CN104894000B - Applications of Providence (Providencia sp.) 2D in dibutyl phthalate contaminated soil remediation - Google Patents
Applications of Providence (Providencia sp.) 2D in dibutyl phthalate contaminated soil remediation Download PDFInfo
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Abstract
The invention belongs to environment pollutant biological treatment technical field, specifically discloses Providence（Providenciasp.）Applications of the 2D in dibutyl phthalate contaminated soil remediation, the bacterium were deposited in China typical culture collection center on January 22nd, 2015（CCTCC）, deposit number is CCTCC M 2015057；Providence is inoculated with DBP contaminated soilsProvidenciasp.2D；In the 10th day DBP degradation rate up to 65%, 46% is improved compared to control；Compost is with the addition of in addition and is connect in the soil of bacterium, and its tenth day DBP degradation rate shows that compost can strengthen the degraded of DBP in soil, incited somebody to action up to 88%ProvidenciaSp.2D bacterium are used in combination with compost, can efficiently repair DBP contaminated soils, are with a wide range of applications to reducing DBP pollutions in Agricultural Soil Environment.
Description
Technical field
The present invention relates to environment pollutant biological treatment technical field, more particularly, to Providence
Applications of (Providencia sp.) 2D in dibutyl phthalate contaminated soil remediation.
Background technology
Dibutyl phthalate (di-n-butylphthalate, DBP) belongs to phthalate compound
(Phthalic acidester, PAEs), is one of most important plasticizer of plastics industry, and agricultural chemicals, dyestuff detergent
Carrier and many cosmetics, the raw materials for production of textile, are widely used in packaging material for food, container, medicine equipment and youngster
The fields such as virgin toy.Due to the extensive application of said products, DBP has become global organic pollution, is widely present in
In air, water body, soil and organism.Particularly in agricultural production, because plastic sheeting and the extensive of vinyl house should
With remaining in DBP in agricultural soil and be easily enriched with by food chain in human body.DBP and its metabolin can cause to body
A variety of damages such as genotoxicity, development toxicity, neurotoxicity, multiple organ canceration, therefore Environmental Protection Agency (EPA) and China national
It has all been classified as priority pollutants by (CNEMC) at environmental monitoring center.
Research shows, all very slowly, it is main that biodegradation is that it is decomposed in the environment for DBP hydrolysis and water splitting
Approach.Therefore, it is the important step for improving its biodegradable efficiency to obtain DBP efficient degrading bacterias.Using microbiological deterioration,
DBP is converted into innocuous substance or permineralization, is the generally acknowledged best means for removing DBP pollutions in environment.Due in environment
DBP generally existing, the frequency of occurrences of DBP degradation bacterias is also very high, and it is dagger-axe to have most of microorganism of degradation to DBP at present
Bordetella, Agrobacterium, acinetobacter and Rhodococcus ruber category etc. are stepped on, but these bacteriums reported drop for DBP biology
Solution is incomplete, can produce the higher mesostate of toxicity, or its degradation rate is not met by actual contamination control
It is required that therefore still need to that screening is more efficient obligate or facultative degradation bacteria.
In agricultural production, the DBP pollutions of soil have obtained people's extensive concern.The microorganism of DBP contaminated soils is repaiied
The matter of utmost importance for needing to solve in recovering technology is screening and the structure of efficient degrading bacterial strain.Providencia
(Providencia) it is a kind of bacterium for being widely present in compost, there is no the report on its PAEs that degrades at present.In addition, heap
It is fertile that there is the advantages such as efficient, environmental protection and low cost in terms of soil texture and protection agroecological environment is improved, obtained with reference to screening
The efficient degrading bacteria obtained, pollute the prospect that is widely used to reducing DBP in Agricultural Soil Environment.
The content of the invention
The present invention is in order to overcome the above-mentioned deficiency of prior art, there is provided Providence Providenciasp.2D is in neighbour
Application in dibatyl phithalate contaminated soil remediation.
