CN107418916A - Screen the method for polycyclic aromatic hydrocarbon efficient degrading bacteria and the efficient degrading bacteria of gained - Google Patents
Screen the method for polycyclic aromatic hydrocarbon efficient degrading bacteria and the efficient degrading bacteria of gained Download PDFInfo
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Abstract
The invention discloses a kind of polycyclic aromatic hydrocarbon efficient degrading bacteria WY6, is mycobacteria WY6 (Mycobacterium sp.WY6), and its preserving number is：CCTCC NO:M 2017373.The present invention also provides above-mentioned polycyclic aromatic hydrocarbon efficient degrading bacteria WY6 purposes simultaneously：Administer PAHs in soil high density pollution；For 100mg L‑1Luxuriant and rich with fragrance degradation rate is up to 99.75%, for 50mg L‑1Pyrene degradation rate is up to 98.73%.The present invention also provides a kind of method for screening polycyclic aromatic hydrocarbon efficient degrading bacteria simultaneously.
Description
Technical field
The invention belongs to environmental organic pollutant microorganism remediation technology field, is related to a kind of high frequency zone polycyclic aromatic hydrocarbon
The method of (Polycyclic Aromatic Hydrocarbons, PAHs) degradation bacteria and one plant of polycyclic aromatic hydrocarbon of gained efficiently drop
Solve bacterium Mycobacterium sp.WY6.
Background technology
Polycyclic aromatic hydrocarbon (Polycyclic Aromatic Hydrocarbons, PAHs) is that a kind of typical persistence is organic
Pollutant is thick and form by the phenyl ring of two and the above.Burning, smelting and carrier of coal and oil etc. are mainly derived from,
Can steadily in the long term exist in the environment, be distributed widely in air, water body and soil, because it has " carcinogenic, teratogenesis, mutagenesis "
Characteristic, receive the extensive concern of the public recently, and one of organic pollution repairing research person problem urgently to be resolved hurrily.
PAHs in air can be attached on finely ground particles, and by migrating, settlement action enters water body or soil, and water
PAHs in body also can flowing into soil through water, and because its stronger hydrophobicity finally forms enrichment in soil.
Atmosphere pollution and water pollution are different from, soil pollution has stronger disguise, is not easy to be noticeable, but close with grain-production
It is related.There are some researches show, the PAHs in soil can enter underground water or enter food chain by plant absorption, and then to people
Body health causes larger harm.
Existing soil organic contamination Treatment process can be divided into peripheral doses (heat treatment technics, abstraction technique, surfactant
Elution method etc.), chemical remediation (Fenton reagent oxidation, ozone oxidation etc.) and microorganism remediation technology.Physics and chemistry are repaiied
There is the problem of cost is high, and reparation is not thorough, easily causes secondary pollution to environment in recovering technology, it is difficult to be widely applied, have
Certain restriction., can be by organic pollution and microorganism remediation technology utilizes microorganism in itself using organic pollution as carbon source
The characteristic of water and carbon dioxide is metabolized as, there is the characteristics of energy consumption is low, accessory substance is few, environment-friendly, is considered as economical and repairs effect
A kind of more efficient method of fruit.
It is that improvement is directly used in environment for a long time by substantial amounts of PAHs degradation bacterias in PAHs contaminated soils, be present
The degradation bacteria warehouse of PAHs pollutions.There are some researches show, the improvement to P in soil AHs be not only limited to low concentration pollution (<5mg
kg-1), using fossil fuel as the high energy consumption enterprise mainly energized such as steel plant, smeltery, paper mill, Electroplate Factory and oil
In the soil on the peripheries such as factory, PAHs pollution concentrations are up to hundreds of thousands of units (mg kg-1).Using efficient screening technique,
Polycyclic aromatic hydrocarbon-degrading bacteria pick-up rate is improved, and therefrom obtains efficient degrading bacteria, it is polycyclic aromatic hydrocarbons contaminated with important to administering soil
Meaning.
Bibliography of the present invention is as follows：
1.Hennessee C T,Seo J S,Alvarez AM,et al.Polycyclic aromatic
hydrocarbon-degrading species isolated from Hawaiian soils:Mycobacterium
crocinum sp.nov.,Mycobacterium pallens sp.nov.,Mycobacterium rutilum sp.nov.,
Mycobacterium rufum sp.nov.and Mycobacterium aromaticivorans sp.nov[J]
.International journal of systematic and evolutionary microbiology,2009,59
(2):(Hennessee C T, Seo J S, Alvarez A M, wait polycyclic aromatic hydrocarbon separated in the soil of Hawaii to 378-387
Degradation bacteria：Mycobacterium crocinum sp.nov.,Mycobacterium pallens sp.nov.,
Mycobacterium rutilum sp.nov., Mycobacterium rufum sp.nov. and Mycobacterium
aromaticivorans sp.Nov[J].《International system and evolution JOURNAL OF MICROBIOLOGY》2009,59(2):378-387)；
2.Wang H,Lou J,Gu H,et al.Efficient biodegradation of phenanthrene by
a novel strain Massilia sp.WF1isolated from a PAH-contaminated soil[J]
.Environmental Science and Pollution Research,2016,23(13):13378-13388(Wang H,
Lou J, Gu H, wait mono- plant of novel strain Massilia sp.WF1 screened from polycyclic aromatic hydrocarbon pollution of and its height to phenanthrene
Effect degraded [J]《Environmental science and Pollution Study》2016,23(13):13378-13388)；
3.Masakorala K,Yao J,Cai M,et al.Isolation and characterization of a
novel phenanthrene(PHE)degrading strain Psuedomonas sp.USTB-RU from petroleum
contaminated soil[J].Journal of hazardous materials,2013,263:493-500
(Masakorala K, Yao J, Cai M, wait from oil-polluted soils it is separated go out one plant of new phenanthrene degradation bacteria
Psuedomonas sp.USTB-RU and its characteristic [J]《Harmful substance periodical》2013,263:493-500)；
4.Sun K,Liu J,Gao Y,et al.Isolation,plant colonization potential,and
phenanthrene degradation performance of the endophytic bacterium Pseudomonas
sp.Ph6-gfp[J].Scientific reports,2014,4(Suppl 1):5462 (Sun K, Liu J, Gao Y, are waited in
Raw bacterium Pseudomonas sp.BZ-3 separation, plant colonize potentiality and luxuriant and rich with fragrance degradation property [J]《Science Report》2014,4
(Suppl 1):5462)；
5.Lin M,Hu X,Chen W,et al.Biodegradation of phenanthrene by
Pseudomonas sp.BZ-3,isolated from crude oil contaminated soil[J]
.International Biodeterioration&Biodegradation,2014,94:176-181(Lin M,Hu X,
