CN105950501A - Pantoea sp. for degrading polycyclic aromatic hydrocarbon organic pollutants - Google Patents
Pantoea sp. for degrading polycyclic aromatic hydrocarbon organic pollutants Download PDFInfo
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Abstract
The invention discloses pantoea sp. PYR16 for degrading polycyclic aromatic hydrocarbon organic pollutants, and belongs to the field of microbial and environmental organic pollutant repair and control. The strain is preserved in China Center for Type Culture Collection, with preservation number of CCTCC NO: M2016227. The strain disclosed by the invention can be used for effectively degrading various polycyclic aromatic hydrocarbons; the strain is broad in environment-adaptive and heavy-metal resistant capacities; and the strain, when implanted in severe environments, can be used for repairing soil or water bodies which are affected by the combined pollution of heavy metals and polycyclic aromatic hydrocarbons.
Description
Technical field
The invention belongs to microorganism and environmental organic pollutant repairing and treating field, be specifically related to a strain fall
Solve the general bacterium (Pantoea sp.) of multiring aromatic hydrocarbon organic pollution, the invention still further relates to answering of this general bacterium
With and a kind of method of degrading polycyclic aromatic hydrocarbons class organic pollution.
Background technology
Polycyclic aromatic hydrocarbon (be called for short PAHs) refer to a class by two or more phenyl ring with wire, horn shape or
The condensed ring Hydrocarbon of tufted arrangement, wherein has the PAHs referred to as height of 4 phenyl ring and more than 4
Molecular weight polycyclic aromatic hydrocarbon (High molecular weight-PAHs is called for short HMW-PAHs).Polycyclic aromatic hydrocarbon
Source mainly include two aspects: be on the one hand the activity of nature self, such as forest fire, dynamic
Plant remains rots, the formation of oil coal, volcanic eruption etc.；Another one aspect is that the mankind pollute row
For, such as the imperfect combustion of Fossil fuel, the burning of municipal solid wastes, oil exploitation, processing
Transport and Chemical Manufacture and the discharge etc. of vehicle exhaust.Mankind's activity is the overwhelming majority to ambient contribution
Polycyclic aromatic hydrocarbons contaminated, the main pollution source of environment polycyclic aromatic hydrocarbon that is well recognized as.Polycyclic aromatic hydrocarbon is generally deposited
It is petroleum chemicals, rubber, plastics, lubricating oil, antirust oil and unburnt organic compound
In the materials such as thing, it can pass through air and the flowing of water body, long-distance migration occurs, the most extensively
It is distributed in the various surrounding mediums such as air, soil, water body, deposit and plant.Due to multi-ring
Virtue is low through water solublity, adsorptivity strong, long half time so that it is can be continuously present in environment the several years,
Generally along with aromatic ring number and the increase of molecular weight, its bioavailability is the poorest, therefore repairs macromolecule
Measure multi-ring fragrance more difficult compared to low-molecular-weight polycyclic aromatic hydrocarbon.The fat-soluble of polycyclic aromatic hydrocarbon makes it
Can be enriched to step by step be in the internal of food chain end people by food chain, and be distributed to in-vivo tissue,
By the catalytic action during protein ketone-dilute alcohol tautomerism, cause the irreversible transformation of albumen,
And then make cell generation canceration.Therefore human health and ecosystem are caused grave danger, many cyclophanes
The pollution amelioration of warp is study hotspot both domestic and external.
Traditional polycyclic aromatic hydrocarbons contaminated reparation relies primarily on physical chemistry reparation (chemical oxidation, visitor
Soil or method of digging, stripping, thermal desorption, burn, the method such as landfill is removed or pollution in fixing soil
Thing), these methods often processing cost is high, repair not thoroughly, easily produce secondary pollution and uncomfortable
Close large-scale application.It addition, though the environmental pollution that single pollutant are constituted in nature happens occasionally,
But in fact the Single Pollution in absolute sense is non-existent, pollute the association of many tools and comprehensive i.e.