The purpose of the present invention is achieved by the following technical programs：
Applications of Providence (Providencia sp.) 2D in DBP contaminated soil remediations, the Providian
This bacterium Providencia sp.2D was deposited in China typical culture collection center (CCTCC), preservation on January 22nd, 2015
Numbering is CCTCC M 2015057, and preservation address is：Wuhan City, Hubei Province Wuhan University.
The matter of utmost importance for needing to solve in the microorganism remediation technology of DBP contaminated soils be efficient degrading bacterial strain screening with
Structure.Providencia (Providencia) is a kind of bacterium being widely present in compost, be there is no at present on its degraded
PAEs report.Present invention separation screening from animal dunghill fertilizer, which obtains one plant, has efficient, degradable performance to DBP
Providence, Providencia sp.2D are named as, and find that the bacterium can effectively reduce the DBP pollutions in soil, be reason
The Soil Environmental Pollution thought repairs microorganism.
The bacterial strain Providencia sp.2D are enriched with from farm-animals excrement compost sample by enrichment culture method and obtained
, the optimum growing condition of the bacterium is：30~40 DEG C of temperature, pH are 7.0~8.5.
The bacterial strain Providencia sp.2D (dusty yeast 5.0g, peptone on beef-protein medium (LB)
10.0g, sodium chloride 10.0g, pH=7.0) culture 7 days of ruling, bacterium colony is in crocus, quality paste, abundant moistening, is easily chosen
Rise, edge is relatively thin；It is in elongated rod shape under SEM, is about 1.5~3.0 μm, it is wide about 0.5~0.8 μm；Contaminated through gram
Color identifies that the bacterium is Gram-negative bacteria.
The Physiology and biochemistry identification experiment result of the bacterial strain Providencia sp.2D is as follows：Methyl red test (positive),
V-P experiments (feminine gender), indole reaction (positive), citrate utilize (positive), inositol production acid experiment (positive), glucose production
Acid experiment (positive), Starch Hydrolysis experiment (feminine gender), urine enzyme test (positive), gelatin liquefaction test (feminine gender), nitrate reduction
(positive), oxidase test (feminine gender) and glucose aerogenesis experiment (feminine gender).
The 16S rDNA gene orders such as SEQ ID NO of the bacterial strain Providencia sp.2D：Shown in 1, pass through NCBI
Official website (http://www.ncbi.nlm.nih.gov/) blast program with other logged in bacterium bacterial strain 16S rDNA
Sequence is compared, the results showed that the bacterial strain and Providencia sp. similitude highests, homology is up to 99%.
Preferably, above-mentioned application is：Providence Providencia sp.2D are inoculated with DBP contaminated soils,
The dosage of the Providence Providencia sp.2D is：Add in contaminated soil per 200g DBP containing 100mg/kg
Add Providence Providencia sp.2D bacteria suspensions (OD600nm=0.8) 50mL.
The application of above-mentioned Providence Providencia sp.2D degraded dibutyl phthalate is specially：Will
The soil that obtained bacteria suspension access is polluted by dibutyl phthalate after Providence Providencia sp.2D activation
In earth.
As a kind of preferred technical scheme, above-mentioned application is to pass through Providence Providencia sp.2D
It is activated overnight, concussion and cultivate to logarithmic phase, collects thalline, will according to being actually needed regulation thalline content and bacteria suspension being made
Obtained bacteria suspension access is in by dibutyl phthalate contaminated soil.
In addition, inventor's research is it has also been found that soil can be further enhanced by adding animal excreta compost simultaneously in DBP contaminated soils
DBP degraded in earth, therefore, during concrete application of the present invention, it can also be added in DBP contaminated soils dynamic
Thing dunghill fertilizer accesses Providence Providencia sp.2D again after mixing；The animal excreta compost and DBP contaminated soils
Mass ratio be 1：15.
The present invention also provides Providence Providencia sp.2D and is preparing DBP contaminated soil remediation modifying agents
In application.
