CN108676732A - Vacation Escherichia WNF15 one plant oval and its application - Google Patents
Vacation Escherichia WNF15 one plant oval and its application Download PDFInfo
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- CN108676732A CN108676732A CN201810805641.2A CN201810805641A CN108676732A CN 108676732 A CN108676732 A CN 108676732A CN 201810805641 A CN201810805641 A CN 201810805641A CN 108676732 A CN108676732 A CN 108676732A
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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
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
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- 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
- B09C1/105—Reclamation of contaminated soil microbiologically, biologically or by using enzymes using fungi or plants
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/347—Use of yeasts or fungi
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Abstract
The invention discloses vacation Escherichia WNF15 one plant oval and its applications.The bacterial strain is preserved in China typical culture collection center on June 15th, 2018, and deposit number is CCTCC NO：M 2018373.Oval vacation Escherichia WNF15 of the present invention is the bacterial strain of efficient degradation sulfonated phenol formaldehyde resin, and resistance is stronger.Bacterial strain WNF15 can effectively degrade sulfonated phenol formaldehyde resin, can be used as the strain excellent of sulfonated phenol formaldehyde resin pollution environmental organism processing, and application prospect is good.
Description
Technical field
The invention belongs to technical field of environmental microorganism, are related to vacation Escherichia WNF15 one plant oval and its application.
Background technology
Sulfonated phenol formaldehyde resin is a kind of linear macromolecule made of phenol, formaldehyde and sodium sulfite are condensed under alkaline condition
Compound has good surface-active, wetting, emulsification, dispersion and anchorage, and thermal stability is good, high temperature resistant, is filtered frequently as drop
It loses agent and is used for gas drilling liquid；It is also widely used in manufacture coating and dyestuff.Thus have the characteristics that length of difficult to degrade and residual life,
If into water environment or soil, seriously affect animal, plant growth, or even be detrimental to health, carry out sulfonation waste resin without
Evilization treatment research is significant.
It is common both at home and abroad either physically or chemically to be handled for environment such as organic pollution contaminated soil, water bodys,
But these processing methods are not thorough in the presence of processing, high energy consumption, easily cause secondary pollution risk.Biologic treating technique has environment friend
Good, at low cost, the contaminant degradation advantages such as thoroughly, are widely used to the processing of organic pollution.Utilize microbial degradation sulphur
Change phenolic resin, is a good pollutant dispelling tactics, but its key is separation screening efficient degradation microorganism.
According to statistics, China's oil gas drilling can generate about 1,500,000 m of the discarded slurry containing sulfonated phenol formaldehyde resin every year3,
Potential environmental hazard is big, therefore there is an urgent need to screen the microorganism of efficient degradation sulfonated phenol formaldehyde resin, to promote contaminant water
Sulfonated phenol formaldehyde resin is degraded in body or soil.Micro- life such as many bacteriums, actinomyces, yeast, mould and algae in nature
Species group has the ability of degradable organic pollutant.For lignin organic matter, fungi can be by secreting extracellular lignin
Degrading enzyme realizes lignin degradation, and degradation efficiency is better than bacterium；The molecular structure of sulfonated phenol formaldehyde resin is similar to lignin,
Therefore fungi can be screened for sulfonated phenol formaldehyde resin of degrading, to eliminate environmental contaminants.
Invention content
The present invention is directed to the biodegradation technique problem of existing sulfonated phenol formaldehyde resin contaminated soil, and screening obtains one plant of ovum
Shape vacation Escherichia WNF15 and its application in sulfonated phenol formaldehyde resin contaminated soil of degrading.
In order to reach above-mentioned technical purpose, the present invention is realized especially by following technical scheme：
The present invention acquires from Sichuan Province Deyang City waste drilling mud disposal field and discards polysulfonate water-based drilling mud cake sample,
Fungal bacterial strain WNF15 is obtained by taming, isolating and purifying.Cultivated in PDA culture medium, bacterial strain WNF15 white myceliums without diaphragm,
Generate arthrospore, oval, spore size 2-3 × 2.5 μm；Bacterium colony is cotton-shaped, quality is loose.
