CN104845890B - Applications of earth mould (Agromyces sp.) the MT E in a variety of phthalic acid esters of degrading - Google Patents
Applications of earth mould (Agromyces sp.) the MT E in a variety of phthalic acid esters of degrading Download PDFInfo
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- CN104845890B CN104845890B CN201510066068.4A CN201510066068A CN104845890B CN 104845890 B CN104845890 B CN 104845890B CN 201510066068 A CN201510066068 A CN 201510066068A CN 104845890 B CN104845890 B CN 104845890B
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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Abstract
The invention belongs to environment pollutant biological treatment technical field, specifically disclose earth mould (AgromycesSp.) applications of the MT E in a variety of phthalic acid esters of degrading, the bacterial strain was deposited in China typical culture collection center on January 22nd, 2015（CCTCC）, deposit number is CCTCC M 2015054.The bacterium can carry out aerobic degradation by sole carbon source of DBP and DEHP, and the degradation rate of 7 days is respectively up to 97.06% and 86.25% in the minimal medium that the DBP and DEHP respectively containing 200 mg/L are sole carbon source.After the bacterium is inoculated in into contaminated soil 10 days, DBP and DEHP degradation rate is respectively up to 77.29% and 55.51% in soil, and its efficient degradation rate illustrates that the bacterium has a good application prospect in PAEs contaminated soil reparations.
Description
Technical field
The present invention relates to environment pollutant biological treatment technical field, in particular it relates to earth mould (Agromycessp.)
Applications of the MT-E in a variety of phthalic acid esters of degrading.
Background technology
Phthalic acid ester, also known as phthalate ester, abridge PAEs, is the general designation of the ester of phthalic acid formation.Wherein, adjacent benzene
Dibutyl carboxylic acid（di-n- butylphthalate, DBP）And diisooctyl phthalate（di-(2-ethylhexyl)
Phthalate, DEHP）It is two kinds of most widely used plastic plasticizers, its content is up to 40 ~ 60% in PVC plastic.It is near several
Over 10 years, China's vinyl house and agricultural film usage amount are sharply increased.A large amount of with plastic sheeting use, increasing DBP
Agricultural land soil is released into DEHP, they turn into the organic pollution being most often detected in agricultural land soil, detects level
Mg/kg is not reached.Research shows, both phthalic acid esters have a high toxicity, teratogenesis, carcinogenicity and mutagenicity, with
And genotoxicity, by Environmental Protection Agency USA（EPA）It is classified as priority pollutants.Therefore, the DBP in China's agricultural soil
It is urgently to be resolved hurrily with DEHP pollution problems.
Because the chemical stability of both phthalic acid esters is fine, pass through the chemistry such as photodissociation, hydrolysis under natural conditions
The speed of event resolves is very slow, and its long half time is for 20 years, and most DBP and DEHP mainly pass through life
Thing degradation pathway disappears from environment.Wherein microbial degradation has cost low, mild condition, secondary pollution etc. is not produced excellent
Point, meets constructing environment friendly and requirement and the development trend of conservation-minded society.Therefore, made using the degraded of microorganism
It is to remove the best means that phthalic acid ester pollutes in environment with DBP and DEHP are converted into innocuous substance.
At present, domestic and foreign scholars filter out many bacterial strains with good degradation property for DBP and DEHP respectively, but
Can wherein degrade simultaneously both phthalic acid esters bacterial strain it is seldom.Earth mould（Agromycessp.）Be in soil extensively
The class Pseudomonas existed, some strains of the Pseudomonas are had been reported that at present, and have can efficient degradation phenol, agricultural chemicals Diacloden and resistance to heavy
The characteristic of metal, but there is not yet the report of its degradable phthalic acid ester.
The content of the invention
There is provided the application of one plant of earth fungal strain in order to overcome the above-mentioned deficiency of prior art by the present invention.
It is a further object to provide one kind degraded dibutyl phthalate and/or phthalic acid two are different pungent
The bacteria suspension of ester.
