CN104845899A - Application of Rhodococcus sp. 2G in degradation of phthalate - Google Patents
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Abstract
The invention specifically discloses application of Rhodococcus sp. 2G in degradation of phthalate, which belongs to the technical field of biological treatment of environmental pollutants. The Rhodococcus sp. 2G is preserved in China Center for Type Culture Collection (CCTCC) on January 22, 2015, with an accession number of CCTCC M2015056. The Rhodococcus sp. 2G can use a plurality of PAEs as a sole carbon source and energy for growth and propagation, and is capable of almost thoroughly degrading mixed PAEs with a concentration of 200 mg/kg (including DMP, DEP, DBP and DEHP respectively with a concentration of 200 mg/kg) in an inorganic salt medium within 7 d under the condition of pure culture, wherein the degradation rates of all the PAEs exceed 97%. The Rhodococcus sp. 2G has great prospects when applied in biological treatment of environmental pollutants.
Description
Technical field
The present invention relates to environment pollutant biological treatment technical field, more specifically, relate to rhodococcus (
rhodococcus sp.) application of 2G in degraded phthalic ester.
Background technology
Phthalic ester (Phthalates, PAEs), also known as phthalate, is fluidizer the most common in plastic cement industry.In the daily and industrial production being widely used in high molecular weight plastic product, as polyvinyl chloride (PVC), polypropylene (PP), foam, also can make an addition in makeup, cementing agent, coating, ink.As softening agent, the content of PAEs in plastics accounts for about 20 ~ 30%, sometimes can up to 50%.In order to improve the plasticity-of plastic prod necessity, PAEs to be also non-covalently aggregated in the matrix of plastic prod, but is connected by hydrogen bond or Van der Waals force with between polyolefins plastic molecules, retains chemical property relatively independent separately each other.Therefore, As time goes on, PAEs can move to external environment by plastics, causes the pollution to environment.Research simultaneously shows, comprises 10 kinds of PAEs such as DMP, DEP, DBP and DEHP and has been considered to similar estrogenic effect, can disturb normal endocrine in humans and animals body thus affect its healthy reproduction.In recent years, due to plastics widely using in the world, cause PAEs ubiquitous in the environment.At present, have existence PAEs being detected in soil, river, lake, bottom soil, tap water, refuse tip and even food, PAEs has become one of the most general pollutent in the whole world.
The speed of PAEs hydrolysis in the environment, photodissociation slowly, belongs to hard-degraded substance.Therefore, microbiological deterioration is considered to the main process of PAEs permineralization in physical environment.In recent years, degradation by bacteria PAEs is utilized to have large quantity research, the bacterial strain of a large amount of efficient degradation PAEs has been separated and has obtained from all kinds of environment such as the active sludge of mangrove forest, soil, ocean, river and waste water treatment plant, but, the degradation bacteria that great majority have been reported is all only have efficient degradation ability to a kind of phthalic ester, and can either efficient degradation short chain PAEs(as DMP, DEP etc.), the long-chain PAEs(that can degrade again is as DBP, DEHP etc.) the germ plasm resource of degradation bacteria be seldom found.
Summary of the invention
The present invention in order to overcome the above-mentioned defect existed in prior art, provide rhodococcus (
rhodococcus sp.) application of 2G in degraded phthalic ester.
Second object of the present invention be to provide rhodococcus (
rhodococcus sp.) application of 2G in the preparation of preparation degraded phthalic ester.
3rd object of the present invention is to provide a kind of bacteria agent of phthalic ester of degrading.
The object of the invention is to be achieved by the following technical programs:
Rhodococcus (
rhodococcus sp.) application of 2G in degraded phthalic ester, described bacterial strain is preserved in China typical culture collection center (CCTCC) on January 22nd, 2015, and deposit number is CCTCC M 2015056, preservation address: Wuhan City, Hubei Province Wuhan University.
Described bacterial strain
rhodococcus sp.2G can carry out aerobic degradation using DMP, DEP, DBP and DEHP as sole carbon source, cultivate in the minimal medium containing 200 mg/L DMP, 200 mg/L DEP, 200 mg/L DBP and 200 mg/L DEHP after 7 days, almost can degradable all PAEs.
