CN103013841A - Scopulariopsis brevicaulis and application thereof - Google Patents
Scopulariopsis brevicaulis and application thereof Download PDFInfo
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- CN103013841A CN103013841A CN2012105631754A CN201210563175A CN103013841A CN 103013841 A CN103013841 A CN 103013841A CN 2012105631754 A CN2012105631754 A CN 2012105631754A CN 201210563175 A CN201210563175 A CN 201210563175A CN 103013841 A CN103013841 A CN 103013841A
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- scopulariopsis brevicaulis
- bacterium
- aromatic compound
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- 241000825258 Scopulariopsis brevicaulis Species 0.000 title claims abstract description 36
- 125000005575 polycyclic aromatic hydrocarbon group Chemical group 0.000 claims abstract description 22
- 150000001491 aromatic compounds Chemical class 0.000 claims abstract description 21
- 230000015556 catabolic process Effects 0.000 claims abstract description 12
- 230000004059 degradation Effects 0.000 claims abstract description 12
- 238000006731 degradation reaction Methods 0.000 claims abstract description 12
- 239000002689 soil Substances 0.000 claims abstract description 11
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 8
- 241000894006 Bacteria Species 0.000 claims description 29
- BBEAQIROQSPTKN-UHFFFAOYSA-N Pyrene Chemical compound C1=CC=C2C=CC3=CC=CC4=CC=C1C2=C43 BBEAQIROQSPTKN-UHFFFAOYSA-N 0.000 claims description 12
- FMMWHPNWAFZXNH-UHFFFAOYSA-N Benz[a]pyrene Chemical compound C1=C2C3=CC=CC=C3C=C(C=C3)C2=C2C3=CC=CC2=C1 FMMWHPNWAFZXNH-UHFFFAOYSA-N 0.000 claims description 10
- GVEPBJHOBDJJJI-UHFFFAOYSA-N Fluoranthene Chemical compound C1=CC(C2=CC=CC=C22)=C3C2=CC=CC3=C1 GVEPBJHOBDJJJI-UHFFFAOYSA-N 0.000 claims description 10
- YNPNZTXNASCQKK-UHFFFAOYSA-N Phenanthrene Chemical compound C1=CC=C2C3=CC=CC=C3C=CC2=C1 YNPNZTXNASCQKK-UHFFFAOYSA-N 0.000 claims description 7
- 230000000593 degrading Effects 0.000 claims description 7
- 238000005067 remediation Methods 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 3
- 238000004321 preservation Methods 0.000 abstract description 5
- 238000003912 environmental pollution Methods 0.000 abstract description 3
- 238000001228 spectrum Methods 0.000 abstract description 2
- 239000000758 substrate Substances 0.000 abstract description 2
- 238000009629 microbiological culture Methods 0.000 abstract 1
- 230000001580 bacterial Effects 0.000 description 19
- 239000000243 solution Substances 0.000 description 8
- 244000005700 microbiome Species 0.000 description 7
- 239000007788 liquid Substances 0.000 description 6
- 239000002609 media Substances 0.000 description 6
- 235000015097 nutrients Nutrition 0.000 description 5
- 229920002483 18S ribosomal RNA Polymers 0.000 description 3
- 241000233866 Fungi Species 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 239000002028 Biomass Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M buffer Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 235000020673 eicosapentaenoic acid Nutrition 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 230000002538 fungal Effects 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 230000002906 microbiologic Effects 0.000 description 2
- 230000000877 morphologic Effects 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- HNBDQABBWNOTRU-UHFFFAOYSA-N thalline Chemical compound C1=CC=[Tl]C=C1 HNBDQABBWNOTRU-UHFFFAOYSA-N 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- 238000010629 Molecular evolutionary genetics analysis Methods 0.000 description 1
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 1
- WEVYAHXRMPXWCK-UHFFFAOYSA-N acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000000711 cancerogenic Effects 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 238000002856 computational phylogenetic analysis Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 238000003810 ethyl acetate extraction Methods 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 239000001963 growth media Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 230000000813 microbial Effects 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 230000003505 mutagenic Effects 0.000 description 1
- 231100000219 mutagenic Toxicity 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000002957 persistent organic pollutant Substances 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 229920005547 polycyclic aromatic hydrocarbon Polymers 0.000 description 1
- -1 pplication Species 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000003252 repetitive Effects 0.000 description 1
- 238000005201 scrubbing Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000001954 sterilising Effects 0.000 description 1
- 230000001131 transforming Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
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Abstract
The invention discloses Scopulariopsis brevicaulis and application thereof. A preservation serial number of Scopulariopsis brevicaulis 4Z is CGMCCNo.6870 (China General Microbiological Culture Collection Center). Scopulariopsis brevicaulis has a wider substrate spectrum, has good degradation ability on organic aromatic compounds, particularly polycyclic aromatic hydrocarbon in an environment, and can be applied to treatment of environmental pollution, for example, Scopulariopsis brevicaulis can act as a bioremediation reagent for soil polluted by the organic aromatic compounds for wide use.
