CN103013841B - Scopulariopsis brevicaulis and application thereof - Google Patents
Scopulariopsis brevicaulis and application thereof Download PDFInfo
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- CN103013841B CN103013841B CN201210563175.4A CN201210563175A CN103013841B CN 103013841 B CN103013841 B CN 103013841B CN 201210563175 A CN201210563175 A CN 201210563175A CN 103013841 B CN103013841 B CN 103013841B
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- pyrene
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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 environment, because its transformation period in environment is subject to people's attention compared with long and carcinogenic, teratogenesis, mutagenic character.EPA is defined as the priority pollutant in environment in early 1980s the PAHs of 16Zhong Weidai branch, and China is also listed in the Black List of environmental pollution.
Microorganism has very strong katabolism ability, and microbiological deterioration is the main approach that in environment, PAHs removes.In recent years, reported the microorganism of a lot of PAHs that can degrade both at home and abroad, but be successfully applied in soil remediation also seldom, this main and character of PAHs is, the degradation capability of microorganism, and the impact of various environmental factorss is relevant.Therefore, can simultaneously the degrade microorganism of polycomponent PAHs of screening, has important practical significance.In recent years, both at home and abroad the microbiological deterioration research of high molecular PAHs is shown, the multiple high molecular PAHs(that can simultaneously degrade is as Fourth Ring and five rings) Microbial resources also seldom.Therefore, can simultaneously the degrade microorganism of multiple PAHs of screening has important practical significance.
Summary of the invention
In view of deficiency of the prior art, one of object 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, depositary institution's preservation of Qi Yi State Intellectual Property Office appointment, specifying information is as follows:
Preservation date: on November 22nd, 2012;
Depositary institution's title: China Committee for Culture Collection of Microorganisms's 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 degraded organic aromatic compound.
Another object of the present invention be to provide aforementioned scopulariopsis brevicaulis bacterium (
scopulariopsis brevicaulis) application of 4Z in preparing the biological degradation reagent of 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 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 at least selected from phenanthrene, pyrene, fluoranthene and benzo [a] pyrene, but is not limited to this.
Scopulariopsis brevicaulis bacterium of the present invention has wider substrate spectrum, polycyclic aromatic hydrocarbons in environment is had to good degradation capability, be suitable for preparing environment remediation preparation, particularly organic aromatic compound contaminated soil reagent, uses and eliminates the environmental pollution that organic aromatic compound causes.
Accompanying drawing explanation
Fig. 1 is the colonial morphology of bacterial strain 4Z on solid medium in embodiment 1;
Fig. 2 a-2d is respectively the growth curve of bacterial strain 4Z in the nutrient solution containing phenanthrene, pyrene, fluoranthene and benzo [a] pyrene in embodiment 2;
Fig. 3 a-3d is respectively in embodiment 2 at bacterial strain 4Z nutrient solution China and Philippines, pyrene, fluoranthene and benzo [a] pyrene concentration curve over time, and CK is the processing contrast that adds 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's 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 a strain energy degrading polycyclic aromatic hydrocarbons.This bacterial strain protruding bacterium colony that is white in color after growing on flat board 4 days, 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), be numbered 4Z, research discovery, 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: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 containing 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. containing the minimal medium of PAHs: the different PAHs(that prepare 1000 mg/L with DMF are luxuriant and rich with fragrance, benzo [a] pyrene, pyrene, fluoranthene) solution, by suitable concentration, add the minimal medium of sterilizing.
2, the isolation and purification of PAHs degradation bacteria
Take the fresh soil of 10 g, add in the triangular flask that 100mL inorganic salt liquid substratum (pyrene concentration is 100.0 mg/L) is housed, 200 r/min shaking culture is 1 week at 28 ℃.By 10% inoculum size, be transferred in fresh pyrene minimal medium, continue to cultivate 1 week 5 times repeatedly.Finally draw 0.1 mL nutrient solution and dilute different gradients and coat on Ma Dingshi culture medium flat plate, picking list bacterium colony, inoculates the single bacterium colony after plate streaking purifying in pyrene inorganic salt liquid substratum, chooses the fastest bacterial strain of the speed of growth as research bacterial strain.