Therefore, rhodococcus
rhodococcus sp.2G also can be used for the preparation preparing degraded phthalic ester, namely provides rhodococcus
rhodococcus sp.the application of 2G in the preparation of preparation degraded phthalic ester.
Preferably, in above-mentioned application, described phthalic ester is dimethyl phthalate and/or diethyl phthalate and/or dibutyl phthalate and/or dimixo-octyl phthalate.
The application of above-mentioned rhodococcus degraded phthalic ester is specially: by rhodococcus
rhodococcus sp.in the medium that bacteria suspension access obtained after 2G activation is polluted by phthalic ester.
As the preferred technical scheme of one, above-mentioned application is by rhodococcus
rhodococcus sp.2G is cultured to logarithmic phase through activated overnight, concussion, collects thalline, regulates thalline content according to actual needs and makes bacteria suspension, is accessed in the water or soil polluted by phthalic ester by obtained bacteria suspension.
Bacterial strain of the present invention
rhodococcus sp.2G is separated also enrichment by enrichment culture method to obtain from the active sludge of sewage work, and this bacterium optimum growing condition is: pH=6 ~ 9, temperature 20 ~ 40 DEG C.
Described bacterial strain
rhodococcus sp.2G is gram-positive microorganism, and the bacterium colony that LB substratum grows is incarnadine, and circular, surface drying is coarse, opaque, and edge is irregular, and Main Biological is oxidase negative, and catalase is positive.Its morphological specificity of scanning electron microscopic observation is shaft-like, is about 1 ~ 3 μm, wide about 0.5 ~ 0.7 μm.
Described bacterial strain
rhodococcus sp.the 16S rDNA sequence of 2G has logged in GenBank database, as shown in SEQ ID NO:1, logged in bacterial isolates 16S rDNA sequence by the blast program of NCBI official website (http://www.ncbi.nlm.nih.gov/) and other to compare, result show this bacterial strain with
rhodococcus sp.similarity is the highest, and homology reaches 99%.
The present invention provides a kind of bacteria agent of phthalic ester of degrading simultaneously, comprises rhodococcus
rhodococcus sp.2G.
The present invention finds that this bacterium is deposited in case in other nutrition (as carbon source), and can strengthen degraded PAEs further, therefore, preferably, the above-mentioned bacteria agent of the present invention also comprises can as rhodococcus
rhodococcus sp.the material of 2G carbon source.
More preferably, described can as rhodococcus
rhodococcus sp.the material of 2G carbon source is peptone, yeast extract, glucose.
Compared with prior art, the present invention has following beneficial effect:
The invention provides rhodococcus
rhodococcus sp.the application of 2G in degraded phthalic ester.Described bacterial strain
rhodococcus sp.2G can utilize dimethyl phthalate, diethyl phthalate, dibutyl phthalate and dimixo-octyl phthalate to carry out growth and breeding as sole carbon source and the energy, under pure culture condition, the mixing PAEs(of 200 mg/kg in minimal medium can be comprised 200 mg/kg DMP, DEP, DBP and DEHP in 7 days by this bacterium respectively) almost degraded is complete, and degradation rate is all more than 97%; Of the present invention being applied in the biological treatment of environmental pollutant has huge prospect.
Accompanying drawing explanation
Fig. 1 is
rhodococcus sp.2G cultivates the growthhabit of 7 days on LB substratum.
Fig. 2 is
rhodococcus sp.the scanning electron microscope (SEM) photograph of 2G.
Fig. 3 is
rhodococcus sp.the phylogenetic tree of the 16SrDNA of 2G.
Fig. 4 is
rhodococcus sp.2G is to the degradation effect of four kinds of mixing PAEs.
Fig. 5 additionally adds the impact of carbon source on four kinds of PAEs degradation effects and growth situation.
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.