The combined pollution that multiple pollutant is formed, can be continuously present in owing to heavy metal is the same with polycyclic aromatic hydrocarbon
In soil environment, therefore heavy metal-polycyclic aromatic hydrocarbon composite pollution is the principal mode of soil pollution.
Biological restoration is to utilize microorganism to repair pollutant, and it not only overcomes physical chemistry repair place
Reason cost is high, repair the defects such as the most thorough, and will not produce secondary pollution, has pollutant and repaiies
The microbial bacterial agent of multiple effect also is adapted for large-scale application.The essence of microorganism remediation is to utilize soil micro-
Organic pollution in biospecificity metabolism soil, be translated into other carbon back macromole of cell and
The process of carbon dioxide.Therefore the core of organic pollution microorganism remediation technology is that accretion rate is fast,
The screening of the degradation bacteria that environmental suitability is strong.And direct enrichment isolation indigenous microorganism from environment, no
Only effectively prevent the biosafety issues that genetic engineering bacterium occurs in actual applications, but also be
A kind of simple and convenient preferable technological means.People have screened from all kinds of ecological environmenies and have obtained at present
A collection of polycyclic aromatic hydrocarbon-degrading bacteria, including pseudomonas (Pseudomonas), arthrobacterium (Arthrobacter),
Rhodococcus fascians (Rhodococcus), mycobacteria (Mycobacterium), bacillus cereus (Bacillus),
Sphingol single-cell (Sphingomonas), Aeromonas (Aeromonas), Bai Yelin kirschner bacterium
(Beijerinckia), nocardia (Nocardioides), bacterium of unlinking (Cycloclasticus), primary
Kirschner bacterium (Burkholderia), comamonas (Polaromonas) and some thermophilic anaerobics
Pseudomonas is as addicted to oil bacterium (Neptunomonas), Janus bacterium (Janibacter), Nocard's bacillus
(Nocardia), degeneration bacterium (Deltaproteobacteria) and Alcaligenes (Alcaligenes) etc..
Summary of the invention
It is an object of the invention to provide the general bacterium of a kind of degrading polycyclic aromatic hydrocarbons class organic pollution.
Applicant separate from the soil of oil pollution obtain a strain can fast degradation multiring aromatic hydrocarbon dirty
The bacterial strain of dye thing, is identified by Physiology and biochemistry and ribosome 16S rDNA gene sequencing is identified
For general Pseudomonas (Pantoea sp.), named Pantoea sp.PYR16.This bacterial strain is in 2016 4
The moon delivers the China typical culture collection center being positioned at Wuhan City, Hubei Province Wuhan University on 26th
(CCTCC) preservation, deposit number is: CCTCC NO:M 2016227.
Result of the test shows:
1) this bacterial strain has an efficient degradation ability to pyrene, and can the pyrene of degrading high concentration.
2) this bacterial strain is in the range of pH is 6-10, best to the degradation effect of pyrene, is therefore suitable for weak acid
Property remove to the polycyclic aromatic hydrocarbon pollutant under the conditions of alkaline environment.
3) this bacterial strain has the ability of NaCl concentration adaptation widely.
4) this bacterial strain has temperature adaptation ability widely, and the polycyclic aromatic hydrocarbon being more suitable under cryogenic conditions has
The removing of organic pollutants, is conducive to carrying out soil in-situ reparation throughout the year with northern area in China.
5) this bacterial strain can the toxic action of heavy metal tolerance in a big way, maintain to polycyclic aromatic hydrocarbon
The efficient degradation of pollutant, has the ability of heavy metal-polycyclic aromatic hydrocarbon composite pollution environment remediation.
6) this bacterial strain has good degradation function, especially to multiple PAHs such as phenanthrene, fluorenes, fluoranthene, pyrene
Be for 4 rings and 4 rings below polycyclic aromatic hydrocarbon degradation effect notable, this is conducive to this bacterium to be applied to
The with serious pollution area of combined pollutant.
7) this bacterial strain has preventing from heavy metal ability widely, and this is conducive to this bacterium to be colonizated in rugged environment
Middle reparation is by heavy metal and the soil of polycyclic aromatic hydrocarbon composite pollution or water body.
More detailed technical scheme is shown in that " specific embodiments " is described.