The total DNA of the extraction bacterial strain WNF15, expands ITS segments, the ITS sequence measured such as SEQ ID NO.1 institutes
Show.Measured ITS sequence is compared in US National Bioinformatics Institute (NCBI) database, WNF15 and GenBank
In oval vacation Escherichia (Pseudallescheria ellipsoidea) CBS 418.73 of type strainTSimilarity most
High (99.7%), so that it is determined that WNF15 belongs to oval false Escherichia (Pseudallescheria in classification
ellipsoidea WNF15)。
Based on features above, the present invention provides false Escherichia (Pseudallescheria one plant oval
Ellipsoidea) WNF15, the bacterial strain have carried out preservation, depositary institution：China typical culture collection center；Address：Hubei
No. 299 Wuhan Universitys of wuchang, wuhan area Bayi Road of province are in the school；Preservation date：On June 15th, 2018, deposit number CCTCC
NO：M 2018373.
Applications of the obtained oval vacation Escherichia WNF15 of the present invention in soil organic pollutant of degrading, especially
Degradation to sulfonated phenol formaldehyde resin.
Preferably, the bacteria suspension of the oval false Escherichia WNF15 or its culture solution or its tunning are dropping
The application solved in sulfonated phenol formaldehyde resin is also within the scope of the present invention.
The present invention also provides the oval vacation Escherichia WNF15 to repair sulfonated phenol formaldehyde resin pollution environment simultaneously
Application in medium, the medium are soil or water.
In addition oval vacation Escherichia WNF15 of the present invention is in the biological agent for preparing degradation sulfonated phenol formaldehyde resin
Application, the biological agent includes oval vacation Escherichia WNF15 or its bacteria suspension or its culture solution or its tunning.
Beneficial effects of the present invention are：
The present invention provides false Escherichia WNF15 one plant oval, which can effectively degrade sulfonated phenol formaldehyde resin, pure training
Under the conditions of supporting, after meeting bacterium culture 14d, to the degradation rate of sulfonated phenol formaldehyde resin up to 37.84%；Under edaphic condition, bacterium processing is connect
After 120d, sulfonated phenol formaldehyde resin degradation rate reaches 98.43%, obvious processing effect.Meanwhile oval false Escherichia WNF15 tools
There is stronger anti-adversity ability, can be used as the biological treatment that excellent degradation bacteria pollutes environment for sulfonated phenol formaldehyde resin, application prospect is good
It is good.
Description of the drawings
Fig. 1 is colonial morphologies of the oval vacation Escherichia WNF15 in PDA culture medium；
Fig. 2 is the systematic growth figure of the ITS sequence of oval false Escherichia WNF15.
Specific implementation mode
Below in conjunction with specific embodiment of the present invention, technical solution of the present invention is clearly and completely described, is shown
So, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on the reality in the present invention
Example is applied, every other embodiment obtained by those of ordinary skill in the art without making creative efforts all belongs to
In the scope of protection of the invention.
Separation, purifying and the preservation of the oval false Escherichia WNF15 of embodiment 1
Polysulfonate waste water-base drilling well mud cake sample is acquired from Sichuan Province Deyang City well drilling waste mud disposal field, low temperature is protected
It deposits and takes back laboratory.
It prepares respectively：
Liquid microelement：MgSO42g, MnSO45g, FeSO4·7H2O 5g, CaCl25g, ZnSO45g, distilled water 1L.
MS culture mediums：NH4Cl 0.6g, K2HPO40.5g, KH2PO40.5g, liquid microelement 16mL, pH are naturally, water
1000mL。
PDA culture medium：Potato 200g, glucose 10g, agar 20g, pH are naturally, water 1000mL.
