CN106544395A - A kind of measure method of the soil microorganism to petroleum hydrocarbon degradation ability - Google Patents
A kind of measure method of the soil microorganism to petroleum hydrocarbon degradation ability Download PDFInfo
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- CN106544395A CN106544395A CN201610957084.7A CN201610957084A CN106544395A CN 106544395 A CN106544395 A CN 106544395A CN 201610957084 A CN201610957084 A CN 201610957084A CN 106544395 A CN106544395 A CN 106544395A
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- hydrocarbon
- soil
- carbon source
- alkane
- petroleum hydrocarbon
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- C—CHEMISTRY; METALLURGY
- 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
Abstract
A kind of method for determining soil petrochina surfactant hydrocarbon degradation bacteria activity of the present invention, with the self-defined carbon source of three kinds of different component hydrocarbon as substrate, carries out soil petrochina surfactant hydrocarbon degradation bacteria determination of activity based on Biolog technologies；Directly specific aim culture can be carried out to feature degradation bacteria in 96 hole microplates using the method, and the absorbance change which causes by real-time monitoring, can metabolic activity of the Efficient Characterization microorganism to petroleum hydrocarbon, method is time saving and energy saving, consumption medicine is micro updating, can be prevented effectively from waste and the pollution on the environment of medicine.
Description
Technical field
The invention belongs to technical field of microbe application, more particularly to a kind of soil microorganism that determines is to petroleum hydrocarbon degradation energy
The method of power.
Background technology
It is considered as highly effective technology using microbial method remedying oil-polluted soils.Repaiied using microbial technique
During multiple oil-polluted soils, mainly petroleum hydrocarbon, alkane and the polycyclic aromatic hydrocarbon three types of Degradation are played to petroleum hydrocarbon
Degradation bacteria.The numbers using maximum possible counting method (most probable number, MPN) three kinds of degradation bacterias of measure many at present
Amount, time-consuming for method (needing to complete in 2-3 weeks once to determine), and required MPN pipes quantity is more, takes up room big, consumes medicine how (every
Pipe chemical feeding quantity is several milliliters), acquired results need artificial statistics, waste time and energy.
The content of the invention
In order to overcome the shortcoming of above-mentioned prior art, it is an object of the invention to provide a kind of soil microorganism that determines is to stone
The method of petroleum hydrocarbon degradation capability, is substrate using self-defined carbon source, the soil petrochina surfactant hydrocarbon degradation bacteria work based on Biolog technologies
Property.
To achieve these goals, the technical solution used in the present invention is：
A kind of measure method of the soil microorganism to petroleum hydrocarbon degradation ability, with petroleum hydrocarbon, alkane and polycyclic aromatic hydrocarbon as base
Matter metabolism carbon source, directly carries out the culture of functional microbial colony in MT2 microplates, and present in soil, degradation bacteria is in profit
When being that substrate carries out metabolism with petroleum hydrocarbon, alkane, polycyclic aromatic hydrocarbon, free electron and the tetrazolium dye in microwell plate of generation occur
Reaction, judges utilization power of the microorganism to three kinds of different component hydrocarbon by the depth of color, then which causes by real-time monitoring
Absorbance change, realize measure to three kinds of different types of surfactant hydrocarbon degradation bacteria metabolic activities.
In the present invention：
Soil Microorganism is extracted in the following way：
Oil-polluted soils sample 1g is taken, 99mL sterile distilled waters are added, 20min is shaken, after sedimentation 30min, on gained
Clear liquid is total bacteria suspension of Soil Microorganism；
The loading methods of the carbon source：
Take 150 μ L of supernatant liquid to be inoculated in the micropore of MT2 plates, be separately added in each hole 10 μ L petroleum hydrocarbons, alkane and
Polycyclic aromatic hydrocarbon is used as carbon source, wherein the 1st hole is the 150 μ L soil supernatants without any carbon source, as control；
Culture and the process for determining are as follows：
The MT2 plates being inoculated with are put into into 25 DEG C of cultures in constant incubator, luminosity is determined every 24h at wavelength 590nm
Value, METHOD FOR CONTINUOUS DETERMINATION 168h；
Computational methods are as follows：
Using formula (1)：AWCD=[∑ (Ci- R)]/average color change rate of 95 calculating per hole
(averagewellcolordevelopment, AWCD), represents the generation of degradation bacteria in each pedotheque with the AWCD values of 120h
Thank to activity, in formula：Ci is the OD values of i-th reacting hole, and R is the OD values of control wells.
