CN104894000A - Application of Providencia sp. 2D in remediation of dibutyl phthalate contaminated soil - Google Patents
Application of Providencia sp. 2D in remediation of dibutyl phthalate contaminated soil Download PDFInfo
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- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
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Abstract
The invention belongs to the technical field of biological treatment of environmental pollutants, and particularly discloses application of Providencia sp. 2D in remediation of dibutyl phthalate (DBP) contaminated soil. The Providencia sp. 2D is preserved in a China center for type culture collection (CCTCC) on January 22, 2015; the preservation number is CCTCC M 2015057. The Providencia sp. 2D is inoculated in the DBP contaminated soil; the DBP degradation rate reaches 65% on the tenth day and is improved by 46% relatively. In addition, the DBP degradation rate reaches 88% on the tenth day in the inoculated soil added with compost, and it shows that the compost can enhance degradation of DBP in the soil. The DBP contaminated soil can efficiently recover by combining the Providencia sp. 2D with the compost; the application has wide application prospects for reducing the DBP pollution in the agricultural soil environment.
Description
Technical field
The present invention relates to environment pollutant biological treatment technical field, more specifically, relate to Providence (
providenciasp.) application of 2D in dibutyl phthalate contaminated soil remediation.
Background technology
Dibutyl phthalate (di-
n-butylphthalate, DBP) phthalate compound (Phthalic acidester is belonged to, PAEs), it is one of topmost softening agent of plastics industry, also be the raw materials for production of agricultural chemicals, the carrier of dyestuff stain remover and many makeup, textiles, be widely used in the fields such as packaging material for food, container, medicine equipment and toy for children.Due to the extensive application of said products, DBP has become global organic pollutant, is extensively present in air, water body, soil and organism.Particularly in agriculture production, due to the widespread use of plastics film and plastic greenhouse, to remain in agricultural soil DBP very easily by food chain enrichment in human body.DBP and metabolite thereof can cause the multiple damage such as genotoxicity, development toxicity, neurotoxicity, multiple organ canceration to body, and therefore it is classified as priority pollutants by Environmental Protection Agency (EPA) and China National Environmental Monitoring Center (CNEMC) all.
Research shows, all slowly, biological degradation is the main path that it decomposes in the environment for the hydrolysis of DBP and water splitting.Therefore, obtaining DBP efficient degrading bacteria is the important step improving its biological degradation efficiency.Utilizing microbiological deterioration, DBP is converted into innoxious substance or permineralization, is the best means that in generally acknowledged removal environment, DBP pollutes.Due to the ubiquity of DBP in environment, the frequency of occurrences of DBP degradation bacteria is also very high, the microorganism great majority of Degradation are had to be Gordona, Agrobacterium, acinetobacter and Rhodococcus ruber genus etc. to DBP at present, but these bacteriums reported are incomplete for the biological degradation of DBP, the mesostate that toxicity is higher can be produced, or its degradation rate can't meet the requirement of actual Environmental capacity, therefore still need screening obligate or facultative degradation bacteria more efficiently.
In agriculture production, the DBP of soil pollutes and has obtained people's extensive concern.The matter of utmost importance solved is needed to be screening and the structure of efficient degrading bacterial strain in the microorganism remediation technology of DBP contaminated soil.Providencia (
providencia) be the bacterioid extensively existed in compost, there is no the report about its degraded PAEs at present.In addition, compost, improving advantages such as having efficient, environmental protection and low cost in Soil structure and protecting agriculture ecotope, combines the efficient degrading bacteria that screening obtains, and to be widely used prospect to DBP pollution in reduction Agricultural Soil Environment.
Summary of the invention
The present invention, in order to overcome the above-mentioned deficiency of prior art, provides Providence
providenciasp. the application of 2D in dibutyl phthalate contaminated soil remediation.
The object of the invention is to be achieved by the following technical programs:
Providence (
providenciasp.) application of 2D in DBP contaminated soil remediation, described Providence
providenciasp. 2D is deposited in China typical culture collection center (CCTCC) on January 22nd, 2015, and deposit number is CCTCC M 2015057, and preservation address is: Wuhan City, Hubei Province Wuhan University.