Description
Technical field
The present invention be more particularly directed to a strain scopulariopsis brevicaulis bacterium and the application in degrading polycyclic aromatic hydrocarbons thereof.
Background technology
Polycyclic aromatic hydrocarbons (Polycyclic aromatic hydrocarbons, PAHs) is a ubiquitous class organic pollutant in the environment, and, teratogenesis long and carcinogenic owing to its transformation period in environment, mutagenic character are subject to people's attention.EPA is defined as priority pollutant in the environment to 16 kinds of PAHs with branch in early 1980s, and China is also listed it in the Black List of environmental pollution.
Microorganism has very strong katabolism ability, and microbiological deterioration is the main approach that PAHs removes in the environment.In recent years, reported the microorganism of a lot of PAHs that can degrade both at home and abroad, but successful Application in the soil remediation also seldom, this main and the character of PAHs, degradation capability of microorganism, the impact that reaches various environmental factorss is relevant.Therefore, can degrade the simultaneously microorganism of polycomponent PAHs of screening has important practical significance.In recent years, both at home and abroad the microbiological deterioration of high molecular PAHs be studies show that can degrade simultaneously multiple high molecular PAHs(such as Fourth Ring and five rings) Microbial resources also seldom.Therefore, can degrade the simultaneously microorganism of multiple PAHs of screening has important practical significance.
Summary of the invention
In view of deficiency of the prior art, one of purpose of the present invention is to provide a strain scopulariopsis brevicaulis bacterium, and it can be realized polycyclic aromatic hydrocarbons, particularly effective degraded of high molecular PAHs.
Scopulariopsis brevicaulis bacterium of the present invention (
Scopulariopsis brevicaulis) be numbered 4Z, in depositary institution's preservation of State Intellectual Property Office's appointment, specifying information is as follows for it:
Preservation date: on November 22nd, 2012;
Depositary institution's title: China Committee for Culture Collection of Microorganisms common micro-organisms center;
Preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, postcode 100101;
Deposit number is CGMCC No. 6870.
Another object of the present invention is to provide aforementioned scopulariopsis brevicaulis bacterium (
Scopulariopsis brevicaulis) application of 4Z in the degraded organic aromatic compound.
Another purpose of the present invention be to provide aforementioned scopulariopsis brevicaulis bacterium (
Scopulariopsis brevicaulis) application of 4Z in the biological degradation reagent of preparation organic aromatic compound.
A further object of the present invention is to provide a kind of organic aromatic compound degrading reagent, it comprise aforementioned scopulariopsis brevicaulis bacterium (
Scopulariopsis brevicaulis) 4Z.
The present invention also provide aforementioned scopulariopsis brevicaulis bacterium (
Scopulariopsis brevicaulis) application of 4Z in repairing the organic aromatic compound contaminated soil.
The present invention also provides a kind of soil remediation reagent, comprise aforementioned scopulariopsis brevicaulis bacterium (
Scopulariopsis brevicaulis) 4Z.
Wherein, aforementioned organic aromatic compound comprises polycyclic aromatic hydrocarbons.
