CN105950473A - Separation and screening method for dimethyl phthalate degradation microorganisms - Google Patents
Separation and screening method for dimethyl phthalate degradation microorganisms Download PDFInfo
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Abstract
The invention provides a separation and screening method for dimethyl phthalate degradation microorganisms. 5g of soil near a wasteyard is taken, 250 mL of inorganic salt medium is added, 10 mL of DMP solution is added, and domestication is conducted for 10 days; DMP-MSM solid plate media are prepared; coating of bacterium liquid is conducted; DMP-MSM liquid media are prepared, the surfaces of the solid plate mediums are observed, selective bacterium picking is conducted on growing bacterial colonies, in other words, 20 or more single colonies growing on different plates are selected at random, the bacterial colonies are picked out through an inoculation needle and transferred into the DMP-MSM liquid media in an inoculation mode, each single colony is inoculated with one bottle of liquid medium, light shielding is conducted in a constant-temperature shake culture box, and culture is conducted for 48 h at the temperature of 30 DEG C and the speed of 100 r/min; 20 or more culture media carrying out culture for 48 h are observed under a microscope, the liquid culture in which a large number of microorganisms grow is subjected to solid-liquid mixed continuous culture repeatedly till the forms of the microorganisms observed under the microscope are consistent, or the microorganisms are authenticated or confirmed as pure strains through a molecular biological method.
Description
Technical field
The present invention relates to the separating screening method of a kind of dimethyl phthalate (DMP) degrading microorganism, belong to micro-
Biological separating screening method technical field.
Background technology
Conventional microorganism separating screening method mainly includes plate streak and spread plate, the most both at home and abroad bolter
Select that the method for dimethyl phthalate degrading microorganism is no exception all uses both approaches.General flow process is: first
Sampled from specific environment before this, such as mud, soil or special contaminated environment etc., after domestication, directly take
The surface that bacteria suspension is coated with the solid plate culture medium containing dimethyl phthalate is cultivated, bacterium colony to be grown
After, carry out repeatedly the isolated and purified strain of plate streaking, finally give the purpose microorganism that a strain is pure.Such screening technique
If applied in being very suitable in the screening process of other microorganisms, successful story also can be found everywhere.But due to neighbour
Dimethyl phthalate is the organic compound of a kind of indissoluble, when making solid plate culture medium, and the adjacent benzene of all additions
Dicarboxylic acid dimethyl ester has all been deposited to the bottom of flat board, and we are coated with streak culture, and the culture medium utilized is all
Being that sub-fraction of the superiors, the microorganism so inoculated above is not readily accessible to dimethyl phthalate substantially,
Coating or the microorganism of streak inoculation can not utilize dimethyl phthalate as sole carbon source, but utilize agar conduct
Nutritional labeling, the most also cannot screen dimethyl phthalate degrading microorganism.
Many times, in order to screen dimethyl phthalate degrading microorganism, when existing way can only extend domestication
Between, such as 30d, 60d or longer time so that in domestication the step for, be enriched with more purpose microorganism, reduce
The quantity of miscellaneous bacteria.In follow-up flat board coating and scratching process, owing to the antibacterial of inoculation is substantially phthalic acid two
Methyl ester degrading microorganism, so purpose microorganism can also be screened.But, this screening technique, due to solid plate
Not containing dimethyl phthalate (reason of precipitation) in culture medium, through the most after purification, degradation characteristic gradually disappears
Losing, the microorganism finally given is without degradation function, it has to repeat domestication and screening, until finishing screen is selected a strain and had
The dimethyl phthalate degrading microorganism of degrading activity.Meanwhile, domestication work is the most dry as dust, also for a long time
Leveraging experiment progress, scientific research personnel's body and mind is also subject to the biggest misery, and these all bring to research work too much
Inconvenience.
Summary of the invention
The invention aims to solve the problem that above-mentioned prior art exists, and then a kind of effective adjacent benzene two is provided
The sharp separation screening technique of formic acid dimethyl ester degrading microorganism.
It is an object of the invention to be achieved through the following technical solutions:
A kind of separating screening method of dimethyl phthalate degrading microorganism, step is as follows:
Step one, take the soil 5g near soot, add the minimal medium (MSM) of 250mL, add
DMP (dimethyl phthalate) solution 10mL, tames 10 days.
Step 2, making DMP-MSM solid plate culture medium: take minimal medium, add the agar powder of 2%,
After high-temp steam sterilizing, being down flat plate, after solidification to be cooled, the DMP adding 10mL on each plating medium surface is molten
Liquid, lucifuge room temperature (25 DEG C) is soaked 24h, then unabsorbed for solid culture primary surface residue DMP solution is moved liquid
Rifle reclaims.
