CN102732429A - Polycyclic aromatic hydrocarbon-degrading bacteria and its application - Google Patents
Polycyclic aromatic hydrocarbon-degrading bacteria and its application Download PDFInfo
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- CN102732429A CN102732429A CN2011100964111A CN201110096411A CN102732429A CN 102732429 A CN102732429 A CN 102732429A CN 2011100964111 A CN2011100964111 A CN 2011100964111A CN 201110096411 A CN201110096411 A CN 201110096411A CN 102732429 A CN102732429 A CN 102732429A
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- fmm
- mucor
- polycyclic aromatic
- aromatic hydrocarbon
- pyrene
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Abstract
The invention belongs to the technical field of organic pollutants bioremediation, specifically relates to a polycyclic aromatic hydrocarbon-degrading bacteria and its application. The degrading bacteria is a strain FMM of Mucor sp., which is preserved in China Center for Type Culture Collection (CCTCC) on March 23rd, 2011, the preservation number is: CCTCC M 2011092. The strain Mucor sp. FMM is taken as a degradation bacteria agent of polycyclic aromatic hydrocarbon. The FMM strain has the advantages of stable degradation performance and wide range of degradation substrates, and is suitable for remediation of acidic PAHs contaminated soil and water (Ph value 5-6.5).
Description
Technical field
The invention belongs to organic pollutant bioremediation technology field, a kind of specifically polycyclic aromatic hydrocarbon-degrading bacteria and application thereof.
Background technology
Polycyclic aromatic hydrocarbons (PAHs) is one type of organic pollutant that is distributed widely in the environment, is the by product of combustion of fossil fuel processes such as oil, coal and energy conversion process, human body is had potential three cause hazardness.Because potential carinogenicity, teratogenecity, the mutagenicity of PAHs have caused the great attention of various countries.
For the existing a lot of researchs of PAHs contaminated soil bioremediation technology, its advantage is 1. cost saving at present, and its expense is about the 1/4-1/3 of burning disposal expense.2. environmental influence is little, and biological prosthetic is the reinforcement of a natural process, and its final product is carbonic acid gas, water and lipid acid etc., does not form secondary pollution or causes pollutent to shift.3. can reduce Pollutant levels etc. to greatest extent.And can improve the soil, increase fertility of land, therefore have and use widely and development prospect.
The existing dirt for many years in irrigated area, Fushun, Shenyang irritates historical; Accumulated a large amount of PAHs in the soil; Because low ring component is easy to volatilization, degraded, and in the natural degradation ability of high loop section relatively low, so accumulated PAHs epipodium component in the soil more; Wherein PAHs content is arranged 4 rings＞5 rings＞3 rings＞6 rings＞2 rings by number of rings, belongs to aged petroleum source contaminated soil.To this research of this situation with 3 the ring phenanthrene, 4 the ring pyrene, 5 the ring benzo (a) pyrene) be main; Is target with advantage degradation bacteria activity with remediation efficiency; Studied the activity and the degradation capability of multiple bacterial strain, wherein the degradation effect of the bacterial strain FMM of Mucor (Mucor sp.) is best.Experimental result is that large-scale original position reparation PAHs contaminated soil engineering provides scientific basis.
Summary of the invention
The purpose of this invention is to provide a kind of polycyclic aromatic hydrocarbon-degrading bacteria and application thereof.
For realizing above-mentioned purpose, the technical scheme that the present invention adopts is:
A kind of polycyclic aromatic hydrocarbon-degrading bacteria: said degradation bacteria is the bacterial strain FMM of Mucor (Mucor sp.), and it is to be preserved in Chinese typical culture collection center (CCTCC) on March 23rd, 2011, and deposit number is: CCTCC M 2011092.
The application of polycyclic aromatic hydrocarbon-degrading bacteria: said bacterial strain Mucor (Mucor sp.) FMM is as the degradation bacterial agent of polycyclic aromatic hydrocarbons.This microbial inoculum is as the degradation bacterial agent of luxuriant and rich with fragrance (3 ring), pyrene (4 ring) and/or benzo (a) pyrene (5 ring).The inoculum size of said microbial inoculum by 5-10 (quality) % is added in contaminated soil or the water body.
The advantage that the present invention had:
Obtained strains screening of the present invention is the PAHs contaminated soil from old dirty irrigated area, and process contains the enrichment culture of the enrichment medium of PAHs pollutent, is minimal medium acclimation shaking culture, screening, separation, the purifying acquisition of sole carbon source again with the PAHs pollutent.Can be sole carbon source and energy growth with phenanthrene, pyrene and benzo (a) pyrene; In minimal medium; When the concentration of luxuriant and rich with fragrance, pyrene and benzo (a) pyrene was 72.84mg/l, 67.49mg/l and 40.34mg/l, the degradation rate of luxuriant and rich with fragrance in 9 days, pyrene and benzo (a) pyrene was respectively 95.33%, 95.16% and 90.36%.FMM strains for degrading according to the invention stable performance, degraded substrate scope is wider, is applicable to the biological prosthetic (pH5-6.5) of acid PAHs contaminated soil and water.
