CN105255753A - Effective degradation strain for phenanthrene Sphingobium sp. Phe-1 and application thereof - Google Patents
Effective degradation strain for phenanthrene Sphingobium sp. Phe-1 and application thereof Download PDFInfo
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- CN105255753A CN105255753A CN201510554197.8A CN201510554197A CN105255753A CN 105255753 A CN105255753 A CN 105255753A CN 201510554197 A CN201510554197 A CN 201510554197A CN 105255753 A CN105255753 A CN 105255753A
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- YNPNZTXNASCQKK-UHFFFAOYSA-N phenanthrene Chemical compound C1=CC=C2C3=CC=CC=C3C=CC2=C1 YNPNZTXNASCQKK-UHFFFAOYSA-N 0.000 title claims abstract description 96
- 230000015556 catabolic process Effects 0.000 title claims abstract description 89
- 238000006731 degradation reaction Methods 0.000 title claims abstract description 89
- 241000927720 Sphingobium sp. Species 0.000 title abstract 2
- 241000894006 Bacteria Species 0.000 claims abstract description 89
- 230000001580 bacterial effect Effects 0.000 claims abstract description 31
- 125000005575 polycyclic aromatic hydrocarbon group Chemical group 0.000 claims abstract description 9
- 238000004321 preservation Methods 0.000 claims abstract description 8
- 239000003205 fragrance Substances 0.000 claims description 62
- 239000007788 liquid Substances 0.000 claims description 22
- 230000000593 degrading effect Effects 0.000 claims description 21
- 239000000725 suspension Substances 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 8
- 238000011081 inoculation Methods 0.000 claims description 7
- 239000002689 soil Substances 0.000 claims description 7
- 239000003795 chemical substances by application Substances 0.000 claims description 6
- 244000005700 microbiome Species 0.000 claims description 6
- -1 polycyclic arene compound Chemical class 0.000 claims description 3
- 239000004480 active ingredient Substances 0.000 claims description 2
- 239000007864 aqueous solution Substances 0.000 claims description 2
- 239000012530 fluid Substances 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 3
- 238000009629 microbiological culture Methods 0.000 abstract 1
- 239000002609 medium Substances 0.000 description 13
- 229910017053 inorganic salt Inorganic materials 0.000 description 11
- 239000000523 sample Substances 0.000 description 10
- 239000002028 Biomass Substances 0.000 description 8
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 8
- 239000012498 ultrapure water Substances 0.000 description 7
- 229910052799 carbon Inorganic materials 0.000 description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 5
- 238000003794 Gram staining Methods 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 4
- 241001052560 Thallis Species 0.000 description 4
- 239000008272 agar Substances 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 230000001954 sterilising effect Effects 0.000 description 4
- 241000589516 Pseudomonas Species 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000003111 delayed effect Effects 0.000 description 3
- 238000004043 dyeing Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000012797 qualification Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 238000005507 spraying Methods 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 108091023242 Internal transcribed spacer Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 241000383837 Sphingobium Species 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000009833 condensation Methods 0.000 description 2
- 230000005494 condensation Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
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- 235000011187 glycerol Nutrition 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 239000002957 persistent organic pollutant Substances 0.000 description 2
- 239000012266 salt solution Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
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- 235000013619 trace mineral Nutrition 0.000 description 2
- 238000004383 yellowing Methods 0.000 description 2
- WWUZIQQURGPMPG-UHFFFAOYSA-N (-)-D-erythro-Sphingosine Natural products CCCCCCCCCCCCCC=CC(O)C(N)CO WWUZIQQURGPMPG-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- 241000607534 Aeromonas Species 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 241000589941 Azospirillum Species 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 241000589565 Flavobacterium Species 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 241000203749 Gordonia bronchialis Species 0.000 description 1
- 241000186359 Mycobacterium Species 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 102000029797 Prion Human genes 0.000 description 1
- 108091000054 Prion Proteins 0.000 description 1
- 241000190932 Rhodopseudomonas Species 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 241000465144 Sphingobium sp. PHE1 Species 0.000 description 1
- 208000031320 Teratogenesis Diseases 0.000 description 1
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- 230000003044 adaptive effect Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 150000004945 aromatic hydrocarbons Chemical class 0.000 description 1
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- 238000004364 calculation method Methods 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