In embodiments of the invention DMP be the abbreviation of dimethyl phthalate, the DEP abbreviation that is diethyl phthalate, the DB abbreviation that is dibutyl phthalate, DEHP is the abbreviation of dimixo-octyl phthalate.
the Isolation and ldentification of embodiment 1 PAEs efficient degrading bacteria
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.
Beef-protein medium (LB): yeast powder 5.0 g, peptone 10.0 g, sodium-chlor 10.0 g, adds ultrapure water to 1 L, 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
Take 5 g activated sludge sample (taking from sewage work) in the 150 mL triangular flasks containing 50 mL sterilized waters, at 30 DEG C, cultivate 3 days under 140 rpm conditions, get 5 mL mud suspensions and add containing 100 mL PAEs(containing 50 mg/L DMP, 50 mg/L DEP, 50 mg/L DBP and 50 mg/L DEHP) above-mentioned MSM substratum in.Through 30 DEG C, after 140 rpm cultivate 7 days, press the continuous enrichment of inoculum size of 1 mL, switching 10 times at every turn, and PAEs content to 800 mg/L(is namely containing 800 mg/L DMP, 800 mg/L DEP, 800mg/L DBP and 800 mg/L DEHP in corresponding raising substratum, in first time switching wild Oryza species, PAEs content is 70mg/L; In second time switching wild Oryza species, PAEs content is 100mg/L; In third time switching wild Oryza species, PAEs content is 150mg/L; In 4th switching wild Oryza species, PAEs content is 200mg/L; After 4th time, in a subculture of often transferring, PAEs content is for improving 100mg/L, and in final substratum, PAEs content is 800mg/L).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 2G.Then by inoculation on LB solid plate 30 DEG C be inverted cultivation 7 days, observe its colonial morphology.
This bacterial strain on LB solid medium streak culture 7 days, bacterium colony is incarnadine, and circular, surface drying is coarse, opaque, edge irregular (Fig. 1), and Main Biological is oxidase negative, and catalase is positive.
Scanning electron microscopic observation is identified: the bacterial classification 2G 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 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 with Isoamyl Acetate FCC, each 20 min, method is with above-mentioned ethanol dehydration process.Through CO
2after dry, film-making is observed; Its morphological specificity of scanning electron microscopic observation is shaft-like, is about 1 ~ 3 μm, wide about 0.5 ~ 0.7 μm (Fig. 2).
The 16S rDNA Molecular Identification of 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 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; With rhodococcus
rhodococcussp. the genomic dna of 2G is template, utilizes bacterial 16 S rDNA universal primer to carry out pcr amplification, obtains the PCR primer that length is 1425 bp.Sequencing result carries out tetraploid rice with GenBank other bacterial sequences listed, this bacterium with
rhodococcussp. the evolutionary tree of the homology of TG14 bacterium (GenBank accession number is KM235732) to be 99%, Fig. 3 be this bacterium.
embodiment 2
rhodococcussp. 2G bacterium is to the degradation effect of PAEs
1, the preparation of bacteria suspension
Bacterial classification 2G after purifying is accessed and is cultured to logarithmic phase containing the LB liquid nutrient medium activated overnight of 10 mL, collect thalline through centrifugal 10 min of 5000 rpm, resuspended after washing bacterium 3 times with PBS, regulate OD
600 nm=0.8 as bacteria suspension.
2,
rhodococcus sp.the degradation property of 2G bacterium measures
To containing 200 mg/L concentration PAEs(namely containing 200 mg/L DMP, 200 mg/L DEP, 200 mg/L DBP and 200 mg/L DEHP) 100mL MSM nutrient solution in inoculate above-mentioned bacteria suspension 1 mL, not connect bacterium in contrast, and regulate pH to be 7.5, often organize three repetitions.At 32 DEG C, 150 rpm constant-temperature tables cultivate 7 days, respectively at 0, and sampling in 1,3,5,7 days also extracting sample, the degraded situation of DMP, DEP, DBP and DEHP in GC/MS working sample.
