CN107118966A - In situ environment microorganism separation method, the separation of soil origin oil degradation microorganism and screening technique - Google Patents
In situ environment microorganism separation method, the separation of soil origin oil degradation microorganism and screening technique Download PDFInfo
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- CN107118966A CN107118966A CN201710312109.2A CN201710312109A CN107118966A CN 107118966 A CN107118966 A CN 107118966A CN 201710312109 A CN201710312109 A CN 201710312109A CN 107118966 A CN107118966 A CN 107118966A
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- microorganism
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/02—Membranes; Filters
- C12M25/04—Membranes; Filters in combination with well or multiwell plates, i.e. culture inserts
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
A kind of in situ environment microorganism separation method and soil origin oil degradation microorganism separation and screening technique, by using micropore, culturing room is cultivated bacterium, the isolation to different types of microorganisms cell and constraint are not only realized, while also achieving the free exchange that the material produced is decomposed in the moisture of in situ environment, soluble substance or microbial metabolism.Using oil degradation functional microorganism separation screening as target, culture enrichment and high flux screening are carried out to objective microbe using this method, relative to conventional method it can be found that and the more bacterial strain species of collection.
Description
Technical field
The invention belongs to technical field of bioengineering, it is related to a kind of microbial culture method, more particularly to a kind of ring in situ
Border microorganism separation method and a kind of separation of soil origin oil degradation microorganism and screening technique.
Background technology
Microorganism is largely present in natural environment, and separation and the culture of microorganism are research and utilize the important of microorganism
Basis.However, there are many defects in traditional microorganism separation and cultural method：One is that can not meet microorganism in natural environment
Growth conditions；During two are natural environment, microorganism be not it is isolated exist, but with other microorganism mutual survivals, by straight
Contact or material exchange formation correlation, and traditional pure culture can not provide the environment of material exchange；Three be not of the same race
The speed of growth of microorganism is different, and traditional dilute, which is applied in plate method, grows fast microorganism rapid multiplying, causes to grow slowly
Microorganism is difficult to be separated；Four be certain micro-organisms increment very little, in the case of environment is unfavorable, it is impossible to form bacterium colony；
Five be that traditional flat board coating microbial culture method is wasted time and energy, and is difficult to high flux screening and the separation of microorganism.
The content of the invention
It is an object of the invention to overcome the defect of prior art to be imitated there is provided a kind of simple operating steps, separation and culture
The really high in situ environment microorganism separation method of good, reliability, and a kind of separation of soil origin oil degradation microorganism and sieve
Choosing method.
To achieve the above object, present invention employs following technical scheme：
A kind of in situ environment microorganism separation method, comprises the following steps：
S1：Prepare in situ environment microorganism Produced Liquid or leachate；
S2：Produced Liquid or leachate are mixed into LB solid mediums to be diluted, make ultimate density bacterium dense to be average each
Only one of which microorganism in micropore culturing room；
S3：The culture medium of mixed bacterium is filling in micropore culturing room, and to be cooled solidify out into seals after agar；
S4：Micropore culturing room is placed in in situ environment and carries out microculture, in incubation in micropore culturing room
Microorganism produces mass exchange with extraneous；
S5：After culture terminates, take out agar and be placed in normal temperature culture on LB flat boards；
S6：Repeat step S5 is repeatedly purified, until obtaining pure bacterial strain.
A kind of soil origin oil degradation microorganism separation and screening technique, comprise the following steps：
T1：Prepare the microorganism leaching liquid of target soil sample；
T2：Produced Liquid or leachate are mixed into LB solid mediums to be diluted, make ultimate density bacterium dense to be average each
Only one of which microorganism in micropore culturing room；
T3：The culture medium of mixed bacterium is filling in micropore culturing room, and to be cooled solidify out into seals after agar；
T4：Choose oil polluted environment；Or soil stirs with oil, simulate oil polluted environment；
T5：Micropore culturing room is placed in oil polluted environment or simulation oil polluted environment and carries out microculture, is trained
Microorganism during supporting in micropore culturing room produces mass exchange with extraneous；
T6：After culture terminates, take out agar and be placed in normal temperature culture on LB flat boards；
T7：Repeat step T5 is repeatedly purified, until obtaining pure bacterial strain；
T7：The single bacterium colony of picking bacterial strain to be screened, is inoculated in LB liquid medium one by one, culture to logarithmic phase, obtains
Seed liquor；
T8：Collect and clean thalline；
T9：Thalline is inoculated in respectively in the MF culture mediums using crude oil, pitch and paraffin as sole carbon source and cultivated；
T10：Judge whether bacterial strain can grow in crude oil, the bacterial strain that can be grown is to possess the micro- life of oil degradation function
Thing.
