CN104673683B - One plant of salt tolerant polycyclic aromatic hydrocarbon-degrading bacteria Tzyjm and its application in marine pollution environment - Google Patents
One plant of salt tolerant polycyclic aromatic hydrocarbon-degrading bacteria Tzyjm and its application in marine pollution environment Download PDFInfo
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- CN104673683B CN104673683B CN201510089660.6A CN201510089660A CN104673683B CN 104673683 B CN104673683 B CN 104673683B CN 201510089660 A CN201510089660 A CN 201510089660A CN 104673683 B CN104673683 B CN 104673683B
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Abstract
The polycyclic aromatic hydrocarbon-degrading bacteria of one plant of salt tolerant, it is characterised in that the bacterial strain is the mould Galactomyces geotrichum Tzyjm in galactolipin ground, is CCTCC NO in the preserving number of China typical culture collection center:M 2014625, the ability for possessing the degrading polycyclic aromatic hydrocarbons under hypersaline environment.The present invention has advantages below：The Tzyjm that the present invention is provided has fast degradation ability to the polycyclic aromatic hydrocarbon in hypersaline environment, and degraded substrate spectrum is wide, and degradation rate is high in a short time.Because the bacterium has resistance to high salt action, make that there is wide application potential in the reparation of its polycyclic aromatic hydrocarbon in the hypersaline environments such as ocean.
Description
Technical field
The invention belongs to the gala of environmental project and microbial engineering field, more particularly to one plant of salt tolerant polycyclic aromatic hydrocarbon
Sugaredly mould (Galactomyces geotrichum) Tzyjm and the application in polycyclic aromatic hydrocarbons contaminated environment remediation
Background technology
Polycyclic aromatic hydrocarbon (PAHs) is a class persistence organic pollutant (POPs).PAHs source is mainly in marine environment
Seabed crude oil seepage, marine transportation, oil tanker accident, oil well seepage, Lu Yuan enter sea pollution etc..With mankind's Activities of Ocean increasingly
Frequently, PAHs levels of pollution present and aggravate trend year by year.The strong carcinogenic HMW PAHs such as BaP, pyrene, benzofluoranthrene is
There is detection.PAHs by attach to marine sediment it is for years accumulated in ocean, be able to can be enriched with marine organisms body
Amplification, causes harm to high nutrition level biology.Fishery cultivating, the edible quality meeting of its product are carried out in the marine site polluted by PAH
It is severely impacted, health can be seriously endangered if being taken in by human body.
Polycyclic aromatic hydrocarbon (PAHs) pollution amelioration is the focus studied in current environment reparation.Microbial degradation is dirty to PAHs
One of important channel that dye soil is repaired.For polycyclic aromatic hydrocarbons contaminated, a large amount of degrading polycyclic aromatic hydrocarbons have been carried out both at home and abroad micro-
Biological screening operation.Up to the present, it is separated go out multiple bacterial strains with degrading polycyclic aromatic hydrocarbons ability, such as pseudomonas
(Pseudomonas putida G7), Mycobacterium (Mycobacterium vanbaalenii PYR-1), bacillus
Belong to (Bacillus sp.P1) etc..
The polycyclic aromatic hydrocarbon-degrading bacteria that tradition is isolated can suppress the metabolism of microorganism when running into hypersaline environment, from
And microorganism is lost degradation capability, and because condition is limited, the hypersaline environment such as general ocean is carrying out degrading polycyclic aromatic hydrocarbons
Shi Buhui carries out desalting processing, therefore in actual applications, it is hypersaline environment polycyclic aromatic hydrocarbon biochemistry drop to filter out salt tolerant degradation bacteria
Solution provides feasibility.
The content of the invention
There is provided one kind degraded is polycyclic for the not halophilic weak point of polycyclic aromatic hydrocarbon-degrading bacteria in the prior art by the present invention
Aromatic hydrocarbons bacterial strain, the bacterial strain can apply to the pollution control of polycyclic aromatic hydrocarbon in the hypersaline environments such as ocean.
To reach above-mentioned purpose, the technical scheme is that：
The galactolipin that the present invention is provided it is mould (Galactomyces geotrichum), be named as Galactomyces
Geotrichum Tzyjm, were preserved in " China typical culture collection center ", its preserving number CCTCC on December 4th, 2014
NO:M 2014625.Address：Wuhan, China Wuhan University.