Chen W, wait degradation bacteria Pseudomonas sp.BZ-3 and its degraded [J] to phenanthrene that is located away from oil-polluted soils
《International bio is degenerated and biodegradation》2014,94:176-181)；
6.Ping L,Zhang C,Zhu Y,et al.Biodegrading of pyrene by a newly
isolated Pseudomonas putida PL2[J].Biotechnology and Bioprocess Engineering,
2011,16(5):(Ping L, Zhang C, Zhu Y, wait mono- plant of strain Pseudomonas newly separated of to 1000-1008
Degraded [J] of putida PL2 to pyrene《Biotechnology and bioprocess engineering》2011,16(5):1000-1008)；
7.Ping L,Guo Q,Chen X,et al.Biodegradation of pyrene and benzo[a]
pyrene in the liquid matrix and soil by a newly identified Raoultella
planticola strain[J].3Biotech,2017,7(1):56 (Ping L, Guo Q, Chen X, wait the new identifications of mono- plant of
Biodegradation [J] in liquid and soil to BaP of Raoultella planticola bacterial strains《3 biotechnologys》
2017,7(1):56.)；
8.Xu H,Li X,Sun Y,et al.Biodegradation of pyrene by free and
immobilized cells of Herbaspirillum chlorophenolicum strain FA1[J].Water,Air
and Soil Pollution,2016,227(4):1 (Xu H, Li X, Sun Y, waits free and solidifies Herbaspirillum
Biodegradation [J] of chlorophenolicum strain FA1 cells to pyrene《Water body, air and soil pollution》2016,
227(4):1.)。
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of side of simple and reliable screening polycyclic aromatic hydrocarbon efficient degrading bacteria
One high-efficiency degradation bacterium of method and gained, the bacterium has the luxuriant and rich with fragrance ability with pyrene of degraded concurrently, and degradation rate is fast, good degrading effect.
In order to solve the above-mentioned technical problem, the present invention provides a kind of polycyclic aromatic hydrocarbon efficient degrading bacteria WY6, is mycobacteria
WY6 (Mycobacterium sp.WY6), its preserving number are：CCTCC NO:M 2017373.
The present invention also provides above-mentioned polycyclic aromatic hydrocarbon efficient degrading bacteria WY6 purposes simultaneously：Administer PAHs in soil
(PAHs) high density pollution.
For 100mg L-1Luxuriant and rich with fragrance degradation rate is up to 99.75%, for 50mg L-1Pyrene degradation rate is up to 98.73%.
The present invention also provides a kind of method for screening polycyclic aromatic hydrocarbon efficient degrading bacteria simultaneously, comprises the following steps：
1), the domestication of P in soil AHs degradation bacterias：
The addition 45mL aqua sterilisas in 5g PAHs contaminated soils, shaken cultivation 2 hours under the conditions of 28 DEG C, 130rpm,
Obtain soil suspension；
The domestication culture of 9mL PAHs degradation bacterias is added in 1mL soil suspensions and is based on 28 DEG C, 130rpm condition constant temperature oscillators
Middle shaken cultivation 14 days, the soil suspension after culture must be tamed；
2), the screening of P in soil AHs degradation bacterias：
Soil suspension after domestication culture obtained by step 1) is diluted 10 respectively3、104、105Times, it is each to draw 200 μ L dilutions
It is coated on PAHs bacterial isolation flat boards, is placed in 28 DEG C of incubators and cultivates；
Select on single bacterium colony streak inoculation to the new PAHs bacterial isolation flat boards with degraded circle, purify number generation, treat
Its colonial morphology is single, after stabilization, obtains polycyclic aromatic hydrocarbon efficient degrading bacteria.
Improvement as the method for the screening polycyclic aromatic hydrocarbon efficient degrading bacteria of the present invention：
In the step 2), identified by 16S rRNA gene pairs bacterium colonies, satisfaction can condition of culture and to degradation bacteria
Screening flat board correspond to PAHs pollutants have degradation capability for polycyclic aromatic hydrocarbon-degrading bacteria.
Further improvements in methods as the screening polycyclic aromatic hydrocarbon efficient degrading bacteria of the present invention：In the step 1)
The compound method of PAHs degradation bacterias domestication culture medium is to follow the steps below successively：
A. 0.50g NaNO are weighed3、1.00g KH2PO4、0.02g CaCl2、0.20g MgSO4、0.50g(NH4)2SO4、
1.00g NaH2PO4·H2O is dissolved in 1L deionized waters, and after stirring and evenly mixing, regulation pH to 6.5 (uses concentration as 6mol L-1
Sodium hydroxide solution be adjusted)；Sterilize (121 DEG C of autoclave sterilization 30min), must sterilize inorganic salt liquid culture medium；
B. the PAHs mother liquors (single PAHs pollutants, acetone are solvent) that 200 μ L concentration are 5000ppm are drawn, treat PAHs
After acetone volatilization in mother liquor, one layer of PAHs pollutant film is formed；
9mL sterilizing inorganic salt liquid culture mediums are added to above-mentioned PAHs pollutants film, obtain the domestication culture of PAHs degradation bacterias
Base.
Further improvements in methods as the screening polycyclic aromatic hydrocarbon efficient degrading bacteria of the present invention：PAHs in the step 2)
Bacterial isolation flat panel configurations method is to follow the steps below successively：
A, 0.50g NaNO are weighed3、1.00g KH2PO4、0.02g CaCl2、0.20g MgSO4、0.50g(NH4)2SO4、
1.00g NaH2PO4·H2O is dissolved in 1L deionized waters, and after stirring and evenly mixing, regulation pH to 6.5 (uses concentration as 6mol L-1
Sodium hydroxide solution be adjusted), then add 15g agar powders, heating dissolve agar powder, after mixing sterilize (121 DEG C of height
Warm autoclaving 30min), obtain sterilising medium I；
Culture medium I subject to sterilization is cooled to 50 ± 5 DEG C, 15 ± 1mL (culture dish of 90mm diameters) in culture dish；Room temperature
Lower placement obtains PAHs bacterial isolation flat boards lower floor to solidification (about 12 ± 2 hours)；
B, 0.125g NaNO are weighed3、0.25g KH2PO4、0.005g CaCl2、0.05g MgSO4、0.125g(NH4)2SO4、0.25g NaH2PO4·H2O is dissolved in 250mL deionized waters, be well mixed after, regulation pH to 6.5 (use concentration for
6mol L-1Sodium hydroxide solution be adjusted)；Then 3.125g agar powders are added, heating is dissolved agar powder, gone out after mixing
Bacterium (121 DEG C of autoclave sterilization 30min), obtains sterilising medium II；
Culture medium II subject to sterilization is cooled to 50 ± 5 DEG C, adds 25mL 5000mg L-1(any PAHs pollutions of PAHs mother liquors
Thing, acetone are solvent)；Uniformly mixing, treat the acetone volatilization in PAHs mother liquors；
C, the table that 5mL step B gains are uniformly distributed in the PAHs bacterial isolation flat boards lower floor obtained by step A is drawn
Face, first place at room temperature to solidification (about 12 ± 2 hours) and be further continued for placing until anhydrous steam produces (about 36~48 in culture dish
Hour)；Obtain PAHs bacterial isolation flat boards.