Accompanying drawing explanation
Fig. 1 is bacterial strain PYR16 growth under 200mg/L pyrene concentration and degradation curve.
Fig. 2 is the degradation curve that bacterial strain PYR16 initiates pyrene concentration to difference.
Fig. 3 is the bacterial strain PYR16 degradation capability to pyrene under condition of different pH.
Fig. 4 is the bacterial strain PYR16 degradation capability to pyrene under the conditions of different Na ion concentrations.
Fig. 5 is the bacterial strain PYR16 degradation capability to pyrene under condition of different temperatures.
Fig. 6 is bacterial strain PYR16 to degradation capability to pyrene under different heavy metal ion existence conditions.
Fig. 7 is the bacterial strain PYR16 degradation capability to variety classes polycyclic aromatic hydrocarbon.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described in detail.Should be noted that the present invention's
Embodiment is only limitted to illustrate for the present invention, and does not limit effect.Involved by embodiment
Zhao Bin, He Shaojiang chief editor is can refer to about screening technique, buffer and common culture medium formula etc.
The content described by " Microbiology Experiment " and " Molecular Cloning: A Laboratory guide " (see J. Pehanorm
Brooker etc., 2002, Molecular Cloning: A Laboratory guide, the third edition, gold winter wild goose etc. (translating), Science Press,
Beijing).Other various experimental implementation involved in the present invention, are the ordinary skill in the art,
The part being not particularly illustrated in literary composition, those of ordinary skill in the art is referred to the present patent application day
Various common tool books, scientific and technical literature or relevant description, handbook etc. before are practiced.
The isolation identification of embodiment 1PYR16 bacterial strain
Employing is screening soil, concrete screening scheme from the soil that Changqing oilfields is the most petroleum-polluted
As follows:
1) take 10g soil, join in the minimal medium MSM containing 200mg/L pyrene, 28 DEG C
Concussion is cultivated 15 days, then receives the above-mentioned minimal medium containing 200mg/L pyrene with the inoculum concentration of 10%
In MSM culture medium；Switching 5 times continuously.
2) take 0.1ml step 1) obtained by enrichment culture liquid, be applied to the MSM solid plate containing pyrene
On, the bacterium colony that flat board grows, after 15 days, is transferred to the MSM solid containing pyrene by 28 DEG C of static gas wave refrigerator
On flat board, continue to cultivate growth, until single bacterium colony occurring on flat board.
3) single bacterium colony of growing out on the picking MSM solid plate containing pyrene, be inoculated into containing
In the MSM fluid medium of 200mg/L pyrene, 28 DEG C of concussions are cultivated 15 days, it is thus achieved that the bacterial strain of the present invention.
Further bacterial strain morphology and molecular biology identification are carried out.
First, the morphological feature of bacterial strain having been carried out preliminary observation, result shows, this bacterial strain is
Gram negative bacteria, rod-short.
Meanwhile, inventor separates the genomic DNA obtaining bacterial strain, then with bacterial 16 S rRNA gene
Universal primer 27F and 1492R primer DNA is carried out PCR amplification；The pcr amplification product that will obtain
Checking order, its sequencing result is as shown in sequence table SEQ ID NO:1.Then, NCBI nucleic acid is utilized
Data base, has carried out BLAST analysis according to bacterial strain 16S rRNA gene order, and result shows bacterial strain
16S rRNA gene order is up to 99%, in conjunction with Gram’s staining and morphology with the homology of general Pseudomonas
Observing, this Strain Designation is Pantoea sp.PYR16 by inventor.
The embodiment 2PYR16 bacterial strain degraded feature to pyrene
With pyrene as sole carbon source, utilizing culture medium based on MSM culture medium, inventor is to PYR16 bacterium
The degradation capability of pyrene is tested by strain.First, take out the bacterial strain of preservation from-80 DEG C, then inoculate
To R2A solid medium activates 1 day；Picking list colony inoculation to 5 milliliters R2A fluid medium is trained
Support to OD600=0.5；Then take 0.5ml bacterium solution and be inoculated into the MSM cultivation of the pyrene that 25ml contains 200mg/L
In base, it is placed in 28 DEG C of concussions and cultivates.In above-mentioned degraded system, the access amount of PYR16 bacterial strain is about
105-106Left and right.Take out one bottle of culture every 24 hours, measure OD respectively600Value, and utilize acetone/
In normal hexane (1:1) extraction triangular flask, the pyrene of all residuals, then utilizes GC-FID to carry out pyrene content and divides
Analysis.Take three bottles every time and carry out parallel analysis.
