CN102732429B

CN102732429B - Polycyclic aromatic hydrocarbon-degrading bacteria and its application

Info

Publication number: CN102732429B
Application number: CN 201110096411
Authority: CN
Inventors: 台培东; 巩宗强; 李晓军; 刘宛; 张春桂; 张海荣
Original assignee: Institute of Applied Ecology of CAS
Current assignee: Institute of Applied Ecology of CAS
Priority date: 2011-04-15
Filing date: 2011-04-15
Publication date: 2013-09-25
Anticipated expiration: 2031-04-15
Also published as: CN102732429A

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

Polycyclic aromatic hydrocarbon-degrading bacteria and application thereof

Technical field

The invention belongs to organic pollutant bioremediation technology field, specifically a kind of polycyclic aromatic hydrocarbon-degrading bacteria and application thereof.

Background technology

Polycyclic aromatic hydrocarbons (PAHs) is that a class is distributed widely in the organic pollutant 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 restoration 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, therefore accumulated PAHs epipodium component more in the soil, wherein PAHs content is arranged 4 rings＞5 rings＞3 rings＞6 rings＞2 rings by number of rings, belongs to aging petroleum source contaminated soil.At phenanthrene, the pyrene of 4 rings, 5 benzo (a) pyrenes that encircle of this research of this situation based on 3 rings), with the advantage degradation bacteria active with remediation efficiency be target, studied 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 repaired PAHs contaminated soil engineering for large-scale original position scientific basis is provided.

Summary of the invention

The purpose of this invention is to provide a kind of polycyclic aromatic hydrocarbon-degrading bacteria and application thereof.

For achieving the above object, the technical solution used in the present invention is:

A kind of polycyclic aromatic hydrocarbon-degrading bacteria: described 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: described 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 described microbial inoculum by 5-10 (quality) % is added in contaminated soil or the water body.

The advantage that the present invention has:

Obtained strains screening of the present invention is from old dirty irrigated area PAHs contaminated soil, and process contains the enrichment culture of the enrichment medium of PAHs pollutent, is minimal medium domestication cultivation, 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 of the present invention stable performance, degraded substrate scope is wider, is applicable to the biological restoration (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 that the bacterial strain FMM of Mucor provided by the invention (Mucor sp.) is to the degradation rate figure of 3 kinds of pollutents.

Fig. 4 is that the bacterial strain FMM of Mucor provided by the invention (Mucor sp.) is to the removal effect figure of 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 aging PAHs contaminated soil in the dirty irrigated area of 10g (village, back, north, Fushun City's Triratna village topsoil soils), shaking culture 7d in 120rpm/min constant temperature (25 ℃) shaking table, stand-by.

The enrichment medium of the described 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 the 90ml that branch is installed is poured in the triangular flask again.Described 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 tap water, and pH 6.0.Sterilising conditions is: 0.1Mpa moist heat sterilization 30min.

Then by 10% (quality) inoculum size 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.In 25 ℃, tame under the 120rpm/min condition and cultivate 7d.Cultivate the bacterium liquid that is degraded PAHs with the above-mentioned enrichment culture of this triplicate and domestication.

Described 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, obtain the bacterial strain FMM of Mucor (Mucor sp.) by plate isolation, purifying.(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, the sporocyst sphere, columella and sporangiophore connecting place do not have apophysis.Colonial morphology is seen Fig. 1.Sporangiophore and sporocyst form are seen Fig. 2.

The suitableeest 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 suitableeest growth pH value is 5-6.

Embodiment 2

The bacterial strain FMM of Mucor behind above-mentioned separation, the purifying (Mucor sp.) is activated, and described activation namely 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.Described PDA substratum: potato 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, with pollutant dissolution, be vented near doing by jolting again, add the sterilized minimal medium of 30ml, 1.5ml (inoculum size 5%) the FMM bacterium liquid that inoculation has activated, 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 colorimetric cylinder.The upper strata is extracting 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, used the chromatographically pure methanol constant volume of film to 1ml, measure with high performance liquid chromatograph.

Analyze the residual concentration of pollutent in the nutrient solution, according to the minimizing of initial dosage, calculate clearance (referring to table 1 and Fig. 3).By table 1 and Fig. 3 residual concentration measurement result after 9 days as can be known, 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 only is 14.02%, 3.08% and 4.64%.

Described 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 methylene dichloride.

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, activate.Described activation namely 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, by 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 colorimetric cylinder.The upper strata is extracting 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, used the chromatographically pure methanol constant volume of film to 1ml, measure with high performance liquid chromatograph.

Analyze the residual concentration of pollutent in the nutrient solution, according to the minimizing of initial dosage, calculate clearance (referring to table 2 and Fig. 4).The result shows that when phenanthrene, 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.

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

1. polycyclic aromatic hydrocarbon-degrading bacteria is characterized in that: described degradation bacteria be Mucor ( Mucor sp.) bacterial strain FMM, it is preserved in Chinese typical culture collection center (CCTCC) on March 23rd, 2011, deposit number is: CCTCC M2011092.

2. application by the described polycyclic aromatic hydrocarbon-degrading bacteria of claim 1 is characterized in that: described bacterial strain Mucor ( Mucor sp.) FMM is used for preparing the degradation bacterial agent of polycyclic aromatic hydrocarbons;

Described polycyclic aromatic hydrocarbons is phenanthrene, pyrene and/or benzo (a) pyrene.

3. by the application of the described polycyclic aromatic hydrocarbon-degrading bacteria of claim 2, it is characterized in that: with described microbial inoculum by mass percentage the inoculum size of 5-10% be added in contaminated soil or the water body.