CN104673683A - Salt-tolerance polycyclic aromatic hydrocarbon degrading bacterium Tzyjm and application of salt-tolerance polycyclic aromatic hydrocarbon degrading bacterium Tzyjm in marine polluted environment - Google Patents
Salt-tolerance polycyclic aromatic hydrocarbon degrading bacterium Tzyjm and application of salt-tolerance polycyclic aromatic hydrocarbon degrading bacterium Tzyjm in marine polluted environment Download PDFInfo
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- CN104673683A CN104673683A CN201510089660.6A CN201510089660A CN104673683A CN 104673683 A CN104673683 A CN 104673683A CN 201510089660 A CN201510089660 A CN 201510089660A CN 104673683 A CN104673683 A CN 104673683A
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Abstract
The invention discloses a salt-tolerance polycyclic aromatic hydrocarbon degrading bacterium. The salt-tolerance polycyclic aromatic hydrocarbon degrading bacterium is characterized in that the strain is Galactomyces geotrichum Tzyjm, is preserved in China Center for Type Culture Collection with the preservation number of CCTCC NO: M2014625 and has the ability of degrading polycyclic aromatic hydrocarbon under the salty environment. The salt-tolerance polycyclic aromatic hydrocarbon degrading bacterium has the advantages that the Tzyjm has the ability of rapidly degrading polycyclic aromatic hydrocarbon under the salty environment, is wide in degrading substrate range and high in degradation rate within a short time, has the salt-tolerance action and has wide application potential of repairing the polycyclic aromatic hydrocarbon in the salty environments including oceans.
Description
Technical field
The invention belongs to environmental engineering and microbial engineering field, particularly relate to semi-lactosi mould (Galactomyces geotrichum) Tzyjm in ground of a strain salt tolerant polycyclic aromatic hydrocarbons and the application in polycyclic aromatic hydrocarbons contaminated environment remediation
Background technology
Polycyclic aromatic hydrocarbons (PAHs) is a class persistence organic pollutant (POPs).In ocean environment, mainly seabed crude oil seepage, sea transport, oil tanker accident, oil well seepage, Lu Yuan enter sea and pollute in the source of PAHs.Day by day frequent along with mankind's Activities of Ocean, PAHs pollution level presents and increases the weight of trend year by year.The high molecular PAHs carcinogenic by force such as benzopyrene, pyrene, benzofluoranthrene detect.PAHs by attach to oceanic sediment for years accumulate in ocean, can in marine organism can enrichment amplify, high trophic level biology is worked the mischief.Carry out fishery cultivating in the marine site of polluting by PAH, the eating quality of its product can be had a strong impact on, once be taken in by human body, and can serious harm health.
Polycyclic aromatic hydrocarbons (PAHs) pollution amelioration is the focus of research during current environment is repaired.Microbiological deterioration is to one of important channel that PAHs contaminated soil is repaired.For polycyclic aromatic hydrocarbons contaminated, carry out the screening operation of a large amount of degrading polycyclic aromatic hydrocarbons microorganism both at home and abroad.Up to the present, isolate multiple bacterial strain with degrading polycyclic aromatic hydrocarbons ability, as Rhodopseudomonas (Pseudomonas putida G7), Mycobacterium (Mycobacterium vanbaalenii PYR-1), bacillus (Bacillus sp.P1) etc.
The isolated polycyclic aromatic hydrocarbon-degrading bacteria of tradition is when running into hypersaline environment, the metabolism of microorganism can be suppressed, thus make microorganism lose degradation capability, and due to condition restriction, the hypersaline environments such as general ocean can not carry out desalting treatment when carrying out degrading polycyclic aromatic hydrocarbons, therefore in actual applications, filtering out salt tolerant degradation bacteria is that hypersaline environment polycyclic aromatic hydrocarbons biochemical degradation provides feasibility.
Summary of the invention
The present invention is directed to the not halophilic weak point of polycyclic aromatic hydrocarbon-degrading bacteria in prior art, provide a kind of degrading polycyclic aromatic hydrocarbons bacterial strain, this bacterial strain can be applied to the Pollution abatement of polycyclic aromatic hydrocarbons in the hypersaline environments such as ocean.
For achieving the above object, the present invention is achieved by the following technical programs:
Semi-lactosi ground mould (Galactomyces geotrichum) provided by the invention, called after Galactomyces geotrichum Tzyjm, be preserved in " China typical culture collection center " on December 4th, 2014, its preserving number CCTCC NO:M 2014625.Address: Wuhan, China Wuhan University.
It is the ability that sole carbon source and the energy carry out growing that described bacterial strain has with polycyclic aromatic hydrocarbons.