As it is shown in figure 1, after inoculation PYR16 bacterial strain the 15th day, the pyrene in culture medium is degraded more than 70%,
By the 20th day, in degraded system, the pyrene of more than 82% was all completely removed.This absolutely proves that the present invention is carried
The bacterial strain of confession has the ability of removal pyrene quick, thorough.
Further, the PYR16 bacterial strain degradation rate to the pyrene of different initial concentrations is inventors tested a larged
And degradation effect.As in figure 2 it is shown, the pyrene of low concentration (less than 200mg/L), PYR16 bacterial strain 20 days
I.e. degradable pyrene reaches more than 80%；And when initial pyrene concentration brings up to 800mg/L when, inoculation
The clearance that after PYR16 bacterial strain, 20 days can complete about more than 80%.This shows, provided by the present invention
PYR16 bacterial strain has the ability quickly removing high concentration pyrene.
The impact on PYR16 bacterial strain pyrene degradation capability of embodiment 3pH, sodium ion and temperature
First MSM culture medium hydrochloric acid or sodium hydroxide solution are adjusted to pH be respectively 4,5,6,7,
8,9,10, then according to the operational approach inoculation 10 of embodiment 25-106The PYR16 bacterial strain of left and right, 28
DEG C concussion is cultivated and within 10 days, is remained the concentration of pyrene in post analysis cultivating system.As it is shown on figure 3, be 6-10 at pH
In the range of, PYR16 bacterial strain is all able to maintain that the degradation effect to pyrene, but when pH is less than 6 when,
Its degradation capability will be significantly inhibited.Therefore, the suitable treatments of pyrene degradation bacteria provided by the present invention
The pH of environment should be adjusted between 6-10, and, when the pH processing sample is adjusted to 6-10 when,
PYR16 bacterial strain can maintain the degradation efficiency of more than 34% to pyrene material.Bacterium the most provided by the present invention
Strain is suitable for faintly acid and removes to the polycyclic aromatic hydrocarbon pollutant under the conditions of alkaline environment.
Being similar to, inventor adds NaCl in above-mentioned MSM culture medium so that it is concentration is respectively 0%,
1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, then according to the operational approach of embodiment 2 connects
Plant 105-106The PYR16 bacterial strain of left and right, 28 DEG C of concussions are cultivated and are remained pyrene in 10 days post analysis cultivating systems
Concentration.As shown in Figure 4, when the NaCl concentration in cultivating system is less than 3% when, degradation rate can
To maintain more than 35%；When NaCl concentration brings up to about 8% when, the PYR16 degraded to pyrene
Rate remains within about 30%.
Additionally, inventor inoculates 10 according to the operational approach of embodiment 25-106The PYR16 bacterial strain of left and right,
Then it is respectively placed in 4 DEG C, 16 DEG C, 28 DEG C, 37 DEG C, 42 DEG C and shakes cultivation 10 days, then analyze cultivation
System remains the concentration of pyrene.As it is shown in figure 5,28 DEG C, under 37 DEG C of cultivation temperature, PYR16 bacterial strain
The degradation rate of pyrene is able to maintain that more than 65%, wherein under the cultivation temperature of 37 DEG C, above-mentioned strain energy
Enough maintain optimal degradation effect；And once temperature rises to 42 DEG C, its degradation efficiency would fall to 60%
Left and right.This shows that PYR16 strain growth adaptive temperature provided by the present invention is relatively wide and is more suitable for low temperature
Under the conditions of the removing of environment pyrene pollutant, this is beneficial to open with northern area throughout the year in China
Exhibition soil pyrene pollutes in-situ immobilization.
