CN102533928A - Screening method for degradation bacteria with polycyclic aromatic hydrocarbon as substrate - Google Patents

Screening method for degradation bacteria with polycyclic aromatic hydrocarbon as substrate Download PDF

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CN102533928A
CN102533928A CN 201010588933 CN201010588933A CN102533928A CN 102533928 A CN102533928 A CN 102533928A CN 201010588933 CN201010588933 CN 201010588933 CN 201010588933 A CN201010588933 A CN 201010588933A CN 102533928 A CN102533928 A CN 102533928A
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polycyclic aromatic
aromatic hydrocarbons
substrate
degradation bacteria
screening method
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苏振成
胡凤钗
李新宇
王秀娟
张惠文
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Institute of Applied Ecology of CAS
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Institute of Applied Ecology of CAS
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Abstract

The invention relates to the technical field of biological remediation of polluted soil, in particular to a screening method for degradation bacteria with polycyclic aromatic hydrocarbon as a substrate. The method comprises the following steps of: performing enrichment culture on the degrading bacteria with the polycyclic aromatic hydrocarbon as the substrate by using a method for gradually increasing the concentration of the polycyclic aromatic hydrocarbon; after diluting enrichment culture solution by 101-107 times, coating the solution with proper concentration on a sorbitol solid culture medium; after additionally forming a pyrene film, placing in a constant-temperature culture box of 28-32 DEG C to culture for 1-3 weeks; picking up single colonies with degrading rings to be dibbled in a beef extract peptone culture medium containing polycyclic aromatic hydrocarbon respectively; placing in the constant-temperature culture box of 28-32 DEG C to culture for 1-2 weeks; dibbling grown strains in an inorganic salt solid culture medium with a polycyclic aromatic hydrocarbon membrane; and placing in the constant-temperature culture box of 28-32 DEG C to culture for 1-3 weeks so as to grow the strain, namely the degrading strain with the polycyclic aromatic hydrocarbon as a unique carbon source. By using the method, the polycyclic aromatic hydrocarbon degrading bacteria can be quickly screened in a laboratory; and a quick and convenient means is provided for developing a new biological remediation microbial resource.

Description

A kind of is the screening method of the degradation bacteria of substrate with the polycyclic aromatic hydrocarbons
Technical field
The present invention relates to contaminated soil bioremediation technology field, a kind of specifically is the screening method of the degradation bacteria of substrate with the polycyclic aromatic hydrocarbons.
Background technology
(polycyclic aromatic hydrocarbons PAHs) is meant the persistence organic pollutant of one type of uniqueness that is made up of with wire, horn shape or cluster form 2 or 2 above phenyl ring to polycyclic aromatic hydrocarbons.A lot of PAHs can accumulate in vivo through cytotoxicity, genetoxic and immunotoxicity organism is produced carcinogenic, teratogenesis, mutagenesis; Nature biotechnology safety and human health are constituted grave danger. EPA is not with ramose PAHs to list in the priority pollutant list with 16 kinds in the eighties in 20th century, and China is also listed PAHs in the Black List of environmental pollutant in.Therefore, the PAHs contaminated soil being repaired is a task that ten minutes is urgent.
The microbiological deterioration polycyclic aromatic hydrocarbons has the cheap characteristics of environment-friendly high-efficiency; Be acknowledged as one of important channel of removing polycyclic aromatic hydrocarbons in the environment; Also inoculate back in the original position contaminated soil through the efficient PAHs degradation bacteria strains of screening from indigenous microorganism group, reach the purpose of repairing polluted soil.Wherein, the screening of efficient degrading bacteria is the key problem in technology that carries out effective biological prosthetic.
The screening of conventional P AHs degradation function bacterium often has certain blindness and randomness, and workload is big, waste time and energy, and cost is high.Develop that a kind of fast screening method is extremely urgent efficiently, similarly research method is also offered reference for the exploitation of other functional microorganism resources.