To achieve these goals, the present invention is achieved by following scheme：
Earth mould (AgromycesSp.) MT-E is in degraded dibutyl phthalate and diisooctyl phthalate
Application；
The bacterial strain was deposited in China typical culture collection center on January 22nd, 2015（CCTCC）, deposit number is
CCTCC M 2015054。
Separation screening obtains one plant of earth mould to inventor first from the long-term agricultural land soil using vinyl house
(AgromycesSp.) bacterial strain MT-E, the bacterial strain can degrade dibutyl phthalate and diisooctyl phthalate simultaneously；
Therefore, the claimed bacterial strain degrade at the same time in dibutyl phthalate and diisooctyl phthalate should
With.
Earth mould (AgromycesSp.) MT-E is in degraded dibutyl phthalate or diisooctyl phthalate
Application；
The bacterial strain was deposited in China typical culture collection center on January 22nd, 2015（CCTCC）, deposit number is
CCTCC M 2015054。
Because earth mould (AgromycesSp.) bacterial strain MT-E is the dibutyl phthalate that can degrade, and the neighbour that can degrade
Phthalic acid di-isooctyl；And dibutyl phthalate and diisooctyl phthalate are two kinds of plastics being most widely present
Pollutant, therefore the claimed bacterial strain is in degraded dibutyl phthalate or diisooctyl phthalate
Using.
Earth mould (AgromycesSp.) MT-E is situated between in dibutyl phthalate and diisooctyl phthalate pollution
Application in matter reparation；
The bacterial strain was deposited in China typical culture collection center on January 22nd, 2015（CCTCC）, deposit number is
CCTCC M 2015054。
Preferably, the medium is soil, water or air.
It is preferred embodiment that bacterial strain MT-E is prepared into bacteria suspension to be used to degrade when protecting the application of above bacterial strain
Dibutyl phthalate and/or diisooctyl phthalate.
Compared with prior art, the invention has the advantages that：
Separation screening obtains one plant of earth mould to the present invention first from the long-term agricultural land soil using vinyl house
(AgromycesSp.) bacterial strain MT-E, the bacterial strain can degrade dibutyl phthalate and diisooctyl phthalate simultaneously；
The germplasm resource bank of phthalic acid ester degradation bacteria is enriched, and the bacterial strain is widely distributed in soil, it is easy to cultivate, adapt to
Ability is strong；To have additionally by soil remediation description of test in the biological treatment of the actual contaminated soil of the bacterium before huge application
Scape.
Brief description of the drawings
Fig. 1 is the growthform feature that MT-E bacterial strains are cultivated 7 days on LB culture mediums.
Fig. 2 is the stereoscan photograph of MT-E bacterial strains.
Fig. 3 is the 16S rDNA of MT-E bacterial strains phylogenetic tree.
Fig. 4 is degradation kinetics and growth curve of the MT-E bacterial strains to DBP and DEHP.
Fig. 5 is degradation effect of the MT-E bacterial strains to DBP in contaminated soil and DEHP.
Embodiment
The present invention is further elaborated on reference to Figure of description and specific embodiment.Following examples of the present invention
For the present invention preferably embodiment, the present invention mainly illustrates the bacterial strain and the application thought based on the bacterial strain, implements
The replacement of simple parameter can not be repeated in embodiment one by one in mode, but therefore not limited the scope of the invention, its
He it is any without departing from Spirit Essence and the change made under principle of the present invention, modification, replacement, combine, simplification, should be considered as
The substitute mode of effect, within protection scope of the present invention.
Culture medium prescription is as follows described in embodiment：
Inorganic salts nutrient solution（MSM, g/L）：K2HPO4：5.8, KH2PO4：4.5, (NH4)2SO4：2.0, MgCl2：0.16,
CaCl2：0.02, Na2MoO4·2H2O：0.0024, FeCl3：0.0018, MnCl2·2H2O：0.0015.Final pH is 7.5.