Further improvements in methods as the screening polycyclic aromatic hydrocarbon efficient degrading bacteria of the present invention：
The PAHs includes naphthalene, acenaphthylene, acenaphthene, fluorenes, phenanthrene, anthracene, fluoranthene, pyrene, benzo [a] anthracene, in the wrong, benzo (b) fluoranthene, benzo
(k) fluoranthene, benzo [a] pyrene, indenes benzene (1,2,3-cd) pyrene, dibenzo (a, h) anthracene, benzo (g h i).
It is above-mentioned to amount to 16 kinds of PAHs pollutants, in the present invention only by taking luxuriant and rich with fragrance, pyrene as an example.
In the present invention, the polycyclic aromatic hydrocarbon efficient degrading bacteria obtained using the inventive method screening is tested as follows：
PAHs degradation bacterias degradation capability is verified
1mL bacteria suspensions (OD is added in 9mL PAHs degradation bacterias domestication culture medium600nm=1.0)；In 28 DEG C, 130rpm bars
Shaken cultivation in part constant temperature oscillator, residual PAHs concentration is measured by sampling per 12h, calculates degradation bacteria PAHs degradation rates.
The efficient degrading bacteria Mycobacterium sp.WY6 finally obtained corresponding information is as follows：
The affiliated Mycobacterium of efficient degrading bacteria WY6, Chinese is mycobacteria, is located away from Sanming, Fujian Province
City's PAHs contaminated soils.Bacterial strain WY6 is gram-positive bacteria, and thalline is long 2-3 μm in shaft-like, in bacterial isolation culture medium
Its upper bacterium colony is rounded, and single bacterium colony is in integrally yellow, and center is crocus, and surface more moistens.Under liquid culture condi, bacterial strain
WY6 is respectively provided with preferable degradation capability to luxuriant and rich with fragrance and pyrene respectively：With 100mg L-1When phenanthrene is sole carbon source, 48h degradation rates are
99.75%；With 50mg L-1When pyrene is sole carbon source, 72h degradation rates are 98.73%.By bacterial strain Mycobacterium sp.WY6
Carry out preservation, preservation title：Mycobacteria WY6Mycobacterium sp.WY6, depositary institution：China typical culture collection
Center, preservation address：Wuhan, China Wuhan University, deposit number：CCTCC NO:M 2017373, in June, 2017 preservation time
26 days.
Technical scheme includes the domestication of P in soil AHs degradation bacterias, the screening of PAHs degradation bacterias and uses this method
The high-efficiency degradation bacterium Mycobacterium sp.WY6 filtered out.It is used to obtain PAHs in contaminated soil by this method to drop
Bacterial strain is solved, is improved to the pick-up rate of PAHs degradation bacterias, the degradation bacteria storehouse for administering PAHs pollutions being enriched, further to administer
P in soil AHs high density pollutions provide may.Bacterial strain Mycobacterium sp.WY6 are obtained according to screening technique of the present invention
One of PAHs degradation bacterias obtained, the bacterium has the luxuriant and rich with fragrance ability with pyrene of degraded concurrently, and degradation rate is fast, good degrading effect.
In summary, the invention provides a kind of complete method for screening polycyclic aromatic hydrocarbon efficient degrading bacteria, it is specially：(1)
PAHs polluted soil degrading bacteria group's acclimation methods are provided；(2) preferably visualization plate screening method is provided, obtains PAHs degradeds
Strain class, quantity are more.(3) to obtain degradation bacteria Mycobacterium sp.WY6 using this method polycyclic aromatic hydrocarbons contaminated to two kinds
Thing (luxuriant and rich with fragrance and pyrene) good degrading effect, has preferable application prospect.The inventive method is simple, reliable, and common laboratory is easily real
Apply, therefore be expected to be applied to a large amount of acquisition PAHs efficient degrading bacterias.Moreover, filter out strains expressed and go out preferable drop
Solution ability, there is higher application value, biodegradable administer for PAHs high density pollutions in environment provides technical support.
Brief description of the drawings
The embodiment of the present invention is described in further detail below in conjunction with the accompanying drawings.
Fig. 1 is the schematic diagram of PAHs bacterial isolation flat boards；
Fig. 2 is the Phenanthrene efficient degrading bacteria 16S rRNA Phylogenetic Trees screened in soil；
Fig. 3 is the polycyclic aromatic hydrocarbon pyrene efficient degrading bacteria 16S rRNA Phylogenetic Trees screened in soil；
Fig. 4 is bacterial strain Mycobacterium sp.WY6 16S rRNA Phylogenetic Trees；
Fig. 5 is bacterial strain Mycobacterium sp.WY6nidA Phylogenetic Trees；
Fig. 6 is bacterial strain Mycobacterium sp.WY6 transmission electron microscope pictures and colonial morphology in screening flat board；
Fig. 7 is bacterial strain Mycobacterium sp.WY6 Liquid Cultures and soil incubation degradation effect figure.
Embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This.
First, PAHs degradation bacterias domestication culture medium (exemplified by taming luxuriant and rich with fragrance efficient degrading bacteria) compound method is following to carry out successively
Step：
1st, inorganic salts (0.50g NaNO are weighed3, 1.00g KH2PO4, 0.02g CaCl2, 0.20g MgSO4, 0.50g
(NH4)2SO4, 1.00g NaH2PO4·H2O) it is dissolved in 1L deionized waters；After stirring and evenly mixing, concentration is used as 6mol L-1's
Sodium hydroxide solution adjusts pH value of solution to 6.5；121 DEG C of autoclave sterilization 30min are stand-by, that is, sterilizing inorganic salt liquid training is made
Support base.