The matter of utmost importance solved is needed to be screening and the structure of efficient degrading bacterial strain in the microorganism remediation technology of DBP contaminated soil.Providencia (
providencia) be the bacterioid extensively existed in compost, there is no the report about its degraded PAEs at present.The present invention's separation screening from animal dunghill fertilizer obtains a strain has efficient, degradable performance Providence to DBP, called after
providenciasp. 2D, and find that the DBP that this bacterium can effectively reduce in soil pollutes, be that desirable Soil Environmental Pollution repairs microorganism.
Described bacterial strain
providenciasp. 2D is obtained by enrichment culture method enrichment from farm-animals excrement compost sample, and the optimum growing condition of this bacterium is: temperature 30 ~ 40 DEG C, and pH is 7.0 ~ 8.5.
Described bacterial strain
providenciasp. 2D goes up (yeast powder 5.0 g, peptone 10.0 g, sodium-chlor 10.0 g, pH=7.0) streak culture 7 days at beef-protein medium (LB), and bacterium colony is safran, quality paste, abundant moistening, and easily provoked, edge is thinner; In elongated rod shape under scanning electronic microscope, be about 1.5 ~ 3.0 μm, wide about 0.5 ~ 0.8 μm; Identify that this bacterium is Gram-negative bacteria through gramstaining.
Described bacterial strain
providenciasp. the Physiology and biochemistry identification experiment result of 2D is as follows: methyl red test (positive), V-P test (feminine gender), indole reaction (positive), Citrate trianion utilization (positive), inositol produce acid test (positive), the sour test (positive) of glucose product, Starch Hydrolysis test (feminine gender), urine enzyme test (positive), gelatin liquification test (feminine gender), nitrate reduction (positive), oxidase test (feminine gender) and glucose aerogenesis and test (feminine gender).
Described bacterial strain
providenciasp. the 16S rDNA gene order of 2D is as shown in SEQ ID NO:1, has logged in bacterial isolates 16S rDNA sequence compare by the blast program of NCBI official website (http://www.ncbi.nlm.nih.gov/) and other, result show this bacterial strain with
providenciasp. similarity is the highest, and homology reaches 99%.
Preferably, above-mentionedly to be applied as: in DBP contaminated soil, inoculate Providence Providencia sp.2D, the consumption of described Providence Providencia sp.2D is: every 200g adds Providence Providencia sp.2D bacteria suspension (OD containing in the contaminated soil of 100mg/kg DBP
600nm=0.8) 50mL.
Above-mentioned Providence
providenciasp. the application of 2D degraded dibutyl phthalate is specially: by Providence
providenciasp. obtained after 2D activation bacteria suspension access is by dibutyl phthalate contaminated soil.
As the preferred technical scheme of one, above-mentioned application is by Providence
providenciasp. 2D is cultured to logarithmic phase through activated overnight, concussion, collects thalline, regulates thalline content according to actual needs and makes bacteria suspension, by the access of obtained bacteria suspension by dibutyl phthalate contaminated soil.
In addition, contriver's research also finds in DBP contaminated soil, add the degraded that animal excreta compost can strengthen DBP in soil further simultaneously, therefore, in the process of embody rule of the present invention, the mixing of animal excreta compost can also be added in DBP contaminated soil after, access Providence again
providenciasp. 2D; The mass ratio of described animal excreta compost and DBP contaminated soil is 1:15.
The present invention also provides Providence
providenciasp. the application of 2D in preparation DBP contaminated soil remediation modifying agent.
Compared with prior art, the present invention has following beneficial effect:
The invention provides Providence
providenciasp. the application of 2D in DBP contaminated soil remediation, specifically inoculates Providence in DBP contaminated soil
providenciasp. 2D; The soil connecing 2D bacterium is 34.46 mg/kg the 10th day DBP residual quantity, and degradation rate reaches 65%, and the contrast soil DBP degradation rate not connecing bacterium is only 19%, compares according to improve 46%; In addition, with the addition of compost and connecing in the soil of bacterium, its DBP degradation rate of the tenth day reaches 88%, shows that compost can strengthen the degraded of DBP in soil, will
providenciasp. 2D bacterium is combined with compost, can efficiently repair DBP contaminated soil, pollutes to DBP in reduction Agricultural Soil Environment the prospect that is widely used.
