CN104845891A - Agromyces sp. bacterial suspension used for degradation of various phthalates and application thereof - Google Patents

Agromyces sp. bacterial suspension used for degradation of various phthalates and application thereof Download PDF

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CN104845891A
CN104845891A CN201510066106.6A CN201510066106A CN104845891A CN 104845891 A CN104845891 A CN 104845891A CN 201510066106 A CN201510066106 A CN 201510066106A CN 104845891 A CN104845891 A CN 104845891A
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bacteria suspension
dehp
dbp
agromyces
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CN104845891B (en
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蔡全英
莫测辉
赵海明
李彦文
李慧
杜欢
向垒
成玉婷
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Jinan University
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Abstract

The invention specifically discloses an Agromyces sp. bacterial suspension used for degradation of a variety of phthalates and application thereof, which belongs to the technical field of biological treatment of environmental pollutants. The bacterial suspension is prepared by inoculating a purified Agromyces sp. MT-E strain into a conventional microbial liquid medium, carrying out overnight culture to a logarithmic phase, then carrying out centrifugation to collect thalli, washing the thalli with PBS, then carrying out resuspension and adjusting OD<600 nm> to 0.6 to 1.0. The Agromyces sp. MT-E strain is preserved in China Center for Type Culture Collection (CCTCC) on January 22, 2015, with an accession number of CCTCC M2015054. The bacterial suspension can simultaneously degrade DBP and DEHP and has good application prospects in restoration of soil contaminated by PAEs.