Summary of the invention
It is the screening method of the degradation bacteria of substrate with the polycyclic aromatic hydrocarbons that the object of the invention is to provide a kind of.
For realizing above-mentioned purpose, the technical scheme that the present invention adopts is:
A kind of is the screening method of the degradation bacteria of substrate with the polycyclic aromatic hydrocarbons:
1) enrichment culture adopts the method progressively improve polycyclic aromatic hydrocarbons concentration to being that the degradation bacteria of substrate is carried out enrichment culture with the polycyclic aromatic hydrocarbons;
2) sub-sieve is cultivated: get enrichment culture liquid dilution 10 in the step 1) 1-10 7After; Coat in the sorbyl alcohol solid medium; Place 28-32 ℃ of constant incubator to cultivate 1-3 week, single bacterium colony that picking has degraded circle dibbling respectively places 28-32 ℃ of constant incubator to cultivate 1-2 week in the beef-protein medium that contains polycyclic aromatic hydrocarbons;
3) the bacterial strain dibbling of growth is on the inorganic salt solid medium that has the polycyclic aromatic hydrocarbons film one-tenth embrane method rapid screening degradation bacteria: with step 2); Place 28-32 ℃ of constant incubator to cultivate 1-3 week, it can be the degradation bacteria strains of sole carbon source with the polycyclic aromatic hydrocarbons that the bacterial strain that can grow is.
Said step 1) enrichment culture: bacterium appearance is inoculated in the cultivation of transferring in the liquid minimal medium that contains polycyclic aromatic hydrocarbons, and the culture condition of at every turn transferring is for placing 180rmin on the 28-32 ℃ of constant temperature shaking table -1Cultivated 30 days, corotation connects 4-5 time, and switching is measured the each nutrient solution of per-cent meter 10% by volume and is inoculated in and contains in the 50ml liquid minimal medium that polycyclic aromatic hydrocarbons increases gradually, and is for use; Wherein contain in the liquid minimal medium of polycyclic aromatic hydrocarbons polycyclic aromatic hydrocarbons add-on 100 ㎎ L first -1, then with each increase by 100 ㎎ L -1Speed increase gradually.
Said polycyclic aromatic hydrocarbons is at first to be dissolved in acetone, presses mass volume ratio (gml -1) for 1:1000 polycyclic aromatic hydrocarbons is dissolved in the acetone, process 1000 ㎎ L -1Mother liquor.
Polycyclic aromatic hydrocarbons in the said beef-protein medium that contains polycyclic aromatic hydrocarbons is dissolved in DMSO 99.8MIN., presses mass volume ratio (gml -1) for 1:500 polycyclic aromatic hydrocarbons is dissolved in the DMSO 99.8MIN., process 2000 ㎎ L -1Mother liquor.
Said sorbyl alcohol solid medium is: add 1ml VITAMINs mother liquor in every liter of sorbyl alcohol minimal medium.Said sorbyl alcohol minimal medium is to add the 9g sorbyl alcohol in every liter of liquid minimal medium, 0.5g yeast powder and press 2% agar strip of substratum gross weight; Said VITAMINs mother liquor prescription is: para-amino benzoic acid 200mg, vitamin H 200mg, folic acid 200mg; Nicotinic acid 200mg, VA 100mg, Y factor 100mg; Vitamin G 100mg, VitB1 100mg, cobalamin 1mg; Be settled to 1000ml with sterilized water, with 0.4 μ m membrane filtration degerming.