The alkane is hexadecane hydrocarbon, and the polycyclic aromatic hydrocarbon is that anthracene, phenanthrene, pyrene arbitrarily compare mixture.
Compared with prior art, the present invention determines soil microorganism by determining soil petrochina surfactant hydrocarbon degradation bacteria activity
It is to petroleum hydrocarbon degradation ability, different by the thinking for determining quantity with tradition.In specific technological means, directly 96
Specific aim culture, and the absorbance change which causes by real-time monitoring are carried out in the microplate of hole to target micro organism popula, can
The physiological feature of Efficient Characterization microorganism.Method is time saving and energy saving, consumption medicine be micro updating, can be prevented effectively from medicine waste and
Pollution on the environment.
Description of the drawings
Fig. 1 is the metabolic activity for representing degradation bacteria in each pedotheque in the embodiment of the present invention with the AWCD values of 120h
Curve chart.
Fig. 2 is total stone that the quantity of total petroleum hydrocarbon degradation bacteria in the soil that maximum possible counting method is obtained is determined with the present invention
Correlative relationship schematic diagram between petroleum hydrocarbon degradation bacteria metabolic activity.
Fig. 3 is the alkane degradation bacteria generation that the quantity of alkane degradation bacteria in the soil that maximum possible counting method is obtained is determined with the present invention
Thank to the correlative relationship schematic diagram between activity.
Fig. 4 is that the quantity of the PAHs in soil degradation bacteria that maximum possible counting method is obtained is multi-ring with what the present invention was determined
Correlative relationship schematic diagram between Biodegradation of PAHs bacterium metabolic activity.
Specific embodiment
Describe embodiments of the present invention with reference to the accompanying drawings and examples in detail.
In embodiment, respectively with petroleum hydrocarbon, hexadecane hydrocarbon, three kinds of Polycyclic Aromatic Hydrocarbon Mixtures (luxuriant and rich with fragrance 0.4g, anthracene 0.4g, pyrenes
0.12g, is dissolved with dichloromethane and is settled to 1000mL) for self-defined carbon source.Using Biolog-MT2 plates to dropping in soil
The metabolic activity of solution hydro carbons microorganism is measured.
(1) Method And Principle
The complex mixture that petroleum hydrocarbon is made up of alkane, polycyclic aromatic hydrocarbon, a small amount of asphalt colloid.Soil petrochina alkane degradation
The quantity and activity of bacterium is closely related with the biodegradation efficiency of petroleum hydrocarbon.The principle of this method is with petroleum hydrocarbon, alkane, multi-ring
Three kinds of different type hydrocarbon of aromatic hydrocarbons are matrix metabolism carbon source, and the culture of functional microbial colony is directly carried out in MT2 microplates.
Degradation bacteria present in soil when metabolism being carried out for substrate using petroleum hydrocarbon, alkane, polycyclic aromatic hydrocarbon, the free electron of generation with
Tetrazolium dye in microwell plate reacts, and the depth of color can reflect utilization power of the microorganism to three kinds of different component hydrocarbon.
The absorbance change which causes by using instrument real-time monitoring, you can to three kinds of different types of petroleum hydrocarbon degradation bacterium metabolism
Activity is measured.