2nd, the luxuriant and rich with fragrance mother liquor that 200 μ L concentration are 5000ppm is drawn (to weigh 0.5g phenanthrene powder to be dissolved in 100mL acetone, produce
5000ppm phenanthrene mother liquor) it is added to 50mL sterilizing conical flasks bottom；Treat that acetone volatilizees completely, i.e., form one in conical flask inner bottom part
The luxuriant and rich with fragrance pollutant film of layer；
Draw the above-mentioned sterilizing inorganic salt liquid culture mediums of 9mL to add in above-mentioned 50mL triangular flasks, produce phenanthrene degradation bacteria domestication
Culture medium (the final concentration of 100mg L of real use state-1)。
Remarks explanation：
During due to actual use, will be added in 1mL soil suspensions in 9mL PAHs degradation bacterias domestication culture medium, it is therefore, fixed
Final concentration of 100mg L of the justice into its use state-1。
Phenanthrene in above-mentioned steps 2 is made into other any pollutants in 16 kinds of PAHs, being somebody's turn to do for the pollutant can be obtained
The degradation bacteria domestication culture medium of final concentration.
The concentration for the luxuriant and rich with fragrance mother liquor changed in step 2, it just can accordingly obtain the phenanthrene degradation bacteria of corresponding use state final concentration
Tame culture medium.
2nd, PAHs bacterial isolations flat board (exemplified by screening luxuriant and rich with fragrance efficient degrading bacteria) collocation method walks for progress successively is following
Suddenly：
1st, inorganic salts (0.50g NaNO are weighed3, 1.00g KH2PO4, 0.02g CaCl2, 0.20g MgSO4, 0.50g
(NH4)2SO4, 1.00g NaH2PO4·H2O) it is dissolved in 1L deionized waters；After stirring and evenly mixing, concentration is used as 6mol L-1's
Sodium hydroxide solution adjusts pH value of solution to 6.5；Weigh 15g agar powders to make an addition in above-mentioned inorganic salt liquid culture medium, heating makes
Agar powder dissolving is obtained, is mixed；121 DEG C of autoclave sterilization 30min, obtain sterilising medium I；
120rpm vibrations mix sterilising medium I and are cooled to 50 DEG C or so (to avoid rocking excessively violent generation gas
Bubble)；It is poured onto in the culture dish that specification is 90mm diameters, about 15mL is toppled in each culture dish；Room temperature left overnight (about 12
Hour) solidification is allowed to cool, obtain PAHs bacterial isolation flat boards lower floor；It is stand-by；
2nd, inorganic salts (0.125g NaNO are weighed3, 0.25g KH2PO4, 0.005g CaCl2, 0.05g MgSO4, 0.125g
(NH4)2SO4, 0.25g NaH2PO4·H2O) it is dissolved in 250mL deionized waters, after being well mixed, uses concentration as 6mol L-1
Sodium hydroxide solution regulation pH to 6.5；Weigh 3.125g agar powders to add into above-mentioned inorganic salt liquid culture medium, heating makes
Agar powder dissolving is obtained, is stirred and evenly mixed；121 DEG C of autoclave sterilization 30min；Obtain sterilising medium II；
120rpm vibrations mix sterilising medium II and are cooled to 50 DEG C or so (to avoid rocking excessively violent generation gas
Bubble), draw and add 25mL 5000mg L-1(0.5g phenanthrene powder is dissolved in 100mL acetone luxuriant and rich with fragrance mother liquor, produces 5000mg L-1
Luxuriant and rich with fragrance mother liquor)；Blown and beaten repeatedly (careful operation avoids producing bubble) with 10mL liquid-transfering guns so that pollutant (that is, luxuriant and rich with fragrance mother liquor) and training
Foster base uniformly mixes, and causes acetone volatilization；
3rd, the gains for drawing 5mL steps 2 are uniformly distributed in the phenanthrene degradation bacteria screening flat board underlying surfaces of step 1 gained；
First room temperature left overnight (about 12 hours) is allowed to cool solidification, is further continued for placing at room temperature until anhydrous steam production in culture dish
It is stand-by after raw (about 36~48 hours)；Phenanthrene degradation bacteria screening flat board is made.
Remarks explanation：Phenanthrene in above-mentioned steps 2 is made into other any pollutants in 16 kinds of PAHs, the dirt can be obtained
Contaminate thing bacterial isolation flat board.
The concentration for the luxuriant and rich with fragrance mother liquor changed in step 2, it just can accordingly obtain the phenanthrene degradation bacteria screening flat board of corresponding final concentration.
Embodiment 1, a kind of method that luxuriant and rich with fragrance efficient degrading bacteria is screened from soil, carry out following steps successively：
1) weigh 5g to adopt in Sanming, Fujian Province city PAHs contaminated soils, be resuspended in 45mL aqua sterilisas, be placed in 250mL sterilizings
(28 DEG C, 130rpm) is vibrated in conical flask to cultivate 2 hours, obtains soil suspension.
2) draw 1mL steps 1) obtained by soil suspension add that (luxuriant and rich with fragrance concentration is 100mg L to 9mL phenanthrene degradation bacteria domestication culture medium-1) in, the shaken cultivation 14 days in 28 DEG C, 130rpm condition constant temperature oscillators；The soil suspension after culture must be tamed.
3) soil suspension that must be tamed after cultivating obtained by step 2) is diluted 10 respectively3、104、105(volume times) again, it is each to inhale
Take 200 μ L dilutions to be coated in phenanthrene degradation bacteria screening flat board, be placed in 28 DEG C of incubators and cultivate, it is daily observe on flat board whether
There is bacterium colony generation.
4) choosing bacterium colony outer ring has the single bacterium colony of transparent degraded circle, and streak inoculation is in new phenanthrene degradation bacteria screening flat board
On, more generation purifying.
5) after its colonial morphology it is single, it is stable after by 16S rRNA gene pairs, it is identified and preserves strain；That is, it is full
Can condition of culture and to the luxuriant and rich with fragrance polycyclic aromatic hydrocarbon-degrading bacteria with degradation capability.
By the methods described of above-described embodiment 1,7 plants of bacterial strains to phenanthrene with degradation capability are obtained altogether from pedotheque,
Identified by 16S rRNA gene sequencing, two plants of bacterium (PHE-1 and PHE-2) and the bacterial strain Mycobacterium reported
Pallens strain czh-8 have higher similitude, two plants of bacterium (PHE-3 and PHE-4) and the bacterial strain reported
Mycobacterium crocinum strain czh-42 have a higher similitude, other three plants of bacterium respectively with
Cupriavidus metallidurans strain NBRC 102507,Dyella kyungheensis strain THG-
B117 and Dokdonella kunshanensis strain DC-3 have higher similitude, as shown in Figure 2 and Table 1.