The bacterial strain has the ability grown using polycyclic aromatic hydrocarbon as sole carbon source and the energy.
The bacterial strain of the degrading polycyclic aromatic hydrocarbons by the hypersaline environments such as polycyclic aromatic hydrocarbons contaminated ocean it is biological prosthetic in
Using.
The ITS sequence of the bacterial strain is as shown in SEQ NO.1.
The biological prosthetic methods of hypersaline environment such as ocean are carried out using the polycyclic aromatic hydrocarbon-degrading bacteria of the salt tolerant, its feature exists
Comprised the steps of in this method：The beach for choosing Taizhou of Zhejiang gulf encloses pool cultivation sedimentary clay 100g, galactolipin it is mould
(Galactomyces geotrichum) Tzyjm is 2.0% uniform with sedimentary clay by mass percentage, and adding water makes soil moisture content
15% is maintained at, is put into constant temperature oscillator, control cultivation temperature is under 20 DEG C of constant temperature, and shaken cultivation repairs 5 days extremely
20 days.
Tzyjm is to enclose during the pool cultivates sedimentary clay to separate and screen from the beach of City of Taizhou to obtain.Tzyjm of the present invention
Can preferably it be grown under 15~30 DEG C, the condition of culture of pH value 5.0~6.5；Bacterial strain cultivates daystart on YEPD flat boards white
Color bacterium colony occurs, and culture is two days later it is observed that mycelia, hyphae colorless, shape of crawling.Conidium is observed that after three days,
Conidiophore is upright, loose, and very short is in villiform, the conidium of apical growth bunchiness.Conidium short cylinder shape, two ends
Truncation, it is colourless.
Preferably, bacterial strain of the present invention can be that sole carbon source and the energy grow using polycyclic aromatic hydrocarbon.
The screening technique of the above-mentioned polycyclic aromatic hydrocarbon-degrading bacteria of resistance to high salt, can implement successively as follows：
(1) pool cultivation sedimentary clay is enclosed away from top layer soil is taken at 5~20cm under earth's surface from beach, enrichment training is carried out as bacterium source
Support；The enriched medium contains：Glucose, beef extract, peptone, MgSO4, polycyclic aromatic hydrocarbon (include but is not limited to naphthalene, phenanthrene,
Fluoranthene, pyrene, benzanthracene, BaP)；
(2) enrichment culture liquid described in (1) is taken to be inoculated in progress selection culture, institute's nothing in liquid inorganic salt Selective agar medium
Machine salt Selective agar medium contains：NaCl、MgCl2﹒ 6H2O、Na2SO4、CaCl2﹒ 2H2O、TAPSO、KCl、NH4Cl、Na2HPO4﹒
7H2O、NaBr、NaHCO3、H3BO3、SrCl2﹒ 6H2O、NaF、FeCl2﹒ 4H2O, pH is adjusted into 7.6, and (this is simulation natural sea-water group
Into culture medium)；Adding polycyclic aromatic hydrocarbon, (polycyclic aromatic hydrocarbon naphthalene, phenanthrene, fluoranthene, pyrene, benzanthracene, the concentration of BaP are 150mg/L
~250mg/L；)；
(3) take and select culture dilution spread on solid inorganic salt Selective agar medium described in (2), progress separation is produced
To polycyclic aromatic hydrocarbon-degrading bacteria Tzyjm.
Above-mentioned polycyclic aromatic hydrocarbon-degrading bacteria Tzyjm can with the high salt environment such as Yu Haiyang polycyclic aromatic hydrocarbon it is biological prosthetic.
Tzyjm of the present invention can preferably grow under 15~30 DEG C, the condition of culture of pH value 5.0~6.5；Bacterial strain is in YEPD flat boards
Upper culture daystart has white colony appearance, and culture is two days later it is observed that mycelia, hyphae colorless, shape of crawling.After three days
It is observed that conidium, conidiophore is upright, loose, very short is in villiform, the conidium of apical growth bunchiness.Point
Raw spore short cylinder shape, two ends truncation is colourless.
Tzyjm is in YEPD (glucose：0.5%~4.0%；Tryptone：0.5%~4.0%；The mould powder in galactolipin ground：
0.5%~3.0%) culture culture daystart have a white colony appearance, culture two days later it is observed that mycelia, mycelia without
Color, shape of crawling.It is observed that conidium, conidiophore is upright, loose after three days, very short is in villiform, apical growth
The conidium of bunchiness.Conidium short cylinder shape, two ends truncation is colourless.