More specifically, described organic aromatic compound is selected from phenanthrene, pyrene, fluoranthene and benzo [a] pyrene at least, but is not limited to this.
Scopulariopsis brevicaulis bacterium of the present invention has wider substrate spectrum, polycyclic aromatic hydrocarbons in the environment there is good degradation capability, be suitable for preparing the environment remediation preparation, particularly organic aromatic compound contaminated soil reagent is used and is eliminated the environmental pollution that organic aromatic compound causes.
Description of drawings
Fig. 1 is the colonial morphology of bacterial strain 4Z on solid medium among the embodiment 1;
Fig. 2 a-2d is respectively the growth curve of bacterial strain 4Z in the nutrient solution that contains phenanthrene, pyrene, fluoranthene and benzo [a] pyrene among the embodiment 2;
Fig. 3 a-3d is respectively among the embodiment 2 at bacterial strain 4Z nutrient solution China and Philippines, pyrene, fluoranthene and benzo [a] pyrene concentration curve over time, and CK is for adding the processing contrast of same amount inactivated strain;
Scopulariopsis brevicaulis bacterium of the present invention (
Scopulariopsis brevicaulis) preservation date of 4Z is on November 22nd, 2012, depositary institution's name is called China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), and deposit number is CGMCC No. 6870.
Embodiment
The present invention adopts enrichment culture method screening and separating from polycyclic aromatic hydrocarbon pollution to obtain the fungal bacterial strain of strain energy degrading polycyclic aromatic hydrocarbons.This bacterial strain is at the flat board growth protruding bacterium colony that is white in color after 4 days, and colony diameter is about 4～10 mm.Morphological specificity is observed and 18S rDNA the sequencing results shows, this bacterial strain be scopulariopsis brevicaulis (
Scopulariopsis brevicaulis), being numbered 4Z, research finds that this bacterial strain has good degraded potentiality to PAHs.
Below in conjunction with some preferred embodiments technical scheme of the present invention is described in more detail.
Embodiment 1:The degrading polycyclic aromatic hydrocarbons fungi (that is aforementioned scopulariopsis brevicaulis (
Scopulariopsis brevicaulis) 4Z, below also can be called for short " bacterial strain 4Z ") separation, purifying, evaluation.
1, substratum: 1. Ma Dingshi substratum; 2. minimal medium (MS): every liter contains K
2HPO
46.0 g, KH
2PO
45.5 g, NH
4NO
32.0 g, Na
2SO
42.0 g, KCl 2.0 g, MgSO
47H
2O 0.2 g, trace metal salts solution 1.0 mL, distilled water, pH 7.0; 3. the minimal medium that contains PAHs: the different PAHs(that prepare 1000 mg/L with DMF are luxuriant and rich with fragrance, benzo [a] pyrene, pyrene, fluoranthene) solution, add the minimal medium of sterilizing by suitable concentration.
2, the isolation and purification of PAHs degradation bacteria
Take by weighing the fresh soil of 10 g, add and 100mL inorganic salt liquid substratum to be housed (pyrene concentration is in the triangular flask of 100.0 mg/L), 28 ℃ of lower 200 r/1 weeks of min shaking culture.Be transferred in the fresh pyrene minimal medium by 10% inoculum size, continue to cultivate 1 week, 5 times repeatedly.Draw at last 0.1 mL nutrient solution and dilute different gradients and coat on the Ma Dingshi culture medium flat plate, picking list bacterium colony inoculates the single bacterium colony behind the plate streaking purifying in the pyrene inorganic salt liquid substratum, chooses the fastest bacterial strain of the speed of growth as the research bacterial strain.
3, morphologic observation and evaluation
The inoculation of fully activation in the Ma Dingshi substratum, was cultivated 4 days for 28 ℃, behind the gramstaining under opticmicroscope observation of cell form, thalline size.With activated spawn streak inoculation on the Ma Dingshi plate, be inverted in 28 ℃ of incubators and cultivated 4 days, observe colonial morphology, and measure the bacterium colony size, specifically see also Fig. 1.