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; OD pH-value determination pH adopts Shimadzu UV-2450 type ultra-violet and visible spectrophotometer.
rhodococcussp. 2G bacterium mixes the degradation effect of PAEs as shown in Figure 4 to four kinds, and this bacterium has significant degradation effect to four kinds of PAEs under 7 days shaking culture.Particularly for short chain PAEs(as DMP, DEP), 2G bacterium to these two kinds of PAEs at the degradation rate of first day all more than 50%, within the 3rd day, degradation rate bacterium is more than 80%.And to long-chain PAEs(as DBP, DEHP) degradation speed relatively slow, first day bacterium lower than 5%, but is improved rapidly the degradation efficiency of DBP and DEHP on the 3rd day, reaches 48.62% and 69.13% respectively.Cultivate the 5th day shaking bacterium, the short chain PAEs degradation rate in nutrient solution, on the contrary lower than long-chain PAEs, may be because long-chain PAEs is hydrolyzed intermediate products such as producing DMP and DEP.Be cultured to the 7th day, four kinds of PAEs are almost degradable, and degradation rate, all more than 97%, describes
rhodococcussp. 2G bacterium has efficient degradation capability to four kinds of PAEs.
Fig. 5 represents that interpolation LB substratum is as Additional carbon sources pair
rhodococcussp. the degradation capability of 2G bacterium and the impact of growing state.As shown in Figure 5, LB substratum is added not obvious on bacterial growth impact; On short chain PAEs(DMP and DEP) degradation effect impact also remarkable, this may be because short chain PAEs is easily utilized as energy substance by 2G bacterium, and extra carbon source of adding can not affect this bacterium to its utilising efficiency.And for long-chain PAEs(DBP and DEHP), extra interpolation LB substratum not only can promote the quick growth and breeding of 2G bacterium, and the degradation effect of DBP and DEHP is also significantly improved, analyzing reason may be that Additional carbon sources adds the utilising efficiency of bacterium initial stage to carbon source, make it reach logarithmic phase fast, thus improve the degradation effect to DBP and DEHP.In a word, extra carbon source of adding can promote
rhodococcussp. the growth of 2G bacterium, improves its degradation rate to PAEs simultaneously.
Above embodiment explanation
rhodococcussp. 2G bacterium can short, the long-chain PAEs of efficient degradation, and deposit in other carbon sources in case still can efficient degradation PAEs simultaneously, gives security for it carries out biological restoration in the contaminate environment of complexity.
SEQUENCE LISTING
<110> Ji'nan University
The application of <120> rhodococcus (Rhodococcus sp.) 2G in degraded phthalic ester
<130>
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1425
<212> DNA
<213> Rhodococcus sp. 2G 16S rDNA sequence
<400> 1
cgccatggcg ggtgcttacc atgcaagtcg aacgatgaag cccagcttgc tgggtggatt 60
agtggcgaac gggtgagtaa cacgtgggtg atctgccctg cactctggga taagcctggg 120
aaactgggtc taataccgga tatgacctct ggctgcatgg ccaggggtgg aaagtttttc 180
ggtgcaggat gagcccgcgg cctatcagct tgttggtggg gtaatggcct accaaggcga 240
cgacgggtag ccggcctgag agggcgaccg gccacactgg gactgagaca cggcccagac 300
tcctacggga ggcagcagtg gggaatattg cacaatgggc gcaagcctga tgcagcgacg 360
ccgcgkgagg gatgacggcc ttcgggttgt aaacctcttt caccccatga cgaagcgcaa 420
gtgacggtag tgggagaaga agcaccggcc aactacgtgc cagcagccgc ggtaatacgt 480
agggtgcgag cgttgtccgg aattactggg cgtaaagagc tcgtaggcgg tttgtcgcgt 540
cgtctgtgaa atcccgcagc tcaactgcgg gcttgcaggc gatacgggca gactcgagta 600
ctgcagggga gactggaatt cctggtgtag cggtgaaatg cgcagatatc aggaggaaca 660