3, morphologic observation and evaluation
The inoculation of fully activation, in Ma Dingshi substratum, is cultivated 4 days for 28 ℃, after gramstaining under opticmicroscope observation of cell form, thalline size.By activated spawn streak inoculation on Ma Dingshi plate, in 28 ℃ of incubators, be inverted and cultivate 4 days, observe colonial morphology, and measure bacterium colony size, specifically refer to 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 completed by Shanghai Sheng Gong Bioisystech Co., Ltd.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 Neighbor-Joining method to carry out Phylogenetic Analysis.
5, by biomass, change and see, bacterial strain 4Z all can grow on the Ma Dingshi substratum that contains phenanthrene, pyrene, fluoranthene and benzopyrene, especially on the substratum containing 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 degradation characteristic of degrading polycyclic aromatic hydrocarbons fungi to 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, 200 r at 28 ℃/min shaking culture 1 week, centrifugal collection thalline, and with phosphate buffered saline buffer repetitive scrubbing 3 times, then it is standby with phosphate buffered saline buffer, bacteria suspension to be adjusted to desired concn.
2, the degraded of bacterial strain 4Z to polycyclic aromatic hydrocarbons: 10% bacteria suspension is added in 50 mL liquid nutrient mediums, with the processing that adds inactivated bacteria suspension in contrast, add 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.150 r at 28 ℃/min lucifuge shaking culture 6 weeks, in different period samplings, 4 ℃ of freezing preservations.Measure concentration and the OD of PAHs in solution
600variation.
3, the extraction of polycyclic aromatic hydrocarbons and analysis in solution
All pour the bacterium liquid in triangular flask into separating funnel, add 50 mL ethyl acetate to extract three times, after united extraction liquid, use anhydrous Na
2sO
4remove moisture.In water temperature, be that in the rotatory evaporator of 36 ℃, rotation is concentrated into dry.Add 2.0 ml acetonitriles and dissolve, with high performance liquid chromatography (HPLC), measure the concentration of polycyclic aromatic hydrocarbons in solution.
4, degradation results: can find out that by Degrading experiment bacterial strain 4Z has good degradation effect to polycyclic aromatic hydrocarbons, in the time of 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 is also with the working method close with previous embodiment 1-2, the PAHs that utilizes bacterial strain 4Z to commonly see to other, and the PAHs of the 16Zhong Weidai branch of EPA's appointment has carried out degradation experiment as the aforementioned, result demonstration, this bacterial strain 4Z all shows higher degradation capability.
Again and, this case contriver is also with the working method close with previous embodiment 1-2, utilize bacterial strain 4Z to carry out repair process to the soil containing pollutents such as phenanthrene, pyrene, fluoranthene, benzopyrenes, and carried out again measuring to repairing the content of rear Pollution of Polycyclic Aromatic Hydrocarbons in Soil thing, its result demonstration, 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.In technical field of the present invention, holding the identical the knowledgeable of knowing can be by 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 (5)
1. scopulariopsis brevicaulis bacterium (Scopulariopsis brevicaulis) Z-4, its deposit number is CGMCC No.6870.
2. the application of scopulariopsis brevicaulis bacterium in degraded organic aromatic compound described in claim 1, described organic aromatic compound is selected from phenanthrene, pyrene, fluoranthene and benzo [a] pyrene.
3. the application of scopulariopsis brevicaulis bacterium in preparing the biological degradation reagent of organic aromatic compound described in claim 1, described organic aromatic compound is selected from phenanthrene, pyrene, fluoranthene and benzo [a] pyrene.
4. an organic aromatic compound degrading reagent, described organic aromatic compound is selected from phenanthrene, pyrene, fluoranthene and benzo [a] pyrene, it is characterized in that, and described degraded pack is containing the scopulariopsis brevicaulis bacterium described in claim 1.
5. the application of scopulariopsis brevicaulis bacterium in repairing organic aromatic compound contaminated soil described in claim 1, described organic aromatic compound is selected from phenanthrene, pyrene, fluoranthene and benzo [a] pyrene.
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| CN109370910B (en) * | 2018-08-03 | 2021-09-07 | 贵州省植物保护研究所 | Scopulariopsis brevicaulis 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 |
| 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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