CN108998498A - Microorganism kind and its quantity determine method on bituminous pavement in operation - Google Patents
Microorganism kind and its quantity determine method on bituminous pavement in operation Download PDFInfo
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- CN108998498A CN108998498A CN201810912938.9A CN201810912938A CN108998498A CN 108998498 A CN108998498 A CN 108998498A CN 201810912938 A CN201810912938 A CN 201810912938A CN 108998498 A CN108998498 A CN 108998498A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/06—Quantitative determination
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Abstract
It is an object of the present invention to provide microorganism kinds on the bituminous pavement in a kind of operation and its quantity to determine method, solve the problems, such as in runing at present bituminous pavement microorganism kind is indefinite and quantity be difficult to it is determining.The present invention is first according to the working environment of bituminous pavement in operation, collection in worksite sample, preparation dilution bacterium solution, with oese picking first time enrichment culture liquid, it is separated in the flat lining out of oil, after being enriched with three times, observes the dominant bacteria formed on plate, the repeatedly scribing line separation on beef-protein medium, to purify obtained single colonie；And analysis is compared by the PCR of 16S rDNA, sequencing and ncbi database Blast, identify microbe sort；By sterile interior flame, the clump count of six different dilutions is measured respectively.Bituminous pavement microbe sort proposed by the present invention and its quantitative approach are that bituminous pavement operation process microorganism lays the foundation to the aging action of pitch, propose the antimicrobial burn-in of bituminous pavement, extend bituminous pavement service life.
Description
Technical field
The present invention is that microorganism kind and its quantity determine method on a kind of bituminous pavement in operation, belongs to bituminous pavement
Technical field.
Background technique
Microorganism is a huge group, can rapid mass propagation under suitable environment.Pay attention to road in current China
Greening, either full green plants is all planted in highway or urban road, median strip and road shoulder two sides.These plants exist
It beautifies the environment, also provides good living environment to microorganism while absorbing noise and tail gas.Soil has various micro-
Nutrition required for biological self reproducing, growth and development, water, air, pH value, osmotic pressure and temperature condition etc..According to statistics, every gram of fertilizer
Several hundred million to tens microorganisms are usually contained in soil, every gram of lean soil also has millions of to several ten million microorganism.
As the extract of petroleum, the wax in pitch has stronger attraction to the microorganism that wax is food.Microorganism is de-
Wax technology has been widely used in oil field.In recent years, domestic and foreign scholars do the microorganism fungus kind for capableing of degraded oil hydro carbons
Numerous studies.Extension products of the pitch as petroleum have similar component with petroleum.There is a large amount of in the natural environment
Using hydro carbons as the microorganism of sole carbon source and the energy, they are extensive in distributed in nature, and about more than 100 belong to, more than 200 kinds, main
There are bacterium, actinomyces, mould, saccharomycete etc..Contain more ribosomes in their cellular component, hydro carbons can be accumulated in
On cytoplasma membrane, more phosphatide can be also synthesized.The bacteria genus of energy degraded oil hydro carbons mainly has achromobacter, non-lever
Pseudomonas, bacillus, false unicellular Pseudomonas, mycobacterium etc..One of the most common is false unicellular bacterium, it to alkane and
Aromatic hydrocarbon has degradation capability, and the alkane that can thoroughly degrade.The actinomyces Pseudomonas of degraded oil hydro carbons mainly have Nocardia,
Actinomyces etc..The fungi Pseudomonas of degraded oil hydro carbons mainly has candida, aspergillus, mucor, Penicillium etc..In soil
In earth environment, the kind of fungi is more than bacterium.Under normal conditions, every kind of microorganism only has a kind of petroleum component stronger
Degradation capability.
With the continuous research to petroleum hydrocarbon degradation bacterium, there are many methods screened and identify petroleum hydrocarbon degradation bacterium.
The most common method is then to be enriched under given conditions, tame progress thallus pure culture, and being carried out according to its physio-biochemical characteristics
It analyzes and identifies.Conventional method is to carry out enrichment culture to sample on the culture medium using petroleum hydrocarbon as sole carbon source, with culture
The extension of time gradually increases the concentration of petroleum hydrocarbon, to reduce the influence of non-purpose bacterium.Then, it is coated with, is chosen using plate
The isolated single bacterium body of single colonie scribing line carries out pure culture, finally from morphology, physiological and biochemical property, immunological characteristic etc.
It is identified.And the method for comparing innovation at present has using polymerase chain reaction (PCR) 16S rDNA Sequence Identification bacterial strain, laser
Mutagenic and breeding highly effective petroleum surfactant hydrocarbon degradation bacteria etc..