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- 230000006866 deterioration Effects 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
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- 235000019319 peptone Nutrition 0.000 description 1
- 230000002688 persistence Effects 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
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- 239000011780 sodium chloride Substances 0.000 description 1
- 150000003408 sphingolipids Chemical class 0.000 description 1
- WWUZIQQURGPMPG-KRWOKUGFSA-N sphingosine Chemical compound CCCCCCCCCCCCC\C=C\[C@@H](O)[C@@H](N)CO WWUZIQQURGPMPG-KRWOKUGFSA-N 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
Abstract
The invention discloses an effective degradation strain for phenanthrene Sphingobium sp. Phe-1 and application thereof. The bacterial strain is preserved at the China General Microbiological Culture Collection Center, the preservation number is CGMCC No.11239, and the preservation address is No.3, No.1 West Beichen Road, Chaoyang District, Beijing. The degradation strain Phe-1 has quite good degradation effect on the polycyclic aromatic hydrocarbon such as phenanthrene, and the degradation rate of 100 mg/L phenanthrene can reach 100% in 48 hours at the temperature of 35 DEG C and the pH of 7.0. The degradation strain is an effective degradation strain, and the degradation conditions are soft, the phenanthrene with the concentration less than 200 mg/L can be degraded at the temperature of 20-35 DEG C and the pH of 5-8. Moreover, the degradation strain has a wide applied range and good application prospect, is suitable for using as a restoration bacterial strain for contaminated land, and has a better degradation capability than the other degradation bacteria.
Description
Technical field
The invention belongs to organic pollutant degradation bacterium technical field.More specifically, the luxuriant and rich with fragrance efficient degrading bacterial strain of a strain is related to
sphingobiumsp.phe-1 and application thereof.
Background technology
Polycyclic arene compound (PAHs) is ubiquitous persistence toxic organic pollutant in environment.Large quantity research confirms, polycyclic aromatic hydrocarbons has chronic toxicity and carcinogenic, teratogenesis, mutagenic " three cause " effect, causes the extensive concern of various countries environmental science worker.Luxuriant and rich with fragrance (molecular formula C14H10), as a kind of thrcylic aromatic hydrocarbon being distributed widely in physical environment, containing chemical structure--K-region and Bay-region in close relations with " three cause " in polycyclic aromatic hydrocarbons, therefore becomes the medelling compound of polycyclic aromatic hydrocarbons contaminated research.To the biodegradable research of phenanthrene, not only contribute to the elimination that environment China and Philippines pollute, people also can be helped to understand the degradation mechanism of other polycyclic aromatic hydrocarbons and biodegradable feasibility, for the biological restoration of polycyclic aromatic hydrocarbons and oil polluted environment provides corresponding theoretical foundation and control measures.
Microbiological deterioration is that lasting administering method is stablized in a kind of environmental protection, and the degradation bacteria of the phenanthrene found at present has Aeromonas
aeromonassp., Rhodopseudomonas
pseudomonassp., Flavobacterium
flavobacteriumsp., sphingosine Pseudomonas
sphingosinesp.deng (JuhaszandNaidu, 2000).As: a strain Sphingol single-cell
sphingosinesp.gY2B, to the phenanthrene of 10mg/L and 60mg/L starting point concentration, needs 24 and 60h degraded complete (Tao Xueqin etal., 2007) respectively; Another strain Sphingol single-cell
sphingosinesp.Hbe that 98%(thunder is joyous to the degradation rate of 50mg/L phenanthrene in 48h, 2008); Also have other Pseudomonas as Gordonia bronchialis (
gordoniasp.), mycobacterium (
mycobacteriumsp.), azospirillum (
azospirillumsp.), be that the clearance of the phenanthrene of 50mg/L is 27.16% ~ 55.13%(Yang Xiu rainbow etc. to initial mass concentration in 10d, 2009).
But these microorganism strains, to the degradation efficiency of phenanthrene, comprise palliating degradation degree and degradation time is also very not enough.
Summary of the invention
The technical problem to be solved in the present invention overcomes defect and the deficiency that existing phenanthrene waits degrading polycyclic aromatic hydrocarbons technology, and object is to provide the luxuriant and rich with fragrance efficient degrading bacterial strain of a strain
sphingobiumsp.phe-1 and application thereof.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
The luxuriant and rich with fragrance efficient degrading bacterial strain Sphingobiumsp.Phe-1 of one strain, China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved on August 14th, 2015, deposit number is CGMCCNO.11239, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
Single bacterium colony of this bacterial strain is in yellow, and bacterium colony is regular circle shapes, neat in edge, and smooth surface is moistening, and cell is shaft-like under the microscope, and gramstaining is positive, easy coloring.