Table 1, WY6 and similar phenanthrene degradation bacteria strain 16S rRNA gene order similarity matrix tables
Note：
PHE-1, i.e. PHE-1 (Mycobacterium sp.WY6)；
NR 043760, i.e. Cupriavidus metallidurans strain NBRC 102507 (NR 043760)；
NR 109582, i.e. Dokdonella kunshanensis strain DC-3 (NR 109582)；
NR 109691, i.e. Dyella kyungheensis strain THG-B117 (NR 109691)；
NR 043901, i.e. Mycobacterium crocinum strain czh-42 (NR 043901)；
NR 042913, i.e. Mycobacterium houstonense strain ATCC 49403 (NR 042913)；
NR 043760, i.e. Mycobacterium pallens strain czh-8 (NR 043760).
Embodiment 2, from soil screen pyrene efficient degrading bacteria method, carry out following steps successively：
1), weigh 5g to adopt in Sanming, Fujian Province city PAHs contaminated soils, be resuspended in 45mL aqua sterilisas, be placed in 250mL and go out
(28 DEG C, 130rpm) is vibrated in bacterium conical flask to cultivate 2 hours, obtains soil suspension.
2) 1mL 1, is drawn) soil suspension obtained by step 1) adds that (pyrene concentration is to 9mL pyrenes degradation bacteria domestication culture medium
100mg L-1) in, the shaken cultivation 14 days in 28 DEG C, 130rpm condition constant temperature oscillators；The soil suspension after culture must be tamed.
3) soil suspension that must be tamed after cultivating obtained by step 2), is diluted 10 respectively3、104、105Times, it is each to draw 200 μ L
Dilution is coated on pyrene bacterial isolation flat board, is placed in 28 DEG C of incubators and is cultivated, daily to observe on flat board whether have bacterium colony
Generation.
4), choosing bacterium colony outer ring has the single bacterium colony of transparent degraded circle, and streak inoculation is in new pyrene bacterial isolation flat board
On, more generation purifying.
5), after its colonial morphology it is single, it is stable after by 16S rRNA gene pairs, it is identified and preserves strain；That is,
Meet condition of culture and there can be the polycyclic aromatic hydrocarbon-degrading bacteria of degradation capability to pyrene.
By the methods described of above-described embodiment 2,9 plants are obtained altogether to the luxuriant and rich with fragrance bacterial strain with degradation capability, by 16S rRNA
Gene sequencing identifies that one plant of bacterium (PYR-1) is with having reported that bacterial strain Mycobacterium pallens strain czh-8 have
Higher similitude, two plants of bacterium (PYR-2 and PYR-3) and the bacterial strain Mycobacterium crocinum strain reported
Czh-42 has higher similitude, one plant of bacterium (PYR-4) and the bacterial strain Mycobacterium chubuense reported
Strain ATCC 27278 have certain similitude, other five plants of bacterium respectively with Pseudonocardia sulfidoxydans
Strain 592, Pseudomonas carboxydohydrogena strain DSM 1083, Cupriavidus
Metallidurans strain CH34, Dyella jiangningensis strain SBZ3-12 and Dokdonella
Kunshanensis strain DC-3 have higher similitude, as shown in figure 3 and table 2.
Table 2, WY6 and similar pyrene degradation bacteria strains 16S rRNA gene order similarity matrix tables
Note：
PYR-1, i.e. PYR-1 (Mycobacterium sp.WY6)；
The NR 024703, i.e. (NR of Pseudomonas carboxydohydrogena strain DSM 1083
024703)；
NR 074704, i.e. Cupriavidus metallidurans strain CH34 (NR 074704)；
NR 029324, i.e. Pseudonocardia sulfidoxydans strain 592 (NR 029324)；
NR 121724, i.e. Dyella jiangningensis strain SBZ3-12 (NR 121724)；
NR 109582, i.e. Dokdonella kunshanensis strain DC-3 (NR 109582)；
NR 043901, i.e. Mycobacterium crocinum strain czh-42 (NR 043901)；
NR 043760, i.e. Mycobacterium pallens strain czh-8 (NR 043760)；
NR 041902, i.e. Mycobacterium chubuense strain ATCC 27278 (NR 041902).
1 found in conjunction with the embodiments with embodiment 2, to the luxuriant and rich with fragrance bacterial strain PHE-1 with degradation capability with having degradation capability to pyrene
Bacterial strain PYR-1 on 16S rRNA genes with bacterial strain Mycobacterium pallens strain czh-8 have it is higher
Similitude.Through comparing, bacterial strain PHE-1 and bacterial strain PYR-1 16S rRNA gene orders are completely the same, with 16S rRNA bases
Because being actually same bacterial strain on taxonomic identification.Compared through 16S rRNA identified for genes, the bacterial strain belongs to Mycobacterium, ordered
Entitled Mycobacterium sp.WY6, with Mycobacterium pallens strain czh-8 bacterial strain 16S rRNA genes
Similitude is higher (Fig. 4).For further difference bacterial strain Mycobacterium sp.WY6 and bacterial strain Mycobacterium
Pallens strain czh-8, bacterial strain Mycobacterium sp.WY6 nidA genes are subjected to amplification and compare discovery (figure
5) be, Mycobacterium sp.KMS with its nidA gene similitude highests bacterial strain, and with bacterial strain Mycobacterium
Pallens strain czh-8 similarities are relatively low, though show that two plants of bacterium similarity on 16S rRNA genes is higher,
It is different on the degradation mechanism to pollutant.Show (Hennessee, et al.2009) according to relevant report,
Mycobacterium pallens strain czh-8 have certain degradation capability, but specific drop to polycyclic aromatic hydrocarbon fluoranthene and pyrene
It is not explained in solution efficiency report, also have with bacterial strain Mycobacterium sp.WY6 has this to luxuriant and rich with fragrance and pyrene degradation capability
Difference in matter.At the same time, bacterial strain Mycobacterium sp.WY6 and bacterial strain Mycobacterium sp.KMS 16S
RRNA genes similarity is less than 97%, falls within the different strain of mycobacteria subordinate, as shown in table 3.
Table 3, WY6 and similar strain 16S rRNA gene order similarity matrix tables
Table 4, WY6 and similar strain nidA gene order similarity matrix tables
Bacterial strain Mycobacterium sp.WY6 are subjected to preservation, preservation title：Mycobacteria WY6Mycobacterium
Sp.WY6, depositary institution：China typical culture collection center, preservation address：Wuhan, China Wuhan University, deposit number：
CCTCC NO:M 2017373, June 26 2017 preservation time.