Description of drawings
Fig. 1 is the bacterial strain FMM colonial morphology figure of Mucor provided by the invention (Mucor sp.).
Fig. 2 is bacterial strain FMM sporangiophore and the sporocyst figure of Mucor provided by the invention (Mucor sp.).
Fig. 3 is the degradation rate figure of the bacterial strain FMM of Mucor provided by the invention (Mucor sp.) to 3 kinds of pollutents.
Fig. 4 is the removal effect figure of the bacterial strain FMM of Mucor provided by the invention (Mucor sp.) to 3 kinds of pollutents.
Embodiment
Embodiment 1
Screening and the colonial morphology of Mucor (Mucor sp.) bacterial strain FMM.
The screening of efficient degrading bacteria Mucor (Mucor sp.) bacterial strain FMM:
The preparation of bacterium liquid: contain in the enrichment medium of PAHs pollutent at 90ml, add the dirty irrigated area of 10g (village, back, north, Fushun City's Triratna village topsoil soils) aged PAHs contaminated soil, shaking culture 7d in 120rpm/min constant temperature (25 ℃) shaking table, for use.
The enrichment medium of the said PAHs of containing pollutent is: 2.5ml PAHs solution is joined in the aseptic 500ml triangular flask, make that polycyclic aromatic hydrocarbons total amount concentration is 200mg/L in the enrichment medium; After treating that solvent evaporates is intact, the aseptic enrichment medium of 90ml that again branch is installed is poured in the triangular flask.Said enrichment medium is by weight percentage: glucose 0.1%, and peptone 0.05%, yeast extract paste 0.05%, NaCl 0.5%, NH
4NO
31.0%, K
2HPO
40.5%, KH
2PO
40.5%, MgSO
4.7H
2O 0.02%, and surplus is a tap water, and pH 6.0.Sterilising conditions is: 0.1Mpa moist heat sterilization 30min.
Then above-mentioned kind of liquid is transferred in the 150ml triangular flask that the 30ml minimal medium is housed, makes that PAHs total amount concentration is about 200mg/L in the substratum by 10% (quality) inoculum size.In 25 ℃, carry out acclimation shaking culture 7d under the 120rpm/min condition.Be the bacterium liquid of degraded PAHs with above-mentioned enrichment culture of this triplicate and acclimation shaking culture.
Said minimal medium is according to weight percent meter (%): NaCl 0.01; NH
4NO
30.1; CaCl2 0.01; MgSO
4.7H
2O 0.02; FeCl
30.002; KH
2PO
40.1; K
2HPO
40.1 water 100ml, pH are 6.0.
In above-mentioned bacterium liquid, pass through the bacterial strain FMM of plate isolation, purifying acquisition Mucor (Mucor sp.).(referring to Fig. 1, Fig. 2).
Above-mentioned bacterial strains is preserved in Chinese typical culture collection center (CCTCC) on March 23rd, 2011, and deposit number is: CCTCC M 2011092.
The bacterial strain FMM thalli morphology of Mucor (Mucor sp.) is no rhizoid and creeper, and sporangiophore is directly born by mycelium, Dan Sheng, and there is not the sporocyst that expands on the branch top, and sporocyst is spherical, and columella and sporangiophore connecting place do not have apophysis.Colonial morphology is seen Fig. 1.Sporangiophore and sporocyst form are seen Fig. 2.
The righttest growth of bacterial strain FMM and best degradation temperature are 23 ℃-28 ℃, are being lower than 10 ℃ or poor growth when being higher than 40 ℃, and the righttest growth pH value is 5-6.
Embodiment 2
The bacterial strain FMM of Mucor behind above-mentioned separation, the purifying (Mucor sp.) is carried out activation, and said activation promptly the test tube of the FMM bacterial strain of a solid inclined-plane preservation, is all transferred in the 150ml triangular flask, every bottled sterilized PDA substratum of 30ml that has.130 commentaries on classics/min, 25 ℃ of shaking culture 7d.Be FMM bacterium liquid.Said PDA substratum: yam 200g, sucrose 20g, water 1000ml, pH6.0.
Degradation effect experiment employing is shaken bottle liquid culture method and is carried out.3 repetitions are established in each processing.Under aseptic condition, draw 0.60mlPAHs solution and place the empty triangular flask of sterilized 150ml, cover the high temperature plastic film of ventilation; Be vented near doing, add 0.5ml acetone again, through jolting with pollutant dissolution; Be vented near doing again, add the sterilized minimal medium of 30ml, inoculate activatory 1.5ml (inoculum size 5%) FMM bacterium liquid; Shaking culture, 130 commentaries on classics/min, 25 ℃ of concussions were cultivated 9 days.