Beef-protein medium（LB）：The g of dusty yeast 5.0, the g of peptone 10.0, the g of sodium chloride 10.0, plus ultra-pure water
To 1L, pH=7.0 are adjusted；121 DEG C of sterilizing 20min.Solid plate then adds 1.5%（w/v）Agar powder.
The separation and identification of the bacterial strain of embodiment 1
Collection using the agricultural land soil of vinyl house, weighs 5 g fresh soil samples in containing 50 mL sterilized waters throughout the year
In 150 mL triangular flasks, at 30 DEG C, 150 rpm take the addition of 5 mL sludge suspensions to contain 100 mL DBP and DEHP after cultivating 3 days
（Respectively 50 mg/L）MSM culture mediums in.It is continuously rich by 1 mL inoculum concentration every time after 150 rpm are cultivated 7 days through 30 DEG C
Collection, switching 10 times, and PAEs contents are accordingly improved in culture medium to 1000 mg/L.Then the nutrient solution for taming 10 times is diluted
103~105It is coated on LB solid plates, 30 DEG C are inverted culture 1 ~ 3 day.After single bacterium colony is grown on flat board, picking single bacterium colony is more
Secondary line purifying, separation obtains one plant of bacterium, numbering MT-E.Then inoculation is inverted in 30 DEG C on LB solid plates and cultivated
7 days, observe its colonial morphology（See Fig. 1）.As shown in Figure 1, bacterium colony is in dark yellow, flat, moistening, edge out-of-flatness, diffusion life
It is long, almost it is paved with flat board after seven days.
Scanning electron microscopic observation is identified：LB fluid nutrient medium of the bacterial strain MT-E accesses containing 10 mL after purification is stayed overnight into work
Change.Draw 800 μ L bacterium solutions and centrifuge 3 ~ 5 min through 8000 rpm, remove supernatant, add 500 μ L PBS solutions and wash bacterium 3 times.
2.5% glutaraldehyde that 1 mL is added in the bacterial sediment of harvest is fully mixed, and 4 DEG C stand overnight.Then again through 8000 rpm from
The min of the heart 3 ~ 5, removes supernatant, adds 500 μ L PBS solutions and washes bacterium 3 times.Then by thalline respectively 30%, 50%, 70%, 85%,
It is dehydrated 2 times in 90% and 100% graded ethanol, each gradient about soaks 15 min, supernatant is removed in then 8000 rpm centrifugations, most
Ethanol is replaced with isoamyl acetate 2 times, every time 20 min, method is with above-mentioned ethanol dehydration process afterwards.By CO2Film-making after drying
Observation.The visible MT-E thalline under ESEM of Fig. 2 are in rod-short, are about 0.8 ~ 1.5 micron, wide about 0.3 ~ 0.5 micron.
MT-E bacterial strains are subjected to physiological and biochemical property identification, qualification result is shown in Table 1.
The physiological and biochemical property identification of table 1.MT-E bacterial strains
| Physiological and biochemical test | As a result |
| Gram stain test | + |
| V-P is tested | - |
| MR is tested | + |
| Starch Hydrolysis is tested | - |
| Oxidase test | - |
| Contact enzyme reaction | + |
| Urea test | + |
| Indole test | - |
Note："+" represents the positive；"-" represents feminine gender
The 16S rDNA Molecular Identifications of MT-E bacterial strains：Bacteria total DNA is extracted, with bacterial 16 S rDNA universal primers to the bacterium
Genome enters performing PCR amplification.PCR primer is through sequencing（By Shanghai, life work completes sequencing）Afterwards, by sequencing result and GenBank
The 16S rDNA sequences of other bacterial strains of report carry out sequence analysis, and choose relevant bacteria species and do phylogenetic analysis（See Fig. 3）.
In addition, its morphological feature and physiological and biochemical index（It is shown in Table 1）Also with earth mould（Agromycessp.）Also it is the most similar, therefore
The degrading strain identification that present invention separation is obtained is earth mould（Agromycessp.）.