Experiment one, degradation bacteria Mycobacterium sp.WY6 Liquid Cultures and soil incubation degradation capability determine
Previous experiments show that Mycobacterium sp.WY6 are luxuriant and rich with fragrance to two kinds of pollutants respectively and pyrene has degradation capability,
Further to explore the bacterial strain to luxuriant and rich with fragrance and pyrene degradation capability, therefore carry out following steps：
1st, Liquid Culture：
1), luxuriant and rich with fragrance or pyrene degradation bacteria domestication culture medium is placed in 50mL conical flasks respectively, its Sino-Philippines and pyrene final concentration difference
For 100mg L-1With 50mg L-1。
By taking the concentration of phenanthrene as an example, luxuriant and rich with fragrance mother liquor (acetone solution) concentration is 5000mg L-1, draw 200 μ L and add 50mL tapers
Bottle, after acetone volatilizees, conical flask China and Philippines net content is 5000mg L-1× (÷ 1000 of 200 μ L ÷ 1000)=1mg, afterwards
9mL sterilizing inorganic salt liquid culture mediums and 1mL bacterium solutions are added, it is 10mL to correspond to culture volume equivalent to 1mg, and concentration is
1mg ÷ (10mL ÷ 1000)=100mg L-1。
2) 1mL Mycobacterium sp.WY6 bacterium solutions (OD, is added600=1.0), to add 1mL sterilized waters as control.
3), per 12h, sampling once, 10mL methanol, ultrasonic 30min is added to 10mL cultivating systems moderate proportions.
4) liquid in 3), is crossed into film (0.22 μm of PTFE filter membrane) and removes thalline.1.5mL filtered fluids are collected in 2mL loadings
It is used for loading in product.
5), it is measured using HPLC to remaining phenanthrene/pyrene in solution.
2nd, soil incubation：
1) 200g soil, is respectively weighed in sterile 1000mL beakers.
2) it is 5000mg L, to be separately added into 2mL concentration-1Luxuriant and rich with fragrance mother liquor and 0.8mL concentration be 5000mg L-1Pyrene mother liquor, makes
Luxuriant and rich with fragrance and final concentration of the pyrene in soil is respectively 50mg kg-1With 20mg kg-1。
3) Mycobacterium sp.WY6 bacterium solutions (OD, is added600=1.0 bacterium solutions about 2mL), make its final concentration of
106CFU g-1, to add isometric sterilized water as control.
4), the lucifuge culture under the conditions of 28 DEG C, sampled respectively at 0,3,7,14,21,28,35 day.
5) 2g soil, is weighed in 50mL brown pipes, adds 10mL n-hexanes：Acetone (V:V=1:1) mixed solution, surpass
Sound extraction 30min (25 DEG C, 53KHz).
6), 1000 × g of centrifuge tube centrifuges 10min, collects supernatant in new 50mL brown pipes.
7) extraction 2 times, is repeated, 6) supernatant is collected in in brown pipe.
8), supernatant organic phase nitrogen is blown to it is dry, be eventually adding 2.0mL methanol dissolving.
9) liquid in 8), is crossed into film (0.22 μm of PTFE filter membrane) and goes the removal of impurity.Collect 1.5mL and be used for loading.
10), it is measured using HPLC to remaining phenanthrene/pyrene in solution.
Shown by examples detailed above, bacterial strain Mycobacterium sp.WY6 are in Liquid Culture and soil incubation to phenanthrene
There is preferable degradation capability with pyrene.In Liquid Culture, for 100mg L-1Luxuriant and rich with fragrance 48h degradation rates just reach 99.75%, for 50mg
L-1Pyrene 72h is up to 98.73%, and compared to the polycyclic aromatic hydrocarbon-degrading bacteria reported in document, WY6 bacterial strains are in degradation rate and degraded PAHs
Larger advantage is shown in terms of species.
And Mycobacterium sp.WY6 are added in luxuriant and rich with fragrance, pyrene contaminated soil respectively, pyrene Sino-Philippines to soil can be accelerated
Degraded.In WY6 control group is not added with, soil indigenous microorganism needs 21 days could be by the luxuriant and rich with fragrance degraded in soil to 5mg
L-1Left and right is, it is necessary to which 28 days could degrade the pyrene in soil to 5mg L-1Below.Compared with control group, addition
Mycobacterium sp.WY6 bacterial strains treatment groups are at 3 days by the luxuriant and rich with fragrance degraded in soil to 5mg L-1Left and right, at 7 days
Pyrene in soil is degraded to 5mg L-1Below.Mycobacterium sp.WY6 bacterial strains can substantially speed up the degraded of pollutant,
The time of pollutant high density pollution is shortened, reduces high concentration PAHs exposures, further to assist soil original inhabitants low
PAHs degradation bacterias degraded phenanthrene, pyrene pollution offer condition are provided, there is actual application value.Detailed degradation rate such as table 5, table 6, Fig. 7
It is shown.
Table 5, Liquid Culture Mycobacterium sp.WY6 are to phenanthrene/pyrene degradation rate
Table 6, soil incubation Mycobacterium sp.WY6 are to phenanthrene/pyrene degradation rate
Contrast experiment 1, Setup Experiments condition and embodiment 1 are consistent, but are cultivated without 14 days, and remaining is equal to reality
Apply example 1.
Acquired results are：The single bacterium colony species and quantity grown in luxuriant and rich with fragrance efficient degrading bacteria screening flat board is considerably less than embodiment
1, and to grow the time longer for single bacterium colony.It is more to be resistant to bacterium, degraded circle unobvious.
Contrast experiment 2, Setup Experiments condition and embodiment 1 are consistent, but are not coated on the invention degraded
In bacterium screening flat board, and it is coated on luxuriant and rich with fragrance film flat board, remaining is equal to example 1.
The preparation of luxuriant and rich with fragrance film flat board：
1) inorganic salts (0.50g NaNO are weighed3, 1.00g KH2PO4, 0.02g CaCl2, 0.20g MgSO4, 0.50g
(NH4)2SO4, 1.00g NaH2PO4·H2O) it is dissolved in 1L deionized waters.
2) pH to 6.5 is adjusted after stirring and evenly mixing.
3) 15g agar powders are weighed to make an addition in the above-mentioned inorganic salt liquid culture mediums of 1L.
4) heating causes agar powder dissolving, mixes.
5) 121 DEG C of autoclave sterilization 30min.
6) 120rpm vibrations mix sterilising medium and are cooled to 50 DEG C or so (to avoid rocking excessively violent generation gas
Bubble).