The impact on the pyrene degradation effect of PYR16 of embodiment 4 heavy metal ion
According to inoculation method described in embodiment 2, PYR16 is inoculated into the MSM training containing 200mg/L pyrene
Support in base, be separately added into simultaneously 150mg/L manganese sulfate, 75mg/L manganese sulfate, 250mg/L copper sulfate,
125mg/L copper sulfate, 28 DEG C of concussions cultivate after 21 days, analyze the dense of the pyrene of residual in degraded system respectively
Degree.As shown in Figure 6: the manganese sulfate of 75mg/L for the pyrene degradation capability of PYR16 bacterial strain almost without pressing down
System, and under the manganese sulfate existence condition of 150mg/L, PYR16 to the degradation rate of 200mg/L pyrene close to 40%
Left and right；And PYR16 degradation capability is affected bigger by copper sulfate, there is bar at 125mg/L copper sulfate
Under part, bacterial strain PYR16 bacterial strain remains able to maintain the degradation rate of about 25%, and when concentration of copper sulfate carries
The when that height being to 250mg/L, its degradation rate drops to about 16%.This shows provided by the present invention
PYR16 bacterial strain can the toxic action of heavy metal tolerance in a big way, maintain to multiring aromatic hydrocarbon
The efficient degradation ability of pollutant, has the ability of heavy metal-polycyclic aromatic hydrocarbon composite pollution environment remediation.
The embodiment 5PYR16 bacterial strain degradation effect to variety classes PAHs
According to inoculation method described in embodiment 2, PYR16 is inoculated into respectively luxuriant and rich with fragrance containing 200mg/L,
In the MSM culture medium of 200mg/L pyrene, 200mg/L fluorenes and 200mg/L fluoranthene, 28 DEG C of concussion cultivations one
Zhou Hou, analyzes the concentration of the pyrene of residual in degraded system respectively.As shown in Figure 7: at identical cultivation bar
Under part, PYR16 has reached more than 98% to luxuriant and rich with fragrance degradation rate, almost by phenanthrene degraded completely, to fluorenes and pyrene
Degradation effect is relatively good, and degradation rate has respectively reached 60% and 35%, and for bioavailability pole
Low fluoranthene also degrades 23%, this also further illustrate polycyclic aromatic hydrocarbon phenyl ring number increase its to life
The toxicity of thing is the biggest, and degradation process can be more complicated, and degradation cycle also can be relatively long.On the whole
Bacterial strain PYR16 has good degradation function to multiple PAHs, especially for many below 4 rings and 4 rings
Aromatic hydrocarbon ring degradation effect is notable, and this is conducive to this bacterium to be applied to the with serious pollution area of combined pollutant.
Embodiment 6PYR16 bacterial strain is to variety classes heavy metal ion resistance
Take out the bacterial strain of preservation from-80 DEG C, be then seeded into R2A solid medium activates 1 day；Picking
Single colony inoculation to 5 milliliters R2A fluid medium is cultivated to OD600=0.5.It is centrifuged 2 milliliters of sterilizings
Pipe is respectively configured containing 50mg/L, 100mg/L, 150mg/L, 200mg/L, 250mg/L five dense
(configuration liquid is the heavy metal solution of degree gradient: 10*106Heavy metal mother solution and R2A culture medium), so
After cultured PYR16 bacterium solution is accessed in this heavy metal species solution according to the inoculum concentration of 2%, be placed in 28
DEG C shaking table is cultivated, and within every two days, observes once and records its upgrowth situation.PYR16 pair is understood by table 1 below
MnCl2And CuSO4There is bigger tolerance, containing 150mg/LMnCl respectively2With
250mg/LCuSO4Culture medium in still can grow, it is to K2CrO4With Cd (NO3)2Maximum suppression
Growth concentration also has 100mg/L, and this can be from different heavy metals pair and PYR16 metabolism Organic substance related enzyme systems
Mechanism of action difference relevant.Showing that this bacterial strain has preventing from heavy metal ability widely, this is conducive to should
Bacterium is colonizated in rugged environment repairs by heavy metal and polycyclic aromatic hydrocarbons contaminated soil or water body.