The bacterial strain of described degrading polycyclic aromatic hydrocarbons is by the application in the biological restoration of the hypersaline environments such as polycyclic aromatic hydrocarbons contaminated ocean.
The ITS sequence of described bacterial strain is as shown in SEQ NO.1.
The polycyclic aromatic hydrocarbon-degrading bacteria of described salt tolerant is utilized to carry out the method for the hypersaline environment biological restoration such as ocean, it is characterized in that the method is made up of following steps: the beach choosing Taizhou of Zhejiang gulf encloses pool cultivation sedimentary clay 100g, semi-lactosi ground mould (Galactomyces geotrichum) Tzyjm by mass percentage 2.0% with sedimentary clay even, add water and make earth water ratio remain on 15%, put into constant temperature oscillator, control culture temperature under the constant temperature of 20 DEG C, shaking culture repairs 5 days to 20 days.
Tzyjm encloses pool cultivation sedimentary clay to be separated and to screen from the beach of City of Taizhou to obtain.Tzyjm of the present invention at 15 ~ 30 DEG C, better can grow under pH value 5.0 ~ 6.5 culture condition; Bacterial strain is cultivated daystart adularescent bacterium colony and is occurred on YEPD flat board, cultivates and can observe mycelia two days later, hyphae colorless, shape of crawling.Can observe conidium after three days, conidiophore is upright, loose, very short in fine hair shape, the conidium of apical growth bunchiness.Conidium short cylinder shape, two ends truncation, colourless.
As preferably, bacterial strain of the present invention can be sole carbon source and energy growth with polycyclic aromatic hydrocarbons.
The screening method of above-mentioned resistance to high salt polycyclic aromatic hydrocarbon-degrading bacteria, can implement successively as follows:
(1) enclose pool cultivation sedimentary clay from beach and get top layer earth apart from 5 ~ 20cm under earth's surface, carry out enrichment culture as bacterium source; Described enrichment medium contains: glucose, extractum carnis, peptone, MgSO
4, polycyclic aromatic hydrocarbons (including but not limited to naphthalene, phenanthrene, fluoranthene, pyrene, benzanthrene, benzopyrene);
(2) get enrichment culture liquid described in (1) be inoculated in liquid inorganic salt Selective agar medium carry out selection cultivate, institute's inorganic salt Selective agar medium contains: NaCl, MgCl
2﹒ 6H
2o, Na
2sO
4, CaCl
2﹒ 2H
2o, TAPSO, KCl, NH
4cl, Na
2hPO
4﹒ 7H
2o, NaBr, NaHCO
3, H
3bO
3, SrCl
2﹒ 6H
2o, NaF, FeCl
2﹒ 4H
2o, is adjusted to 7.6 (these are the substratum of simulation natural sea-water composition) by pH; (concentration of polycyclic aromatic hydrocarbons naphthalene, phenanthrene, fluoranthene, pyrene, benzanthrene, benzopyrene is 150mg/L ~ 250mg/L to add polycyclic aromatic hydrocarbons; );
(3) getting described in (2) selects culture dilution spread on solid inorganic salt Selective agar medium, carries out being separated namely obtaining polycyclic aromatic hydrocarbon-degrading bacteria Tzyjm.
Above-mentioned polycyclic aromatic hydrocarbon-degrading bacteria Tzyjm can be used for the biological restoration of polycyclic aromatic hydrocarbons in the hypersaline environments such as ocean.
Tzyjm of the present invention at 15 ~ 30 DEG C, better can grow under pH value 5.0 ~ 6.5 culture condition; Bacterial strain is cultivated daystart adularescent bacterium colony and is occurred on YEPD flat board, cultivates and can observe mycelia two days later, hyphae colorless, shape of crawling.Can observe conidium after three days, conidiophore is upright, loose, very short in fine hair shape, the conidium of apical growth bunchiness.Conidium short cylinder shape, two ends truncation, colourless.
Tzyjm is at YEPD (glucose: 0.5% ~ 4.0%; Tryptones: 0.5% ~ 4.0%; The mould powder in semi-lactosi ground: 0.5% ~ 3.0%) cultivate daystart adularescent bacterium colony and occur, cultivate and can observe mycelia two days later, hyphae colorless, shape of crawling.Can observe conidium after three days, conidiophore is upright, loose, very short in fine hair shape, the conidium of apical growth bunchiness.Conidium short cylinder shape, two ends truncation, colourless.
The pcr amplification of the ITS gene of Tzyjm, checking order is completed by Shanghai Mei Ji biotech firm, obtains the sequence that length is 584bp.The gene order selecting homology to be greater than 97% carries out Phylogenetic Analysis, show bacterial strain and Galactomyces geotrichum LMA-317 homology the highest, in conjunction with strain morphology, colony characteristics, qualification bacterial strain belongs to semi-lactosi ground mould (Galactomyces geotrichum).