PCR amplifications, the sequencing of Tzyjm ITS genes are completed by Shanghai Mei Ji biotech firms, have obtained length for 584bp
Sequence.The gene order for selecting homology to be more than 97% carries out Phylogenetic Analysis, shows bacterial strain and Galactomyces
Geotrichum LMA-317 homology highests, with reference to strain morphology, colony characteristicses, identification bacterial strain is with belonging to galactolipin mould
(Galactomyces geotrichum)。
Tzyjm of the present invention both can be in nutrient medium, such as：In YEPD, potato dextrose medium, czapek's medium
Growth, can grow by sole carbon source of polycyclic aromatic hydrocarbon, in the inorganic salts basal medium of the energy again.
In summary, the present invention has advantages below：The Tzyjm that the present invention is provided has to the polycyclic aromatic hydrocarbon in hypersaline environment
Fast degradation ability, and degraded substrate spectrum is wide, degradation rate is high in a short time.Because the bacterium has resistance to high salt action, make it
There is wide application potential in the hypersaline environments such as ocean in the reparation of polycyclic aromatic hydrocarbon.
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.
Brief description of the drawings
Fig. 1 is the degradation rate that Tzyjm encloses the polycyclic aromatic hydrocarbon in pool cultivation sedimentary clay to pollution beach
Embodiment
Below by specific embodiment, technical scheme is described in further detail, but the present invention is simultaneously
It is not limited to these embodiments.
Embodiment 1
The separating screening method for the Tzyjm that the present invention is provided.
Pool cultivation sedimentary clay is enclosed away from top layer soil is taken at 5~20cm under earth's surface from beach, and enrichment culture is carried out as bacterium source,
By glucose 10g, tryptone 10g, naphthalene 60mg, phenanthrene 60mg, fluoranthene 60mg, pyrene 60mg, benzanthracene 60mg, BaP 60mg；
It is added in a certain amount of distilled water, filter membrane is degerming, is cultivated two days on this culture medium.Simulation natural sea-water composition is prepared again
Culture medium, by 22.79g NaCl, 11.18g MgCl2﹒ 6H2O、3.98g Na2SO4、1.46g CaCl2﹒ 2H2O、1.30g
TAPSO、0.72gKCl、0.27g NH4Cl、89.00mg Na2HPO4﹒ 7H2O、83.00mg NaBr、31.00mg NaHCO3、
27.00mg H3BO3、24.00mg SrCl2﹒ 6H2O、2.60mg NaF、2.00mg FeCl2﹒ 4H2O is added in 800mL distilled water,
Naphthalene, phenanthrene, fluoranthene, pyrene, benzanthracene, the polycyclic aromatic hydrocarbon compounds of BaP are added, distilled water is added and is settled to 1000mL, above-mentioned mould
The above-mentioned addition naphthalene of culture medium of plan natural sea-water composition, phenanthrene, fluoranthene, pyrene, benzanthracene, the amount of BaP will make the culture prepared
Naphthalene, phenanthrene, fluoranthene, pyrene, benzanthracene, the ultimate density of BaP are 200mg/mL in liquid, pH are adjusted into 7.6, filter membrane is degerming, i.e.,
Obtain required nutrient solution.Enrichment culture liquid is taken to be cultivated three days on this culture medium, dilution spread is isolated one plant of bacterium and is named as
Tzyjm, the bacterium was preserved in " China typical culture collection center " its preserving number CCTCC NO on December 9th, 2014:
M2014625。
PCR amplifications, the sequencing of Tzyjm ITS genes are completed by Shanghai Mei Ji biotech firms.Homology is selected to be more than 97%
Gene order carry out Phylogenetic Analysis, show bacterial strain and Galactomyces geotrichum LMA-317 homologys most
Height, with reference to strain morphology, colony characteristicses, identification bacterial strain is with belonging to galactolipin mould (Galactomyces geotrichum).
Embodiment 2
The clean beach of Tzyjm degradeds encloses the polycyclic aromatic hydrocarbon manually added in pool cultivation sedimentary clay.