The nutrient solution of above-mentioned cultivation after 9 days handled, adopted dichloromethane extraction, in triangular flask, add methylene dichloride 10ml, cover the sealed plastic film, the 180 commentaries on classics/min 5min that vibrates, after the static layering, lower floor's (dichloromethane layer) puts into is equipped with anhydrous Na
2SO
4In the funnel, be filled in the 25ml tube comparison tubes.The upper strata is extracted twice again, and step is the same, is settled to 25.0ml at last, gets 2ml and crosses the 0.22um organic membrane.The liquid 40ul that accurately drew behind the film places sample bottle, volatilization is near naturally do after, the chromatographically pure methanol constant volume of using film is measured with high performance liquid chromatograph to 1ml.
Analyze the residual concentration of pollutent in the nutrient solution,, calculate clearance (referring to table 1 and Fig. 3) according to the minimizing of initial dosage.Can know that by table 1 and Fig. 3 the residual concentration after 9 days measures the result, and bacterial strain Mucor-FMM is to the degradation rate of 3 kinds of pollutents.The result shows, when luxuriant and rich with fragrance, pyrene and benzo [a] pyrene starting point concentration were 72.84mg/l, 67.49mg/l and 40.34mg/l, the degradation rate of luxuriant and rich with fragrance in 9 days, pyrene and benzo (a) pyrene was respectively 96.86%, 95.38% and 90.70%.
The degradation rate of not inoculating contrast phenanthrene, pyrene and benzo (a) pyrene of FMM bacterium liquid under the same terms is merely 14.02%, 3.08% and 4.64%.
Said PAHs solution is phenanthrene, pyrene and benzo (a) pyrene mixed solution, and luxuriant and rich with fragrance, pyrene concentration is 4mg/ml, and benzo [a] pyrene is 2mg/ml, and 3 kinds of Pollutant levels total amounts are about 10mg/ml, and solvent is a methylene dichloride.
Said minimal medium is according to weight percent meter (%): NaCl 0.01; NH
4NO
30.1; CaCl2 0.01; MgSO
4.7H
2O 0.02; FeCl
30.002; KH
2PO
40.1; K
2HPO
40.1 water 100ml, pH are 6.0.
Residual concentration (mgL behind table 1 bacterial strain Mucor-FMM degraded PAHs 9d
-1)
Embodiment 3
With above-mentioned FMM bacterium liquid, from refrigerator, take out and be placed to room temperature, carry out activation.Said activation promptly under aseptic condition, is seeded in the sterilized PDA substratum by 10% (quality).Use the 150ml triangular flask, every bottled sterilized PDA substratum of 30ml that has.130 commentaries on classics/min, 25 ℃ of shaking culture 7d.Be FMM bacterium liquid.
The experiment employing is shaken bottle liquid culture method and is carried out.3 repetitions are established in each processing.Under aseptic condition, draw 0.60mlPAHs solution and place the empty triangular flask of sterilized 150ml, cover the high temperature plastic film of ventilation; Be vented near doing, add 0.5ml acetone again, through jolting with pollutant dissolution; Be vented near doing again, add in the sterilized 30ml minimal medium.30ml culture medium inoculated 3ml microbial inoculum (inoculum size 10%), shaking culture, 130 commentaries on classics/min, 28 ℃ of concussions were cultivated 10 days.
The nutrient solution of above-mentioned cultivation after 9 days handled, adopted dichloromethane extraction, in triangular flask, add methylene dichloride 10ml, cover the sealed plastic film, the 180 commentaries on classics/min 5min that vibrates, after the static layering, lower floor's (dichloromethane layer) puts into is equipped with anhydrous Na
2SO
4In the funnel, be filled in the 25ml tube comparison tubes.The upper strata is extracted twice again, and step is the same, is settled to 25.0ml at last, gets and crosses the 0.22um organic membrane about 2ml.The liquid 40ul that accurately drew behind the film places sample bottle, volatilization is near naturally do after, the chromatographically pure methanol constant volume of using film is measured with high performance liquid chromatograph to 1ml.
Analyze the residual concentration of pollutent in the nutrient solution,, calculate clearance (referring to table 2 and Fig. 4) according to the minimizing of initial dosage.The result shows, when luxuriant and rich with fragrance, pyrene and benzo [a] pyrene starting point concentration were 93.94mg/ml, 95.26mg/ml and 44.44mg/ml, clearance was 90.80%, 89.83% and 85.70% after 10 days.Do not add the contrast clearance difference 31.15%, 26.97% and 17.03% of microbial inoculum.Experimental result shows that the FMM microbial inoculum has very significantly removal effect.