Said liquid minimal medium is: NaNO 30.5 g, (NH 4) 2SO 41.0g, Na 2HPO 42.5g, KH 2PO 41.0g, trace element solution 1ml, calcium magnesium solution 1ml is settled to 1000ml with zero(ppm) water; Wherein, the trace element solution prescription is: Al 2(SO 4) 318H 2O 108 mg, CoSO 47H 2O 56 mg, CuSO 45H 2O 56 mg, FeSO 47H 2O 3.0g, H 3BO 3611mg, KBr 28 mg, KI 56 mg, LiCl 28mg, MnCl 24H 2O 389mg, Na 2MoO 42H 2O 28mg, Na 2WO 42H 2O 28mg, NiCl 26H 2O 58mg, SnCl 22H 2O 28mg, ZnSO 4H 2O 34mg is settled to 1000ml with zero(ppm) water; Calcium magnesium solution prescription is: CaCl 230g, MgCl 220g is settled to 1000ml with zero(ppm) water.
Said polycyclic aromatic hydrocarbons film: make acetone volatilization for the polycyclic aromatic hydrocarbons acetone soln with configuration places the ventilation, the polycyclic aromatic hydrocarbons film.Said polycyclic aromatic hydrocarbons acetone soln: press mass volume ratio (gml -1) for 1:100 polycyclic aromatic hydrocarbons is dissolved in the acetone, process 10000 ㎎ L -1Mother liquor.The said polycyclic aromatic hydrocarbons acetone soln of getting places the ventilation to make the acetone volatilization clean; Integral body to be filmed is put into more than the fusing point that is heated to corresponding polycyclic aromatic hydrocarbons in the sand-bath; Above flat board, put ice bag, make polycyclic aromatic hydrocarbons distillation back meet dull and stereotyped cooling and attached on the beef extract-peptone solid medium.
Beneficial effect of the present invention is following:
1, the present invention provides a kind of improved one-tenth embrane method; On solid medium, form the macroscopic polycyclic aromatic hydrocarbons film of one deck; Can make with the polycyclic aromatic hydrocarbons is that the degradation bacteria of substrate can make clear being prone to of degraded circle distinguish, can from soil, isolate the bacterial strain with the PAHs function of degrading fast.The present invention is directed to the blindness of traditional degradation bacteria strains screening; Randomness and workload are big; Waste time and energy, shortcoming such as waste resource etc., exploitation be sublimed into embrane method; For rapid screening in the laboratory goes out with the polycyclic aromatic hydrocarbons is the degradation bacteria of substrate, develops new biological prosthetic Microbial resources a kind of means quickly and easily are provided.
2, it is clean that screening method step of the present invention makes the acetone volatilization in stink cupboard, can prevent that the staff from sucking.Two plate make-ups, and with sealing film phonograph seal, evaporate in the time of can avoiding the PAHs heating, thereby health harm reduced to experiment operator.
Adopt plate when 3, the present invention screens, but not the beaker of 500ml not only is convenient to sealing, and can effectively shortens the climb of PAHs, the time saving while, reduce the waste of PAHs.
4, laboratory experiment of the present invention shows, adopts one-tenth embrane method provided by the invention, the bacterial strain that from oil-polluted soils, separates degraded phenanthrene and pyrene that can be a large amount of fast, thus further adopt the liquid shaking bottle method to sieve again.
Description of drawings
The degraded circle that the primary dcreening operation degradation bacteria that Fig. 1 provides for the embodiment of the invention forms on the pyrene film.
The degraded circle that the multiple sieve degradation bacteria that Fig. 2 provides for the embodiment of the invention forms on the pyrene film.
The degradation bacteria that Fig. 3 provides for the embodiment of the invention is at fluorenes, the degraded circle that forms on the luxuriant and rich with fragrance and fluoranthene film.
