CN109777747A - One plant of greasy filth oil degradation bacterial strain and its cultural method and application - Google Patents
One plant of greasy filth oil degradation bacterial strain and its cultural method and application Download PDFInfo
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Abstract
The present invention relates to one plant of greasy filth oil degradation bacterial strain and its cultural method and applications.One plant of greasy filth oil degradation bacteria ECO-17, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 06 01st, 2018, culture presevation number: CGMCC No.15837.Present invention firstly discloses the greasy filth oil degradation bacteria ECO-17 that one plant of screening obtains, the bacterial strain does not need that the surfactants such as microemulsion additionally are added, the limitation for avoiding the secondary environmental pollution caused by chemical reagent is added and its processing greasy filth later cycles being utilized, while greatly reducing processing cost.The bacterial strain can not only handle the storage tank greasy filth waste that oil content is higher than 10%~50%, and also very good lower than 10% oil-polluted soils, water body treatment effect to oil content.
Description
Technical field
The present invention relates to one plant of greasy filth oil degradation bacterial strain and its cultural method and applications, belong to microorganisms technical field.
Background technique
With being continuously increased for oil product demand, petroleum and its product enter environment through a variety of ways, to soil
Serious pollution is caused with water body, and threatens the health of the mankind, the research and application of oil-polluted soils technology are just increasingly
In widespread attention and concern.Current Treatment process mainly has physical method, chemical method, biological method, since physics is repaired
The multiple component and structure for also destroying soil while destroying pollutant in soil (as being heat-treated), and it is expensive；Change
It is preferable to learn repairing effect, but used chemical reagent can generate secondary pollution, limit its scope of application；Wherein with biology drop
The method advantage of solution is no residual hazard rapidly, and low cost receives significant attention.
Bioremediation technology be considered as it is a kind of it is environmentally protective, without secondary pollution, efficient, can thorough degradation of contaminant tool
Promising oil pollution restorative procedure.Studies have shown that the activity of oil degradation bacteria enzyme, environment, inorganic nutrients, fertilizer,
Type of microorganism etc. plays an important role to the reparation in oily pollution place.It is carried out using petroleum race full constituent in petroleum geology micro-
Biodegrade analysis is a kind of unique angle, needs to do comprehensive analysis to full race's substance, have in research noted earlier to positive structure
The spectrum analysis of alkane and pristane, the Study on degradation of phytane, but hopance, gonane race ingredient is not easy to identify, and content is low, research
Seldom.
Biodegrade refers to by the decomposable process of the complex compound of biocatalysis.And microorganism is first in oil degradation
Catabolic enzyme is generated by the metabolism of itself, the hydro carbons and crude oil of heavy is cracked, reduces the viscosity of petroleum, in addition in its growth and breeding
In the process, the active compounds such as solvent, acids, gas, surfactant and biopolymer can be generated conducive to the displacement of reservoir oil, so
Small molecule is become by the further oxygenolysis of other microorganisms afterwards and achievees the purpose that degradation.
It can be seen that it is the key that bioremediation technology that the microorganism of selective degradation petroleum is capable of in screening.
International patent documents WO9905392A1 (application number WOGB98002217) discloses one kind and removes from waste drilling mud
The method deoiled, this method include waste drilling mud being formed surfactant with microemulsion to mix, and be separated into mixture
Phase, wherein the interfacial tension between micro emulsion liquid phase and conjugation polarity phase is less than 10-4mNm-1.The oil (can such as be belonged to by inoculation
Rhodococcus Pseudomonas, Gordona Pseudomonas or Tsukamurella Pseudomonas) hydrocarbon degradation microorganism and be biodegradable.
In petroleum storing process, crude oil precipitates petrochemical industry by oil storage tank, generates a large amount of danger wastes
Greasy filth, greasy filth Central Plains oil content are stacked at the serious pollution for causing the ecology such as air, soil in environment and break up to 10% or more
It is bad.Oil composition complicated composition, the persistence organic pollutants such as component polycyclic aromatic hydrocarbon (POPs) have high toxicity and move at a distance
Shifting property, and can persistently exist in the environment, the crude oil pollution difficulty for repairing high concentration by environmentally protective biological method is huge
Greatly.It is living to need to be added microemulsion formation surface for waste drilling mud deoiling method disclosed in international patent documents WO9905392A1
Property agent, the addition of chemical reagent will cause secondary environmental pollution, and the storage tank greasy filth for oil content higher than 10%~50% is useless
Object processing, the application effect and addition cost of microemulsion are unknown, meanwhile, contained using what chemical surfactant microemulsion treatment was crossed
The later cycles of oil sludge and sand are utilized and are also accordingly restricted.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides one plant of greasy filth oil degradation bacterial strain and its cultural method and applications.