Compared with prior art, the invention has the advantages that：
The invention provides applications of the Providence Providencia sp.2D in DBP contaminated soil remediations, tool
Body is that Providence Providencia sp.2D are inoculated with DBP contaminated soils；The soil of 2D bacterium is connect at the 10th day
DBP residual quantities are 34.46mg/kg, and degradation rate has reached 65%, and the control soil DBP degradation rates for not connecing bacterium are only 19%, phase
46% is improved than control；In addition, in it with the addition of compost and connect the soil of bacterium, its tenth day DBP degradation rate reaches
88%, show that compost can strengthen the degraded of DBP in soil, Providenciasp.2D bacterium are used in combination with compost, can be efficient
DBP contaminated soils are repaired, pollute the prospect that is widely used to reducing DBP in Agricultural Soil Environment.
Brief description of the drawings
Fig. 1 is the growthform that Providencia sp.2D are cultivated 7 days on LB culture mediums.
Fig. 2 is Providencia sp.2D scanning electron microscope (SEM) photograph.
Fig. 3 is Providencia sp.2D 16SrDNA phylogenetic tree.
Fig. 4 is Providencia sp.2D to DBP and the degradation kinetics curve of phthalic acid.
Fig. 5 is degradation effects of the Providencia sp.2D to DBP in different soils processing.
Embodiment
Present disclosure is further illustrated with reference to Figure of description and specific embodiment, but should not be construed as to this
The limitation of invention.Without departing from the spirit and substance of the case in the present invention, the inventive method, step or condition are made simple
Modifications or substitutions, belong to the scope of the present invention；Unless otherwise specified, technological means used in embodiment is art technology
Conventional meanses known to personnel.
The separation and identification of the dibutyl phthalate degradation bacterium of embodiment 1
1st, culture medium prescription
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；Adjust inorganic salts culture
The final pH of liquid is 7.5.DBP is added, it is 50mg/L to make its concentration in inorganic salts nutrient solution, and DBP is as sole carbon source.
Beef-protein medium (LB)：Dusty yeast 5.0g, peptone 10.0g, sodium chloride 10.0g, add ultra-pure water extremely
1L, adjust pH=7.0.121 DEG C sterilize 20 minutes.Solid plate then adds 1.5% (W/V) agar powder.
2nd, separation and identification
Separation method uses sample suspension fask oscillating method：5g compost samples (being derived from farm-animals compost) are weighed in containing 50mL
In the 150mL triangular flasks of sterilized water, at 30 DEG C, take 5mL compost suspension to add 100mL after being cultivated 3 days under the conditions of 140rpm and contain
In DBP (50mg/L) above-mentioned MSM culture mediums.Through 30 DEG C, after being cultivated 7 days under 140rpm, the inoculum concentration for pressing 1mL every time connects
Continuous enrichment, switching 10 times, and accordingly improve in MSM culture mediums DBP contents and (transfer for second, in MSM culture mediums to 1000mg/L
DBP content is 100mg/L, is often transferred once afterwards, and DBP content improves 100mg/L in MSM culture mediums).Then will switching
The nutrient solution dilution 10 of 10 times3~105It is coated on LB solid plates, 30 DEG C are inverted culture 1~3 day.Treat to grow single bacterium on flat board
Fall behind, picking single bacterium colony is repeatedly rule purifying, separation one plant of bacterium of acquisition, numbering 2D.Then by inoculation in LB solids
Culture 7 days is inverted on flat board for 30 DEG C, observes its colonial morphology.The bacterium colony of LB flat boards culture 7 days is in crocus, and quality paste is rich
Thickness moistening, is easily provoked, edge is relatively thin (see Fig. 1).
Scanning electron microscope example prepares and observation：Strain 2D LB fluid nutrient medium of the single bacterium colony access containing 10mL is stayed overnight
Activation.Draw 800 μ L bacterium solutions and centrifuge 3~5min through 8000rpm, remove supernatant, add 500 μ L PBS and wash bacterium 3 times.Harvesting
Bacterial sediment in add 1mL 2.5% (v/v) glutaraldehyde fully mix, 4 DEG C stand overnight.Then again through 8000rpm from
3~5min of the heart, supernatant is removed, add 500 μ L PBS and wash 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 15min, and supernatant is removed in then 8000 rpm centrifugations,
Finally ethanol 2 times, each 20min, the same ethanol dehydration of method are replaced with isoamyl acetate.Thalline passes through CO2Film-making is seen after drying
Examine.It is observed that the bacterium is in elongated rod shape under ESEM, 1.5~3.0 μm are about, wide about 0.5~0.8 μm (see Fig. 2).