ccggtggcga aggcgggtct ctgggcagta actgacgctg aggagcgaaa gcgtgggtag 720
cgaacaggat tagataccct ggtagtccac gccgtaaacg gtgggcgcta ggtgtgggtt 780
tccttccacg ggatccgtgc cgtagccaac gcattaagcg ccccgcctgg ggagtacggc 840
cgcaaggcta aaactcaaag gaattgacgg gggcccgcac aagcggcgga gcatgtggat 900
taattcgatg caacgcgaag aaccttacct gggtttgaca tgtaccggac gactgcagag 960
atgtggtttc ccttgtggcc ggtagacagg tggtgcatgg ctgtcgtcag ctcgtgtcgt 1020
gagatgttgg gttaagtccc gcaacgagcg caacccttgt cctgtgttgc cagcacgtga 1080
tggtggggac tcgcaggara ctgccggggt caactcggag gaagktgggg acgacgtcaa 1140
gtcatcatgc cccttatgtc cagggcttca cacatgctac aatggtcggt acagagggct 1200
gcgataccgt gaggtggagc gaatccctta aagccggtct cagttcggat cggggtctgc 1260
aactcgaccc cgtgaagtcg gagtcgctag taatcgcaga tcagcaacgc tgcggtgaat 1320
acgttcccgg gccttgtaca caccgcccgt cacgtcatga aagtcggtaa cacccgaagc 1380
cggtggccta accccttgtg ggagggagcc gtcgaagtga tcgct 1425
Claims (8)
1. rhodococcus (
rhodococcus sp.) application of 2G in degraded phthalic ester, it is characterized in that, described bacterial strain is preserved in China typical culture collection center (CCTCC) on January 22nd, 2015, and deposit number is CCTCC M 2015056.
2. rhodococcus
rhodococcus sp.the application of 2G in the preparation of preparation degraded phthalic ester, it is characterized in that, described bacterial strain is preserved in China typical culture collection center (CCTCC) on January 22nd, 2015, and deposit number is CCTCC M 2015056.
3. application according to claim 1 and 2, is characterized in that, described phthalic ester is dimethyl phthalate and/or diethyl phthalate and/or dibutyl phthalate and/or dimixo-octyl phthalate.
4. application according to claim 1, is characterized in that, by rhodococcus
rhodococcus sp.in the medium that bacteria suspension access obtained after 2G activation is polluted by phthalic ester.
5. application according to claim 4, is characterized in that, described medium is water, soil.
6. to degrade the bacteria agent of phthalic ester, it is characterized in that, comprise rhodococcus
rhodococcus sp.2G, described rhodococcus
rhodococcus sp.2G is preserved in China typical culture collection center (CCTCC) on January 22nd, 2015, and deposit number is CCTCC M 2015056.
7. bacteria agent according to claim 6, is characterized in that, also comprising can as rhodococcus
rhodococcus sp.the material of 2G carbon source.
8. bacteria agent according to claim 6, is characterized in that, described can as rhodococcus
rhodococcus sp.the material of 2G carbon source is peptone, yeast extract or glucose.
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| CN108410771A (en) * | 2018-04-08 | 2018-08-17 | 宁夏大学 | The application of Rhodococcus sp YC915 and its absorption microbial inoculum in soil phthalic acid ester of degrading |
| CN110195026A (en) * | 2019-03-04 | 2019-09-03 | 暨南大学 | A kind of method that DEHP degradation bacterial agent prepared and its be effectively reduced DEHP pollution in vegetables production |
| CN111575197A (en) * | 2020-04-08 | 2020-08-25 | 暨南大学 | PAEs degrading microbial inoculum, method for preventing and treating vegetable PAEs pollution and application |
| CN113151093A (en) * | 2021-04-25 | 2021-07-23 | 武汉轻工大学 | Strain capable of degrading 3, 5-dimethylphenol and application thereof |
| CN113151093B (en) * | 2021-04-25 | 2022-03-18 | 武汉轻工大学 | Strain capable of degrading 3, 5-dimethylphenol and application thereof |
| WO2024130870A1 (en) * | 2022-12-24 | 2024-06-27 | 南京农业大学 | Glutamicibacter a4 with phthalic acid ester-degrading ability and use thereof |
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