) to being equipped with 500mL seawater meat soup, be placed in constant-temperature table, 28 DEG C, 160rpm, cultivate 48h.
The bacterium liquid getting enrichment culture in the sterilizing centrifugal bottle of 500mL, with the centrifugal 10min of the rotating speed of 6000r/min.Use stroke-physiological saline solution repetitive scrubbing afterwards and centrifugal 2 ~ 3 times, be made into certain density bacteria suspension, by bacterial suspension inoculation in decabromodiphenyl oxide degraded substratum, the concentration of decabromodiphenyl oxide is 50mg/L.Erlenmeyer flask tinfoil wraps up, and avoids illumination on experiment impact.Sample measures through GC-MS, analysis axis to seamount Halomonas (Halomonas axialensis) SWIR-CL71 to the degradation effect of decabromodiphenyl oxide.
GC-MS model and optimum configurations:
Model: GC-MSD.
Chromatographic column: superelevation inertia GC capillary column, DB-5MS UI (15m × 0.250mm × 0.25 μm), article No. 122-5512UI, Agilent company of the U.S..
Instrument working conditions: injector temperature: 290 DEG C; Input mode: pulse Splitless injecting samples; Sampling volume: 1 μ L; Flow and pattern: 1mL/min, constant current mode; Level Four bar temperature: 150 DEG C; Ion source temperature: 250 DEG C; Temperature programming: initial temperature 120 DEG C, maintenance 2min, 40 DEG C/min is raised to 250 DEG C, and 10 DEG C/min is raised to 310 DEG C, retains 10min.
Decabromodiphenyl oxide appearance time: concentration is that the decabromodiphenyl oxide standard substance of 50mg/L (are bought in China National Measuring Science Research Inst., mark thing numbering GBW 08709) upper machine testing, obtain decabromodiphenyl oxide total ion current figure (as Fig. 2), its retention time is 17.834.The mass-to-charge ratio of the characteristic ion of decabromodiphenyl oxide has 207,398,797,959 etc. (as Fig. 3), and wherein selected 398 as quota ion.
Decabromodiphenyl oxide working curve makes: compound concentration is 30,40,50,60, the decabromodiphenyl oxide-methylene dichloride standardized solution of 80mg/L, draw decabromodiphenyl oxide concentration-peak area typical curve, this typical curve is y=4109.1x – 69881, R 2=0.9991.
Axial seamount Halomonas is to the degradation rate of decabromodiphenyl oxide: not inoculate the substratum of axial seamount Halomonas (Halomonas axialensis) SWIR-CL71 as a control group, to inoculate the substratum of axial seamount Halomonas (Halomonas axialensis) SWIR-CL71 (connect bacterium amount and be about 3g/L) as experimental group, experimental group and control group all arrange 3 Duplicate Samples.Be placed in constant-temperature table, 28 DEG C, 160r/min concussion cultivation 8d.Utilize decabromodiphenyl oxide in GC-MS test experience group and control group and calculated the residual quantity of decabromodiphenyl oxide by decabromodiphenyl oxide working curve.Axial seamount Halomonas (Halomonas axialensis) SWIR-CL71 is 87% to 8 of decabromodiphenyl oxide days degradation rates.

Claims (4)

1. axial seamount Halomonas (Halomonas axialensis) SWIR-CL71, be preserved in China typical culture collection center on April 8th, 2015, preservation center deposit number is CCTCC NO.M 2015211.
2. the enrichment screening method of axial seamount Halomonas (Halomonas axialensis) SWIR-CL71 as claimed in claim 1, it is characterized in that the sole carbon source adopting isoconcentration interpolation pollutent to grow as it, decabromodiphenyl oxide-dichloromethane solution and trace element is added in artificial seawater substratum MMC, decabromodiphenyl oxide concentration is 100mg/L, pH7.4; This bacterium colony is circular, and regular edges is neat, and oyster white is smooth, moistening, protruding; Thalline is microbot shape, Gram-negative; Described artificial seawater substratum MMC consists of NaCl 24g/L, NH 4nO 31g/L, KCl 0.7g/L, KH 2pO 42.0g/L, Na 2hPO 43.0g/L, pH 7.4, MgSO 47g/L; Described trace element comprises CaCl 2mg/L, FeCl 36H 2o 50mg/L, CuSO 40.5mg/L, MnCl 2.H 2o 0.5mg/L, ZnSO 4.7H 2o 10mg/L.
3. axial seamount Halomonas (Halomonas axialensis) SWIR-CL71 applies in degrading decabromodiphenyl ether as claimed in claim 1.
4. apply as claimed in claim 3, it is characterized in that the method for described degrading decabromodiphenyl ether is: the method for degrading decabromodiphenyl ether can be: it is in the liquid nutrient medium of sole carbon source that axial seamount Halomonas (Halomonas axialensis) SWIR-CL71 is inoculated in decabromodiphenyl oxide, is placed in shaking table enrichment culture under lucifuge condition; The condition of degraded is pH7.4, and temperature is 28 DEG C, and the slewing rate of shaking table is 160rpm.
CN201510266347.5A 2015-05-22 2015-05-22 Axial seamount Halomonas and its application in degrading decabromodiphenyl ether Expired - Fee Related CN104894016B (en)

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