4, the structure of the pcr amplification of 18S rDNA, sequential analysis and phylogenetic tree
The order-checking of the DNA extraction of fungal gene, PCR and PCR product is given birth to worker Bioisystech Co., Ltd by Shanghai and is finished.Fungi bacterium 18S rDNA sequence compares by Blast program and GenBank amplifying nucleic acid data.Then utilize MEGA 4.0 software building phylogenetic trees, adopt the Neighbor-Joining method to carry out Phylogenetic Analysis.
5, see by the biomass variation, bacterial strain 4Z all can grow containing on the Ma Dingshi substratum of phenanthrene, pyrene, fluoranthene and benzopyrene, especially on the substratum that contains fluoranthene and benzopyrene, biomass is larger, illustrates that bacterial strain 4Z has good degraded or tolerance to these two kinds of pollutents.
Embodiment 2:The degrading polycyclic aromatic hydrocarbons fungi is to the degradation characteristic of polycyclic aromatic hydrocarbons
1, under aseptic condition, bacterial strain 4Z is inoculated in the Ma Dingshi liquid nutrient medium that contains 100. mg/L polycyclic aromatic hydrocarbonss, 28 ℃ of lower 200 r/1 weeks of min shaking culture, centrifugal collection thalline, and with phosphate buffered saline buffer repetitive scrubbing 3 times, it is for subsequent use with phosphate buffered saline buffer bacteria suspension to be transferred to desired concn again.
2, bacterial strain 4Z is to the degraded of polycyclic aromatic hydrocarbons: the bacteria suspension with 10% adds in the 50 mL liquid nutrient mediums, with the processing that adds the inactivated bacteria suspension in contrast, add the polycyclic aromatic hydrocarbons mother liquor, make the ultimate density of phenanthrene, pyrene, fluoranthene be about 100 mg/L, the ultimate density of benzo [a] pyrene is about 20 mg/L.3 repetitions are established in each processing.28 ℃ of lower 150 r/6 weeks of min lucifuge shaking culture are in different period samplings, 4 ℃ of freezing preservations.Measure concentration and the OD of PAHs in the solution
600Variation.
3, the extraction of polycyclic aromatic hydrocarbons and analysis in the solution
All pour the bacterium liquid in the triangular flask into separating funnel, add 50 mL ethyl acetate extraction three times, use anhydrous Na behind the united extraction liquid
2SO
4Remove moisture.Be that rotation is concentrated into dried in 36 ℃ the rotatory evaporator in water temperature.Add the dissolving of 2.0 ml acetonitriles, measure the concentration of polycyclic aromatic hydrocarbons in the solution with high performance liquid chromatography (HPLC).
4, degradation results: can find out that by Degrading experiment bacterial strain 4Z has good degradation effect to polycyclic aromatic hydrocarbons, in the time in 6 weeks, bacterial strain 4Z is respectively 60.0%, 64.3%, 61.9% and 82.1% to the degradation rate of phenanthrene, pyrene, fluoranthene, benzopyrene.
In addition, this case contriver utilizes bacterial strain 4Z to other PAHs that commonly sees also with the working method close with previous embodiment 1-2, and 16 of the EPA appointment kinds of PAHs with branch have carried out degradation experiment as the aforementioned, the result shows that this bacterial strain 4Z all shows higher degradation capability.
Reach again, this case contriver is also with the working method close with previous embodiment 1-2, utilize bacterial strain 4Z that the soil that contains the pollutents such as phenanthrene, pyrene, fluoranthene, benzopyrene has been carried out repair process, and the content of repairing rear Pollution of Polycyclic Aromatic Hydrocarbons in Soil thing carried out again measuring, its result shows that contaminated soil is able to effective reparation.
It is pointed out that above explanation and preferred embodiment may not be interpreted as limiting design philosophy of the present invention.Holding the identical the knowledgeable of knowing in technical field of the present invention can be with technical thought of the present invention with various form improvement change, and such improvement and change are interpreted as belonging in protection scope of the present invention.