Step 3, coating bacterium solution: take the soil supension 100uL tamed, be uniformly coated on the DMP-that step 2 makes
MSM solid plate media surface, is not inverted, in incubator lucifuge, cultivate 48h under the conditions of 30 DEG C.
Step 4, making DMP-MSM fluid medium: take sterilized minimal medium 10mL, add 100uL
DMP solution, adds 5 μ L tween 80s, makes DMP solution be uniformly dispersed in minimal medium rather than heavy
Form sediment bottom culture medium.
Step 5, observation have cultivated the solid plate media surface of 48h, carry out selectivity for the bacterium colony grown
Choose bacterium, i.e. randomly choose 20 or single bacterium colony of greater number of different plated growth, with Inoculating needle, these bacterium colonies are chosen
Go out, in the DMP-MSM fluid medium that step 4 of transferring makes, each one bottle of fluid medium of single colony inoculation,
In constant-temperature table incubator lucifuge, 30 DEG C, cultivate 48h under the conditions of 100r/min.
Step 6,20 or the greater number of culture medium having cultivated 48h to step 5, carry out microscope observation, right
Follow-up strain separating purification is carried out in the fluid medium growing a large amount of microorganism.
Step 7, microorganism isolated and purified: will step 6 grow a large amount of microorganism fluid medium take out
100uL, according to the method in step 3, the DMP-MSM solid culture primary surface being applied in step 2；Repeat step
The experimental procedure of rapid three to step 6 carries out solid-liquid and is mixed continuously, until the microbial morphology of microexamination
Unanimously, or through molecular biology method identify and be defined as pure culture.
Beneficial effects of the present invention: the present invention devises a whole set of for the micro-life of degraded of separation screening dimethyl phthalate
The method of thing.The separating screening method of the application of the invention, it is possible to separation screening is to phthalic acid fast and effectively
Dimethyl ester degrading microorganism.
In the present invention, DMP infusion method is applied to overcome DMP to precipitate when making DMP-MSM solid medium
Problem, so when coating and streak plate, the microorganism inoculated can sufficiently contact DMP, substantially increase
The efficiency of separation screening.But, simply use flat board coating and plate streak still have certain probability to make to screen
Microorganism be not purpose microorganism.There is the phthalic acid two of degrading activity to a strain in order to enable separation screening more accurately
Methyl ester degrading microorganism, in the present invention, after the coating of each solid plate is cultivated, has all carried out a fluid medium
Cultivate, the single bacterium colony that will grow on flat board, be inoculated into respectively in DMP-MSM fluid medium, so, if
Fluid medium has a large amount of microbial reproduction, it may be determined that it is exactly the dimethyl phthalate degraded of degrading activity
Microorganism.
And when making fluid medium, the most commonly used DMP is added directly into the method in MSM culture medium, though
So the microorganism of inoculation also can be bred in such fluid medium, but due to DMP aggregate and precipitate, can touch
The microorganism of DMP is considerably less, and the time that result causes fluid medium to cultivate DMP degradation bacteria is greatly prolonged, and typically trains
Time once of supporting is more than 10d.So be insoluble in fluid medium to solve DMP and reduce the problem of incubation time,
The present invention adds in liquid medium within the tween 80 of trace so that DMP is dispersed in MSM fluid medium
In, thus substantially reduce incubation time.
Above-mentioned this " solid-liquid " mixing continuous culture method solve in the industry about indissoluble organogenous sediment as only
The variety of problems run into during the separation screening of the microorganism of one nutrient substance, successfully avoid screening using agar as
The microorganism of nutrient substance, and substantially reduce the separation screening time, especially separation screening is with phthalate
Material is more suitable for as the degrading microorganism of sole carbon source, this method.
Accompanying drawing explanation
Fig. 1 is the HPLC measurement result schematic diagram of 60 μ L/L DMP standard solution.
Fig. 2 is the HPLC testing result schematic diagram of degradation bacteria experimental group residue DMP.
Detailed description of the invention
The present invention is described in further detail below: the present embodiment is carried out under premised on technical solution of the present invention
Implement, give detailed embodiment, but protection scope of the present invention is not limited to following embodiment.