Embodiment
Embodiment 1
1) gather Shen and comfort top layer, irrigated area 0-20 ㎝ soil as sample, 4 ℃ of refrigerators are preserved.Take by weighing and supply examination pedotheque 5g, insert 45ml and contain in the liquid minimal medium of Fourth Ring polycyclic aromatic hydrocarbon pyrene, wherein pyrene concentration is 100 ㎎ L -1, add an amount of granulated glass sphere, place 180 rmin on the 28-32 ℃ of constant temperature shaking table -1Enrichment culture 1 month after the enrichment culture, pipettes nutrient solution for the first time and inserts 45ml by 10% volume ratio and contain in the liquid minimal medium of pyrene, carries out enrichment culture second time, and gross weight is answered 4 times, and revolution connects once, and the concentration of pyrene improves 100 ㎎ L -1, promptly the concentration of last pyrene is increased to 400 ㎎ L -1
2) above-mentioned enrichment culture liquid is carried out 10 times of serial gradient dilutions to 10 with sterilized water under aseptic condition -7, get 10 respectively -5, 10 -6With 10 -7The bacterium liquid 100 μ l of three concentration are coated on the sorbyl alcohol solid medium, place 28-32 ℃ of constant temperature culture 1-3 week, and picking has each some strain of single bacterium colony that the degraded circle is arranged of different shape, and dibbling is in containing 10 ㎎ L respectively -1The beef extract-peptone solid medium of pyrene on, and bacterium colony carried out serial number.Place 28-32 ℃ of constant temperature culture 1-2 week.
Contain the preparation of the liquid minimal medium of pyrene: getting concentration is 1000 ㎎ L -1With the pyrene mother liquor of acetone preparation, with 0.22 μ m filter membrane Sterile Filtration, measure 5-20ml pyrene acetone mother liquor, add in the sterilized 250ml triangular flask, treat that the acetone volatilization finishes, add the liquid minimal medium of 50ml sterilization, the final concentration that makes pyrene is 100-400 ㎎ L -1
In addition, contain the preparation of the beef extract-peptone solid medium of pyrene: in the beef extract-peptone solid medium, add 2000 ㎎ L -1The DMSO 99.8MIN. mother liquor of pyrene, final concentration is 10 ㎎ L -1
Wherein, liquid minimal medium prescription is: NaNO 30.5 g, (NH 4) 2SO 41.0g, Na 2HPO 42.5g, KH 2PO 41.0g, trace element solution 1ml, calcium magnesium solution 1ml is settled to 1000ml with zero(ppm) water; Wherein, the trace element solution prescription is: Al 2(SO 4) 318H 2O 108 mg, CoSO 47H 2O 56 mg, CuSO 45H 2O 56 mg, FeSO 47H 2O 3.0g, H 3BO 3611mg, KBr 28 mg, KI 56 mg, LiCl 28mg, MnCl 24H 2O 389mg, Na 2MoO 42H 2O 28mg, Na 2WO 42H 2O 28mg, NiCl 26H 2O 58mg, SnCl 22H 2O 28mg, ZnSO 4H 2O 34mg is settled to 1000ml with zero(ppm) water.Calcium magnesium solution prescription is: CaCl 230g, MgCl 220g is settled to 1000ml with zero(ppm) water.
Beef extract-peptone solid culture based formulas is: Carnis Bovis seu Bubali cream 5g, and peptone 10g, NaCl 5g, agar strip 20g is settled to 1000ml with zero(ppm) water, and pH 7.2 ~ 7.4.
The sorbyl alcohol solid medium is: add 1ml VITAMINs mother liquor in every liter of sorbyl alcohol minimal medium.
The sorbyl alcohol minimal medium is to add 9g sorbyl alcohol, the agar strip of the weight percent 2% of 0.5g yeast powder and substratum gross weight in every liter of liquid minimal medium; Said VITAMINs mother liquor prescription is: para-amino benzoic acid 200mg, vitamin H 200mg, folic acid 200mg; Nicotinic acid 200mg, VA 100mg, Y factor 100mg; Vitamin G 100mg, VitB1 100mg, cobalamin 1mg; Be settled to 1000ml with sterilized water, with 0.4 μ m membrane filtration degerming.