Further, the micropore culturing room is provided by situ environment microbial cultivation device；The in situ environment microorganism
Culture apparatus includes upper cover plate, lower cover and clamping plate；The middle part of the upper cover plate, lower cover and clamping plate is more provided with position consistency
Individual longitudinal hole；Miillpore filter, the miillpore filter are respectively equipped between the upper cover plate and clamping plate, between lower cover and clamping plate
Multiple independent micropore culturing room are cooperatively formed with through hole.
A kind of in situ environment microorganism separation method that the present invention is provided and the separation of soil origin oil degradation microorganism with
Screening technique, by using micropore, culturing room is cultivated bacterium, not only realize to different types of microorganisms cell every
From with constraint, while also achieving the moisture of in situ environment, soluble substance or microbial metabolism decomposes the freedom of the material produced
Exchange.Using oil degradation functional microorganism separation screening as target, using this method to objective microbe carry out culture enrichment and
High flux screening, relative to conventional method it can be found that and the more bacterial strain species of collection.
Brief description of the drawings
A kind of explosive view in situ environment microbial cultivation device that Fig. 1 uses for the present invention；
A kind of overall structure diagram in situ environment microbial cultivation device that Fig. 2 uses for the present invention；
A kind of assembly in situ environment microbial cultivation device that Fig. 3 uses for the present invention uses schematic diagram.
Embodiment
The embodiment provided below in conjunction with accompanying drawing 1 to 3, further illustrates a kind of in situ environment microorganism separation side of the invention
Method and the separation of soil origin oil degradation microorganism and the embodiment of screening technique.The invention is not restricted to following examples
Description.
Embodiment 1：
The present embodiment mainly introduces a kind of structure composition and operation principle of in situ environment microbial cultivation device.Such as Fig. 1
Shown in 3, the invention provides a kind of in situ environment microbial cultivation device, including upper cover plate 1, lower cover 3 and clamping plate 2.It is excellent
Choosing, the upper cover plate 1, lower cover 3, the horizontal plane projection of shape of clamping plate 2 and miillpore filter 4 are pros of the same size
Shape, the one side of the upper cover plate 1 and lower cover 3 is plane, and the thickness at another side edge is more than middle part and set at lead to the hole site
Thickness, so can effectively lift the intensity at edge, so as to improve the bulk strength and reliability of device.
The middle part of the upper cover plate 1, lower cover 3 and clamping plate 2 is provided with multiple longitudinal holes 5 of position consistency；The upper lid
The miillpore filter 4 that aperture is 0.22 micron, the micropore filter are respectively equipped between plate 1 and clamping plate 2, between lower cover 3 and clamping plate 2
Film 4 cooperatively forms multiple independent micropore culturing room with through hole 5.As seen in Figure 1, the micropore culturing room is cylinder
Shape, cylindrical circumferencial direction is the side wall of the through hole 5 of clamping plate 2, is sealing structure；The upper/lower terminal of cylinder is filtered by micropore
Film 4 is covered, and is Semi-seal structure.When device is placed in situ environment, different types of microorganisms cell is bound in different
Grown in micropore culturing room, moisture, in situ environment soluble substance or microbial metabolism are decomposed the material produced and can passed freely through
Miillpore filter 4, but microorganism can not but pass through, so as to realize the purpose of microorganism isolation culture.
It is preferred that, after the completion of culture, during device for opening, in order to prevent miillpore filter 4 and upper cover plate 1, lower cover 3 or clamping plate
Adhesion is produced between 2, it is broken, and then cause exterior materials to enter and contaminated microvia culturing room, the upper cover plate 1 and clamping plate 2 it
Between, the miillpore filter 4 between lower cover 3 and clamping plate 2 be 2 layers.
The upper cover plate 1, lower cover 3, the relevant position at the edge of clamping plate 2 and miillpore filter 4, are used for assembly provided with multiple
Fixed screw 6.The upper cover plate 1 is identical with the structure of lower cover 3 or is mirror image.Pin-connected panel, successively by cover plate 1,
Two layers miillpore filter 4, clamping plate 2, two layers miillpore filter 4, lower cover 3 are stitched together, and fixed bolt is passed through into screw 6 simultaneously
It is fixed, you can to complete assembled.
In view of the scale tested every time in actual applications it is different, it is necessary to micropore culturing room quantity also have difference, be
The versatility of the present apparatus is improved, the present apparatus can also be used by multiple units (see accompanying drawing 2) split as assembly (see accompanying drawing 3).