(2) key instrument and equipment
96 microwell plates of Biolog-MT2, Biolog, Hayward, CA；
Z-36HK type High speed refrigerated centrifuges, German Harmer；
250HG type digital display illumination shaken cultivation casees, golden Altar cities essence reach instrument manufacturing factory；
ELX808BLG type Biolog microbial identification instrument, Biolog companies of the U.S..
(3) the step of method
The extraction of (a) soil microorganism：Oil-polluted soils sample 1g is taken, 99mL sterile distilled waters, shake is added
20min, after sedimentation 30min, gained supernatant is total bacteria suspension of Soil Microorganism.
The selection of (b) self-defined carbon source and loading methods：Take 150 μ L of supernatant liquid to be inoculated in the micropore of MT2 plates, at each
10 μ L total petroleum hydrocarbons, hexadecane hydrocarbon, anthracene, phenanthrene, three kinds of Polycyclic Aromatic Hydrocarbon Mixtures of pyrene are separately added in hole as carbon source.Wherein
1 hole is the 150 μ L soil supernatants without any carbon source, used as control.
C () is cultivated and is determined：The MT2 plates being inoculated with are put into in constant incubator (25 DEG C) cultures, every 24h in wavelength
Shading value, METHOD FOR CONTINUOUS DETERMINATION 168h are determined at 590nm.
D () calculates：Average color change rate (the average well color per hole are calculated using formula (1)
Development, AWCD), the metabolic activity of degradation bacteria in each pedotheque is represented with the AWCD values of 120h, as a result such as Fig. 1
It is shown.
The checking of method
Using maximum possible counting method (most probable number, MPN) to soil petrochina hydrocarbon, alkane is multi-ring
The quantity of Biodegradation of PAHs bacterium is counted, the pass between the degradation bacteria metabolic activity that the quantity and said method to degradation bacteria is determined
System carry out correlation analysiss (Microsoft office excel, 2007).Acquired results as shown in figs 2-4, as a result show,
Linear relationship between the degradation bacteria activity that the degradation bacteria quantity and this method that maximum possible counting method is measured is determined is good,
Illustrate that the method can be used to characterize the microbial activity of petroleum-polluted soil petrochina surfactant hydrocarbon degradation bacteria.
Claims (3)
1. a kind of measure method of the soil microorganism to petroleum hydrocarbon degradation ability, it is characterised in that with petroleum hydrocarbon, alkane and multi-ring
Aromatic hydrocarbons is matrix metabolism carbon source, directly carries out the culture of functional microbial colony in MT2 microplates, is dropped present in soil
Tetrazolium of the solution bacterium when metabolism is carried out for substrate using petroleum hydrocarbon, alkane, polycyclic aromatic hydrocarbon, in the free electron and microwell plate of generation
Dyestuff reacts, and judges utilization power of the microorganism to three kinds of different component hydrocarbon by the depth of color, then by prison in real time
Its absorbance change for causing is surveyed, the measure to three kinds of different types of surfactant hydrocarbon degradation bacteria metabolic activities is realized.
2. method of the soil microorganism to petroleum hydrocarbon degradation ability is determined according to claim 1, it is characterised in that by such as
Under type extracts Soil Microorganism：
Oil-polluted soils sample 1g is taken, 99mL sterile distilled waters are added, 20min is shaken, after sedimentation 30min, gained supernatant
As total bacteria suspension of Soil Microorganism；
The loading methods of the carbon source：
Take 150 μ L of supernatant liquid to be inoculated in the micropore of MT2 plates, 10 μ L petroleum hydrocarbons, alkane and multi-ring are separately added in each hole
Aromatic hydrocarbons is used as carbon source, wherein the 1st hole is the 150 μ L soil supernatants without any carbon source, as control；
Culture and the process for determining are as follows：
The MT2 plates being inoculated with are put into into 25 DEG C of cultures in constant incubator, shading value are determined at wavelength 590nm every 24h, even
It is continuous to determine 168h；
Computational methods are as follows：
Using formula AWCD=[∑ (Ci- R)]/average color change rate of 95 calculating per hole
(averagewellcolordevelopment, AWCD), represents the generation of degradation bacteria in each pedotheque with the AWCD values of 120h
Thank to activity, in formula：Ci is the OD values of i-th reacting hole, and R is the OD values of control wells.