Accompanying drawing explanation
Fig. 1 is the growthhabit that Providencia sp. 2D cultivates 7 days on LB substratum.
Fig. 2 is
providenciasp. the scanning electron microscope (SEM) photograph of 2D.
Fig. 3 is
providenciasp. the phylogenetic tree of the 16SrDNA of 2D.
Fig. 4 is
providenciasp. 2D is to the degradation kinetics curve of DBP and phthalic acid.
Fig. 5 is
providenciasp. 2D is to the degradation effect of DBP in different soils process.
Embodiment
Further illustrate content of the present invention below in conjunction with Figure of description and specific embodiment, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, the simple modification do the inventive method, step or condition or replacement, all belong to scope of the present invention; If do not specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art.
the Isolation and ldentification of embodiment 1 dibutyl phthalate degradation bacterium
1, culture medium prescription
Inorganic salt nutrient solution (MSM, g/L): K
2hPO
4: 5.8; KH
2pO
4: 4.5; (NH
4)
2sO
4: 2.0; MgCl
2: 0.16; CaCl
2: 0.02; Na
2moO
42H
2o:0.0024; FeCl
3: 0.0018; MnCl
22H
2o:0.0015; The final pH regulating inorganic salt nutrient solution is 7.5.Add DBP, make its concentration in inorganic salt nutrient solution be 50 mg/L, DBP is as sole carbon source.
Beef-protein medium (LB): yeast powder 5.0 g, peptone 10.0 g, sodium-chlor 10.0 g, adds ultrapure water to 1L, regulates pH=7.0.121 DEG C of sterilizings 20 minutes.Solid plate then adds 1.5%(W/V) agar powder.
2, Isolation and ldentification
Separation method adopts sample suspension fask oscillating method: take 5 g compost samples (taking from farm-animals compost) in the 150 mL triangular flasks containing 50 mL sterilized waters, at 30 DEG C, cultivate under 140 rpm conditions and get 5 mL compost suspensions after 3 days and add 100 mL containing DBP(50 mg/L) above-mentioned MSM substratum in.Through 30 DEG C, cultivate under 140 rpm after 7 days, press the continuous enrichment of inoculum size of 1 mL, switching 10 times at every turn, and DBP content to 1000 mg/L(second time switching in corresponding raising MSM substratum, in MSM substratum, the content of DBP is 100mg/L, often transfer once afterwards, in MSM substratum, the content of DBP improves 100mg/L).Then by the nutrient solution of switching 10 times dilution 10
3~ 10
5coat on LB solid plate, be inverted cultivation 1 ~ 3 day for 30 DEG C.After flat board growing single bacterium colony, picking list bacterium colony is repeatedly rule purifying, is separated acquisition one strain bacterium, is numbered 2D.Then by inoculation on LB solid plate 30 DEG C be inverted cultivation 7 days, observe its colonial morphology.The LB slat chain conveyor bacterium colony of 7 days is safran, and quality paste is abundant moistening, is easily provoked, the thinner (see figure 1) in edge.
Scanning electron microscope example Preparation and survey: by the LB liquid nutrient medium activated overnight of single bacterium colony access containing 10 mL of bacterial classification 2D.Draw 800 μ L bacterium liquid through centrifugal 3 ~ 5 min of 8000 rpm, remove supernatant liquor, add 500 μ L PBS and wash bacterium 3 times.1 mL 2.5%(v/v is added in the bacterial sediment of results) glutaraldehyde fully mixes, 4 DEG C of hold over night.And then through centrifugal 3 ~ 5 min of 8000 rpm, remove supernatant liquor, add 500 μ L PBS and wash bacterium 3 times.Subsequently by thalline respectively 30%, 50%, 70%, 85%, dewater 2 times in the graded ethanol of 90% and 100%, each gradient about soaks 15 min, and then 8000 rpm are centrifugal removes supernatant liquor, finally replace ethanol 2 times, each 20 min, the same ethanol dehydration of method with Isoamyl Acetate FCC.Thalline is through CO
2after dry, film-making is observed.Can observe this bacterium under scanning electron microscope is elongated rod shape, is about 1.5 ~ 3.0 μm, wide about 0.5 ~ 0.8 μm of (see figure 2).