A kind of luxuriant and rich with fragrance efficient degradation agent, with above-mentioned luxuriant and rich with fragrance efficient degrading bacterial strain
sphingobiumsp.phe-1 or its bacteria suspension are main active ingredient.
Above-mentioned luxuriant and rich with fragrance efficient degrading bacterial strain
sphingobiumsp.phe-1, or above-mentioned luxuriant and rich with fragrance efficient degradation agent, the application in degrading polycyclic aromatic hydrocarbons compounds (PAHs) is also within protection scope of the present invention.
Preferably, described polycyclic arene compound is luxuriant and rich with fragrance.Specifically refer to containing the phenanthrene in luxuriant and rich with fragrance solution.
More specifically, the described solution containing phenanthrene refers to the aqueous solution into luxuriant and rich with fragrance contaminating fluid or phenanthrene-polluted soil.Namely degradation bacteria strains Phe-1 of the present invention can realize the treatment and purification of any medium polluted by phenanthrene.
Still more preferably, described phenanthrene concentration is in the solution below 200mg/L.Namely degradation bacteria strains Phe-1 can realize the degraded of the phenanthrene below to 200mg/L concentration very efficiently.
Specifically preferably, the condition of above-mentioned application is the degraded realized under pH5 ~ 8,20 ~ 35 DEG C of conditions phenanthrene.
More preferably, the condition of above-mentioned application is the degraded realized under pH6 ~ 7,30 ~ 35 DEG C of conditions phenanthrene.
Most preferably, the condition of above-mentioned application is the degraded realized under pH7,35 DEG C of conditions phenanthrene.
Preferably can embodiment as one, the concrete grammar of above-mentioned application is: according to the volume ratio of 1 ~ 5%, by degradation bacteria strains
sphingobiumsp.the bacterial suspension inoculation of Phe-1 is in the contaminated liquid containing phenanthrene.
Preferably, degradation bacteria strains in described bacteria suspension
sphingobiumsp.the number of cells of Phe-1 is 10
9/ mL.
Preferably, the inoculation volume ratio of bacteria suspension is 1%.
The present invention is from Qingyuan City's refinery soil, the efficient degrading bacteria of a strain phenanthrene is filtered out by the mode of enrichment culture and separation and purification, gramstaining is positive, identify through 16SrRNA, this bacterial strain is under the jurisdiction of sphingolipid Pseudomonas (Sphingobium), called after Sphingobiumsp.Phe-1, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on August 14th, 2015, deposit number is CGMCCNO.11239, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
This bacterial strain is cultivate 48h in the constant incubator of 30 DEG C in temperature, and can see single bacterium colony clearly, be faint yellow during beginning, slowly yellowing; Bacterium colony is regular circle shapes, neat in edge, and smooth surface is moistening.Cell is shaft-like under the microscope, and gramstaining is positive, easily painted.
Degradation bacteria strains Phe-1 of the present invention 35 DEG C, under pH7.0, luxuriant and rich with fragrance concentration is the condition of 100mg/L, the degradation rate of growth and very high phenanthrene preferably can be obtained, can the degraded of the phenanthrene of 100mg/L completely at 48h, illustrate that degradation bacteria strains Phe-1 is the efficient degrading bacterial strain of a strain phenanthrene, be suitable as contaminated site and repair bacterial strain, contrast other degradation bacteria and there is good degraded advantage.
The present invention has following beneficial effect:
The present invention has screened a strain first can the luxuriant and rich with fragrance bacterial strain of colleges and universities' degraded
sphingobiumsp.phe-1, this bacterial strain has extraordinary degradation effect to phenanthrene, can the degraded of the phenanthrene of 100mg/L completely (degradation rate reaches 100%), be the efficient degrading bacterial strain of a strain phenanthrene at 48h, be suitable as contaminated site and repair bacterial strain, contrast other degradation bacteria and there is good degraded advantage.