But bituminous pavement microbe species are indefinite in operation at present and quantity is difficult to determine, it is difficult to further research
Aging action of the microorganism to asphalt material.Therefore, the present invention is according to the different brackets highway and urban road pitch in operation
Road surface actual working environment condition, field sampling, screening, separation bituminous pavement on microorganism, using the PCR of 16S rDNA,
Sequencing identification strain, the quantity of various microorganisms in sample is determined based on viable bacteria counting method, thus the asphalt road clearly in operation
Microbe sort and quantity on face provide fundamental basis to the function and effect of bituminous pavement for further microorganisms.
Summary of the invention
(1) technical problem
Microorganism kind and its quantity determine method on the bituminous pavement that the present invention is to provide in a kind of operation, solve current
Bituminous pavement microbe species in operation are indefinite and quantity is difficult to determining problem, on the bituminous pavement in clearly runing
Microorganism lay the foundation to the aging action of asphalt material.
(2) technical solution
In order to solve the problems, such as in runing at present bituminous pavement microorganism kind is indefinite and quantity is difficult to determining, be bright
The aging action effect of true bituminous pavement operation process microorganism provides foundation.The present invention is according to the different brackets road in operation
Bituminous pavement actual working environment condition, field sampling, screening, separate microorganism are identified using the PCR of 16S rDNA, sequencing
Strain, the quantity of each microorganism in sample is determined using viable bacteria counting method, to identify microorganism on the bituminous pavement in operation
Classification and its quantity.Technical solution of the present invention is as follows: first according to the real work ring of the different brackets bituminous pavement in operation
Border condition, 20 parts of samples of collection in worksite air-dry after removing sundries, are prepared into the identical sample of size；Take 3 grams of samples that culture is added
In base, it is placed on 28 DEG C of constant-temperature tables enrichment culture 7 days；With oese picking first time enrichment culture liquid, drawn on oily plate
Line separation after being enriched with three times, observes the dominant bacteria formed on plate, the consistent single colonie of form is chosen, in beef extract egg
Repeatedly scribing line separation on white peptone culture medium, to purify obtained single colonie；Pass through the PCR of 16S rDNA, sequencing and NCBI data
The Blast in library compares analysis, and identification obtains the classification of specific microorganism；Cultured stable bacterium solution is taken, different times is diluted to
Number measures the clump count of six different dilutions respectively, formulates six concentration gradients, inhaled with sterilizing by sterile interior flame
Manage 1 milliliter of injection plate of each absorption.In incubator after culture 24 hours, bacterium colony counting is carried out, finally determines the pitch in operation
Microbe sort and its quantity on road surface.
(3) beneficial effect
With China's rapid economic development, the mileage of highways and streets, tunnel, forest zone and road in scenic area, rural highway
Also it is continuously increased.Part of road, the bituminous pavement such as woodland and road in scenic area, tunnel, rural highway and urban road are long
Phase is in dark, how wet, pollution environment, this provides advantage for microorganism mass propagation, breeds on bituminous pavement
A large amount of microorganisms.Microorganism using in pitch light components and wax as food, decompose pitch in light components, destroy pitch
Originally stable colloform texture makes pitch be hardened, become fragile, and cohesive force decline causes asphalt membrane to peel off, bituminous pavement is made pine occur
The Failure types such as scattered, cracking accelerate bituminous pavement to destroy under vehicular load repeated action.It is an object of the present invention to provide one kind
Microbe sort and its quantity determine method on bituminous pavement in operation, solve bituminous pavement microbe sort in operation at present
Indefinite and quantity is difficult to determining problem, runs process microorganism for bituminous pavement and lays the foundation to the aging action of pitch,
To propose the antimicrobial burn-in of bituminous pavement, extends bituminous pavement service life, be of great practical significance.
Specific embodiment
The present invention provides microbe sort and its quantity on the bituminous pavement in a kind of operation and determines method, specific implementation step
It is rapid as follows:
(1) the typical section for choosing different brackets road samples on road shoulder part bituminous pavement surface layer, by the sample of acquirement
It is fitted into sterilized sample sack, seals, record sample number into spectrum, acquisition time, place, every a sample of 20 meters of acquisitions, often
A section acquires 10 parts of samples；
(2) it is air-dried after indoor removing sundries, is prepared into the identical sample of size, certain mass sample is taken to be put into Liquid Culture
In base, the enrichment culture 7 days on 28 DEG C of constant-temperature tables, preparing dilution is 10-1、10-2、10-3、10-4、10-5、10-6Solution；
(3) 0.1 milliliter of bacterium solution for using aseptic straw to draw each dilution respectively three times, corresponds to instill and has write number
Three plating mediums in, coated plating medium is inverted in 30 DEG C of constant-temperature tables and is cultivated 4 days；
(4) oese picking first time enrichment culture liquid is used, is separated in the flat lining out of oil, after being enriched with three times, observation exists
The dominant bacteria formed on plate chooses the consistent single colonie of form, the repeatedly scribing line separation on beef-protein medium,
To purify obtained single colonie, is compared and divided by the Blast of the polymerase chain reaction of 16S rDNA, sequencing and ncbi database
Analysis, identifies the kind of specific microorganism；
(5) clump count for measuring different dilutions respectively by sterile interior flame respectively draws 1 milliliter of note with sterilized straw
Enter plate, inject 15 milliliters of nutrient agars, rotate plate, after mixing, is placed in culture 24 hours in incubator, into
Row bacterium colony statistics, determines different genera micro organism quantity.