Said PAHs solution is phenanthrene, pyrene and benzo (a) pyrene mixed solution, and luxuriant and rich with fragrance, pyrene concentration is 4mg/ml, and benzo [a] pyrene is 2mg/ml, and 3 kinds of Pollutant levels total amounts are about 10mg/ml, and solvent is a methylene dichloride.
The table 2 bacterial strain Mucor-residual concentration (mgL of FMM degraded PAHs after 10 days
-1)
| Handle | Luxuriant and rich with fragrance | Pyrene | Bap | Total amount |
| Initially | 93.94 | 95.26 | 44.44 | 233.64 |
| Contrast | 64.68 | 69.57 | 36.87 | 171.12 |
| FMM | 8.64 | 9.69 | 6.35 | 24.68 |
Claims (4)
1. polycyclic aromatic hydrocarbon-degrading bacteria, it is characterized in that: said degradation bacteria is the bacterial strain FMM of Mucor (Mucorsp.), and it is to be preserved in Chinese typical culture collection center (CCTCC) on March 23rd, 2011, and deposit number is: CCTCC M 2011092.
2. application by the described polycyclic aromatic hydrocarbon-degrading bacteria of claim 1, it is characterized in that: said bacterial strain Mucor (Mucor sp.) FMM is as the degradation bacterial agent of polycyclic aromatic hydrocarbons.
3. by the application of the described polycyclic aromatic hydrocarbon-degrading bacteria of claim 2, it is characterized in that: this microbial inoculum is as the degradation bacterial agent of luxuriant and rich with fragrance (3 ring), pyrene (4 ring) and/or benzo (a) pyrene (5 ring).
4. by the application of claim 2 or 3 said polycyclic aromatic hydrocarbon-degrading bacterias, it is characterized in that: the inoculum size of said microbial inoculum by 5-10 (quality) % is added in contaminated soil or the water body.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103013841A (en) * | 2012-12-24 | 2013-04-03 | 中国科学院苏州纳米技术与纳米仿生研究所 | Scopulariopsis brevicaulis and application thereof |
| CN105483026A (en) * | 2015-12-31 | 2016-04-13 | 广东省生态环境与土壤研究所 | Mucor circinelloides H3 and application thereof in pyrene degradation |
| CN107684686A (en) * | 2017-08-03 | 2018-02-13 | 天津大学 | Biodegrading process using Rhodococcus sp JZX 01 to polycyclic aromatic hydrocarbon pollutant pyrene |
| CN110468065A (en) * | 2019-07-18 | 2019-11-19 | 中国科学院城市环境研究所 | One polycyclic aromatic hydrocarbon degrading bacteria Q3 and application thereof |
| CN114703085A (en) * | 2022-01-28 | 2022-07-05 | 中国科学院沈阳应用生态研究所 | Compound microbial agent for degrading polycyclic aromatic hydrocarbon |
| CN114807111A (en) * | 2022-03-30 | 2022-07-29 | 中国科学院沈阳应用生态研究所 | Composite immobilized microbial agent and preparation method and application thereof |
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Patent Citations (5)
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| JP2003052367A (en) * | 2001-08-20 | 2003-02-25 | Daiwa Kasei Kk | Method of decomposition treatment for organic contaminant |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103013841A (en) * | 2012-12-24 | 2013-04-03 | 中国科学院苏州纳米技术与纳米仿生研究所 | Scopulariopsis brevicaulis and application thereof |
| CN105483026A (en) * | 2015-12-31 | 2016-04-13 | 广东省生态环境与土壤研究所 | Mucor circinelloides H3 and application thereof in pyrene degradation |
| CN105483026B (en) * | 2015-12-31 | 2019-01-29 | 广东省生态环境与土壤研究所 | Roll up branch Mucor H3 and its application in degradation pyrene |
| CN107684686A (en) * | 2017-08-03 | 2018-02-13 | 天津大学 | Biodegrading process using Rhodococcus sp JZX 01 to polycyclic aromatic hydrocarbon pollutant pyrene |
| CN110468065A (en) * | 2019-07-18 | 2019-11-19 | 中国科学院城市环境研究所 | One polycyclic aromatic hydrocarbon degrading bacteria Q3 and application thereof |
| CN114703085A (en) * | 2022-01-28 | 2022-07-05 | 中国科学院沈阳应用生态研究所 | Compound microbial agent for degrading polycyclic aromatic hydrocarbon |
| CN114807111A (en) * | 2022-03-30 | 2022-07-29 | 中国科学院沈阳应用生态研究所 | Composite immobilized microbial agent and preparation method and application thereof |
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