Meanwhile, degradation bacteria Phe-1 of the present invention is when the degraded application to phenanthrene, and condition is relatively loose, and can realize the degraded of the phenanthrene below to 200mg/L concentration under pH5 ~ 8,20 ~ 35 DEG C of conditions, the even phenanthrene of degradable greater concn, range of application is wider.
Accompanying drawing explanation
Fig. 1 is degradation bacteria strains
sphingobiumsp.the colonial morphology of Phe-1.
Fig. 2 is degradation bacteria strains
sphingobiumsp.the microscopic examination of Phe-1.
Fig. 3 is degradation bacteria strains
sphingobiumsp.the 16SrRNA electrophoretogram of Phe-1.
Fig. 4 is degradation bacteria strains
sphingobiumsp.the phylogenetic tree of Phe-1.
Fig. 5 is the impact of temperature on degradation bacteria strains Phe-1 increment.
Fig. 6 is the impact of pH on degradation bacteria strains Phe-1 increment.
Fig. 7 is the impact of luxuriant and rich with fragrance concentration on degradation bacteria strains Phe-1 increment.
Fig. 8 be under different incubation time luxuriant and rich with fragrance concentration on the impact of degradation bacteria strains Phe-1 increment.
Fig. 9 is the growth curve of degradation bacteria strains Phe-1.
Figure 10 is the degradation curve of degradation bacteria strains Phe-1 to phenanthrene.
Embodiment
Further illustrate the present invention below in conjunction with Figure of description and specific embodiment, but embodiment does not limit in any form to the present invention.Unless stated otherwise, the present invention adopts reagent, method and apparatus are the art conventional reagent, method and apparatus.
Unless stated otherwise, following examples agents useful for same and material are commercial.
Following examples agents useful for same is as follows:
(1) inorganic salt liquid substratum: by adding trace element solution 1mL/L in inorganic salt solution, be settled to 1000mL with high purity water, regulates pH7.0,121 DEG C of sterilizing 30min, for subsequent use.
Wherein, inorganic salt solution comprises: (NH4)
2sO
4(analytical pure) 3g/L, KH
2pO
4(analytical pure) 0.5g/L, Na
2hPO
4(analytical pure) 0.5g/L, MgSO
4(analytical pure) 0.2609g/L;
Trace element solution comprises: FeCl
3(analytical pure) 0.3g/L, FeSO
4 .7H
2o(analytical pure) 0.3g/L, MnSO
4 .h
2o(analytical pure) 0.15g/L, ZnSO
4(analytical pure) 0.14g/L, COCl
2(analytical pure) 0.2g/L.
(2) inorganic salt solid medium: add agar 20g/L in inorganic salt liquid substratum, takes out after autoclaving and is inverted flat board, for subsequent use after condensation.
(3) LB liquid nutrient medium: peptone (analytical pure) 10g/L, yeast extract (analytical pure) 5g/L, NaCl(analytical pure) 10g/L.
(4) LB solid medium: add agar 20g/L in LB liquid nutrient medium, takes out after autoclaving and is inverted flat board, for subsequent use after condensation.
(5) screening culture medium: add phenanthrene in inorganic salt liquid substratum, make its final concentration reach 10,50,100,150,250mg/L.
Luxuriant and rich with fragrance reference liquid preparation: take the luxuriant and rich with fragrance dissolution of solid of 0.4g in acetone, be settled to 50mL, make the luxuriant and rich with fragrance reference liquid that concentration is 4000mg/L.
Above-mentioned agar used (analytical pure) 20g/L, NaOH(analytical pure) 40g/L, H
2sO
4(analytical pure) 161g/L, luxuriant and rich with fragrance (>98%) 4g/L.
the screening of the luxuriant and rich with fragrance efficient degrading bacterial strain of embodiment 1
1, pedotheque
Pedotheque picks up from Long Tang town, Qingyuan City Qingcheng District refinery (north latitude N23 ° 36 ' 20.86 " east longitude E113 ° 04 ' 19.34) soil, removes foreign material, is loaded in sealed bag, in 4 DEG C of Refrigerator stores.
Prepared by soil supension: take 10g soil in 90mL high purity water, and at 30 DEG C, 180rpm vibrates 30min.