As one of specific embodiment, the upper cover plate 1 and/or lower cover 3 be provided with other unit upper cover plates 1 and/or under
Cover plate 3 connect to insert slot, hook, magnet suction buckle or thread gluing.User of service easily can will according to the actual requirements
Multiple units are applied in combination.
A kind of in situ environment microbial cultivation device that the present embodiment is provided, by by upper cover plate, lower cover, clamping plate and micro-
The micropore culturing room that hole filter membrane is constituted, not only realizes the isolation to different types of microorganisms cell and constraint, while also realizing
The free exchange of the material produced is decomposed in the moisture of in situ environment, soluble substance or microbial metabolism, it is achieved thereby that in situ
The function of environmental microorganism culture enrichment.
Embodiment 2：
The present embodiment mainly introduces a kind of oil Produced Liquid microorganism separation method.For the oil gathered from Jilin Oil Field
Produced Liquid, separation function microorganism in situ is carried out using the culture apparatus described in embodiment 1.Step is as follows：
1st, appropriate Produced Liquid is taken to prepare the solid medium containing 2% agar；
2nd, taking part oil Produced Liquid to count record microorganism concn with blood counting chamber, (actual measurement concentration is about 7.6*
107Individual/mL)；
3rd, gradient dilution.Part of dilution liquid is taken, (is cooled in the 2% Produced Liquid solid medium for being mixed into the preparation of the 1st step
About 45 DEG C), it is 10 to make ultimate density bacterium dense3Individual/mL (i.e. 1/μ L, make only one of which cell in average individual hole)；
4th, clamping plate is put into the culture medium of above-mentioned mixed bacterium, is cooled to its solidification；Clamping plate is quickly removed, makes the fine jade containing bacterium
Fat fritter is stayed in clamping plate through hole；
5th, it is careful to remove agar unnecessary on clamping plate, assembling culture apparatus and good seal；
6th, culture apparatus is placed in room temperature in oil Produced Liquid (or becoming temperature state) Culture in situ；
7th, culture apparatus Culture in situ took out, taken apart after 20 days；
8th, take out in the agar in clamping plate, 12 orifice plates for being placed on the Produced Liquid solid medium containing 2% agar, normal temperature
Culture extremely forms bacterium colony；
Whether the 9th, picking colony, is inoculated on 2%LB flat boards, can be grown before judging on LB；
10th, the bacterial strain that can be grown on LB flat boards is subjected to three purifying using LB, obtains pure bacterial strain；
11st, strain gene group DNA is extracted；
12nd, 16s rDNA fragments (genetic fingerprints), sequencing identification strain classification are obtained with universal primer PCR.
Embodiment 3：
The present embodiment mainly introduces a kind of culture apparatus using described in embodiment 1 and carries out edaphon separation method.
For the pedotheque gathered near Peking University's Wei Ming Lake, micro- life in culture apparatus original position separation pedotheque is utilized
Thing.Step is as follows：
1st, 1g soil is taken to insert in 10mL sterilized waters, vibration 10min obtains soil extract；
2nd, taking part soil extract to count record microorganism concn with blood counting chamber, (actual measurement concentration is about 4.8*
107Individual/mL)；
3rd, gradient dilution.Part of dilution liquid is taken, is mixed into 2% LB solid mediums and (is cooled to about 45 DEG C), is made final
Dense concentration bacterium is 103Individual/mL (i.e. 1/μ L, make only one of which cell in average individual hole)；
4th, clamping plate is put into the culture medium of above-mentioned mixed bacterium, is cooled to its solidification；Culture apparatus is quickly removed, is made containing bacterium
Agar fritter stay in hole；
5th, agar unnecessary on clamping plate, assembling culture apparatus and good seal are removed；
6th, culture apparatus is placed in soil situ culture, enough water is added in soil, soil is fully soaked, with
Increase material diffusivity in environment, more fully with the external world material exchange can occur for the microorganism allowed in culture apparatus；
7th, culture apparatus Culture in situ took out, taken apart after 20 days；
8th, the agar in clamping plate is taken out, is placed on 1.5%LB flat boards, normal temperature culture, waits to grow a certain size clone；
9th, picked clones, three purifying are carried out on LB flat boards, pure bacterial strain is obtained；
10th, strain gene group DNA is extracted；
11st, 16s rDNA fragments (genetic fingerprints), sequencing identification strain classification are obtained with universal primer PCR.