Table 1 bacterial strain PYR16 growing state under different heavy metal concentrations
Note :+can grow；--can not grow.
Claims (5)
1. general Pseudomonas (Pantoea sp.) PYR16 of a strain degrading polycyclic aromatic hydrocarbons class organic pollution,
It is characterized in that: be deposited in China typical culture collection center, deposit number is: CCTCC NO:
M 2016227。
2. the general Pseudomonas PYR16 described in claim 1 is in degrading polycyclic aromatic hydrocarbons class organic pollution
Application.
Application the most according to claim 2, it is characterised in that: the organic dirt of described multiring aromatic hydrocarbon
Dye thing is phenanthrene, fluorenes, fluoranthene, pyrene.
4. a method for degrading polycyclic aromatic hydrocarbons class organic pollution, connects including in contaminated sample
The step of the kind general Pseudomonas PYR16 described in claim 1.
5., for a microbial bacterial agent for degrading polycyclic aromatic hydrocarbons class organic pollution, its active component is
General Pseudomonas PYR16 described in claim 1.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107306532A (en) * | 2017-06-13 | 2017-11-03 | 南京农业大学 | A kind of method for removing USEPA PAHs in plant simultaneously using compound PAHs degradation bacterias |
| CN110408562A (en) * | 2019-07-12 | 2019-11-05 | 南京农业大学 | A kind of preparation method and application of cadmium pollution soil repair and the complex micro organism fungicide for promoting plant growth |
| CN110773562A (en) * | 2019-11-05 | 2020-02-11 | 北京高能时代环境技术股份有限公司 | Microbial remediation method for polycyclic aromatic hydrocarbon in heavy metal-polycyclic aromatic hydrocarbon combined contaminated soil |
| CN113528371A (en) * | 2021-04-25 | 2021-10-22 | 中国石油大学(华东) | Complex microbial inoculant and restoration of pyrene-heavy metal complex polluted environment by complex microbial inoculant |
| CN115820494A (en) * | 2022-12-02 | 2023-03-21 | 华南理工大学 | Pantoea dispersa for efficiently degrading polycyclic aromatic hydrocarbon and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107306532A (en) * | 2017-06-13 | 2017-11-03 | 南京农业大学 | A kind of method for removing USEPA PAHs in plant simultaneously using compound PAHs degradation bacterias |
| CN107306532B (en) * | 2017-06-13 | 2021-09-07 | 南京农业大学 | Method for simultaneously removing USEPA PAHs in plant body by using composite PAHs degrading bacteria |
| CN110408562A (en) * | 2019-07-12 | 2019-11-05 | 南京农业大学 | A kind of preparation method and application of cadmium pollution soil repair and the complex micro organism fungicide for promoting plant growth |
| CN110408562B (en) * | 2019-07-12 | 2021-05-04 | 南京农业大学 | Preparation method and application of compound microbial agent for repairing cadmium-polluted soil and promoting plant growth |
| CN110773562A (en) * | 2019-11-05 | 2020-02-11 | 北京高能时代环境技术股份有限公司 | Microbial remediation method for polycyclic aromatic hydrocarbon in heavy metal-polycyclic aromatic hydrocarbon combined contaminated soil |
| CN113528371A (en) * | 2021-04-25 | 2021-10-22 | 中国石油大学(华东) | Complex microbial inoculant and restoration of pyrene-heavy metal complex polluted environment by complex microbial inoculant |
| CN115820494A (en) * | 2022-12-02 | 2023-03-21 | 华南理工大学 | Pantoea dispersa for efficiently degrading polycyclic aromatic hydrocarbon and application thereof |
| CN115820494B (en) * | 2022-12-02 | 2024-03-12 | 华南理工大学 | Pantoea dispersa capable of efficiently degrading polycyclic aromatic hydrocarbon and application thereof |
| CN116197229A (en) * | 2023-01-17 | 2023-06-02 | 浙江工商大学 | Thermal desorption restoration prediction method and system for polycyclic aromatic hydrocarbon contaminated soil |
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| CN105950501B (en) | 2019-08-20 |
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