2, efficient degrading bacterial strain screening
(1) enrichment culture
Get luxuriant and rich with fragrance reference liquid 0.5mL in the triangular flask of the sterilizing of drying, treat acetone volatilization completely, measure 90mL inorganic salt liquid and cultivate based in triangular flask, then measure soil supension 10mL in triangular flask, make luxuriant and rich with fragrance concentration be 10mg/L, in 25 DEG C, lucifuge cultivates 5d in 160rpm incubator.Improving luxuriant and rich with fragrance concentration is successively 50mg/L, 100mg/L, 200mg/L, 250mg/L, and switching liquid is 10mL, and culture cycle is 5d.
(2) separation and purification
Get last enrichment culture bacterium liquid and be diluted to 10
-2, 10
-4, 10
-6mg/L, get 0.1mL and be applied to (luxuriant and rich with fragrance concentration is 50mg/L, adopts methylene dichloride spraying, after methylene dichloride volatilization completely, inoculate bacterium liquid) on LB solid medium, three parallel, flat board is placed in 30 DEG C of incubators, observes colony growth situation.
Single bacterium colony larger for grow on plates is chosen, is inoculated in inorganic salt liquid substratum (luxuriant and rich with fragrance concentration is 50mg/L) and cultivates 2d, cultivation bacterium liquid is diluted to 10
-2, 10
-4, 10
-6mg/L, get 0.1mL and be applied to (luxuriant and rich with fragrance concentration is 50mg/L, adopts methylene dichloride spraying, after methylene dichloride volatilization completely, inoculate bacterium liquid) on LB solid medium, three parallel, flat board is placed in 30 DEG C of incubators, repeats until purifying agaric.
By the fungi preservation after purifying on the luxuriant and rich with fragrance inorganic salt inclined-plane of spraying, wait to grow that to be placed on 4 DEG C of Storage in refrigerator compared with macrocolony for subsequent use.
(3) collecting cells
By the strain inoculation after purifying to containing in luxuriant and rich with fragrance inorganic salt liquid substratum, in 30 DEG C, in 160rpm incubator, lucifuge cultivates 2d, the centrifugal 20min of 4000r/min, abandon supernatant liquor and add recentrifuge after phosphate buffered saline buffer shaken well, repeat 2 ~ 3 times, cell concn is adjusted to 10
9individual/mL is for subsequent use.
(4) fungi preservation
Inclined-plane is preserved: be inoculated on LB inclined-plane, cultivates 1 ~ 2 day, is stored in 4 DEG C of refrigerators for 30 DEG C, transfer after 1 ~ February (be forwarded on the slant medium of newly joining, preserve after cultivating 1 ~ 2d by upper method).
Glycerine is preserved: the bacterium liquid of the glycerine+85% of 15% sterilizing, and vibration, makes it to mix, be placed in-85 DEG C of refrigerators and preserve.After 0.5 ~ 1 year, with transfering loop scrape freeze culture surface, streak inoculation on LB flat board, 30 DEG C of overnight incubation, be placed in inorganic salt liquid substratum (luxuriant and rich with fragrance concentration is 50mg/L) with the bacterial cultures that transfering loop picking flat board has been grown, preserve by upper method after cultivating 1 ~ 2d.
Morphologic observation: target degradation bacteria is cultivate 48h in the constant incubator of 30 DEG C just can see single bacterium colony clearly in temperature, is faint yellow during beginning, slowly yellowing; Bacterium colony is regular circle shapes, neat in edge, smooth surface moistening (as shown in Figure 1).
2, gramstaining
(1) method
Smear: get clean slide glass, drip high purity water, diameter is about 2mm, and the bacterial strain of purifying on plate of making even, selects half bacterium colony, coats on slide glass, makes it to form uniform thin film, dries under natural condition;
Fixing: slide glass is passed through flame 2 ~ 3 times, and fixed cell, is advisable with non-scald on hand;
Dyeing: drip ammonium oxalate-crystal violet solution, dyeing 1min, cleans residual dye liquor with high purity water, paper using suck dry moisture;
Mordant dyeing: drip 1 iodine liquid, cleans with high purity water after 1min, paper using suck dry moisture;
Decolouring: drip 95% ethanol decolorization 20-30s continuously to effluent liquid without purple, clean with high purity water immediately;
Redye: drip luxuriant red dye liquor, redye 3-5min, clean with high purity water;
Observe: in basis of microscopic observation cellular prion protein.
(2) target degradation bacteria under the microscope cell be shaft-like, gramstaining is positive, easily painted (as shown in Figure 2).