The Physiology and biochemistry identification of indicator of bacterial strain includes 12：Methyl red test, V-P experiments, indole reaction, citrate profit
Tested with the experiment of, inositol production acid, the experiment of glucose production acid, Starch Hydrolysis, urine enzyme test, gelatin liquefaction test, nitrate reduction,
Oxidase test and the experiment of glucose aerogenesis.Specific experiment the results are shown in Table 1.
The physiological and biochemical property of the 2D bacterium of table 1
| Physiological and biochemical test | As a result | Physiological and biochemical test | As a result |
| Methyl red | + | Starch Hydrolysis | - |
| V-P is tested | - | Enzyme is urinated to produce | + |
| Indole reaction | + | Gelatin liquefaction (20 DEG C) | - |
| Citrate utilizes | + | Nitrate reduction | + |
| Inositol production acid | + | Oxidase test | - |
| Glucose production acid | + | Glucose aerogenesis | - |
+：It is positive；-：It is negative
The 16S rDNA identifications of bacterial strain：Bacteria total DNA is extracted, the bacterium genome is entered with bacterial 16 S rDNA universal primers
Performing PCR expands.PCR primer is after sequencing (being completed by Shanghai life work), the 16S that will report in sequencing result and GenBank
RDNA sequences carry out sequence analysis, and if choose dry strain do phylogenetic analysis.As shown in figure 3, the present invention isolates and purifies
(Providencia stuartii, culture presevation number are NBRC to the bacterial strain 2D arrived 16S rDNA sequences with Providence
12930) for homology up to 99%, evolutionary distance is nearest.Other strains also have compared with high homology with the Pseudomonas.Therefore, present invention sieve
The bacterial strain that choosing obtains is accredited as Providencia, is named as Providenciasp.2D.
Embodiment 2Providenciasp.2D is to DBP and its degradation experiment of intermediate product phthalic acid
1st, prepared by bacteria suspension
LB fluid nutrient medium of the strain Providenciasp.2D accesses containing 10mL after purification is activated overnight culture
To logarithmic phase, thalline is collected through 5000rpm centrifugations 10min, is resuspended after washing bacterium 3 times with PBS, adjusts OD600nm=0.8 is used as strain
Suspension.
2nd, degradation capability determines
Respectively to containing various concentrations DBP (50,100,200,500,1000mg/L) and phthalic acid (25,50,100,
200th, 500mg/L) MSM nutrient solutions (inorganic salts nutrient solution 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) connect in
Bacterium 1mL, not connect bacterium as control, and it is 8.0 to adjust pH, every group of three repetitions.At 30 DEG C, 140rpm constant-temperature tables culture 6
My god, periodically sampling, the degraded situation through GC/MS measure DBP and phthalic acid.
Chromatographic condition：Using Shimadzu Corporation QP2010Plus type GC/MS tandem mass spectrometers.Chromatographic column is Agilent HP-5 posts
Sub (0.25 μ m 0.25mm × 30m), injector temperature are 250 DEG C, and ion gun (EI) temperature is 220 DEG C, using Splitless injecting samples 1
μ L, carrier gas are high-purity helium.Heating schedule is：Initial temperature is 100 DEG C, keeps 2min, and 15 DEG C/min gradients rise to 129
DEG C, 280 DEG C then are warming up to 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 92.5~110.2% to mark average recovery rate, and relative deviation is less than 10.5%, and instrument detection is limited to 0.12~0.45ug/g.Should
Method meets trace organic substance quantitative analysis requirement.