The Physiology and biochemistry identification of indicator of bacterial strain comprises 12: methyl red test, V-P test, indole reaction, Citrate trianion utilization, inositol produce acid test, the sour test of glucose product, Starch Hydrolysis test, urine enzyme test, gelatin liquification test, nitrate reduction, oxidase test and glucose aerogenesis and test.Specific experiment the results are shown in Table 1.
The 16S rDNA of bacterial strain identifies: extract bacteria total DNA, carry out pcr amplification with bacterial 16 S rDNA universal primer to this bacterium genome.The 16S rDNA sequence reported in sequencing result and GenBank, after order-checking (being completed by the raw work in Shanghai), is carried out sequence analysis by PCR primer, and chooses some bacterial classifications and do phylogenetic analysis.As shown in Figure 3, the 16S rDNA sequence of bacterial strain 2D that obtains of separation and purification of the present invention and Providence (
providencia stuartii, culture presevation number is NBRC 12930) and homology reaches 99%, and evolutionary distance is nearest.Also higher homology is had with other bacterial classifications of this Pseudomonas.Therefore, the identification of strains that the present invention screens acquisition is Providencia, called after
providenciasp. 2D.
embodiment 2
providenciasp. 2D is to the degradation experiment of DBP and intermediate product phthalic acid thereof
1, collecting cells
By the bacterial classification after purifying
providenciasp. 2D access is cultured to logarithmic phase containing the LB liquid nutrient medium activated overnight of 10 mL, collects thalline through centrifugal 10 min of 5000 rpm, resuspended after washing bacterium 3 times with PBS, regulates OD
600 nm=0.8 as bacteria suspension.
2, degradation capability measures
Respectively to containing different concns DBP(50,100,200,500,1000 mg/L) and MSM nutrient solution (the inorganic salt nutrient solution g/L:K of phthalic acid (25,50,100,200,500 mg/L)
2hPO
4: 5.8; KH
2pO
4: 4.5; (NH
4)
2sO
4: 2.0; MgCl
2: 0.16; CaCl
2: 0.02; Na
2moO
42H
2o:0.0024; FeCl
3: 0.0018; MnCl
22H
2o:0.0015) meet bacterium 1 mL in, not connect bacterium in contrast, and regulate pH to be 8.0, often organize three repetitions.At 30 DEG C, 140 rpm constant-temperature tables cultivate 6 days, regularly sample, and measure the degraded situation of DBP and phthalic acid through GC/MS.
Chromatographic condition: adopt Shimadzu Corporation QP2010 Plus type GC/MS tandom mass spectrometer.Chromatographic column is that (m), injector temperature is 250 DEG C to Agilent HP-5 pillar in 0.25 μm × 0.25 mm × 30, and ion source (EI) temperature is 220 DEG C, and adopt Splitless injecting samples 1 μ L, carrier gas is high-purity helium.Heating schedule is: initial temperature is 100 DEG C, keeps 2 min, and 15 DEG C/min gradient rises to 129 DEG C, is then warming up to 280 DEG C with 40 DEG C/min, keeps 5min.
Quality control: adopt external standard method and six point calibration reference material production standard curves.It is 92.5 ~ 110.2% that 6 kinds of PAEs mix target matrix mark-on average recovery rate, and relative deviation is lower than 10.5%, and instrument detects and is limited to 0.12 ~ 0.45 ug/g.The method meets trace organic substance quantitative analysis requirement.
Result is as shown in Figure 4:
providenciasp. 2D bacterium is at the DBP(1000 mg/L of 72 h to high density) degradation efficiency is more than 80%, and when DBP concentration is lower than 200 mg/L, in 72 h, DBP is almost degradable, shows that this bacterium has efficient degradation capability to DBP.To the degradation capability analysis of DBP intermediate product, can find out that the degradation capability to high density phthalic acid of this bacterium is significantly lower than DBP, but to lower concentration phthalic acid (≤100 mg/L) degradation capability comparatively strong (almost degradable at 144 h); When O-phthalic acid concentration is 200 mg/L, at the degradation rate of 144 h more than 90%.Illustrate that the main metabolites phthalic acid of this bacterium to DBP also has stronger degradation capability, DBP permineralization the most at last.
embodiment 3
providenciasp. 2D is to the Study on degradation of DBP in contaminated soil
1, for examination soil processing
Soil is Agricultural University Of South China's Farm Rice soil, and pH is 6.7, C/N is 5.98, air-dry rear mistake 60 mesh sieve.Animal excreta compost picks up from this farm, and pH is 8.8, C/N is 17.49.Commonly use adding proportion (compost: soil=1:15, w/w) according to farm and add compost in rice soil, mixing obtains pH=7.54, and C/N is the conditioned soil of 10.8.Do not add composting soil (conventional soil) and conditioned soil adds DBP respectively by above-mentioned, make its concentration all reach 100 mg/kg.