The separating screening method of a kind of dimethyl phthalate degrading microorganism involved by the present embodiment, by following steps
Implement:
Step one, take the soil 5g near soot, add the minimal medium of 250mL, add DMP (adjacent benzene
Dicarboxylic acid dimethyl ester, a kind of plasticiser) solution 10mL, tames 10 days；
Described minimal medium (MSM) formula is as follows:
A, a great number of elements solution: NH4NO3、2g；MgSO4·7H2O、0.5g；NaCl、0.5g；
K2HPO4·3H2O、2g；KH2PO4, 0.4g and H2O、1000mL。
B, trace element solution: FeSO4·7H2O、300mg；CoCl2·6H2O、100mg；ZnSO4·7H2O、
80mg；CaCl2, 100mg and H2O、1000mL。
Before using, taking B solution 2mL and join in solution A, adjusting pH value is 6.5.
The domestication process of described step one: the soil liquid to be tamed is placed in 500mL sterilizing triangular flask, uses sterilizing
Tampon seals, and is positioned over by triangular flask in the constant-temperature table incubator of lucifuge, and cultivation temperature 30 DEG C, 100r/min shakes
Bottle, continuous domestication 10 days.
Step 2, making DMP-MSM solid plate culture medium: take minimal medium, add the agar powder of 2%,
After high-temp steam sterilizing, it is down flat plate, after solidification to be cooled, adds the DMP of 10mL on each plating medium surface
Solution, lucifuge room temperature (25 DEG C) is soaked 24h, is then used by unabsorbed for solid culture primary surface residue DMP solution
Liquid-transfering gun reclaims.
The condition of high-temp steam sterilizing in described step 2: 121 DEG C, sterilizing 25min.
Described step 2 is down flat the condition of plate: plate is 90mm diameter glass material, pour in flat board culture medium be 15～
20mL。
DMP is insoluble in minimal medium, is also insoluble in agar culture medium, as oil, and Direct precipitation after addition,
And it is gathered into one.Write inside domestic and international all papers be all in agar culture medium, be directly added into DMP after, system
Make solid plate.But experiment finds, owing to DMP precipitates, the DMP of addition can not be from the culture medium that sterilizing is good
Pour out；If can pour out, also it is precipitation in plate.And when being coated with bacterium solution, all it is coated onto solid culture base table
Face, so, coating or the microorganism of streak inoculation are actually not readily accessible to DMP, so screen obtain micro-
Biology in theory, is not the most the antibacterial of degraded DMP, and major part is to utilize agar as the microorganism of nutrient.
It addition, have people by after the most miscible to DMP and acetone and other organic solvent, it is then added in above-mentioned culture medium, but
Experiment finds, DMP is still insoluble in solid or fluid medium.
Step 3, coating bacterium solution: take the soil supension 100uL tamed, be uniformly coated on the DMP-that step 2 makes
MSM solid plate media surface, is not inverted, lucifuge, 30 DEG C of cultivation 48h in incubator.
Owing to, in the solid plate that soaked through DMP, DMP is present in the upper strata at solid medium substantially, if
It is inverted and cultivates, have part DMP to get off by aggregate flow, affect experiment effect.
General culture medium is inverted the reason cultivated: the ambient temperature owing to cultivating is high, has steam in culture dish, meets
Condense into water droplet to planar surface, drop onto media surface, cause local bacterial not grow.And in the present invention, by
There is one layer of DMP to cover in solid culture primary surface, during cultivating, seldom produce steam, therefore will not produce
Above-mentioned water droplet, cultivates so need not be inverted.Meanwhile, experimentation also confirms that, through cultivating for a long time, does not has
Producing steam, culture medium does not the most dry up.
Step 4, making DMP-MSM fluid medium: take sterilized minimal medium 10mL, add 100uL
DMP solution, adds 5 μ L tween 80s, makes DMP solution be uniformly dispersed in minimal medium.
Owing to DMP is insoluble in MSM culture medium and agar culture medium, so directly adding DMP in both culture medium
If, before said, can precipitate.If joined by antibacterial in such culture medium, antibacterial is not owing to reaching
DMP, purpose microorganism would not grow, or growth is the slowest.So, the present invention adds a kind of surface
Activating agent: tween 80, it can make DMP be dispersed in MSM culture medium.So, antibacterial just can connect completely
Contact DMP so that the purpose rapid microbial growth in fluid medium, substantially reduce incubation time.