Claims (8)
- The scopulariopsis brevicaulis bacterium ( Scopulariopsis brevicaulis) 4Z, its deposit number is CGMCC No. 6870.
- 2. the application of the described scopulariopsis brevicaulis bacterium of claim 1 in the degraded organic aromatic compound.
- 3. the as claimed in claim 2 application of scopulariopsis brevicaulis bacterium in the degraded organic aromatic compound is characterized in that described organic aromatic compound comprises polycyclic aromatic hydrocarbons.
- 4. such as the application of scopulariopsis brevicaulis bacterium as described in claim 2 or 3 in the degraded organic aromatic compound, it is characterized in that described organic aromatic compound is selected from phenanthrene, pyrene, fluoranthene and benzo [a] pyrene at least.
- 5. the application of the described scopulariopsis brevicaulis bacterium of claim 1 in the biological degradation reagent of preparation organic aromatic compound, described organic aromatic compound comprises polycyclic aromatic hydrocarbons.
- 6. organic aromatic compound degrading reagent, described organic aromatic compound comprises polycyclic aromatic hydrocarbons, it is characterized in that, described degraded pack contains scopulariopsis brevicaulis bacterium claimed in claim 1.
- 7. the application of the described scopulariopsis brevicaulis bacterium of claim 1 in repairing the organic aromatic compound contaminated soil, described organic aromatic compound comprises polycyclic aromatic hydrocarbons.
- 8. a soil remediation reagent is characterized in that, comprises scopulariopsis brevicaulis bacterium claimed in claim 1.
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| CN103013841B CN103013841B (en) | 2014-04-16 |
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Cited By (5)
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| CN108774621A (en) * | 2018-06-15 | 2018-11-09 | 四川农业大学 | One plant of scopulariopsis brevicaulis WNF22 and its application |
| CN108949587A (en) * | 2018-08-14 | 2018-12-07 | 北京绿安创华环保科技有限公司 | Microbial bacterial agent, organic waste prone soil renovation agent and the preparation method and application thereof |
| CN109370910A (en) * | 2018-08-03 | 2019-02-22 | 贵州省植物保护研究所 | A kind of scopulariopsis brevicaulis and application thereof |
| CN110024807A (en) * | 2019-05-15 | 2019-07-19 | 贵州省植物保护研究所 | A kind of composition, the preparation method and the usage of biocontrol fungi and sulfoxaflor |
| CN113755337A (en) * | 2021-08-30 | 2021-12-07 | 中国科学院广州地球化学研究所 | Polycyclic aromatic hydrocarbon degrading fungi GIG-1 and GIG-2 in petroleum-polluted soil, mixed microbial inoculum thereof and application thereof |
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| CN108774621A (en) * | 2018-06-15 | 2018-11-09 | 四川农业大学 | One plant of scopulariopsis brevicaulis WNF22 and its application |
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| CN108949587A (en) * | 2018-08-14 | 2018-12-07 | 北京绿安创华环保科技有限公司 | Microbial bacterial agent, organic waste prone soil renovation agent and the preparation method and application thereof |
| CN108949587B (en) * | 2018-08-14 | 2022-01-14 | 北京绿安创华环保科技有限公司 | Microbial agent, organic garbage sandy soil remediation agent, preparation method and application thereof |
| CN110024807A (en) * | 2019-05-15 | 2019-07-19 | 贵州省植物保护研究所 | A kind of composition, the preparation method and the usage of biocontrol fungi and sulfoxaflor |
| CN113755337A (en) * | 2021-08-30 | 2021-12-07 | 中国科学院广州地球化学研究所 | Polycyclic aromatic hydrocarbon degrading fungi GIG-1 and GIG-2 in petroleum-polluted soil, mixed microbial inoculum thereof and application thereof |
| CN113755337B (en) * | 2021-08-30 | 2022-06-10 | 中国科学院广州地球化学研究所 | Polycyclic aromatic hydrocarbon degrading fungi GIG-1 and GIG-2 in petroleum-polluted soil, mixed microbial inoculum thereof and application thereof |
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