3) with aseptic toothpick with step 2) in cultured bacterial strain dibbling on the flat board of the beef-protein medium that the pyrene film is arranged, place 28-32 oIn C constant temperature culture 1-3 week, periphery of bacterial colonies the degraded circle occurs and shows that then substrate is degraded, and it can be the degradation bacteria strains of sole carbon source with the pyrene that this bacterium is.
The preparation of pyrene film:
1) presses mass volume ratio (gml -1) for 1:100 pyrene is dissolved in the acetone, process 10000 ㎎ L -1Mother liquor.
2) draw 10000 ㎎ L -1The acetone mother liquor 2ml of pyrene in a plate; In stink cupboard, make acetone volatilization clean, form a uniform skim in the plate bottom, tile the plate of solid pyrene to postvaccinal dull and stereotyped back-off to the bottom then on; And interface is sealed with sealing film; Integral body is put in the sand-bath and heats, and above flat board, puts ice bag, makes pyrene distillation back meet dull and stereotyped cooling and attached on the solid medium.
4) carry out multiple sieve according to degradation rate: choose 3) the bigger part bacterial strain of middle degraded circle, be inoculated in the beef extract-peptone liquid nutrient medium, be cultured to OD 600=0.6-0.8 cleans with phosphate buffered saline buffer, inserts by 10% inoculum size to contain pyrene 100 ㎎ L -1-400 ㎎ L -1The liquid minimal medium in, place 180rmin on the 28-32 ℃ of constant temperature shaking table -1Cultivated 10 days.Extract residue pyrene component in the substratum, adopt the HPLC method to measure degradation rate.
Embodiment 2
1) gather Shen and comfort top layer, irrigated area 0-20 ㎝ soil as sample, 4 ℃ of refrigerators are preserved.Take by weighing and supply examination pedotheque 5g, insert 45ml contain the trinucleated polycyclic aromatic hydrocarbons luxuriant and rich with fragrance (acenaphthene, fluorenes, anthracene, methods such as fluoranthene with) the liquid minimal medium in, its Sino-Philippines concentration is 200 ㎎ L -1, add an amount of granulated glass sphere, place 180rmin on the 28-32 ℃ of constant temperature shaking table -1Enrichment culture 1 month after the enrichment culture, pipettes nutrient solution for the first time and inserts 45ml by 10% volume ratio and contain in the luxuriant and rich with fragrance liquid minimal medium, carries out enrichment culture second time, multiple 4 times of gross weight, and revolution connects once, and the concentration of phenanthrene improves 100 ㎎ L -1, promptly last luxuriant and rich with fragrance concentration is increased to 500 ㎎ L -1
2) above-mentioned enrichment culture liquid is carried out 10 times of serial gradient dilutions to 10 with sterilized water under aseptic condition -7, get 10 respectively -5, 10 -6With 10 -7The bacterium liquid 100 μ l of three concentration are coated on the sorbyl alcohol solid medium, place 28-32 ℃ of constant temperature culture 1-3 week, and picking has each some strain of single bacterium colony that the degraded circle is arranged of different shape, and dibbling is in containing 10 ㎎ L respectively -1The beef extract-peptone solid medium of phenanthrene on, and bacterium colony carried out serial number.Place 28-32 ℃ of constant temperature culture 1-2 week.