Therefore, inventor by earth mould (AgromycesSp.) bacterial strain MT-E is deposited in China typical culture collection center
（CCTCC）Preservation, preservation date is on January 22nd, 2015, and deposit number is CCTCC M 2015054；The Strain Designation isAgromycessp. MT-E.Preservation address is：Wuhan City, Hubei Province Wuhan University.
The earth mould of embodiment 2 (AgromycesSp.) bacterial strain MT-E is tested to DBP and DEHP degradation effect
S1. the preparation of bacteria suspension：MT-E bacterial strains after purification are accessed into the LB fluid nutrient medium activated overnights containing 10 mL
Culture centrifuges 10 min to logarithmic phase, through 5000 rpm and collects thalline, is resuspended after washing bacterium 3 times with PBS, adjusts OD600 nm =
0.8 is used as bacteria suspension.
S2. to containing 200 mg/L concentration DBP and DEHP（Respectively contain 200 mg/L）100 mL MSM nutrient solutions in be inoculated with
The above-mentioned mL of bacteria suspension 1, not connect bacterium as negative control, and it is 7.5, every group of three repetitions to adjust pH.In 30 DEG C, 150 rpm
Constant-temperature table culture 7 days, sampled respectively at 0,1,3,5,7 day and extracts sample, through DBP and DEHP in GC/MS determination samples
Degraded situation, while with the bacterial growth situation OD at spectrophotometric determination each time point600 nm。
Sample treatment：The mL of sample 100 after being cultivated in shaking flask is transferred to separatory funnel, 20 mL dichloros are added
The min of methane oscillation extraction 10, organic phase is collected with 150 mL triangular flasks（Lower phase）, the aqueous phase in separatory funnel adds 20
ML dichloromethane same procedure oscillation extraction 3 times, merges organic phase, then crosses the anhydrous Na dried containing 18 cm2SO4Glass
Glass post（1.5 cm×35 cm）It is dried, collects filtrate with heart bottle, rotated to be evaporated the rear mL of constant volume 10 to be measured.
Chromatographic condition：Using Shimadzu Corporation's QP2010 Plus type GC/MS tandem mass spectrometers.Chromatographic column is Agilent HP-5
Pillar (0.25 μm × 0.25 mm × 30 m), injector temperature is 250 DEG C, ion gun（EI）Temperature is 220 DEG C, using not
The μ L of split sampling 1, carrier gas is high-purity helium.Heating schedule is：Initial temperature is 100 DEG C, keeps 2 min, 15 DEG C/min ladders
Degree rises to 129 DEG C, is then warming up to 280 DEG C with 40 DEG C/min, keeps 5min.
Quality control：Standard curve is made using external standard method and six point calibration standard substances.6 kinds of PAEs mix target matrix and added
It is 90.5 ~ 107.6% to mark average recovery rate, and relative deviation is less than 10.7%, and instrument detection is limited to 0.62-1.22 ug/mL.
From accompanying drawing 4, with the extension of incubation time, DBP and DEHP residual quantity is significantly reduced in sample.Particularly
DBP, its degradation rate is higher than DEHP, is respectively 8.40% in the 1st, 3,5,7 day DBP degradation rate, and 40.64%, 89.30%,
97.06%；And DEHP degradeds are relatively slow, but with the extension of incubation time, remain to reach preferable degradation effect.The 1st,
3,5,7 days DEHP degradation rate is respectively 5.31%, 30.92%, 73.12%, 86.25%.In addition, as incubation time increases, MT-
The growth of E bacterial strains is gradually vigorous, illustrates that the bacterium can be good at carrying out growth and breeding using DBP and DEHP, in phthalic acid
Ester pollution environment it is biological prosthetic in have good application potential.