1, in soil, DBP degradation effect is analyzed
Process below Setup Experiments: connect bacterium routine soil and conditioned soil as experimental group; Do not connect routine soil and the conditioned soil as a control group of bacterium; The conditioned soil that sterilized conditioned soil and first sterilizing connect 2D bacterium more as a control group two, often organizes three repetitions.Take above-mentioned soil 200 g containing 100 mg/kg DBP respectively in triangular flask, in soil, add bacteria suspension (OD
600 nm=0.8) 50 mL, fully mix.Soil is adjusted to field capacity (water content of about 30%), in 30 DEG C of thermostat containers, lucifuge is cultivated, and regularly sampling also measures DBP residual quantity in soil through GC/MS.
Through 10 days
providenciasp. the biological reinforced repair process of 2D, in each process, Fig. 5 is shown in the dynamic change of contaminated soil DBP residual quantity.Connect DBP degradation effect in the routine soil of 2D bacterium significantly to strengthen, at the 0th, 2,4,6,8,10 day, in soil, DBP residual quantity is respectively 98.41 mg/kg, 88.13 mg/kg, 75.40 mg/kg, 61.25 mg/kg, 44.85 mg/kg and 34.46 mg/kg, degradation rate at the 10th day reaches 65%, and the contrast soil DBP degradation rate not connecing bacterium is only 19%, explanation
providenciasp. 2D bacterium significantly can strengthen the degraded of DBP in soil.In addition, with the addition of compost and connecing in the conditioned soil of bacterium, connect bacterium soil relative to what do not add compost, the degradation effect of DBP strengthens further, and its DBP degradation rate of the tenth day reaches 88%, shows that compost can strengthen the degraded of DBP in soil, will
providenciasp. 2D bacterium is combined with compost, can efficiently repair DBP contaminated soil.
In addition, non-sterilizing and in the conditioned soil of inoculation 2D bacterium the degradation efficiency of DBP be significantly higher than conditioned soil that is sterilized and inoculation 2D bacterium, the indigenous microorganism illustrating in 2D bacterium and conditioned soil is formed and acts synergistically, and facilitates the degraded of the DBP in soil.
SEQUENCE LISTING
<110> Ji'nan University
<120> Providence (Providencia
Sp.) application of 2D in dibutyl phthalate contaminated soil remediation
<130>
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1445
<212> DNA
<213> 16S rDNA sequence
<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.) application of 2D in DBP contaminated soil remediation, is characterized in that, this bacterium is deposited in China typical culture collection center (CCTCC) on January 22nd, 2015, and deposit number is CCTCC M 2015057.
2. Providence
providenciasp. the application of 2D in preparation DBP contaminated soil remediation modifying agent, it is characterized in that, this bacterium is deposited in China typical culture collection center (CCTCC) on January 22nd, 2015, and deposit number is CCTCC M 2015057.
3. application according to claim 1, is characterized in that, described in be applied as Providence
providenciasp. in obtained after 2D activation bacteria suspension access DBP contaminated soil.
4. application according to claim 3, is characterized in that, adds animal excreta compost and access Providence again after mixing in DBP contaminated soil
providenciasp. 2D bacteria suspension.
5. application according to claim 4, is characterized in that, the mass ratio of described animal excreta compost and DBP contaminated soil is 1:15.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| 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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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| 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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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107446856A (en) * | 2017-08-30 | 2017-12-08 | 西安罗格斯生物科技有限公司 | Occupy the application of larva Providence bacterial strain, enzyme preparation and its degrading pesticide residues |
| CN107446856B (en) * | 2017-08-30 | 2020-08-11 | 侨康生物科技(广东)有限公司 | Providencia juveniles strain, enzyme preparation and application thereof in degrading pesticide residue |
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