Polysulfonate waste water-base drilling well mud cake sample 5.0g is taken, is added in 95mL MS culture mediums, 25 DEG C and 140r/min items
Shaken cultivation 7d under part；1mL culture solutions are drawn, continuously add fresh sterilized 99mL containing 1% sulfonated phenol formaldehyde resin MS cultures
Base, 25 DEG C, 140r/min continuation shaken cultivation 7d, repeats this operation 3 times.1mL culture solutions are finally drawn, using gradient dilution
Method, in being coated in PDA culture medium, 25 DEG C of constant temperature incubations.After growing bacterium colony, the different bacterium colony of form is selected from tablet and is existed
Point connects culture on PDA plate.It waits for that bacterium colony generates spore, chooses a circling point and connect culture, repeat the operation and combine microexamination, directly
Until obtaining pure bacterial strain.Bacterial strain access PDA slant mediums after purification are preserved.
As shown in Figure 1, the bacterial strain WNF15 isolated and purified of the present embodiment is cultivated in PDA culture medium, white mycelium
No diaphragm generates arthrospore, oval, spore size 2-3 × 2.5 μm, and bacterium colony is cotton-shaped shape, quality is loose.
The amplification of the ITS of 2 bacterial strain WNF15 of embodiment and Phylogenetic Analysis
Bacterial strain total DNA is extracted, is primer amplification ITS segments with ITS4 and ITS5 using total DNA as template, PCR reactions are used
Bio-RADMyCyclerTM instruments.
Reaction system (50 μ l)：2 × PCRMix25 μ l, primer I TS4 and ITS5 (10 μM) each 1 μ l, 1 μ l of DNA profiling add
Ultra-pure water complements to 50 μ l；The nucleotide sequence of primer I TS4 and ITS5 are as shown in SEQID No.2 and SEQID No.3.
PCR reaction conditions：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 1min, 54 DEG C of annealing 1min, 72 DEG C extend 2min, follow
Ring 30 times；72 DEG C of final extension 8min.
After pcr amplification product detects on 1.0% agarose gel electrophoresis, it is sent to the Shanghai life limited public affairs of work bioengineering
Department carries out sequencing.The calculating of gene order similarity is carried out with software DNAman6.0.Surveyed sequence results such as SEQID
No.1。
The sequence results of gained are compared in US National Bioinformatics Institute (NCBI) database, are found
The ITS gene orders of WNF15 and the oval false Escherichia (Pseudallescheria of type strain in database
ellipsoidea)CBS 418.73TSimilarity highest, be 99.7%.According to NCBI comparison results, similitude highest is selected
Type strain as reference strains, with adjacent method (Neighbor-joining) phylogenetic tree construction of MEGA6.0 softwares
(Fig. 2), value of bootstrapping (bootstrap) 1000.
Based on features above, bacterial strain WNF15 is accredited as oval false Escherichia (Pseudallescheria
ellipsoidea).The bacterial strain is stored in China typical culture collection center on June 15th, 2018, and deposit number is
CCTCC NO：M 2018373.
The oval false Escherichia WNF15 degradation sulfonated phenol formaldehyde resin abilities of embodiment 3 measure
Mycelium growth vigor measures：WNF15 is inoculated in activation culture 3d on PDA solid mediums, in colony edge internal diameter
1cm card punch punches, 4 fungus blocks of picking, is inoculated in MS culture solutions of the 100mL containing 1% sulfonated phenol formaldehyde resin, 25 DEG C, 140r/
Min shaken cultivations are control not connect bacterium containing 1% sulfonated phenol formaldehyde resin MS culture solutions, are repeated 3 times.It is double after shaken cultivation 14d
Layer nylon net filter mycelium, distillation water washing mycelium 3 times, 80 DEG C dry to constant weight, and weigh and record.
Strains for degrading sulfonated phenol formaldehyde resin ability measures：The filtrate in above-mentioned experiment is collected, its TOC content is measured and is recorded
(T1), the initial TOC contents for the treatment of fluid are measured in the same method and record (T2).
Measurement result shows that WNF15 contains in the culture solution that sulfonated phenol formaldehyde resin is only carbon source after culture 14d, mycelia
Body biomass is 0.147g, is significantly higher than the 0.006g of control treatment, illustrates that the bacterial strain has good degradation sulfonated phenolic tree
The ability of fat.The TOC values of T2, which are 0.305g/L, to be shown to TOC measurement results, the TOC values of T1 are 0.189g/L, are computed, connect
Kind of WNF15 is to the degradation rate of sulfonated phenol formaldehyde resin up to 37.84%.