Technical solution of the present invention is as follows:
One plant of greasy filth oil degradation bacteria (Tsukamurella pulmonis) ECO-17, is preserved on 06 01st, 2018
China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Institute of Microorganism, Academia Sinica, culture presevation number: CGMCC No.15837.
The cultural method of above-mentioned greasy filth oil degradation bacteria (Tsukamurella pulmonis) ECO-17, steps are as follows:
(1) greasy filth oil degradation bacteria (Tsukamurella pulmonis) ECO-17 is taken to line solid activation medium
On, bacterial strain after activation is made in activation culture；
(2) for strain inoculated into fluid nutrient medium, seed liquor is made in shaking table culture after taking step (1) activation obtained；
(3) seed liquor made from step (2) is taken, is forwarded to and expands in culture medium by percent by volume 2%~10%, is expanded
Bacterium solution is made in culture.
Preferred according to the present invention, in the step (1), solid activation medium is LB solid medium, and component is as follows:
Peptone 10g, yeast extract 5g, sodium chloride 10g, agar 20g, water are settled to 1L, and pH is natural.
It is preferred according to the present invention, in the step (1), activation condition are as follows: 28~37 DEG C of inversions are cultivated 1~2 day.
Preferred according to the present invention, the fluid nutrient medium in the step (2) is with the expansion culture medium in step (3)
LB liquid medium, component are as follows:
Peptone 10g, yeast extract 5g, sodium chloride 10g, water are settled to 1L, and pH is natural.
It is preferred according to the present invention, the shaking table culture condition in the step (2) are as follows: 28~37 DEG C of revolving speeds are 100~200
Under conditions of rev/min, shaking table culture 1~2 day.
It is preferred according to the present invention, the expansion condition of culture in the step (3) are as follows: 28~37 DEG C, dissolved oxygen 20~70%
Under conditions of, expand culture 1~2 day.
Above-mentioned greasy filth oil degradation bacteria (Tsukamurella pulmonis) ECO-17 answering in greasy filth oil degradation
With.
Preferred according to the present invention, the application, steps are as follows:
(i) by greasy filth and minimal medium according to mass volume ratio 1:(5~30) ratio mixing, unit g/ml, system
Obtain greasy filth to be degraded；
(ii) obtained wait be inoculated with mud oil degradation bacteria (Tsukamurella in greasy filth of degrading to step (i)
Pulmonis) the bacterium solution of ECO-17, bacterium solution are inoculated in the ratio of volume mass percentage 1~10%, and units/ml/g is in temperature
28~37 DEG C, under conditions of dissolved oxygen 20~70%, cultivate 3~5 days to get.
Preferred according to the present invention, in the step (i), every liter of component of minimal medium is as follows:
KNO31.5g, (NH4)SO41.5g, K2HPO41g, KH2PO41g, MgSO4·7H2O 0.5g, NaCl 0.5g,
FeSO4·7H2O 0.01g, dH2O is settled to 1L.
Beneficial effect
Present invention firstly discloses the greasy filth oil degradation bacterias (Tsukamurella pulmonis) that one plant of screening obtains
The hydrocarbon degradation microorganism that ECO-17, the bacterial strain and the existing known Tsukamurella for waste drilling mud oil removing belong to
(such as in international patent documents WO9905392A1) is compared, and is not needed that the surfactants such as microemulsion additionally are added, is avoided chemistry
The limitation that reagent is added caused secondary environmental pollution and its utilizes to processing greasy filth later cycles, greatly reduces simultaneously
Processing cost.The bacterial strain can not only handle the storage tank greasy filth waste that oil content is higher than 10%~50%, and low to oil content
In 10% oil-polluted soils, water body treatment effect it is also very good.