Contain the preparation of luxuriant and rich with fragrance liquid minimal medium: getting concentration is 1000 ㎎ L -1With the luxuriant and rich with fragrance mother liquor of acetone preparation, with 0.22 μ m filter membrane Sterile Filtration, measure the 5-20ml mother liquor, add in the sterilized 250ml triangular flask, treat that the acetone volatilization finishes, add the liquid minimal medium of 50ml sterilization, making final concentration is 200-500 ㎎ L -1
In addition, contain the preparation of luxuriant and rich with fragrance beef extract-peptone solid medium: in the beef extract-peptone solid medium, add 2000 ㎎ L -1The luxuriant and rich with fragrance mother liquor of DMSO 99.8MIN., final concentration is 10 ㎎ L -1
Wherein, liquid minimal medium prescription is: NaNO 30.5 g, (NH 4) 2SO 41.0g, Na 2HPO 42.5g, KH 2PO 41.0g, trace element solution 1ml, calcium magnesium solution 1ml is settled to 1000ml with zero(ppm) water; Wherein, the trace element solution prescription is: Al 2(SO 4) 318H 2O 108 mg, CoSO 47H 2O 56 mg, CuSO 45H 2O 56 mg, FeSO 47H 2O 3.0g, H 3BO 3611mg, KBr 28 mg, KI 56 mg, LiCl 28mg, MnCl 24H 2O 389mg, Na 2MoO 42H 2O 28mg, Na 2WO 42H 2O 28mg, NiCl 26H 2O 58mg, SnCl 22H 2O 28mg, ZnSO 4H 2O 34mg is settled to 1000ml with zero(ppm) water.Calcium magnesium solution prescription is: CaCl 230g, MgCl 220g is settled to 1000ml with zero(ppm) water.
Beef extract-peptone solid culture based formulas is: Carnis Bovis seu Bubali cream 5g, and peptone 10g, NaCl 5g, agar strip 20g is settled to 1000ml with zero(ppm) water, pH 7.2-7.4.
The sorbyl alcohol solid medium is: add 1ml VITAMINs mother liquor in every liter of sorbyl alcohol minimal medium.
The sorbyl alcohol minimal medium is to add 9g sorbyl alcohol, the agar strip of the weight percent 2% of 0.5g yeast powder and substratum gross weight in every liter of liquid minimal medium; Said VITAMINs mother liquor prescription is: para-amino benzoic acid 200mg, vitamin H 200mg, folic acid 200mg; Nicotinic acid 200mg, VA 100mg, Y factor 100mg; Vitamin G 100mg, VitB1 100mg, cobalamin 1mg; Be settled to 1000ml with sterilized water, with 0.4 μ m membrane filtration degerming.
3) with aseptic toothpick with step 2) in cultured bacterial strain dibbling on the flat board of the beef-protein medium that luxuriant and rich with fragrance film is arranged, place 28-32 oIn C constant temperature culture 1-3 week, periphery of bacterial colonies the degraded circle occurs and shows that then substrate is degraded, and it can be the degradation bacteria strains of sole carbon source with the phenanthrene that this bacterium is.
The preparation of luxuriant and rich with fragrance film:
1) presses mass volume ratio (gml -1) for 1:100 phenanthrene is dissolved in the acetone, process 10000 ㎎ L -1Mother liquor.
2) draw 10000 ㎎ L -1Luxuriant and rich with fragrance acetone soln 2ml in a plate; In stink cupboard, make acetone volatilization clean, form a uniform skim in the plate bottom, tile the luxuriant and rich with fragrance plate of solid to postvaccinal dull and stereotyped back-off to the bottom then on; And interface is sealed with sealing film; Integral body is put in the sand-bath and heats, and above flat board, puts ice bag, makes polycyclic aromatic hydrocarbons distillation back meet dull and stereotyped cooling and attached on the solid medium.
4) carry out multiple sieve according to degradation rate: choose 3) the bigger part bacterial strain of middle degraded circle, be inoculated in the beef extract-peptone liquid nutrient medium, be cultured to OD 600=0.6-0.8 cleans with phosphate buffered saline buffer, inserts by 10% inoculum size to contain luxuriant and rich with fragrance 100 ㎎ L -1-400 ㎎ L -1The liquid minimal medium in, place 180rmin on the 28-32 ℃ of constant temperature shaking table -1Cultivated 10 days.Extract residue PAHs component in the substratum, adopt the HPLC method to measure degradation rate.
 
Application examples 1
Gathering the topsoil of comforting the irrigated area in Shen and try soil sample as supplying, is the medelling compound with Fourth Ring PAHs pyrene, the degradation bacteria of screening pyrene from this soil.