7) it is poured onto in the culture dish that specification is 90mm diameters.Topple over about 20mL in each culture dish.
8) cooled and solidified is stayed overnight, it is stand-by after placing to anhydrous steam in culture dish.
9) luxuriant and rich with fragrance mother liquor (the 5000mg L of 2mL acetone solutions are drawn-1), inorganic salts solid culture primary surface uniformly is added to,
After acetone volatilization after luxuriant and rich with fragrance film inorganic salts solid medium.
Acquired results are：It is longer that plated growth goes out the single bacterium colony time, and circle of degrading is not easy to observe, doubtful degradation bacteria compared with
It is more, but show that most of bacterium has no degradation capability after empirical tests so that judgement is directly perceived not as PAHs screening and culturing mediums, increases work
Measure, and it is less actually to obtain degradation bacteria number of species.
Contrast experiment 3, searching document, compare screening obtained strains Mycobacterium sp.WY6 and existing reported bacterium
Strain is in minimal medium to the degradation effect of pollutant.
Degradation effect compares with report bacterial strain in table 7, Mycobacterium SP.WY6 inorganic salt liquid culture mediums
Acquired results are as follows：As shown in table 7, for the degradation capability of phenanthrene, the degradable 100mg L of other bacterial strains-1Phenanthrene,
But reach identical degradation rate, other bacterial strains such as Massilia sp WF1, Psuedomonas sp.USTB-RU are respectively necessary for
The time of 3 days and 7 days, and degrade be not as thorough as Mycobacterium sp.WY6；Pseudomonas sp.Ph6-gfp and BZ-
3 degradable maximum concentrations and degradation rate are not so good as Mycobacterium sp.WY6.For the degradation capability of pyrene, bacterial strain
Pseudomonas putida PL2 and Mycobacterium sp.WY6 have identical tolerable concentration, degradable concentration
50mg L-1Pyrene, but strain Pseudomonas putida PL2 at 6 days degradation rate just up to 50.00%, degradation effect is not so good as
Mycobacterium sp.WY6.And bacterial strain Raoultella planticola PL7, Herbaspirillum
Chlorophenolicum Strain FA1, Pseudomonas sp.BZ-3 can tolerate concentration and degradation rate is not so good as bacterial strain
Mycobacterium sp.WY6.As can be seen here, bacterial strain Mycobacterium sp.WY6 not only have degradable a variety of pollutions
The ability of thing, and have higher degradation rate and tolerable concentration to two kinds of pollutants phenanthrene and pyrene, and verified in soil
Its degradation capability, theoretical foundation can be provided for in-situ immobilization Phenanthrene, pyrene pollution, there is higher answer in actual applications
Use prospect.
Finally, it is also necessary to it is noted that listed above is only several specific embodiments of the invention, it is clear that be not limited to
Above example, there can also be many deformations.One of ordinary skill in the art can directly export from present disclosure
Or all deformations associated, it is considered as protection scope of the present invention.
Claims (7)
1. polycyclic aromatic hydrocarbon efficient degrading bacteria WY6, it is characterised in that：For mycobacteria WY6 (Mycobacterium sp.WY6), its
Preserving number is：CCTCC NO:M 2017373.
2. polycyclic aromatic hydrocarbon efficient degrading bacteria WY6 as claimed in claim 1 purposes, it is characterised in that：Administer polycyclic in soil
Aromatic hydrocarbons high density pollution.
3. the method for polycyclic aromatic hydrocarbon efficient degrading bacteria is screened, it is characterized in that comprising the following steps：
1), the domestication of P in soil AHs degradation bacterias：
45mL aqua sterilisas are added in 5g PAHs contaminated soils, shaken cultivation 2 hours under the conditions of 28 DEG C, 130rpm, are obtained
Soil suspension；
9mL PAHs degradation bacterias domestication culture is added in 1mL soil suspensions based on being shaken in 28 DEG C, 130rpm condition constant temperature oscillators
Culture 14 days is swung, the soil suspension after culture must be tamed；
2), the screening of P in soil AHs degradation bacterias：
Soil suspension after domestication culture obtained by step 1) is diluted 10 respectively3、104、105Times, it is each to draw the coating of 200 μ L dilutions
In on PAHs bacterial isolation flat boards, it is placed in 28 DEG C of incubators and cultivates；
Select on single bacterium colony streak inoculation to the new PAHs bacterial isolation flat boards with degraded circle, purify number generation, treat its bacterium
Fall form it is single, it is stable after, obtain polycyclic aromatic hydrocarbon efficient degrading bacteria.
4. the method for screening polycyclic aromatic hydrocarbon efficient degrading bacteria according to claim 3, it is characterized in that：
In the step 2), identified by 16S rRNA gene pairs bacterium colonies, satisfaction can condition of culture and to bacterial isolation
Flat board correspond to PAHs pollutants have degradation capability for polycyclic aromatic hydrocarbon-degrading bacteria.
5. the method for the screening polycyclic aromatic hydrocarbon efficient degrading bacteria according to claim 3 or 4, it is characterized in that in the step 1)
PAHs degradation bacterias domestication culture medium compound method to follow the steps below successively：
A. 0.50g NaNO are weighed3、1.00g KH2PO4、0.02g CaCl2、0.20g MgSO4、0.50g(NH4)2SO4、1.00g
NaH2PO4·H2O is dissolved in 1L deionized waters, after stirring and evenly mixing, adjusts pH to 6.5；Sterilizing, the inorganic salt liquid that must sterilize training
Support base；
B. the PAHs mother liquors that 200 μ L concentration are 5000ppm are drawn, after the acetone volatilization in PAHs mother liquors, form one layer of PAHs
Pollutant film；
9mL sterilizing inorganic salt liquid culture mediums are added to above-mentioned PAHs pollutants film, obtain PAHs degradation bacterias domestication culture medium.