Detailed description of the invention
16S rDNA expands when Fig. 1 is greasy filth oil degradation bacteria (Tsukamurella pulmonis) ECO-17 strain idenfication
Electrophoresis result photo after increasing；
In figure: swimming lane M, D2000DNA Marker；The 16S of swimming lane 17, Tsukamurella pulmonis ECO-17
RDNA amplified fragments.
Specific embodiment
Technical solution of the present invention is further elaborated below with reference to embodiment, but institute's protection scope of the present invention is not limited to
This.
Biological material source
One plant of greasy filth oil degradation bacteria (Tsukamurella pulmonis) ECO-17, is preserved on 06 01st, 2018
China Committee for Culture Collection of Microorganisms's common micro-organisms center, in the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 of address
Institute of microbiology, the academy of sciences, state, culture presevation CGMCC No.15837.
Culture medium
Minimal medium, every liter of component are as follows:
KNO31.5g, (NH4)SO41.5g, K2HPO41g, KH2PO41g, MgSO4·7H2O 0.5g, NaCl 0.5g,
FeSO4·7H2O 0.01g, dH2O is settled to 1L；
Petroleum-inorganic salts solid medium, every liter of component are as follows:
KNO31.5g, (NH4)SO41.5g, K2HPO41g, KH2PO41g, MgSO4·7H2O 0.5g, NaCl 0.5g,
FeSO4·7H2O 0.01g, 10% petroleum, agar 20g, dH2O is settled to 1L.
Petroleum-inorganic salt liquid culture medium, every liter of component are as follows:
KNO31.5g, (NH4)SO41.5g, K2HPO41g, KH2PO41g, MgSO4·7H2O 0.5g, NaCl 0.5g,
FeSO4·7H2O 0.01g, 10% petroleum, dH2O is settled to 1L.
Embodiment 1
It acquires Gudong field oil storage tank and precipitates bed mud sample 2g, be placed in the sterile triangular flask of 150mL, sterile inorganic salts are added
Culture medium 50mL, sterile glass beads 2g cultivate 3d under the conditions of 30 DEG C, 150rpm, draw bacterium solution, it is dilute to carry out gradient with sterile water
It releases, is diluted to 10 respectively-1, 10-2, 10-3, 10-4, 10-5Times, each 100 μ L that are coated with are to sterile petroleum-inorganic salts solid culture
Base, stationary culture 3d under the conditions of 30 DEG C, picking growth is fast, bacterium colony is big is cloned into 50mL petroleum-inorganic salt liquid culture medium,
3d is cultivated under the conditions of 30 DEG C, 150rpm, that is, is drawn 100 μ L bacterium solutions and be applied to sterile petroleum-inorganic salts solid medium, choose
Take monoclonal.
Take monoclonal that Shandong Sheng Qiangde Biotechnology Co., Ltd is sent to be sequenced, through detecting, 16S rDNA sequence contains
1385bp, nucleotide sequence, through strain idenfication, belong to Tsukamurella pulmonis as shown in SEQ ID NO.1, name
For greasy filth oil degradation bacteria (Tsukamurella pulmonis) ECO-17, it is preserved in China Microbiological on 06 01st, 2018
Culture presevation administration committee common micro-organisms center, the address Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences are micro-
Biological study institute, culture presevation CGMCC No.15837.
Strain idenfication process is as follows:
Sample: bacterial solution；
Bacterial genomes DNA extraction kit: TIANGEN Biotech (Beijing) Co., Ltd., DP302；
TAE buffer (50 ×, 1L): Tris 242g, glacial acetic acid 57.1ml, Na2EDTA.2H2O 37.2g, add water to
1L；
Agarose: BIOWET, AGAROSE G-10；
2 × Pfu PCR MasterMix, D2000DNA Marker, nucleic acid dye, loading buffer etc.: Tiangeng is raw
Change scientific and technological (Beijing) Co., Ltd
DNA purification and recovery kit: TIANGEN Biotech (Beijing) Co., Ltd., DP214
The consumptive materials such as centrifuge tube, pipette tips: Gene Era Biotech company, the U.S. (GEB)
Primer: by Suzhou, Jin Weizhi Biotechnology Co., Ltd is synthesized, and ddH2O is added according to synthesis is single, be made 10 μM it is molten
Liquid.
1, extracting genome DNA is operated by DP302 kit.