1) gather Shen and comfort top layer, irrigated area 0-20 ㎝ soil as sample, 4 ℃ of refrigerators are preserved.Take by weighing and supply examination pedotheque 5g, insert 45ml and contain in the liquid minimal medium of Fourth Ring PAHs pyrene, wherein Fourth Ring PAHs pyrene concentration is 100 ㎎ L -1, add an amount of granulated glass sphere, place 180rmin on the 28-32 ℃ of constant temperature shaking table -1Enrichment culture 1 month after the enrichment culture, pipettes nutrient solution for the first time and inserts in the liquid minimal medium of 45ml Fourth Ring PAHs pyrene by 10% volume ratio; Carry out the enrichment culture second time; Multiple 4 times of gross weight, revolution connects once, and the concentration of Fourth Ring PAHs pyrene improves 100 ㎎ L -1, the concentration of promptly last Fourth Ring PAHs pyrene is increased to 400 ㎎ L -1
2) above-mentioned enrichment culture liquid is carried out 10 times of serial gradient dilutions to 10 with sterilized water under aseptic condition -7, get 0 respectively -5, 10 -6With 10 -7The bacterium liquid 100 μ l of three concentration are coated on the sorbyl alcohol solid medium, place 28-32 ℃ of constant temperature culture 1-3 week, and picking has each some strain of single bacterium colony that the degraded circle is arranged of different shape, and dibbling is in containing Fourth Ring PAHs pyrene 10 ㎎ L respectively -1The beef extract-peptone solid medium on, and bacterium colony carried out serial number.Place 28-32 ℃ of constant temperature culture 1-2 week.
The preparation of the liquid minimal medium of Fourth Ring PAHs pyrene: getting concentration is 1000 ㎎ L -1With the Fourth Ring PAHs pyrene mother liquor of acetone preparation,, measure 5-20mlPAHs acetone mother liquor with 0.22 μ m filter membrane Sterile Filtration; Add in the sterilized 250ml triangular flask; Treat that the acetone volatilization finishes, add the liquid minimal medium of 50ml sterilization, the final concentration that makes Fourth Ring PAHs pyrene is 100-400 ㎎ L -1
In addition, contain the preparation of the beef extract-peptone solid medium of Fourth Ring PAHs pyrene: in the beef extract-peptone solid medium, add 2000 ㎎ L -1DMSO 99.8MIN. pyrene mother liquor, final concentration is 10 ㎎ L -1
3) with aseptic toothpick with step 2) in cultured bacterial strain dibbling on the flat board of the beef-protein medium that Fourth Ring PAHs pyrene film is arranged, place 28-32 oIn C constant temperature culture 1-3 week, periphery of bacterial colonies the degraded circle occurs and shows that then substrate is degraded, and it can be the degradation bacteria strains (referring to Fig. 1) of sole carbon source with Fourth Ring PAHs pyrene that this bacterium is.
The preparation of Fourth Ring PAHs pyrene film:
1) presses mass volume ratio (gml -1) for 1:100 Fourth Ring PAHs pyrene is dissolved in the acetone, process 10000 ㎎ L -1Mother liquor.
2) draw 10000 ㎎ L -1Fourth Ring PAHs pyrene acetone soln 2ml in a plate; In stink cupboard, make acetone volatilization clean, form a uniform skim in the plate bottom, tile the plate of solid polycyclic aromatic hydrocarbons to postvaccinal dull and stereotyped back-off to the bottom then on; And interface is sealed with sealing film; Integral body is put in the sand-bath and heats, and above flat board, puts ice bag, makes polycyclic aromatic hydrocarbons distillation back meet dull and stereotyped cooling and attached on the solid medium.