In order to verify advantage of the culture apparatus method relative to conventional microbiological separation method, we respectively with this method and
Traditional painting flat board separation method, carries out a point bacterium to same pedotheque, individually separated 136 and 118 plants of bacterium.As a result show
Show, compared to traditional separation method, in category level, culture apparatus method can isolate microorganism with a greater variety, flat board side
The bacterium of method separation comes from 9 category, and the bacterial strain of culture apparatus method separation is from 21 category.
Embodiment 4：
The present embodiment mainly introduces a kind of manual simulation's oil-polluted soils, is entered using the culture apparatus described in embodiment 1
The method of row separation soil origin oil degradation microorganism.For the pedotheque gathered near Peking University's Wei Ming Lake, people
Work simulates oil pollution process, is enriched with and screens the oil degradation microorganism of origin in environment with high throughput.
1st, the step 1 of reference implementation example 3 is to 5, by the microbial inoculant in pedotheque in culture apparatus；
2nd, manual simulation's oil pollution, per 1kg soil addition 20g crude oil, and soil is uniform with churning up the raw oil；
3rd, culture apparatus is placed in the oil pollution soil of manual simulation, Culture in situ 3 weeks；
4th, take agar in culture apparatus, picking chip apart, be placed in the soil extract solid training containing 2% agar and 5%LB
In 12 orifice plates for supporting base, normal temperature culture is to forming bacterium colony；
5th, purify three times, carry out bacterial strain identification.
From the point of view of the result of genetic fingerprints similitude, most of bacterial strain and identified bacterial strain similitude all 99% with
On, new microbial species are not found, reason is probably that selected crude oil sample diversity is relatively low.
Embodiment 5：
The present embodiment mainly introduces a kind of culture apparatus using described in embodiment 1 and carries out oil degradation functional microorganism
Screening technique.In order to verify in manual simulation's oil pollution soil, whether more stones can be obtained using culture apparatus separation method
The oily micro- life of degradation function, while the microorganism of these oil degradation functions is filtered out, using more difficult quilt in crude oil, and crude oil
The pitch of degraded, paraffin prepare MF minimal mediums respectively as sole carbon source, and screening can utilize the micro- of these carbon source for growth
Biology, step is as follows：
1st, the single bacterium colony of picking bacterial strain to be screened, is inoculated in LB liquid medium, culture to logarithmic phase, obtains seed
Liquid；
2nd, thalline is collected by centrifugation, and thalline is cleaned three times with not carbonaceous sources MF culture mediums；
3rd, adjust bacterial strain OD600 with not carbonaceous sources MF culture mediums, be inoculated in respectively 50mL with crude oil, or 25mL with pitch and
Paraffin for sole carbon source MF culture mediums in, Shaking culture；
4th, after 10 days, observe and determine OD600 values, judge whether bacterial strain can grow in crude oil；
5th, acquisition is subjected to secondary screening, culture using crude oil and pitch, the microbial strains of paraffin, every kind of bacterial strain does 3
It is individual parallel, determine the growth curve and utilization of carbon source efficiency of bacterial strain.
Further, edaphon is inoculated in culture apparatus (using soil extract as base before pollution occurs
Matter), then manual simulation's soil petroleum pollution, will be inoculated with culture apparatus chip and has been placed in Polluted Soil situ culture.
The abundant oil carbon source due to having in environment, therefore the edaphon of degradable oil has growth vigor, because
This can grow faster and more in culture, on the contrary due to carbon source lack, it is impossible to the growth of the microorganism of degraded oil by
Suppress.In this way, it can be enriched with and screen with high throughput the oil degradation microorganism of origin in soil.As control,
We have separated the microorganism in same untainted pedotheque with culture apparatus simultaneously.As a result show, compared to direct
Point bacterium in soil in the original location, the microorganism kind separated after oil pollution there occurs significant change, Acinetobacter,
The microbial strains amount showed increased of Pseudomonas, Aeromonas category, the category that some original position soils can be isolated does not divide
(it may out be suppressed by oil), and some new category can be separated, these microorganisms may oil preferably
Contaminated soil environment.These edaphons selected by oil pollution may have stronger oil degradation potentiality, together
When can well adapt to soil environment again, it is thus possible to be the function stem of potential petroleum pollution.
Above content is to combine specific preferred embodiment further description made for the present invention, it is impossible to assert
The specific implementation of the present invention is confined to these explanations.For general technical staff of the technical field of the invention,
On the premise of not departing from present inventive concept, some simple deduction or replace can also be made, should all be considered as belonging to the present invention's
Protection domain.