2, PCR amplification
2.1 universal primer information
2.2 PCR amplification system components and composition
2.3 PCR cycle parameters
Initial denaturation: 94 DEG C, 3min；94 DEG C, 30s of denaturation, annealing 55,30s extend, and 72 DEG C, (totally 35 are followed 1.5min
Ring)；Extend 72 DEG C, 10min；4 DEG C of preservations.
3, agarose gel electrophoresis detects
The Ago-Gel of preparation 1.0%, voltage is set as 18V/cm, electrophoresis time 20min when electrophoresis.Using nucleic acid
Dyestuff carries out agarose electrophoresis dyeing, is taken pictures using ultraviolet gel imaging system.As a result as shown in Figure 1.
4, purification and recovery
Ago-Gel recycling, recovery product are carried out to target fragment using plain agar sugar gel DNA QIAquick Gel Extraction Kit
Suzhou Jin Weizhi Biotechnology Co., Ltd is sent to be sequenced.
Sequencing splicing sequence and blast are compared:
Sequence alignment is carried out by sequence 16S rDNA, the discovery immediate strain number of genetic affinity is NR_
029302.1。
Embodiment 2
The cultural method of above-mentioned greasy filth oil degradation bacteria (Tsukamurella pulmonis) ECO-17, steps are as follows:
(1) greasy filth oil degradation bacteria (Tsukamurella pulmonis) ECO-17 is taken to line on LB solid medium,
35 DEG C inversion activation culture 2 days, be made activation after bacterial strain；
(2) for strain inoculated into LB liquid medium, 35 DEG C of revolving speeds are 150 revs/min after taking step (1) activation obtained
Under conditions of, shaking table culture 2 days, seed liquor is made；
(3) seed liquor made from step (2) is taken, is forwarded in LB liquid medium by percent by volume 2%~10%, 35
DEG C, under conditions of dissolved oxygen 20%~70%, expand culture 2 days, bacterium solution is made.
Through detecting, the cell concentration of bacterium solution is 109cfu/ml。
Embodiment 3
Application of greasy filth oil degradation bacteria (Tsukamurella pulmonis) ECO-17 in greasy filth oil degradation, step
It is rapid as follows:
(i) greasy filth of oil content 10% and minimal medium are mixed according to the ratio of mass volume ratio 1:5, unit g/
Greasy filth to be degraded is made in ml；
(ii) obtained wait be inoculated with greasy filth oil degradation bacteria (Tsukamurella in greasy filth of degrading to step (i)
Pulmonis) the bacterium solution of ECO-17, bacterium solution are inoculated in the ratio of volume mass percentage 5%, units/ml/g, are 37 in temperature
DEG C, revolving speed be 200 revs/min under conditions of, cultivate 5 days to get.
Using the degradation rate of the petroleum in gravimetric method detection greasy filth, greasy filth oil degradation bacteria (Tsukamurella
Pulmonis) ECO-17 to oil content be 10% greasy filth petroleum degradation rate in 5d up to 21.9%.
Comparative example
According to the method for embodiment 3, identical greasy filth oil degradation is carried out using known bacterial strain NR_029302.1 and is tested,
Through detecting, it is known that the greasy filth petroleum degradation rate that bacterial strain NR_029302.1 is 10% to oil content in 5d is only 0.35%.
Interpretation of result
By can be seen that the present invention to oil content in embodiment 3 and comparative example for 10% greasy filth oil degradation data
Disclosed greasy filth oil degradation bacteria (Tsukamurella pulmonis) ECO-17 is directed to the stone that can directly carry out in greasy filth
Oil degradation, without using chemical surfactant to be handled, greatly reduces the environmental risk of processes composition and processing,
More existing known oil degradation bacteria is in terms of handling greasy filth pollution with the advantage of highly significant.
Claims (10)
1. one plant of greasy filth oil degradation bacteria (Tsukamurella pulmonis) ECO-17, it is preserved on 06 01st, 2018
State's Microbiological Culture Collection administration committee common micro-organisms center, address: in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Institute of microbiology, the academy of sciences, state, culture presevation number: CGMCC No.15837.
2. the cultural method of greasy filth oil degradation bacteria described in claim 1 (Tsukamurella pulmonis) ECO-17, special
Sign is that steps are as follows:
(1) greasy filth oil degradation bacteria (Tsukamurella pulmonis) ECO-17 is taken to line on solid activation medium, it is living
Change culture, bacterial strain after activation is made；
(2) for strain inoculated into fluid nutrient medium, seed liquor is made in shaking table culture after taking step (1) activation obtained；
(3) seed liquor made from step (2) is taken, is forwarded to and expands in culture medium by percent by volume 2%~10%, expands training
It supports, bacterium solution is made.