Tzyjm of the present invention both can at nutritional medium, as: grow in YEPD, potato dextrose medium, Cha Shi substratum, can take polycyclic aromatic hydrocarbons as sole carbon source, grow in the inorganic salt basic medium of the energy again.
In sum, the present invention has the following advantages: Tzyjm provided by the invention has fast degradation ability to the polycyclic aromatic hydrocarbons in hypersaline environment, and degraded substrate spectrum is wide, and degradation rate is high at short notice.Because this bacterium has resistance to high salt action, make, in the reparation of its polycyclic aromatic hydrocarbons in the hypersaline environments such as ocean, there is wide application potential.
Latin literary fame: Galactomyces geotrichum Tzyjm
Depositary institution: China typical culture collection center
Address: Wuhan, China Wuhan University
Preservation date: on December 4th, 2014
Deposit number: CCTCC NO:M 2014625.
Accompanying drawing explanation
Fig. 1 is that Tzyjm encloses to polluting beach the degradation rate that the pool cultivates the polycyclic aromatic hydrocarbons in sedimentary clay
Embodiment
Below by specific embodiment, technical scheme of the present invention is described in further detail, but the present invention is not limited to these embodiments.
Embodiment 1
The separating screening method of Tzyjm provided by the invention.
Enclose pool cultivation sedimentary clay from beach and get top layer earth apart from 5 ~ 20cm under earth's surface, carry out enrichment culture as bacterium source, by glucose 10g, Tryptones 10g, naphthalene 60mg, luxuriant and rich with fragrance 60mg, fluoranthene 60mg, pyrene 60mg, benzanthrene 60mg, benzopyrene 60mg; Join in a certain amount of distilled water, filter membrane is degerming, and this substratum is cultivated two days.The substratum of preparation simulation natural sea-water composition again, by 22.79g NaCl, 11.18g MgCl
2﹒ 6H
2o, 3.98g Na
2sO
4, 1.46g CaCl
2﹒ 2H
2o, 1.30g TAPSO, 0.72gKCl, 0.27g NH
4cl, 89.00mg Na
2hPO
4﹒ 7H
2o, 83.00mg NaBr, 31.00mg NaHCO
3, 27.00mg H
3bO
3, 24.00mg SrCl
2﹒ 6H
2o, 2.60mg NaF, 2.00mg FeCl
2﹒ 4H
2o adds in 800mL distilled water, add the polycyclic aromatic hydrocarbon compounds of naphthalene, phenanthrene, fluoranthene, pyrene, benzanthrene, benzopyrene, add distilled water and be settled to 1000mL, the substratum of above-mentioned simulation natural sea-water composition is above-mentioned add naphthalene, phenanthrene, fluoranthene, pyrene, benzanthrene, benzopyrene amount the ultimate density of naphthalene, phenanthrene, fluoranthene, pyrene, benzanthrene, benzopyrene in the nutrient solution prepared will be made to be 200mg/mL, pH is adjusted to 7.6, filter membrane is degerming, obtains required nutrient solution.Get enrichment culture liquid to cultivate three days on this substratum, dilution spread isolates a strain bacterium called after Tzyjm, and this bacterium is preserved in " China typical culture collection center " its preserving number CCTCC NO:M2014625 on December 9th, 2014.
The pcr amplification of the ITS gene of Tzyjm, checking order is completed by Shanghai Mei Ji biotech firm.The gene order selecting homology to be greater than 97% carries out Phylogenetic Analysis, show bacterial strain and Galactomyces geotrichum LMA-317 homology the highest, in conjunction with strain morphology, colony characteristics, qualification bacterial strain belongs to semi-lactosi ground mould (Galactomyces geotrichum).
Embodiment 2
Tzyjm clean beach of degrading encloses the artificial polycyclic aromatic hydrocarbons added in pool cultivation sedimentary clay.
Choose and add pyrene, benzanthrene, benzopyrene concentration respectively for the clean beach of 200mg/kg encloses pool cultivation sedimentary clay 100g, Tzyjm mixes with earth by 2.0% (mass percent) consumption, add water and make water ratio remain on 15%, put into constant temperature oscillator, control culture temperature under the constant temperature of 20 DEG C, shaking culture repairs 5 days, can realize enclosing pool cultivation sedimentary clay to beach to repair, obtain the soil after repairing, the degradation rate of pyrene, benzanthrene, benzopyrene is more than 75.2%.