Claims (4)
1. a kind of in situ environment microorganism separation method, it is characterised in that：Comprise the following steps：
S1：Prepare in situ environment microorganism Produced Liquid or leachate；
S2：Produced Liquid or leachate are mixed into LB solid mediums to be diluted, make ultimate density bacterium dense for average each micropore
Only one of which microorganism in culturing room；
S3：The culture medium of mixed bacterium is filling in micropore culturing room, and to be cooled solidify out into seals after agar；
S4：Micropore culturing room is placed in in situ environment and carries out microculture, micro- life in incubation in micropore culturing room
Thing produces mass exchange with extraneous；
S5：After culture terminates, take out agar and be placed in normal temperature culture on LB flat boards；
S6：Repeat step S5 is repeatedly purified, until obtaining pure bacterial strain.
2. in situ environment microorganism separation method according to claim 1, it is characterised in that：The micropore culturing room is by original
Position environmental microorganism culture apparatus is provided；The in situ environment microbial cultivation device includes upper cover plate, lower cover and clamping plate；Institute
The middle part for stating upper cover plate, lower cover and clamping plate is provided with multiple longitudinal holes of position consistency；Between the upper cover plate and clamping plate, under
Miillpore filter is respectively equipped between cover plate and clamping plate, the miillpore filter cooperatively forms multiple independent micropore cultures with through hole
Room.
3. a kind of soil origin oil degradation microorganism separation and screening technique, it is characterised in that：Comprise the following steps：
T1：Prepare the microorganism leaching liquid of target soil sample；
T2：Produced Liquid or leachate are mixed into LB solid mediums to be diluted, make ultimate density bacterium dense for average each micropore
Only one of which microorganism in culturing room；
T3：The culture medium of mixed bacterium is filling in micropore culturing room, and to be cooled solidify out into seals after agar；
T4：Choose oil polluted environment；Or soil stirs with oil, simulate oil polluted environment；
T5：Micropore culturing room is placed in oil polluted environment or simulation oil polluted environment and carries out microculture, was cultivated
Microorganism in Cheng Zhong micropores culturing room produces mass exchange with extraneous；
T6：After culture terminates, take out agar and be placed in normal temperature culture on LB flat boards；
T7：Repeat step T5 is repeatedly purified, until obtaining pure bacterial strain；
T7：The single bacterium colony of picking bacterial strain to be screened, is inoculated in LB liquid medium one by one, culture to logarithmic phase, obtains seed
Liquid；
T8：Collect and clean thalline；
T9：Thalline is inoculated in respectively in the MF culture mediums using crude oil, pitch and paraffin as sole carbon source and cultivated；
T10：Judge whether bacterial strain can grow in crude oil, the bacterial strain that can be grown is to possess oil degradation functional microorganism.
4. soil origin oil degradation microorganism separation method according to claim 3, it is characterised in that：The micropore training
Room is supported to be provided by situ environment microbial cultivation device；The in situ environment microbial cultivation device includes upper cover plate, lower cover
And clamping plate；The middle part of the upper cover plate, lower cover and clamping plate is provided with multiple longitudinal holes of position consistency；The upper cover plate and folder
Miillpore filter is respectively equipped between plate, between lower cover and clamping plate, the miillpore filter cooperatively forms multiple independent with through hole
Micropore culturing room.
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| CN108048326A (en) * | 2018-01-30 | 2018-05-18 | 内蒙古农业大学 | The device and method of biology in situ directional separation culture plant growth-promoting rhizobacteria |
| CN108441422A (en) * | 2018-04-08 | 2018-08-24 | 郭庆 | A kind of soil bacteria screening system |
| CN108587910A (en) * | 2018-04-24 | 2018-09-28 | 郭庆 | A method of separation bacterium solution bacterium |
| CN108687131A (en) * | 2017-12-07 | 2018-10-23 | 苏州市环科环保技术发展有限公司 | Chemical pollution site remediation method |
| CN109825419A (en) * | 2019-03-27 | 2019-05-31 | 山东科技大学 | The tower cultivation reactor and its application method of wastewater treatment are used for based on list/Hybrid NC machine tool induction mineralising |
| CN110423673A (en) * | 2019-08-15 | 2019-11-08 | 北京大学 | A kind of method of Culture in situ device and high-throughput isolation and screening microorganism |
| CN112143617A (en) * | 2020-09-11 | 2020-12-29 | 铜仁职业技术学院 | Target bacterium screening device, preparation method and application method thereof |
| CN112375687A (en) * | 2020-11-11 | 2021-02-19 | 云南省烟草农业科学研究院 | Soil microorganism trapping method |
| CN112689669A (en) * | 2018-09-14 | 2021-04-20 | 克亚诺斯生物技术公司 | Method for cultivating a microorganism of interest and related apparatus |
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