3. cultural method as claimed in claim 2, which is characterized in that in the step (1), solid activation medium is solid for LB
Body culture medium, component are as follows:
Peptone 10g, yeast extract 5g, sodium chloride 10g, agar 20g, water are settled to 1L, and pH is natural.
4. cultural method as claimed in claim 2, which is characterized in that in the step (1), activation condition are as follows: 28~37 DEG C
It is inverted culture 1~2 day.
5. cultural method as claimed in claim 2, which is characterized in that fluid nutrient medium and step (3) in the step (2)
In expansion culture medium be LB liquid medium, component is as follows:
Peptone 10g, yeast extract 5g, sodium chloride 10g, water are settled to 1L, and pH is natural.
6. cultural method as claimed in claim 2, which is characterized in that the shaking table culture condition in the step (2) are as follows: 28~
Under conditions of 37 DEG C of revolving speeds are 100~200 revs/min, shaking table culture 1~2 day.
7. cultural method as claimed in claim 2, which is characterized in that the expansion condition of culture in the step (3) are as follows: 28~
37 DEG C, under conditions of dissolved oxygen 20~70%, expand culture 1~2 day.
8. greasy filth oil degradation bacteria described in claim 1 (Tsukamurella pulmonis) ECO-17 is in greasy filth oil degradation
In application.
9. application as claimed in claim 8, which is characterized in that steps are as follows:
(i) by greasy filth and minimal medium according to mass volume ratio 1:(5~30) ratio mixing, unit g/ml, be made to
Degradation greasy filth；
(ii) obtained wait be inoculated with mud oil degradation bacteria (Tsukamurella pulmonis) in greasy filth of degrading to step (i)
The bacterium solution of ECO-17, bacterium solution in volume mass percentage 1~10% ratio be inoculated with, units/ml/g, temperature be 28~37 DEG C,
Under conditions of dissolved oxygen 20~70%, cultivate 3~5 days to get.
10. application as claimed in claim 9, which is characterized in that in the step (i), every liter of component of minimal medium is such as
Under:
KNO31.5g, (NH4)SO41.5g, K2HPO41g, KH2PO41g, MgSO4·7H2O 0.5g, NaCl 0.5g,
FeSO4·7H2O 0.01g, dH2O is settled to 1L.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111139189A (en) * | 2020-01-14 | 2020-05-12 | 浙江工业大学 | Aspergillus WBX-38 and application thereof in production of cyclopiazonic acid |
| CN113025524A (en) * | 2021-03-18 | 2021-06-25 | 生态环境部南京环境科学研究所 | Crude oil degrading bacteria SS-21NJ and application thereof |
| CN114989998A (en) * | 2021-04-30 | 2022-09-02 | 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) | Halophilic petroleum hydrocarbon degrading bacterium and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102703348A (en) * | 2012-05-25 | 2012-10-03 | 武汉科技大学 | Alkane degrading bacteria and application thereof |
-
2018
- 2018-07-09 CN CN201810747534.9A patent/CN109777747B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102703348A (en) * | 2012-05-25 | 2012-10-03 | 武汉科技大学 | Alkane degrading bacteria and application thereof |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111139189A (en) * | 2020-01-14 | 2020-05-12 | 浙江工业大学 | Aspergillus WBX-38 and application thereof in production of cyclopiazonic acid |
| CN111139189B (en) * | 2020-01-14 | 2022-04-19 | 浙江工业大学 | Aspergillus WBX-38 and application thereof in production of cyclopiazonic acid |
| CN113025524A (en) * | 2021-03-18 | 2021-06-25 | 生态环境部南京环境科学研究所 | Crude oil degrading bacteria SS-21NJ and application thereof |
| CN113025524B (en) * | 2021-03-18 | 2021-09-21 | 生态环境部南京环境科学研究所 | Crude oil degrading bacteria SS-21NJ and application thereof |
| CN114989998A (en) * | 2021-04-30 | 2022-09-02 | 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) | Halophilic petroleum hydrocarbon degrading bacterium and application thereof |
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