The adverse-resistant characteristic of the oval false Escherichia WNF15 of embodiment 4
In order to obtain the anti-adversity ability of oval false Escherichia WNF15, its alkaline-resisting, salt tolerant and growth temperature are mainly determined
Range.By the PDA slant cultures of above-mentioned oval false Escherichia WNF15 on fresh PDA tablet activation culture 3d.
It is inoculated on the tablet for trying culture medium, is repeated 3 times with one ring mycelia of oese picking using an inoculation method.
It is basic culture medium with PDA, pH to 7.0,8.0,9.0,10.0 and 11.0 is adjusted respectively with 1% NaOH solution.Equally, salt tolerant
Property to measure with PDA be basic culture medium, it is 1%, 2%, 4%, 6%, 8% and 10% to adjust NaC1 mass volume fractions.Alkaline-resisting,
The tablet of Salt tolerance is 25 DEG C of culture 5d, measures colony diameter, calculates mycelial growth rate.Growth temperature range measures,
It will be inoculated with the PDA plate of strains tested, is individually positioned in 10 DEG C, 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C and 35 DEG C of biochemical cultivation case
Middle constant temperature incubation 5d measures colony diameter, calculates mycelial growth rate.
The result shows that oval vacation Escherichia WNF15 has good adverse-resistant characteristic, initial pH=7.0~11.0 of growth；
Its salt resistance ability is stronger, can be grown on the PDA plate containing 6%NaCl；Growth temperature range is wider, can be in 10~35 DEG C of temperature
Growth in range.
5 oval vacation Escherichia WNF15 of embodiment is to sulfonated phenol formaldehyde resin contaminated soil TOC removal abilities
It is naturally native to weigh 5kg, is added to distill the spare sulfonated phenol formaldehyde resin stock solution of water dissolution, adjusts sulfonation in soil
Phenolic resin content is 1%, is mixed well spare；By the oval false Escherichia WNF15 of contaminated soil amount 0.3% (m/m) inoculation
Microbial inoculum (bacterium number >=2.0 × 108Cfu/g, auxiliary material are fine rice bran and wheat bran), it mixes well, is control not connect bacterium, is repeated 3 times.
120d (in June, 2017 in October, 2017) is handled under natural conditions, during which pays attention to keeping system for handling humidity 25% or so.Point
Not Ce Ding nature soil background, initial contamination soil and processing 120d connect bacterium and do not meet bacterium processing contaminated soil sample leaching liquor TOC
Content, calculates TOC removal rates, and formula is as follows：
In formula, W1 represents background soil extraction TOC values, and W2 represents initial contamination soil extraction TOC values, W3 representative office
Reason 120d's does not connect soil bacteria or connects soil bacteria leaching liquor TOC values.
The result shows that (table 1), by 120d processing, control soil extraction TOC removal rates are only 11.38%；And it is inoculated with
Strains tested processing, soil extraction TOC removal rates are up to 98.43%, it is seen that WNF15 has degradation sulfonated phenol formaldehyde resin well
Ability.
Treatment effects of the 1 bacterial strain WNF15 of table to sulfonated phenol formaldehyde resin contaminated soil
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
Understanding without departing from the principles and spirit of the present invention can carry out these embodiments a variety of variations, modification, replace
And modification, the scope of the present invention is defined by the appended.
