CN104805035A - Rhodococcus sp. 2G used for simultaneous degradation of plurality of phthalic acid esters - Google Patents
Rhodococcus sp. 2G used for simultaneous degradation of plurality of phthalic acid esters Download PDFInfo
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Abstract
The invention belongs to the technical field of environmental pollutant biological treatment, and specifically discloses rhodococcus sp. 2G used for simultaneous degradation of a plurality of phthalic acid esters (PAEs). The rhodococcus sp. 2G is preserved at China Center for Type Culture Collection (CCTCC) on 22th, Jan., 2015, and preservation number is CCTCC No.M 2015056. The rhodococcus sp. 2G is high in activity, and is capable of realizing growth and breeding by taking a plurality of PAEs as the only carbon source and energy; complete degradation of PAEs mixed with a inorganic salt culture medium at a ratio of 200mg/kg can be almost realized in 7 days by the rhodococcus sp. 2G under pure culture conditions; the rhodococcus sp. 2G is widely distributed in the natural world; adaptability is high; culture collection is enlarged greatly, and the rhodococcus sp. 2G possesses a promising application prospect in biological treatment of environmental pollutants.
Description
Technical field
The present invention relates to environment pollutant biological treatment technical field, more specifically, relate to a strain can degrade simultaneously multiple phthalic ester rhodococcus (
rhodococcus sp.) 2G.
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 defect existing for above-mentioned prior art, provide a kind of multiple phthalic ester of can simultaneously degrading rhodococcus (
rhodococcus sp.) 2G.
The object of the invention is to be achieved by the following technical programs:
A kind of rhodococcus of multiple phthalic ester of can simultaneously degrading (
rhodococcus sp.) 2G, described bacterial strain is preserved in China typical culture collection center (CCTCC) on January 22nd, 2015, and deposit number is CCTCC NO:M 2015056, and preservation address is: Wuhan, China Wuhan University.
Described bacterial strain
rhodococcus sp.2G can carry out aerobic degradation using DMP, DEP, DBP and DEHP as sole carbon source, cultivates after 7 days, bacterial strain in the minimal medium containing 200 mg/L DMP, 200 mg/L DEP, 200 mg/L DBP and 200 mg/L DEHP
rhodococcus sp.2G almost can degradable all PAEs.
Therefore, further, phthalic ester of the present invention refers to dimethyl phthalate and/or diethyl phthalate and/or dibutyl phthalate and/or dimixo-octyl phthalate.
Described bacterial strain
rhodococcus sp.2G is separated from the active sludge of sewage work by enrichment culture method and enrichment obtains, 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%.
Compared with prior art, the present invention has following beneficial effect:
The invention provides a strain can to degrade the rhodococcus of multiple phthalic ester simultaneously
rhodococcus sp.2G, this bacterial strain vigor is high, multiple phthalic ester can be utilized to carry out growth and breeding as sole carbon source and the energy, and under pure culture condition, the mixing PAEs(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; Described rhodococcus
rhodococcus sp.2G is widely distributed at occurring in nature, adaptable, has expanded microorganism resource storehouse greatly, in the biological treatment of environmental pollutant, have huge application 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.
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.
embodiment
1 PAEs
the Isolation and ldentification of 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 pair
pAEs
degradation effect
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.
SEQUENCE LISTING
<110> Ji'nan University
<120> mono-strain can be degraded rhodococcus (Rhodococcus sp.) 2G of multiple phthalic ester simultaneously
<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 (4)
1. a multiple phthalic ester of can simultaneously degrading rhodococcus (
rhodococcus sp.) 2G, 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 NO:M 2015056.
2. rhodococcus according to claim 1
rhodococcus sp.2G, is characterized in that, described phthalic ester is dimethyl phthalate and/or diethyl phthalate and/or dibutyl phthalate and/or dimixo-octyl phthalate.
3. rhodococcus according to claim 1
rhodococcus sp.2G, is characterized in that, described bacterial strain
rhodococcus sp.the 16S rDNA sequence of 2G is as shown in SEQ ID NO:1.
4. rhodococcus according to claim 1
rhodococcus sp.2G, is characterized in that, described bacterial strain is gram-positive microorganism, and bacterium colony is incarnadine, and circular, surface drying is coarse, opaque, and edge is irregular.
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Cited By (5)
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| 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 |
| CN114107092A (en) * | 2021-11-02 | 2022-03-01 | 暨南大学 | Plant endophyte Gordonia L191 for degrading phthalate 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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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN110195026B (en) * | 2019-03-04 | 2022-10-11 | 暨南大学 | Preparation of DEHP degrading microbial inoculum and method for effectively reducing DEHP pollution in vegetable 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 |
| CN114107092A (en) * | 2021-11-02 | 2022-03-01 | 暨南大学 | Plant endophyte Gordonia L191 for degrading phthalate and application thereof |
| CN114107092B (en) * | 2021-11-02 | 2023-11-24 | 暨南大学 | Endophyte Gordonia L191 for degrading phthalate 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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