Embodiment 3
Tzyjm degraded encloses the polycyclic aromatic hydrocarbons in pool cultivation sedimentary clay by polycyclic aromatic hydrocarbons contaminated beach.
Get City of Taizhou one beach and enclose pool cultivation sedimentary clay, HPLC analyzes and measures main polycyclic aromatic hydrocarbon pollutant phenanthrene, naphthalene, fluoranthene, pyrene and PAHs total amount are respectively 197.0, 60.3, 264.4, 180.5, 983.7ng/g, Tzyjm mixes with earth by 2.0% (mass percent) consumption, add water and make water ratio remain on 15%, put into constant temperature oscillator, control culture temperature under the constant temperature of 25 DEG C, shaking culture repairs 20 days, measure residual luxuriant and rich with fragrance, naphthalene, fluoranthene, pyrene and PAHs total amount are respectively 66.2, 20.8, 130.6, 97.8, 472.5ng/g, degradation rate is respectively 64.8%, 68.2%, 50.6%, 45.8%, 51.2%.
Method of the present invention is utilized to repair contaminated soil under high salt condition, show that the repairing effect of polycyclic aromatic hydrocarbons is good, obvious to the degradation effect of polycyclic aromatic hydrocarbons, bacterial strain salt tolerant is described and reaches the effect of good degrading polycyclic aromatic hydrocarbons, compared with prior art, improve the repairing effect to polycyclic aromatic hydrocarbons contaminated environment, be more conducive to the reparation to polycyclic aromatic hydrocarbons in the hypersaline environments such as ocean in reality.
Specific embodiment described in the present invention is only to the explanation for example of the present invention's spirit.Those skilled in the art can make various amendment or supplement or adopt similar mode to substitute to described specific embodiment; but spirit of the present invention can't be departed from or surmount the scope that appended claims defines; within the spirit and principles in the present invention all; any amendment of doing, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (6)
1. the polycyclic aromatic hydrocarbon-degrading bacteria of a strain salt tolerant, it is characterized in that, this bacterial strain is the mould Galactomyces geotrichum Tzyjm in semi-lactosi ground, is CCTCC NO:M 2014625, possesses the ability of degrading polycyclic aromatic hydrocarbons under hypersaline environment at the preserving number of China typical culture collection center.
2. the polycyclic aromatic hydrocarbon-degrading bacteria of salt tolerant according to claim 1, it is characterized in that described bacterial strain has with polycyclic aromatic hydrocarbons is the ability that sole carbon source and the energy carry out growing.
3. the polycyclic aromatic hydrocarbon-degrading bacteria of salt tolerant according to claim 1, is characterized in that the ITS sequence of described bacterial strain is as shown in SEQ NO.1.
4. the polycyclic aromatic hydrocarbon-degrading bacteria of salt tolerant according to claim 1, is characterized in that Selective agar medium contains: NaCl, MgCl
2﹒ 6H
2o, Na
2sO
4, CaCl
2﹒ 2H
2o, TAPSO, KCl, NH
4cl, Na
2hPO
4﹒ 7H
2o, NaBr, NaHCO
3, H
3bO
3, SrCl
2﹒ 6H
2o, NaF, FeCl
2﹒ 4H
2o, is adjusted to 7.6 by pH.
5. the application of the polycyclic aromatic hydrocarbon-degrading bacteria of salt tolerant in the biological restoration of the polycyclic aromatic hydrocarbons contaminated environment in ocean according to claim 1.
6. utilize the polycyclic aromatic hydrocarbon-degrading bacteria of salt tolerant described in claim 1 to carry out the method for contaminate environment biological restoration, it is characterized in that the method is made up of following steps: choose polycyclic aromatic hydrocarbons contaminated beach and enclose pool cultivation sedimentary clay 100g, the mould Galactomyces geotrichum Tzyjm in semi-lactosi ground 2.0% mixes with earth by mass percentage, add water and make earth water ratio remain on 15%, put into constant temperature oscillator, control culture temperature under the constant temperature of 20 DEG C, shaking culture repairs 5 days to 20 days.
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CN110760449A (en) * | 2019-11-15 | 2020-02-07 | 浙江工业大学 | Geotrichum galactose ZJPH1810 and application thereof in preparation of (S) -1- (2, 6-dichloro-3-fluorophenyl) ethanol |
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Effective date of registration: 20190926 Address after: Room 2321, 4 Wanda Plaza, Taizhou City, Zhejiang Province, 318000 (office only) Patentee after: Zhejiang Taicheng Environmental Technology Co., Ltd. Address before: 318000 No. 788, Xueyuan Road, Zhejiang, Taizhou Patentee before: Taizhou Vocational & Technical College |