Claims (1)
1. microorganism kind and its quantity determine method on the bituminous pavement in a kind of operation, it is characterised in that this method it is specific
Steps are as follows:
(1) the typical section for choosing different brackets road samples on road shoulder part bituminous pavement surface layer, the sample of acquirement is packed into
In sterilized sample sack, sealing records sample number into spectrum, acquisition time, place, every a sample of 20 meters of acquisitions, Mei Gelu
Section 10 parts of samples of acquisition；
(2) it is air-dried after indoor removing sundries, is prepared into the identical sample of size, certain mass sample is taken to be put into fluid nutrient medium
In, the enrichment culture 7 days on 28 DEG C of constant-temperature tables, preparing dilution is 10-1、10-2、10-3、10-4、10-5、10-6Solution；
(3) 0.1 milliliter of bacterium solution for using aseptic straw to draw each dilution respectively three times, corresponds to instill and has write the three of number
In a plating medium, coated plating medium is inverted in 30 DEG C of constant-temperature tables and is cultivated 4 days；
(4) oese picking first time enrichment culture liquid is used, separates in the flat lining out of oil, after being enriched with three times, observes in plate
The dominant bacteria of upper formation chooses the consistent single colonie of form, the repeatedly scribing line separation on beef-protein medium, with pure
Change obtained single colonie, analysis, mirror are compared by the Blast of the polymerase chain reaction of 16S rDNA, sequencing and ncbi database
The kind of fixed specific microorganism；
(5) it is flat respectively to draw 1 milliliter of injection with sterilized straw for the clump count for measuring different dilutions respectively by sterile interior flame
Ware injects 15 milliliters of nutrient agars, rotates plate, after mixing, is placed in culture 24 hours in incubator, carries out bacterium
Statistics is fallen, determines different genera micro organism quantity.
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| CN201810912938.9A CN108998498A (en) | 2018-08-06 | 2018-08-06 | Microorganism kind and its quantity determine method on bituminous pavement in operation |
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| CN201810912938.9A CN108998498A (en) | 2018-08-06 | 2018-08-06 | Microorganism kind and its quantity determine method on bituminous pavement in operation |
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| CN108998498A true CN108998498A (en) | 2018-12-14 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109576186A (en) * | 2018-12-27 | 2019-04-05 | 天津天丰泽田生物科技有限公司 | A kind of petroleum hydrocarbon degradation composite bacteria agent and preparation method thereof |
| CN112662727A (en) * | 2020-12-16 | 2021-04-16 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | High-throughput analysis and breeding method for functional microorganisms based on extinct dilution culture group |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102399719A (en) * | 2011-10-21 | 2012-04-04 | 中国人民解放军海军医学研究所 | Bacterium DW3 capable of degrading marine diesel oil pollutants |
| CN104877930A (en) * | 2015-04-24 | 2015-09-02 | 南开大学 | Separating and screening method for saline-alkaline-resistant bacteria degrading petroleum hydrocarbon |
-
2018
- 2018-08-06 CN CN201810912938.9A patent/CN108998498A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102399719A (en) * | 2011-10-21 | 2012-04-04 | 中国人民解放军海军医学研究所 | Bacterium DW3 capable of degrading marine diesel oil pollutants |
| CN104877930A (en) * | 2015-04-24 | 2015-09-02 | 南开大学 | Separating and screening method for saline-alkaline-resistant bacteria degrading petroleum hydrocarbon |
Non-Patent Citations (1)
| Title |
|---|
| 李金江: "沥青路面材料离散与微生物的作用", 《云南交通科技》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109576186A (en) * | 2018-12-27 | 2019-04-05 | 天津天丰泽田生物科技有限公司 | A kind of petroleum hydrocarbon degradation composite bacteria agent and preparation method thereof |
| CN112662727A (en) * | 2020-12-16 | 2021-04-16 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | High-throughput analysis and breeding method for functional microorganisms based on extinct dilution culture group |
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