3,16SrDNA qualification
(1) entrust Guangzhou Hua Nuo Science and Technology Ltd., use the general primers designed ITS1/ITS4 of eucaryon bacterium respectively, the general primers designed 27F/1492R of protokaryon bacterium, identifies bacterial classification.
Following system (50 μ L) is configured: PCRMix25 μ L, ddH in PCR pipe
2o21 μ L, upstream primer 2 μ L, downstream primer 2 μ L, DNA profiling is single bacterium colony.
In the enterprising performing PCR reaction of PCR instrument, undertaken by following response procedures: 95 DEG C, 15min; 95 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 1min30s, 30 circulations; 72 DEG C of 10min; 4 DEG C of ∞.
Qualification: the agarose gel (1g agar Icing Sugar adds 100mLTAE electrophoretic buffer) of configuration 1% carries out electrophoresis to reacted PCR primer, and PCR primer applied sample amount is 3 μ L.Result shows, and eucaryon dientification of bacteria primer (ITS1/ITS4) is without amplified band, and protokaryon dientification of bacteria primer (27F/1492R) occurs band at about 1500bp.
(2) correct to stripe size PCR primer checks order, and according to checking order, the sequence of having spliced carries out Blast sequence analysis at NCBI.As shown in Figure 3, its phylogenetic tree as shown in Figure 4 for 16SrRNA qualification result.
Identified by 16SrRNA, target degradation bacteria belongs to Sphingobium and belongs to, called after Sphingobiumsp.Phe-1, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on August 14th, 2015, deposit number is CGMCCNO.11239, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
the luxuriant and rich with fragrance efficient degrading bacterial strain of embodiment 2
sphingobiumsp.the growth characteristics research of Phe-1
1, temperature is on the impact of degradation bacteria bulk-growth
(1) be sole carbon source with phenanthrene, probe into the impact that differing temps grows degradation bacteria strains Phe-1.
By finite concentration, (number of cells is about 10
9/ mL) bacteria suspension in 1% ratio be inoculated in 20mL minimal medium (luxuriant and rich with fragrance content is 50mg/L), 160rpm shaking table cultivate 24h.Culture temperature is respectively 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C.3, each sample is parallel, measures OD600 value, calculating mean value.Select the suitableeest thalli growth temperature.
(2) result as shown in Figure 5.From experimental result, the susceptibility of degradation bacteria strains Phe-1 to temperature is higher, and from 20 DEG C to 35 DEG C, the biomass of degradation bacteria strains Phe-1 constantly increases, and 35 DEG C time, degradation bacteria strains Phe-1 biomass reaches maximum value 0.096; And 40 DEG C time, the biomass of degradation bacteria strains Phe-1 is worsened, biomass is 0.02, grows hardly.
2, pH value is on the impact of thalli growth amount
(1) be sole carbon source with phenanthrene, probe into the impact that different pH grows degradation bacteria strains Phe-1.
By finite concentration, (number of cells is about 10
9the bacteria suspension of/mL is inoculated in 20mL minimal medium (luxuriant and rich with fragrance content is 50mg/L), 160rpm in the ratio of 1%, and 24h cultivated by 35 DEG C of shaking tables.Initial pH value is respectively 5.0, and 6.0,7.0,8.0,9.0.3, each sample is parallel, measures OD600 value, calculating mean value.Determine the optimum pH of thalli growth.
(2) result as shown in Figure 6.From Fig. 6, from pH=3.0 to PH=7.0, degradation bacteria strains Phe-1 raises along with pH, increment constantly increases, when pH=7.0, biomass reaches maximum value 0.177, pH increases again, and the biomass of degradation bacteria strains Phe-1 reduces, to sum up, the scope that degradation bacteria strains Phe-1 can be 5.0 ~ 8.0 at pH has one to increase preferably.
3, luxuriant and rich with fragrance concentration is on the impact of thalli growth amount
(1) be sole carbon source with phenanthrene, probe into different luxuriant and rich with fragrance concentration to the impact of microorganism growth.
By finite concentration, (number of cells is about 10
9) bacteria suspension in 1% ratio be inoculated in 20mL minimal medium, pH is 7.0, luxuriant and rich with fragrance concentration gradient 0,20,50,100,200mg/L temperature is 35 DEG C, rotating speed is that under 160rpm, 24h cultivated by shaking table.3, each sample is parallel, measures OD600 value, calculating mean value.Determine the luxuriant and rich with fragrance concentration of the best that degradation bacteria grows.