As a result it is as shown in Figure 4：DBP (1000mg/L) degradation efficiency of Providenciasp.2D bacterium in 72h to high concentration
More than 80%, when DBP concentration is less than 200mg/L, DBP is almost degradable in 72h, and it is efficient to show that the bacterium has to DBP
Degradation capability.The degradation capability of DBP intermediate products is analyzed, it can be seen that the degraded energy to high concentration phthalic acid of the bacterium
Power is substantially less than DBP, but low concentration phthalic acid (≤100mg/L) degradation capability relatively (is almost dropped by force completely in 144h
Solution)；When O-phthalic acid concentration is 200mg/L, 144h degradation rate more than 90%.Illustrate main metabolic of the bacterium to DBP
Product phthalic acid also has stronger degradation capability, most DBP permineralizations at last.
Study on degradation of the embodiment 3Providenciasp.2D to DBP in contaminated soil
1st, for trying soil processing
Soil is Agricultural University Of South China's Farm Rice soil, pH 6.7, C/N 5.98,60 mesh sieves is crossed after air-drying.Animal excreta
Compost picks up from the farm, pH 8.8, C/N 17.49.Adding proportion (compost is often used according to farm：Soil=1：15, w/w)
Compost is added in rice soil, mixes and obtains pH=7.54, C/N is 10.8 conditioned soil.Composting soil (routine is not added with by above-mentioned
Soil) and conditioned soil add DBP respectively, its concentration is reached 100mg/kg.
1st, DBP degradation effects are analyzed in soil
Handled below Setup Experiments：The conventional native and conditioned soil of bacterium is connect as experimental group；Conventional soil and the improvement of bacterium are not connect
Soil as a control group one；Sterilized conditioned soil and first sterilizing connects the conditioned soil of 2D bacterium as a control group two, every group of three weights again
It is multiple.The above-mentioned DBP containing 100mg/kg soil 200g is weighed respectively in triangular flask, and bacteria suspension (OD is added into soil600nm=
0.8) 50 mL, fully mix.Soil is adjusted to field capacity (about 30% water content), lucifuge is trained in 30 DEG C of insulating boxs
Support, periodically sampling simultaneously determines DBP residual quantities in soil through GC/MS.
By the Providenciasp.2D of 10 days biological reinforced repair process, contaminated soil DBP is remained in each processing
Amount dynamic change is shown in Fig. 5.Connect DBP degradation effects in the conventional soil of 2D bacterium to significantly increase, at the 0th, 2,4,6,8,10 day, in soil
DBP residual quantities be respectively 98.41mg/kg, 88.13mg/kg, 75.40mg/kg, 61.25mg/kg, 44.85mg/kg and
34.46mg/kg, the degradation rate at the 10th day has reached 65%, and the control soil DBP degradation rates for not connecing bacterium are only 19%, are said
Bright Providenciasp.2D bacterium can significantly increase the degraded of DBP in soil.In addition, it with the addition of compost and connecing the improvement of bacterium
In soil, bacterium soil is connect relative to be not added with compost, DBP degradation effect further enhances, and its tenth day DBP degradation rate reaches
88%, show that compost can strengthen the degraded of DBP in soil, Providenciasp.2D bacterium are used in combination with compost, can be high
Effect repairs DBP contaminated soils.
In addition, the degradation efficiency for not sterilizing and being inoculated with DBP in the conditioned soil of 2D bacterium is significantly higher than sterilized and inoculation 2D bacterium
Conditioned soil, illustrate that the indigenous microorganism in 2D bacterium and conditioned soil is formed and act synergistically, promote the degraded of the DBP in soil.