Step 5, observation have cultivated the solid plate media surface of 48h, carry out selectivity for the bacterium colony grown
Choose bacterium, i.e. randomly choose 20 or single bacterium colony of greater number of different plated growth, with Inoculating needle, these bacterium colonies are chosen
Go out, in the DMP-MSM fluid medium made in step 4 of transferring.One bottle of liquid culture of each single colony inoculation
Base, in constant-temperature table incubator lucifuge, 30 DEG C, 100r/min cultivate 48h.Whole range request sterile working excessively.
Step 6, to above-mentioned 20 or the greater number of fluid medium having cultivated 48h, carry out microscope observation, right
Follow-up strain separating purification can be carried out in the fluid medium growing a large amount of microorganism.
To this step, if DMP-MSM fluid medium has a large amount of microbial reproduction, then it is assumed that this bacterium can utilize
DMP is as sole carbon source nutrient substance.Because in the formula of culture medium, carbon source only one, it is simply that DMP.If
If bacterial reproduction growth, it is necessarily required to the necessary nutrient substance that carbon source, nitrogen source both are basic.So, as long as at this
Being found that substantial amounts of antibacterial in one step, just explanation, DMP degrading microorganism has screened.
But there is a problem to need explanation: the carbon source in solid medium is except DMP, it is also possible to agar；And liquid
Except DMP in body culture medium, also tween 80.The antibacterial grown out from solid medium turn be linked into liquid training
Supporting in base, the carbon source that they own together is but DMP, so proving the above results further.
Step 7, microorganism isolated and purified: will step 6 grow a large amount of microorganism fluid medium take out
100uL, according to the method for step 3, the DMP-MSM solid culture primary surface being applied in step 2.Repeat step
The experimental procedure of three to step 6 carries out " solid-liquid " and is mixed continuously, until the microorganism shape of microexamination
State is consistent, or is defined as pure culture through additive method qualifications such as molecular biology.
If not repeating step 3 to the such incubation of step 6, simple according to common " culture of isolated " bacterium
The method planted, i.e. coating+line, is all to carry out on solid medium, it is possible to make to utilize agar as carbon source
Microorganism screened out, and probability is the biggest.Therefore it is necessary in twice solid medium separation screening operation
Between add a fluid medium incubation, eliminate the impact of agar microorganism with this.
The microorganism how determining isolated is exactly DMP degrading microorganism？
The DMP-MSM fluid medium containing pure culture being finally separating to obtain is centrifuged with the rotating speed of 5000r/min
15min, DMP degrading microorganism can be deposited to bottom centrifuge tube.Carefully outwell supernatant, hang with a small amount of sterile purified water
Drift along the cell formed sediment.Through " suspended centrifugal precipitation " (being typically washed cell) of 3 times, finally give is thin
Born of the same parents' precipitate is pure purpose microorganism, namely do not have other impurity (this step primarily to wash off tween 80 and
There is no the DMP of degraded, in order to avoid false positive occurs in subsequent experimental).
Again with the cell of aseptic distillation aqueous suspension precipitation, after obtaining pure cell suspension, the concentration of diluting cells suspension,
Spectrophotometer is made to detect OD600=0.2.Then the DMP standard solution of 60uL/L is configured.
The above-mentioned pure bacterial solution of 0.5mL is joined in 10mL above-mentioned DMP standard solution, at constant-temperature table incubator
Middle lucifuge, 30 DEG C, the DMP surplus cultivated after 12h in bioassay standard solution of 100r/min.Meanwhile, additionally take
10mLDMP standard solution, adds the sterile purified water of 0.5mL, is put into together in same incubator, by same
Condition is cultivated, i.e. operation repetitive, in this, as matched group.
Determining instrument: Dalian Erie spy high performance liquid chromatograph EC2000.High performance liquid chromatography testing conditions: ODS-
C18 post；Flowing phase: methanol/water (1:1)；Flow velocity: 1.0mL/min；Column temperature: 25 DEG C；Detection wavelength: 228
nm；Sample introduction: 20uL.
Table 1 testing result
The result obtained according to table 1, the DMP peak area of degradation bacteria experimental group does ratio with the peak area of DMP standard solution
Relatively, it can be seen that decrease 29.98%, in i.e. 12 hours, DMP is by this microbial degradation 29.98%.So entering
One step demonstrates, and this bacterium has the ability of DMP degraded.
And in the flow process of above separation screening degrading microorganism, it has been described that only with DMP as sole carbon in culture medium
Source.
To sum up, illustrating according to both sides, this bacterium has DMP degradation capability, is DMP degrading microorganism.