Description

A kind of earth mould bacteria suspension of multiple phthalic ester of degrading and application thereof
Technical field
The present invention relates to environment pollutant biological treatment technical field, particularly, relate to a kind of earth mould bacteria suspension and application thereof of multiple phthalic ester of degrading.
Background technology
Phthalic ester, also known as phthalate, abbreviation PAEs is the general designation of the ester that phthalic acid is formed.Wherein, dibutyl phthalate (di- n-butylphthalate, DBP) and dimixo-octyl phthalate (di-(2-ethylhexyl) phthalate, DEHP) be two kinds of plastic plasticizers the most widely used, in PVC plastic, its content can reach 40 ~ 60%.In recent decades, China's plastic greenhouse and plastic sheeting for farm use usage quantity sharply increase.Along with a large amount of uses of plastics film, increasing DBP and DEHP is released into agricultural land soil, and they become the organic pollutant be the most often detected in agricultural land soil, detects rank and reaches mg/kg.Research shows, these two kinds of phthalic esters have high toxicity, teratogenecity, carinogenicity and mutagenicity, and genotoxicity, are classified as priority pollutants by USEPA (EPA).Therefore, DBP and the DEHP pollution problem in China's agricultural soil is urgently to be resolved hurrily.
Because the chemical stability of these two kinds of phthalic esters is fine; under natural condition by the speed of the chemical action such as photodissociation, hydrolysis disappearance slowly; its transformation period reaches 20 years, DBP and DEHP of the overwhelming majority is mainly disappeared from environment by biodegradation pathway.Wherein to have cost low in microbiological deterioration, and mild condition, does not produce the advantages such as secondary pollution, meets requirement and the development trend of constructing environment friendly and conservation-minded society.Therefore, utilizing the Degradation of microorganism DBP and DEHP to be converted into innoxious substance is remove the best means that in environment, phthalic ester pollutes.
At present, Chinese scholars filters out many bacterial strains with good degradation property for DBP and DEHP respectively, but the bacterial strain of these two kinds of phthalic esters of wherein can simultaneously degrading is little.Earth mould ( agromycessp.) be the class Pseudomonas extensively existed in soil, having some bacterial classification of this Pseudomonas of report to have at present can the characteristic of efficient degradation phenol, agricultural chemicals Diacloden and resistance to heavy metal, but there is not yet the report of its degradable phthalic ester.
Summary of the invention
The present invention, in order to overcome the above-mentioned deficiency of prior art, provides a kind of earth mould bacteria suspension of multiple phthalic ester of degrading.
Another object of the present invention is to provide a kind of purposes of earth mould bacteria suspension of multiple phthalic ester of degrading.
To achieve these goals, the present invention is achieved by following scheme:
To degrade a bacteria suspension of DBP and/or DEHP, prepare by the following method: by after purifying earth mould ( agromycessp.) bacterial strain MT-E accesses conventional microbiological liquid nutrient medium incubated overnight to logarithmic phase, collected by centrifugation thalline, resuspended adjustment OD after washing bacterium with PBS 600 nm=0.6 ~ 1.0 as bacteria suspension;
Described bacterial strain is deposited in China typical culture collection center (CCTCC) on January 22nd, 2015, and deposit number is CCTCC M 2015054.
Preferably, OD is regulated 600 nm=0.8 as bacteria suspension.
Preferably, described conventional microbiological liquid nutrient medium is LB liquid nutrient medium, NA liquid nutrient medium or PDA liquid nutrient medium.
Preferably, bacterium is washed 2 ~ 4 times with PBS.
Bacteria suspension described above is repairing the application in DBP and/or DEHP pollution medium.
Preferably, described medium is soil, water or air.
To degrade a biotechnological formulation of DBP and/or DEHP, containing bacteria suspension described above and assistant agent.
Compared with prior art, the present invention has following beneficial effect:
The present invention to use the agricultural land soil of plastic greenhouse throughout the year separation screening first obtain a strain earth mould ( agromycessp.) bacterial strain MT-E, this bacterial strain can be degraded dibutyl phthalate and dimixo-octyl phthalate simultaneously; Enriched the germplasm resource bank of phthalic ester degradation bacteria, and this bacterial strain is widely distributed in soil, is easy to cultivate, adaptable; Huge application prospect will be had in the biological treatment of the actual contaminated soil of this bacterium additionally by soil remediation description of test.
Accompanying drawing explanation
Fig. 1 is the growthhabit feature that MT-E bacterial strain is cultivated 7 days on LB substratum.
Fig. 2 is the stereoscan photograph of MT-E bacterial strain.
Fig. 3 is the phylogenetic tree of the 16S rDNA of MT-E bacterial strain.
Fig. 4 is that MT-E bacterial strain is to the degradation kinetics of DBP and DEHP and growth curve.
Fig. 5 is the degradation effect of MT-E bacterial strain to DBP and DEHP in contaminated soil.
Embodiment
The present invention is elaborated further below in conjunction with Figure of description and specific embodiment.Following examples of the present invention are the present invention's preferably embodiment; the present invention mainly sets forth described bacterial strain and the application thought based on described bacterial strain; in embodiment, the replacement of simple parameter can not repeat one by one in an embodiment; but therefore do not limit the scope of the invention; other are any do not deviate from spirit of the present invention and principle under do change, modification, substitute, combine, simplify; the substitute mode of equivalence should be regarded as, be included within protection scope of the present invention.
Described in embodiment, culture medium prescription is as follows:
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.Final pH is 7.5.
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 sterilizing 20min.Solid plate then adds 1.5%(w/v) agar powder.
the Isolation and ldentification of embodiment 1 bacterial strain
Gather the agricultural land soil throughout the year using plastic greenhouse, take 5 g fresh soil samples in the 150 mL triangular flasks containing 50 mL sterilized waters, at 30 DEG C, 150 rpm get after cultivating 3 days 5 mL mud suspensions add be respectively 50 mg/L containing 100 mL DBP and DEHP() MSM substratum in.Through 30 DEG C, after 150 rpm cultivate 7 days, each continuous enrichment of inoculum size by 1 mL, switching 10 times, and PAEs content to 1000 mg/L in corresponding raising substratum.Then by the nutrient solution of domestication 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, numbering MT-E.Then by inoculation on LB solid plate 30 DEG C be inverted cultivation 7 days, observe its colonial morphology (see figure 1).As shown in Figure 1, bacterium colony is dark yellow, flat, moistening, edge unfairness, diffusion growth, is almost paved with flat board after seven days.