Repairing effects of the bacterial strain MT-E of embodiment 3 to DBP and DEHP contaminated soils
S1. for examination soil processing：Soil is Agricultural University Of South China's Farm Rice soil, crosses 60 mesh sieves after air-drying, pH is 6.7,
DBP and DEHP is added thereto, its content in soil is respectively reached 100 mg/kg, and add distilled water to be adjusted to field water holding
Amount（About 30%）, lucifuge aging 15 days.
S2. weigh respectively and above-mentioned contain 100 mg kg-1 The DBP and DEHP g of soil 200 adds in triangular flask into soil
Plus the mL of bacteria suspension 50, fully mix, the processing for not connecing bacterium in addition is control group.Soil is adjusted to field capacity（About 30%
Water content）, the lucifuge culture in 30 DEG C of insulating boxs, respectively at 0,2,4,6,8,10 day, periodically sampling extracting, was then surveyed through GC/MS
Determine DBP and DEHP residual quantities in soil.
Sample extraction：Weigh 1 g and air-dry the pedotheque ground in 35 mL glass centrifuge tubes, add 20 mL dichloro
Methane and the min of ultrasonic extraction 10, are then collected by centrifugation supernatant through 3500 rpm.20 mL dichloro is added in soil sample precipitation
Methane same procedure ultrasonic extraction 3 times, merges supernatant.Then rotated evaporation and concentration supernatant, clear with dichloromethane ultrasound
Pillar is crossed after washing（The cm of 1.0 cm × 35, filler is anhydrous Na from top to bottom2SO4, silica gel and aluminum oxide）, collected and filtered with heart bottle
Liquid is through N2After drying, dissolved again with chromatographic pure dichloromethane and be settled to 1 mL, treat that GC/MS is determined.
GC/MS chromatographic conditions are same as above.
Quality control：Standard curve is made using external standard method and six point calibration standard substances.6 kinds of PAEs mix target matrix and added
It is 92.5 ~ 110.2% to mark average recovery rate, and relative deviation is less than 10.5%, and instrument detection is limited to 0.12 ~ 0.45 ug g-1.The party
Method meets trace organic substance quantitative analysis requirement.
As shown in figure 5, compared with not connecing the control group of bacterium,AgromycesSp. MT-E bacterium are significantly enhanced in soil
DBP and DEHP degradation effect.The 0th after bacterium is connect, 2,4,6,8,10 days, the DBP residuals in soil are respectively 95.13 mg/
kg、92.46 mg/kg、82.45 mg/kg、63.49 mg/kg、34.08 mg/kg、21.16 mg/kg；DEHP's is residual in soil
Allowance is respectively 97.06 mg/kg, 94.28 mg/kg, 87.15 mg/kg, 76.36 mg/kg, 55.21 mg/kg, 43.18
mg/kg.Connecing the 10th day that bacterium is cultivated, DBP degradation rate improves about 77%, DEHP than control group and then improved about
55%.DBP and DEHP contents in soil are gradually reduced with the extension of repair time.It can be seen that,Agromyces sp. MT-E
Bacterium can significantly improve the elimination of DBP and DEHP in contaminated soil, can be used as DBP and DEHP contaminated soil bioremediation technologies
Preferable microorganism.