Result as shown in Figure 7.As can be seen from Figure 7, along with the increase of luxuriant and rich with fragrance concentration, the growth of degradation bacteria strains Phe-1 occurs first raising the trend reduced afterwards, and degradation bacteria strains Phe-1 is when luxuriant and rich with fragrance concentration is 100mg/L, and biomass reaches maximum value 0.17; Subsequently, along with the further raising of luxuriant and rich with fragrance concentration, degradation bacteria strains growth be affected, there is delayed growth phenomenon.
(2) in addition, above-mentioned shaking table continues to be cultured to 72h after cultivating 24h, and result as shown in Figure 8.
In addition, as can be seen from Figure 8, when incubation time is 24h, along with the increase of luxuriant and rich with fragrance concentration, the growth of degradation bacteria strains Phe-1 occurs first raising the trend reduced afterwards, and degradation bacteria strains Phe-1 biomass when luxuriant and rich with fragrance concentration is 100mg/L reaches maximum value, subsequently along with the further raising of luxuriant and rich with fragrance concentration, the growth of degradation bacteria strains Phe-1 is affected, and occurs delayed growth phenomenon.But after incubation time reaches 48h, even if luxuriant and rich with fragrance concentration reaches 200mg/L, the growth sustainable growth on the contrary of degradation bacteria, show the prolongation along with the time, degradation bacteria can tolerate the phenanthrene of greater concn.
4, degradation bacteria growth curve measures
(1) take phenanthrene as sole carbon source, under optimum condition (pH be 7.0, rotating speed 160rpm, temperature 35 DEG C), measure the growth curve of degradation bacteria strains Phe-1.
Specifically: by finite concentration, (number of cells is about 10
9) bacteria suspension in 1% ratio be inoculated in 20mL minimal medium (pH be 7.0, luxuriant and rich with fragrance content be 100mg/L), cultivate 0 in 160rpm, 35 DEG C of shaking tables, 12,24,36,48,72,96,120h measures OD600 value.3, each sample is parallel, calculating mean value, draws the growth curve of degradation bacteria.
(2) result as shown in Figure 9.Go out from the experimental results, 0 ~ 12h is the adaptive phase of degradation bacteria strains Phe-1, and 12 ~ 24h is degradation bacteria strains Phe-1 fast growing period, and 24 ~ 48h is the stationary phase of degradation bacteria strains Phe-1, then just enters decline phase.So best Inoculating date is this period of 24 ~ 48h.
the luxuriant and rich with fragrance efficient degrading bacterial strain of embodiment 3
sphingobiumsp.the degradation capability of Phe-1 measures
1, take phenanthrene as sole carbon source, luxuriant and rich with fragrance concentration is 100mg/L, by 1% inoculation degradation bacteria strains Phe-1 bacteria suspension, under optimum condition (pH be 7.0, rotating speed 160rpm, temperature 35 DEG C), measures degradation bacteria strains Phe-1 degradation curve.
Concrete grammar is: by finite concentration, (number of cells is about 10
9/ mL) bacteria suspension in 1% ratio be inoculated in 20mL minimal medium (luxuriant and rich with fragrance content is 100mg/L, pH is 7.0), cultivate 0 in 160rpm, 35 DEG C of shaking tables, 1,2,3,4, whole bottle extraction after 5d, measure luxuriant and rich with fragrance residual quantity.3, each sample is parallel, calculating mean value, draws the degradation curve of degradation bacteria.Control group: add NaN
3(200mg/L) sterilization.
Degradation rate calculation formula: degradation rate=(control sample-degraded sample)/control sample * 100%.
2, result as shown in Figure 10.
As can be seen from Figure 10, after 2d, luxuriant and rich with fragrance degradation rate, just close to 100%, shows that this degradation bacteria strains Phe-1 is a kind of luxuriant and rich with fragrance efficient degrading bacteria, has very high degradation capability to phenanthrene.
In sum, luxuriant and rich with fragrance efficient degrading bacteria of the present invention
sphingobiumsp.phe-1 can tolerate the phenanthrene up to 200mg/L concentration, all has Degradation to the phenanthrene below 200mg/L concentration.When luxuriant and rich with fragrance concentration is 100mg/L, in 48h, degraded completely.