Scanning electron microscopic observation is identified: the bacterial strain MT-E after purifying is accessed the LB liquid nutrient medium activated overnight containing 10 mL.Draw 800 μ L bacterium liquid through centrifugal 3 ~ 5 min of 8000 rpm, remove supernatant liquor, add 500 μ L PBS solution and wash bacterium 3 times.2.5% glutaraldehyde adding 1 mL in the bacterial sediment of results 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 solution 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 with Isoamyl Acetate FCC, each 20 min, method is with above-mentioned ethanol dehydration process.Through CO 2after dry, film-making is observed.Fig. 2 as seen under scanning electron microscope MT-E thalline be rod-short, be about 0.8 ~ 1.5 micron, wide about 0.3 ~ 0.5 micron.
MT-E bacterial strain is carried out physiological and biochemical property qualification, and qualification result is in table 1.
The physiological and biochemical property qualification of table 1. MT-E bacterial strain
Physiological and biochemical test Result
Gram stain test ＋
V-P tests －
MR tests ＋
Starch Hydrolysis is tested －
Oxidase test －
Catalase reacts ＋
Urea test ＋
Indole test －
Note: "+" represents the positive; "-" represents feminine gender
The 16S rDNA Molecular Identification of MT-E bacterial strain: extract bacteria total DNA, with bacterial 16 S rDNA universal primer, pcr amplification is carried out to this bacterium genome.The 16S rDNA sequence of other bacterial strains reported in sequencing result and GenBank, after order-checking (completing order-checking by the raw work in Shanghai), is carried out sequence analysis by PCR primer, and chooses relevant bacteria species and do phylogenetic analysis (see figure 3).In addition, its morphological specificity and physiological and biochemical index (see table 1) also with earth mould ( agromycessp.) also similar, therefore the present invention be separated the degrading strain identification of acquisition be earth mould ( agromycessp.).
Therefore, contriver by earth mould ( agromycessp.) bacterial strain MT-E is deposited in China typical culture collection center (CCTCC) preservation, and preservation date is on January 22nd, 2015, and deposit number is CCTCC M 2015054; Described Strain Designation is agromycessp. MT-E.Preservation address is: Wuhan City, Hubei Province Wuhan University.
embodiment 2 earth mould ( agromycessp.) bacterial strain MT-E tests the degradation effect of DBP and DEHP
S1. the preparation of bacteria suspension: the MT-E bacterial strain access after purifying 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.
S2. to containing 200 mg/L concentration DBP and DEHP(respectively containing 200 mg/L) 100 mL MSM nutrient solutions in inoculate above-mentioned bacteria suspension 1 mL, not connect bacterium as negative control, and regulate pH to be 7.5, often organize three repetitions.At 30 DEG C, 150 rpm constant-temperature tables cultivate 7 days, and respectively at 0, sampling in 1,3,5,7 days also extracting sample, the degraded situation of DBP and DEHP in GC/MS working sample, simultaneously with the bacterial growth situation OD of each time point of spectrophotometric determination 600 nm.
Sample preparation: sample 100 mL after cultivating in shaking flask is transferred to separating funnel, add 20 mL methylene dichloride oscillation extraction 10 min, organic phase (lower phase) is collected with 150 mL triangular flasks, aqueous phase in separating funnel adds the methylene dichloride same procedure oscillation extraction 3 times of 20 mL again, merge organic phase, then cross the anhydrous Na of having dried containing 18 cm 2sO 4glass column (1.5 cm × 35 cm) carries out drying, collects filtrate with heart bottle, and after rotating evaporate to dryness, constant volume 10 mL is to be measured.
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 90.5 ~ 107.6% that 6 kinds of PAEs mix target matrix mark-on average recovery rate, and relative deviation is lower than 10.7%, and instrument detects and is limited to 0.62-1.22 ug/mL.
From accompanying drawing 4, along with the prolongation of incubation time, in sample, the residual quantity of DBP and DEHP significantly reduces.Particularly DBP, its degradation rate, higher than DEHP, is respectively 8.40%, 40.64%, 89.30%, 97.06% at the degradation rate of the the the 1st, 3,5,7 day DBP; And DEHP degraded is relatively slow, but along with the prolongation of incubation time, still can reach good degradation effect.5.31% is respectively, 30.92%, 73.12%, 86.25% at the degradation rate of the the the 1st, 3,5,7 day DEHP.In addition, along with incubation time increases, the growth of MT-E bacterial strain is vigorous gradually, illustrates that this bacterium can be good at utilizing DBP and DEHP to carry out growth and breeding, in the biological restoration of phthalic ester contaminate environment, has good application potential.
embodiment 3 bacterial strain MT-E is to the repairing effect of DBP and DEHP contaminated soil
S1. for examination soil processing: soil is Agricultural University Of South China's Farm Rice soil, air-dry rear mistake 60 mesh sieve, and pH is 6.7, add DBP and DEHP wherein, make its content in soil reach 100 mg/kg respectively, and add distilled water and be adjusted to field capacity (about 30%), aging 15 days of lucifuge.
S2. take respectively and above-mentionedly contain 100 mg kg -1soil 200 g of DBP and DEHP, in triangular flask, adds bacteria suspension 50 mL, fully mixes in soil, and the process not connecing bacterium is in addition control group.Soil is adjusted to field capacity (water content of about 30%), in 30 DEG C of thermostat containers, lucifuge is cultivated, respectively at 0, and 2,4,6,8, within 10 days, regularly sample extracting, then measure DBP and DEHP residual quantity in soil through GC/MS.
Sample extraction: take the air-dry pedotheque ground of 1 g in 35 mL glass centrifuge tube, adds the methylene dichloride of 20 mL and ultrasonic extraction 10 min, then through 3500 rpm collected by centrifugation supernatant liquors.Add the methylene dichloride same procedure ultrasonic extraction 3 times of 20 mL in soil sample precipitation again, merge supernatant liquor.Then through rotary evaporation concentrated supernatant, with crossing pillar after methylene dichloride ultrasonic cleaning, (1.0 cm × 35 cm, filler is anhydrous Na from top to bottom 2sO 4, silica gel and aluminum oxide), collect filtrate through N with heart bottle 2after drying up, again dissolve with chromatographic pure dichloromethane and be settled to 1 mL, treating that GC/MS measures.
GC/MS chromatographic condition is same as above.
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 -1.The method meets trace organic substance quantitative analysis requirement.
As shown in Figure 5, compared with the control group not connecing bacterium, agromycessp. MT-E bacterium significantly enhances the degradation effect of DBP and DEHP in soil.After connecing bacterium the 0th, 2,4,6,8,10 day, the DBP in soil is residual was 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; In soil, the residual quantity of DEHP 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.Connect bacterium cultivate the 10th day, the degradation rate of DBP improves about 77%, DEHP than control group and then improves about 55%.DBP and DEHP content in soil all progressively reduces with the prolongation of repair time.It is visible, agromycessp. MT-E bacterium can significantly improve the elimination of DBP and DEHP in contaminated soil, can be used as the desirable microorganism of DBP and DEHP contaminated soil bioremediation technology.
SEQUENCE LISTING
 