The above, the only present invention preferably detailed description of the invention, it is whole that these detailed description of the invention are all based on the present invention
Different implementations under body design, and protection scope of the present invention is not limited thereto, and any is familiar with the art
Technical staff in the technical scope that the invention discloses, the change that can readily occur in or replacement, all should contain in the present invention
Protection domain within.Therefore, protection scope of the present invention should be as the criterion with the protection domain of claims.
Claims (5)
1. the separating screening method of a dimethyl phthalate degrading microorganism, it is characterised in that
Step one, take the soil 5g near soot, add the minimal medium of 250mL, add DMP solution
10mL, tames 10 days；
Step 2, making DMP-MSM solid plate culture medium: take minimal medium, add the agar powder of 2%,
After high-temp steam sterilizing, being down flat plate, after solidification to be cooled, the DMP adding 10mL on each plating medium surface is molten
Liquid, lucifuge soaking at room temperature 24h, then reclaims unabsorbed for solid culture primary surface residue DMP solution liquid-transfering gun；
Step 3, coating bacterium solution: take the soil supension 100uL tamed, be uniformly coated on the DMP-that step 2 makes
MSM solid plate media surface, is not inverted, in incubator lucifuge, cultivate 48h under the conditions of 30 DEG C；
Step 4, making DMP-MSM fluid medium: take sterilized minimal medium 10mL, add 100uL
DMP solution, adds 5 μ L tween 80s, makes DMP solution be uniformly dispersed in minimal medium；
Step 5, observation have cultivated the solid plate media surface of 48h, carry out selectivity for the bacterium colony grown
Choose bacterium, i.e. randomly choose 20 or single bacterium colony of greater number of different plated growth, with Inoculating needle, these bacterium colonies are chosen
Go out, in the DMP-MSM fluid medium that step 4 of transferring makes, each one bottle of fluid medium of single colony inoculation,
In constant-temperature table incubator lucifuge, 30 DEG C, cultivate 48h under the conditions of 100r/min；
Step 6,20 or the greater number of culture medium having cultivated 48h to step 5, carry out microscope observation, right
Follow-up strain separating purification is carried out in the fluid medium growing a large amount of microorganism；
Step 7, microorganism isolated and purified: will step 6 grow a large amount of microorganism fluid medium take out
100uL, according to the method in step 3, the DMP-MSM solid culture primary surface being applied in step 2；Repeat step
The experimental procedure of rapid three to step 6 carries out solid-liquid and is mixed continuously, until the microbial morphology of microexamination
Unanimously, or through molecular biology method identify and be defined as pure culture.
The separating screening method of dimethyl phthalate degrading microorganism the most according to claim 1, its feature
Being, described minimal medium formula is as follows:
A, a great number of elements solution: NH4NO3、2g；MgSO4·7H2O、0.5g；NaCl、0.5g；
K2HPO4·3H2O、2g；KH2PO4, 0.4g and H2O、1000mL；
B, trace element solution: FeSO4·7H2O、300mg；CoCl2·6H2O、100mg；ZnSO4·7H2O、
80mg；CaCl2, 100mg and H2O、1000mL；
Before using, taking B solution 2mL and join in solution A, adjusting pH value is 6.5.
The separating screening method of dimethyl phthalate degrading microorganism the most according to claim 1, its feature
It is, the domestication process of described step one: the soil liquid to be tamed is placed in 500mL sterilizing triangular flask, with going out
Bacterium tampon seals, and is positioned over by triangular flask in the constant-temperature table incubator of lucifuge, and cultivation temperature 30 DEG C, 100r/min shakes
Bottle, continuous domestication 10 days.
The separating screening method of dimethyl phthalate degrading microorganism the most according to claim 1, its feature
It is, the condition of high-temp steam sterilizing in described step 2: 121 DEG C, sterilizing 25min.
The separating screening method of dimethyl phthalate degrading microorganism the most according to claim 1, its feature
Being, being down flat the condition of plate in described step 2: plate is 90mm diameter glass material, pouring culture medium in flat board into is
15～20mL.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113750437A (en) * | 2021-09-23 | 2021-12-07 | 天津工业大学 | Method for enhancing interface activation to efficiently biodegrade PET (polyethylene terephthalate) |
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| CN103602619A (en) * | 2013-11-21 | 2014-02-26 | 南京理工大学 | Thauera sp. capable of degrading triethylamine as well breeding method and application of Thauera sp. |
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| CN113750437A (en) * | 2021-09-23 | 2021-12-07 | 天津工业大学 | Method for enhancing interface activation to efficiently biodegrade PET (polyethylene terephthalate) |
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Application publication date: 20160921 |