In addition, the luxuriant and rich with fragrance maximum concentration of this experimental verification is 200mg/L, but contriver considers from the angle of those skilled in the art, for the phenanthrene of greater concn, and degradation bacteria
sphingobiumsp.phe-1 also still should have Degradation, just there will be delayed growth phenomenon.
Claims (10)
1. the luxuriant and rich with fragrance efficient degrading bacterial strain Sphingobiumsp.Phe-1 of a strain, it is characterized in that, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on August 14th, 2015, deposit number is CGMCCNO.11239, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
2. a luxuriant and rich with fragrance efficient degradation agent, is characterized in that, with efficient degrading bacterial strain luxuriant and rich with fragrance described in claim 1
sphingobiumsp.phe-1 or its bacteria suspension are main active ingredient.
3. luxuriant and rich with fragrance efficient degrading bacterial strain described in claim 1
sphingobiumsp.the application of luxuriant and rich with fragrance efficient degradation agent in degrading polycyclic aromatic hydrocarbons compounds described in Phe-1 or claim 2.
4. apply according to claim 3, it is characterized in that, described polycyclic arene compound is luxuriant and rich with fragrance.
5. apply according to claim 4, it is characterized in that, described phenanthrene is containing the phenanthrene in luxuriant and rich with fragrance solution.
6. apply according to claim 5, it is characterized in that, described phenanthrene concentration is in the solution below 200mg/L.
7. apply according to claim 5, it is characterized in that, described solution is the aqueous solution of luxuriant and rich with fragrance contaminating fluid or phenanthrene-polluted soil.
8. apply according to claim 3, it is characterized in that, the condition of application is the degraded realized under pH5 ~ 8,20 ~ 35 DEG C of conditions phenanthrene.
9. according to the arbitrary described application of claim 3 ~ 7, it is characterized in that, the method for application is: according to the volume ratio of 1 ~ 5%, by degradation bacteria strains
sphingobiumsp.the bacterial suspension inoculation of Phe-1 is in the contaminated liquid containing phenanthrene.
10. apply according to claim 9, it is characterized in that, degradation bacteria strains in described bacteria suspension
sphingobiumsp.the cell concn of Phe-1 is 10
9individual/mL, the inoculation volume ratio of described bacteria suspension is 1%.
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| CN109929781A (en) * | 2019-03-26 | 2019-06-25 | 中国矿业大学 | One plant of degradation luxuriant and rich with fragrance bacterial strain and its application in soil remediation |
| CN110317745B (en) * | 2019-05-20 | 2021-03-12 | 中国科学院广州地球化学研究所 | Ralstonia pickettii M1 strain and application thereof in degrading phenanthrene and biphenyl |
| CN110317745A (en) * | 2019-05-20 | 2019-10-11 | 中国科学院广州地球化学研究所 | Ralstonia pickettii M1 bacterial strain and its application in degradation phenanthrene and biphenyl |
| JP2021016385A (en) * | 2019-07-22 | 2021-02-15 | 肇▲慶▼学院 | PAHs-HEAVY METAL COMPLEX POLLUTANT-DEGRADING/ADSORBING BACTERIUM AND APPLICATION THEREOF IN ENVIRONMENTAL POLLUTION RESTORATION |
| CN110317760B (en) * | 2019-07-22 | 2020-09-29 | 肇庆学院 | PAHs-heavy metal combined pollution degrading/adsorbing bacterium and application thereof in environmental pollution remediation |
| CN110317760A (en) * | 2019-07-22 | 2019-10-11 | 肇庆学院 | One plant of PAHs- heavy-metal composite pollution degradation/adhered bacteria and its application in environmental pollution reparation |
| CN110734882A (en) * | 2019-11-25 | 2020-01-31 | 上海交通大学 | phenanthrene efficient degradation bacterial strains and application thereof in environmental remediation |
| CN110734882B (en) * | 2019-11-25 | 2022-07-05 | 上海交通大学 | Phenanthrene efficient degradation strain and application thereof in environmental remediation |
| CN113980856A (en) * | 2021-11-17 | 2022-01-28 | 中国农业科学院研究生院 | Novel pseudomonas strain and application thereof |
| CN113980856B (en) * | 2021-11-17 | 2023-08-29 | 中国农业科学院研究生院 | New pseudomonas strain and application thereof |
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