6. the method for the screening polycyclic aromatic hydrocarbon efficient degrading bacteria according to claim 3 or 4, it is characterized in that in the step 2)
PAHs bacterial isolation flat panel configurations method is to follow the steps below successively：
A, 0.50g NaNO are weighed3、1.00g KH2PO4、0.02g CaCl2、0.20g MgSO4、0.50g(NH4)2SO4、1.00g
NaH2PO4·H2O is dissolved in 1L deionized waters, after stirring and evenly mixing, is adjusted pH to 6.5, is then added 15g agar powders, heating makes
Agar powder dissolves, and is sterilized after mixing, obtains sterilising medium I；
Culture medium I subject to sterilization is cooled to 50 ± 5 DEG C, and 15 ± 1mL is in culture dish；Place at room temperature to solidifying, obtain PAHs drops
Xie Jun screening flat boards lower floor；
B, 0.125g NaNO are weighed3、0.25g KH2PO4、0.005g CaCl2、0.05g MgSO4、0.125g(NH4)2SO4、
0.25g NaH2PO4·H2O is dissolved in 250mL deionized waters, after being well mixed, adjusts pH to 6.5；Then 3.125g is added
Agar powder, heating dissolve agar powder, are sterilized after mixing, obtain sterilising medium II；
Culture medium II subject to sterilization is cooled to 50 ± 5 DEG C, adds 25mL 5000mg L-1PAHs mother liquors；Uniformly mixing, treat PAHs mothers
Acetone volatilization in liquid；
C, the surface that 5mL step B gains are uniformly distributed in the PAHs bacterial isolation flat boards lower floor obtained by step A is drawn, first
Place at room temperature to solidifying, be further continued for placing until anhydrous steam produces in culture dish；Obtain PAHs bacterial isolation flat boards.
7. the method for the screening polycyclic aromatic hydrocarbon efficient degrading bacteria according to claim 5 or 6, it is characterized in that：
The PAHs includes naphthalene, acenaphthylene, acenaphthene, fluorenes, phenanthrene, anthracene, fluoranthene, pyrene, benzo [a] anthracene, in the wrong, benzo (b) fluoranthene, benzo (k)
Fluoranthene, benzo [a] pyrene, indenes benzene (1,2,3-cd) pyrene, dibenzo (a, h) anthracene, benzo (g h i).
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Cited By (7)
| 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 |
| CN108300674A (en) * | 2017-12-28 | 2018-07-20 | 永清环保股份有限公司 | A kind of oil degradation bacteria and its preparation method and the application in degrading crude oil |
| CN108546666A (en) * | 2018-05-18 | 2018-09-18 | 温州大学 | One plant of Raoul bacterium and it is total to the application in detoxification in pyrene-Cr (VI) combined pollution |
| CN108823142A (en) * | 2018-07-05 | 2018-11-16 | 天津城建大学 | It is a kind of using rainwater containing naphthalene as the screening technique of the degradation bacteria of substrate |
| CN110241065A (en) * | 2019-05-18 | 2019-09-17 | 南京农业大学 | A method of utilizing USEPA PAHs in original inhabitants PAHs degradation bacteria group removal agricultural land soil |
| CN111748494A (en) * | 2020-06-30 | 2020-10-09 | 武汉大学 | Nitrogen-fixing campylobacter for degrading polycyclic aromatic hydrocarbon and application thereof |
| CN113462609A (en) * | 2021-07-30 | 2021-10-01 | 中国科学院南海海洋研究所 | Mycobacterium for efficiently degrading polycyclic aromatic hydrocarbon and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101423806A (en) * | 2007-11-02 | 2009-05-06 | 中国科学院沈阳应用生态研究所 | Organic aromatic compound degrading bacteria and use thereof |
| CN102776135A (en) * | 2011-05-12 | 2012-11-14 | 中国科学院沈阳应用生态研究所 | Mixed inoculant for degrading polycyclic aromatic hydrocarbon pollutants |
| CN102899271A (en) * | 2012-09-29 | 2013-01-30 | 轻工业环境保护研究所 | Mycobacterium 16F for efficiently degrading polycyclic aromatic hydrocarbons and benzene organic matters and application thereof |
| CN103045507A (en) * | 2012-12-12 | 2013-04-17 | 轻工业环境保护研究所 | Bacteria separation method for efficiently degrading high molecular weight polycyclic aromatic hydrocarbons and application thereof |
-
2017
- 2017-08-01 CN CN201710645578.6A patent/CN107418916B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101423806A (en) * | 2007-11-02 | 2009-05-06 | 中国科学院沈阳应用生态研究所 | Organic aromatic compound degrading bacteria and use thereof |
| CN102776135A (en) * | 2011-05-12 | 2012-11-14 | 中国科学院沈阳应用生态研究所 | Mixed inoculant for degrading polycyclic aromatic hydrocarbon pollutants |
| CN102899271A (en) * | 2012-09-29 | 2013-01-30 | 轻工业环境保护研究所 | Mycobacterium 16F for efficiently degrading polycyclic aromatic hydrocarbons and benzene organic matters and application thereof |
| CN103045507A (en) * | 2012-12-12 | 2013-04-17 | 轻工业环境保护研究所 | Bacteria separation method for efficiently degrading high molecular weight polycyclic aromatic hydrocarbons and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| HAIPING GU: "The effective migration of Massilia sp. WF1 by Phanerochaete chrysosporium and its phenanthrene biodegradation in soil", 《SCIENCE OF THE TOTAL ENVIRONMENT》 * |
| HAIZHENWANG: "Efficient biodegradation of phenanthrene by a novel strain Massilia sp. WF1 isolated from a PAH-contaminated soil", 《ENVIRON SCI POLLUT RES》 * |
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| CN108300674A (en) * | 2017-12-28 | 2018-07-20 | 永清环保股份有限公司 | A kind of oil degradation bacteria and its preparation method and the application in degrading crude oil |
| CN108300674B (en) * | 2017-12-28 | 2020-08-28 | 永清环保股份有限公司 | Petroleum degrading bacteria, obtaining method thereof and application of petroleum degrading bacteria in crude oil degradation |
| CN108546666A (en) * | 2018-05-18 | 2018-09-18 | 温州大学 | One plant of Raoul bacterium and it is total to the application in detoxification in pyrene-Cr (VI) combined pollution |
| CN108546666B (en) * | 2018-05-18 | 2021-06-01 | 温州大学 | Lauraria and application thereof in pyrene-Cr (VI) combined pollution co-detoxification |
| CN108823142A (en) * | 2018-07-05 | 2018-11-16 | 天津城建大学 | It is a kind of using rainwater containing naphthalene as the screening technique of the degradation bacteria of substrate |
| CN110241065A (en) * | 2019-05-18 | 2019-09-17 | 南京农业大学 | A method of utilizing USEPA PAHs in original inhabitants PAHs degradation bacteria group removal agricultural land soil |
| CN110241065B (en) * | 2019-05-18 | 2020-07-21 | 南京农业大学 | Method for removing USEPA (ultra-small animal plant artificial plant) PAHs (polycyclic aromatic hydrocarbons) in farmland soil by utilizing indigenous PAHs degrading flora |
| CN111748494A (en) * | 2020-06-30 | 2020-10-09 | 武汉大学 | Nitrogen-fixing campylobacter for degrading polycyclic aromatic hydrocarbon and application thereof |
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