SEQUENCE LISTING
<110>Ji'nan University
<120>Providence（Providencia
Sp.）Applications of the 2D in dibutyl phthalate contaminated soil remediation
<130>
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1445
<212> DNA
<213>16S rDNA sequences
<400> 1
gagcatggcg gcagctacac atgcaagtcg agcggtaaca ggagaagctt gcttctcgct 60
gacgagcggc ggacgggtga gtaatgtatg gggatctgcc cgatagaggg ggataactac 120
tggaaacggt agctaatacc gcataatctc taaggagcaa agcaggggaa cttcggtcct 180
tgcgctatcg gatgaaccca tatgggatta gctagtaggt gaggtaatgg ctcacctagg 240
cgacgatccc tagctggtct gagaggatga tcagccacac tgggactgag acacggccca 300
gactcctacg ggaggcagca gtggggaata ttgcaccaat gggcgcaagc ctgatgcagc 360
catgccgcgt gtatgaagaa ggccctaggg ttgtaaagta ctttcagtcg ggaggaaggc 420
gttgatgcta atatcatcaa cgattgacgt taccgacaga agaagcaccg gctaactccg 480
tgccagcagc cgcggtaata cggagggtgc aagcgttaat cggaattact gggcgtaaag 540
cgcacgcagg cggttaatta agttagatgt gaaatccccg ggcttaacct gggaatggca 600
tctaaaactg gttagctaga gtcttgtaga ggggggtaga attccatgtg tagcggtgaa 660
atgcgtagag atgtggagga ataccggtgg cgaaggcggc cccctggaca aagactgacg 720
ctcaggtgcg aaagcgtggg gagcaaacag gattagatac cctggtagtc cacgctgtaa 780
acgatgtcga tttgaaggtt gttcccttga ggagtggctt tcggagctaa cgcgttaaat 840
cgaccgcctg gggagtacgg ccgcaaggtt aaaactcaaa tgaattgacg ggggcccgca 900
caagcggtgg agcatgtggt ttaattcgat gcaacgcgaa gaaccttacc tactcttgac 960
atccagagaa cttagcagag atgctttggt gccttcggga actctgagac aggtgctgca 1020
tggctgtcgt cagctcgtgt tgtgaawtgt tgggttaagt cccgcaacga gcgcaaccct 1080
tatcctttgt tgccagcgat tcggtcsgga actcaaagga gactgccggk gatamaccgg 1140
aggaaggtgg ggatgacgtc aagtcatcat ggcccttacg agtagggcta cacacgtgct 1200
acaatggcgt atacaaagag aagcgacctc gcgagagcaa gcggaactca taaagtacgt 1260
cgtagtccgg attggagtct gcaactcgac tccatgaagt cggaatcgct agtaatcgta 1320
gatcagaatg ctacggtgaa tacgttcccg ggccttgtac acaccgcccg tcacaccatg 1380
ggagtgggtt gcaaaagaag taggtagctt aaccttcggg agggcgctac cactttgatc 1440
gtgtt 1445
Claims (5)
1. Providence（Providenciasp.）Applications of the 2D in DBP contaminated soil remediations, it is characterised in that should
Bacterium was deposited in China typical culture collection center on January 22nd, 2015（CCTCC）, deposit number is CCTCC M
2015057。
2. Providence（Providenciasp.）Applications of the 2D in DBP contaminated soil remediation modifying agents are prepared, it is special
Sign is that the bacterium was deposited in China typical culture collection center on January 22nd, 2015（CCTCC）, deposit number CCTCC
M 2015057。
3. application according to claim 1, it is characterised in that the application is by Providence（Providencia
sp.）After 2D activation in obtained bacteria suspension access DBP contaminated soils.
4. application according to claim 3, it is characterised in that animal excreta compost is added in DBP contaminated soils and is mixed
Providence is accessed after even again（Providenciasp.）2D bacteria suspensions.
5. application according to claim 4, it is characterised in that the mass ratio of the animal excreta compost and DBP contaminated soils
For 1：15.
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| CN201510066107.0A CN104894000B (en) | 2015-02-09 | 2015-02-09 | Applications of Providence (Providencia sp.) 2D in dibutyl phthalate contaminated soil remediation |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1370227A (en) * | 1999-08-20 | 2002-09-18 | 李性器 | Bacterial consortium EBC1000 and method using bacterial consortium EBC 1000 for remedying biologically recalcitrant toxic chemicals contained in industrial wastewater etc. |
| CN102703347A (en) * | 2012-05-25 | 2012-10-03 | 武汉科技大学 | Dibutyl phthalate degrading bacteria and application of dibutyl phthalate degrading bacteria |
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