SEQUENCE LISTING
<110>Ji'nan University
<120>Earth mould (AgromycesSp.) applications of the MT-E in a variety of phthalic acid esters of degrading
<130>
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1408
<212> DNA
<213>The 16S rDNA sequences of MT-E bacterial strains
<400> 1
tgctttacca tgcagtcgaa cgatgaactt ggagcttgct ctgggggatt agtggcgaac 60
gggtgagtaa cacgtgagta acctgccctg gactctggga taacttcgag aaatcggagc 120
taataccgga taggaccttg caccgcatgg tgtggggtgg aaagtttttc ggtttgggat 180
ggactcgcgg cctatcagct tgttggtgag gtaatggctc accaaggcgt cgacgggtag 240
ccggcctgag agggtgaccg gccacactgg gactgagaca cggcccagac tcctacggga 300
ggcagcagtg gggaatattg cacaatgggc gcaagcctga tgcagcaacg ccgcgtgcgg 360
gatgacggcc ttcgggttgt aaaccgcttt tagtagggaa gaagccttcg ggtgacggta 420
cctgcagaaa aaggaccggc taactacgtg ccagcagccg cggtaatacg tagggtccga 480
gcgttgtccg gaattattgg gcgtaaagag ctcgtaggcg gtttgtcgcg tctgctgtga 540
aaactagagg ctcaacctct agcctgcagt gggtacgggc agacttgagt ggtgtagggg 600
agactggaat tcctggtgta gcggtggaat gcgcagatat caggaggaac accgatggcg 660
aaggcaggtc tctgggcact tactgacgct gaggagcgaa agcgtgggga gcgaacagga 720
ttagataccc tggtagtcca cgccgtaaac gttgggcgct agatgtgggg acctttccac 780
ggtttccgtg tcgtagctaa cgcattaagc gccccgcctg gggagtacgg ccgcaaggct 840
aaaactcaaa ggaattgacg ggggcccgca caagcggcgg agcatgcgga ttaattcgat 900
gcaacgcgaa gaaccttacc aaggcttgac atacacgaga acgggccaga aatggtcaac 960
tctttggaca ctcgtgaaca ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt 1020
gggttaagtc ccgcaacgag cgcaaccctc gtcgcatgtt gccagcacgt tatggtgggg 1080
actcatgtga gactgccggg gtcaactcgg aggaaggtgg ggatgacgtc aaatcatcat 1140
gccccttatg tcttgggctt cacgcatgct acaatggccg gtacaaaggg ctgcgatgtc 1200
gtaaggcgga gcgaatccca aaaagccggt ctcagttcgg attgaggtct gcaactcgac 1260
ctcatgaagt cggagtcgct agtaatcgca gatcagcaac gctgcggtga atacgttccc 1320
gggccttgta cacaccgccc gtcaagtcat gaaagtcggt aacacccgaa gccggtggcc 1380
taacccttgt gaggagccgt agaaggga 1408
Claims (5)
1. earth mould (Agromyces Sp.) MT-E degrades dibutyl phthalate and diisooctyl phthalate at the same time
In application；The bacterial strain was deposited in China typical culture collection center on January 22nd, 2015（CCTCC）, deposit number
For CCTCC M 2015054.
2. earth mould (Agromyces Sp.) MT-E is in degraded dibutyl phthalate or diisooctyl phthalate
Using；
The bacterial strain was deposited in China typical culture collection center on January 22nd, 2015（CCTCC）, deposit number is
CCTCC M 2015054。
3. earth mould (Agromyces Sp.) MT-E is repairing dibutyl phthalate and/or diisooctyl phthalate
Application in pollution medium；The bacterial strain was deposited in China typical culture collection center on January 22nd, 2015（CCTCC）,
Deposit number is CCTCC M 2015054.
4. application according to claim 3, it is characterised in that the medium is soil.
5. the application according to claim 1,2 or 3, it is characterised in that bacterial strain MT-E is prepared into bacteria suspension is used to degrade
Dibutyl phthalate and/or diisooctyl phthalate.
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| CN201510066068.4A CN104845890B (en) | 2015-02-09 | 2015-02-09 | Applications of earth mould (Agromyces sp.) the MT E in a variety of phthalic acid esters of degrading |
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| CN114107092B (en) * | 2021-11-02 | 2023-11-24 | 暨南大学 | Endophyte Gordonia L191 for degrading phthalate and application thereof |
| CN115261248B (en) * | 2022-02-18 | 2023-07-14 | 浙江工业大学 | Long side chain PAEs efficient degradation strain Gordonisp.GZ-YC 7 and application thereof |
| CN114535277B (en) * | 2022-03-04 | 2023-09-15 | 上海大学 | Application of iron-containing steel slag combined with thermal desorption technology in repairing phthalate polluted soil and repairing method thereof |
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