Choose addition pyrene, benzanthracene, BaP concentration and respectively enclose pool cultivation sedimentary clay 100g for 200mg/kg clean beach,
Tzyjm is well mixed by 2.0% (mass percent) consumption with soil, and adding water makes moisture content be maintained at 15%, is put into constant temperature and shakes
Swing in device, control cultivation temperature is under 20 DEG C of constant temperature, and shaken cultivation is repaired 5 days, can realize and pool cultivation is enclosed to beach
Sedimentary clay is repaired, the soil after being repaired, and pyrene, benzanthracene, the degradation rate of BaP are more than 75.2%.
Embodiment 3
The polycyclic aromatic hydrocarbon in pool cultivation sedimentary clay is enclosed in Tzyjm degradeds by polycyclic aromatic hydrocarbons contaminated beach.
Take the beach of City of Taizhou one to enclose pool cultivation sedimentary clay, HPLC analyses determine main polycyclic aromatic hydrocarbon pollutant it is luxuriant and rich with fragrance,
Naphthalene, fluoranthene, pyrene and PAHs total amounts are respectively 197.0,60.3,264.4,180.5,983.7ng/g, Tzyjm presses 2.0% (quality
Percentage) consumption is well mixed with soil, and adding water makes moisture content be maintained at 15%, is put into constant temperature oscillator, control culture temperature
Degree is under 25 DEG C of constant temperature, and shaken cultivation is repaired 20 days, determines residual phenanthrene, naphthalene, fluoranthene, pyrene and PAHs total amounts and is respectively
66.2nd, 20.8,130.6,97.8,472.5ng/g, degradation rate is respectively 64.8%, 68.2%, 50.6%, 45.8%,
51.2%.
Contaminated soil under high salt conditions is repaired using the method for the present invention, shows the repairing effect of polycyclic aromatic hydrocarbon
It is good, illustrate bacterial strain salt tolerant and reach the effect of preferable degrading polycyclic aromatic hydrocarbons to the degradation effect of polycyclic aromatic hydrocarbon substantially, it is and existing
Technology is compared, and improves the repairing effect to polycyclic aromatic hydrocarbons contaminated environment, is more beneficial in practice in the hypersaline environments such as ocean
The reparation of polycyclic aromatic hydrocarbon.
Specific embodiment described in the present invention is only to spirit explanation for example of the invention.Technology neck belonging to of the invention
The technical staff in domain can be made various modifications or supplement to described specific embodiment or be replaced using similar mode
Generation, but without departing from the spiritual of the present invention or surmount scope defined in appended claims, all essences in the present invention
God is with principle, and any modification, equivalent substitution and improvements made etc. should be included in the scope of the protection.
Claims (3)
1. the polycyclic aromatic hydrocarbon-degrading bacteria of one plant of salt tolerant, it is characterised in that the bacterial strain is the mould Galactomyces in galactolipin ground
Geotrichum Tzyjm, are CCTCC NO in the preserving number of China typical culture collection center:M 2014625, possesses
The ability of degrading polycyclic aromatic hydrocarbons under hypersaline environment.
2. according to claim 1 the polycyclic aromatic hydrocarbon-degrading bacteria of salt tolerant the polycyclic aromatic hydrocarbons contaminated environment in ocean it is biological prosthetic in
Application.
3. carry out the method that pollution environmental organism is repaired, its feature using the polycyclic aromatic hydrocarbon-degrading bacteria of salt tolerant described in claim 1
It is that this method is comprised the steps of：Choose polycyclic aromatic hydrocarbons contaminated beach and enclose pool cultivation sedimentary clay 100g, galactolipin it is mould
Galactomyces geotrichum Tzyjm 2.0% are well mixed with sedimentary clay by mass percentage, and adding water contains sedimentary clay
Water rate is maintained at 15%, is put into constant temperature oscillator, and control cultivation temperature is under 20 DEG C of constant temperature, and shaken cultivation repairs 5
It was to 20 days.
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| CN110760449B (en) * | 2019-11-15 | 2021-04-06 | 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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| CN102864086B (en) * | 2012-10-12 | 2013-11-06 | 台州职业技术学院 | Strain capable of degrading polycyclic aromatic hydrocarbons (PAHs) and application thereof in soil remediation |
| CN102864112B (en) * | 2012-10-12 | 2014-03-12 | 台州职业技术学院 | Heavy metal resistant strain for degrading polycyclic aromatic hydrocarbons (PAHs) and application thereof in combined contaminated soil remediation |
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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 |
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