<110> Ji'nan University
 
The earth mould bacteria suspension of the multiple phthalic ester of <120> mono-kind degraded and application thereof
 
<130>
 
<160> 1
 
<170> PatentIn version 3.3
 
<210> 1
<211> 1408
<212> DNA
The 16S rDNA sequence of <213> MT-E bacterial strain
 
<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 (7)

1. to degrade a bacteria suspension of DBP and/or DEHP, it is characterized in that, prepare by the following method: by after purifying earth mould ( agromycessp.) bacterial strain MT-E accesses conventional microbiological liquid nutrient medium and is cultured to logarithmic phase, collected by centrifugation thalline, resuspended adjustment OD after washing bacterium with PBS 600 nm=0.6 ~ 1.0 as bacteria suspension;
Described bacterial strain is deposited in China typical culture collection center (CCTCC) on January 22nd, 2015, and deposit number is CCTCC M 2015054.
2. bacteria suspension according to claim 1, is characterized in that, regulates OD 600 nm=0.8 as bacteria suspension.
3. bacteria suspension according to claim 1, is characterized in that, described conventional microbiological liquid nutrient medium is LB liquid nutrient medium, NA liquid nutrient medium or PDA liquid nutrient medium.
4. bacteria suspension according to claim 1, is characterized in that, washes bacterium 2 ~ 4 times with PBS.
5. bacteria suspension described in any one of Claims 1-4 is repairing the application in DBP and/or DEHP pollution medium.
6. application according to claim 5, is characterized in that, described medium is soil, water or air.
7. to degrade a biotechnological formulation of DBP and/or DEHP, it is characterized in that, containing bacteria suspension and assistant agent described in any one of Claims 1-4.
CN201510066106.6A 2015-02-09 2015-02-09 A kind of earth mould bacteria suspension of a variety of phthalic acid esters of degrading and its application Active CN104845891B (en)

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