3. method according to claim 1 and 2, it is characterised in that the alkane is hexadecane hydrocarbon, the polycyclic aromatic hydrocarbon
Arbitrarily compare mixture for anthracene, phenanthrene, pyrene.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107796906A (en) * | 2017-11-15 | 2018-03-13 | 天津大学 | Rhodococcus sp degradation condition is improved based on metabolism group to improve the method for polycyclic aromatic hydrocarbon pyrene degradation rate |
| CN109868234A (en) * | 2018-12-28 | 2019-06-11 | 江西理工大学 | A kind of polycyclic aromatic hydrocarbon-degrading bacteria and its polycyclic aromatic hydrocarbons contaminated methods and applications of detection |
| CN111394419A (en) * | 2019-01-03 | 2020-07-10 | 中国石油天然气集团有限公司 | Method for detecting petroleum hydrocarbon degrading ability of petroleum hydrocarbon degrading bacteria in micro-scale mode |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101186890A (en) * | 2007-12-14 | 2008-05-28 | 华南理工大学 | Diesel oil alkane component degradation bacterium and application thereof |
| CN102794296A (en) * | 2012-08-01 | 2012-11-28 | 中国石油天然气股份有限公司 | Complex microbial inoculant for restoring petroleum-contaminated soil in loess highlands and restoring method thereof |
| CN104726370A (en) * | 2015-02-16 | 2015-06-24 | 湖南农业大学 | Enterobacter with crude oil degradation effect and application of enteric bacilli |
-
2016
- 2016-11-03 CN CN201610957084.7A patent/CN106544395A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101186890A (en) * | 2007-12-14 | 2008-05-28 | 华南理工大学 | Diesel oil alkane component degradation bacterium and application thereof |
| CN102794296A (en) * | 2012-08-01 | 2012-11-28 | 中国石油天然气股份有限公司 | Complex microbial inoculant for restoring petroleum-contaminated soil in loess highlands and restoring method thereof |
| CN104726370A (en) * | 2015-02-16 | 2015-06-24 | 湖南农业大学 | Enterobacter with crude oil degradation effect and application of enteric bacilli |
Non-Patent Citations (2)
| Title |
|---|
| 刘玉华 等: "不动杆菌属（Acinetobacter）细菌降解石油烃的研究进展", 《微生物学通报》 * |
| 郑华 等: "BIOLOG在土壤微生物群落功能多样性研究中的应用", 《土壤学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107796906A (en) * | 2017-11-15 | 2018-03-13 | 天津大学 | Rhodococcus sp degradation condition is improved based on metabolism group to improve the method for polycyclic aromatic hydrocarbon pyrene degradation rate |
| CN109868234A (en) * | 2018-12-28 | 2019-06-11 | 江西理工大学 | A kind of polycyclic aromatic hydrocarbon-degrading bacteria and its polycyclic aromatic hydrocarbons contaminated methods and applications of detection |
| CN109868234B (en) * | 2018-12-28 | 2020-06-09 | 江西理工大学 | Polycyclic aromatic hydrocarbon degrading bacteria, method for detecting polycyclic aromatic hydrocarbon pollution by polycyclic aromatic hydrocarbon degrading bacteria and application of polycyclic aromatic hydrocarbon degrading bacteria |
| CN111394419A (en) * | 2019-01-03 | 2020-07-10 | 中国石油天然气集团有限公司 | Method for detecting petroleum hydrocarbon degrading ability of petroleum hydrocarbon degrading bacteria in micro-scale mode |
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Application publication date: 20170329 |