Sequence table
<110>Sichuan Agricultural University
<120>Vacation Escherichia WNF15 one plant oval and its application
<141> 2018-07-20
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 660
<212> DNA
<213>Oval vacation Escherichia (Pseudallescheria ellipsoidea)
<400> 1
ctccggactg gcccagagag gtgggcaact accactcagg gccggaaagt tgtccaaact 60
cggtcattta gaggaagtaa aagtcgtaac aaggtctccg ttggtgaacc agcggaggga 120
tcattacaga gttactactc caaacccatt gtgaacctta cctatgttct gttgcctcgg 180
cggcgtcggt cagcgcccct ctgaaaagag gacgatgccc ctcccgccgg cagcaccaaa 240
ctcttgaatt ttacagcgga tacagttctg atttgaaaac aaaaaacaag ttaaaacttt 300
caacaacgga tctcttggtt ctggcatcga tgaagaacgc agcgaaatgc gataagtaat 360
gtgaattgca gaattcagtg aatcatcgaa tctttgaacg cacattgcgc ccggcagtaa 420
tctgccgggc atgcctgtcc gagcgtcatt tcaaccctcg aacctccgtt tcctcaggga 480
agcccagggt cggtgttggg gcgctacggc gagtcctcgc gacccccgta ggccctgaaa 540
tacagtggcg gtcccgccgc ggttgccttc tgcgtagtaa atctcttttg caagctcgca 600
ttgggtcccg gcggaggcct gccgtcaaac cacctaacaa ctccagatgg ttgacctcgg 660
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence (ITS4)
<400> 2
tcctccgctt attgatatgc 20
<210> 3
<211> 22
<212> DNA
<213>Artificial sequence (ITS5)
<400> 3
ggaagtaaaa gtcgtaacaa gg 22
Claims (6)
1. vacation Escherichia WNF15 one plant oval, which is characterized in that the bacterial strain is preserved in Chinese allusion quotation on June 15th, 2018
Type culture collection, deposit number are CCTCC NO：M 2018373.
2. oval false Escherichia WNF15 according to claim 1, which is characterized in that the oval false Escherichia
WNF15 is cultivated on PDA, and white mycelium generates arthrospore, oval, spore size 2-3 × 2.5 μm without diaphragm；Bacterium colony is wadding
Shape, quality are loose.
3. oval false Escherichia WNF15 according to claim 1, which is characterized in that the oval false Escherichia
The ITS sequence of WNF15 is as shown in SEQ ID NO.1.
4. applications of the oval vacation Escherichia WNF15 described in claim 1 in sulfonated phenol formaldehyde resin contaminated soil of degrading.
5. oval vacation Escherichia WNF15 described in claim 1 or its bacteria suspension or its culture solution or its tunning are dropping
Solve the application in sulfonated phenol formaldehyde resin contaminated soil.
6. oval vacation Escherichia WNF15 described in claim 1 is in the biology for preparing degradation sulfonated phenol formaldehyde resin contaminated soil
Application in preparation.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111304099A (en) * | 2020-03-18 | 2020-06-19 | 中国石油天然气集团有限公司 | Composite microbial inoculum, preparation method thereof and method for treating drilling solid waste by using composite microbial inoculum |
| US20210274790A1 (en) * | 2020-03-05 | 2021-09-09 | Fraunhofer-Gesellschaft Zur Foerderung Der Angewandten Forschung E.V. | Methods of multi-species insect pest control |
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| JP2002155036A (en) * | 2000-11-17 | 2002-05-28 | Yamanouchi Pharmaceut Co Ltd | New antifungal compound and method for producing the same |
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| CN104056857A (en) * | 2013-03-22 | 2014-09-24 | 东北林业大学 | Electrically-assistant microbial remediation method of oil polluted soil |
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| JP2002155036A (en) * | 2000-11-17 | 2002-05-28 | Yamanouchi Pharmaceut Co Ltd | New antifungal compound and method for producing the same |
| CN1649996A (en) * | 2002-01-04 | 2005-08-03 | 宝洁公司 | Cellulose-active microorganisms |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US20210274790A1 (en) * | 2020-03-05 | 2021-09-09 | Fraunhofer-Gesellschaft Zur Foerderung Der Angewandten Forschung E.V. | Methods of multi-species insect pest control |
| CN111304099A (en) * | 2020-03-18 | 2020-06-19 | 中国石油天然气集团有限公司 | Composite microbial inoculum, preparation method thereof and method for treating drilling solid waste by using composite microbial inoculum |
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