4) carry out multiple sieve according to degradation rate: choose 3) the bigger part bacterial strain of middle degraded circle, be inoculated in the NR liquid nutrient medium, be cultured to OD 600=0.6-0.8 cleans with phosphate buffered saline buffer, inserts by 10% inoculum size to contain Fourth Ring PAHs pyrene 100-400 ㎎ L -1The liquid minimal medium in, place 180rmin on the 28-32 ℃ of constant temperature shaking table -1Cultivated 10 days.Extract residue Fourth Ring PAHs pyrene component in the substratum, adopt the HPLC method to measure degradation rate.Choose the maximum bacterial strain of wherein 1 strain degraded circle through liquid shaking bottle measuring pyrene degradation rate, concentration of substrate is 100 ㎎ L -1, incubation time is 7 days, degradation rate is 94.4%.
Obtaining strain more than 30 through this method screening is the degradation bacteria strains of sole carbon source with Fourth Ring PAHs pyrene, extracts the DNA of bacterial strain, and its 16S rDNA sequence increases.Increase used preceding primer 2 7F (5 '-AGAGTTTGATCMTGGCTCAG-3 ') and back primer 1492R (5 '-TA CGGHTACCTTGTTACGAC TT-3 ') derives from 8-27 and 1492-1513 gene fragments of intestinal bacteria 16S rDNA respectively.The amplified reaction program is following: 96 ℃ of preparatory sex change 10 minutes, and 95 ℃ of sex change 1 minute, 55 ℃ of annealing 1 minute, 72 ℃ were extended 1.5 minutes, 35 circulations, last 72 ℃ were extended 10 minutes.Reaction product detects with the agarose gel electrophoresis of 1 ﹪, send the order-checking of the big genome company of China through cutting glue purification.The sequence that records is landed GeneBank and is carried out the blast analysis, mycobacterium is arranged, Gordon Salmonella, pseudomonas, rhodococcus etc.
Application examples 2
Gather the topsoil of comforting the irrigated area in Shen and try soil sample as supplying, the degradation bacteria of screening pyrene adopts improved one-tenth embrane method in the bacterium that from this soil, obtains.With Fourth Ring PAHs pyrene is substrate, puts each bacterial strain respectively and receives on the sorbyl alcohol solid medium, makes the pyrene film, and the bacterial strain that the degraded circle occurs can be confirmed as the degradation bacteria (see figure 2) of pyrene.
Application examples 3
Gather the topsoil of comforting the irrigated area in Shen and try soil sample as supplying, screen fluorenes in the bacterium that from this soil, obtains, the degradation bacteria of phenanthrene and fluoranthene polycyclic aromatic hydrocarbons adopts improved one-tenth embrane method.Be respectively substrate with these three kinds of polycyclic aromatic hydrocarbonss, put each bacterial strain respectively and receive on the sorbyl alcohol solid medium, make film forming, the bacterial strain that the degraded circle occurs can be confirmed as the degradation bacteria (see figure 3).

Claims (10)

1. one kind is the screening method of the degradation bacteria of substrate with the polycyclic aromatic hydrocarbons, it is characterized in that:
1) enrichment culture adopts the method progressively improve polycyclic aromatic hydrocarbons concentration to being that the degradation bacteria of substrate is carried out enrichment culture with the polycyclic aromatic hydrocarbons;
2) sub-sieve is cultivated: get enrichment culture liquid dilution 10 in the step 1) 1-10 7After; Coat in the sorbyl alcohol solid medium; Place 28-32 ℃ of constant incubator to cultivate 1-3 week, single bacterium colony that picking has degraded circle dibbling respectively places 28-32 ℃ of constant incubator to cultivate 1-2 week in the beef-protein medium that contains polycyclic aromatic hydrocarbons;
3) the bacterial strain dibbling of growth is on the inorganic salt solid medium that has the polycyclic aromatic hydrocarbons film one-tenth embrane method rapid screening degradation bacteria: with step 2); Place 28-32 ℃ of constant incubator to cultivate 1-3 week, it can be the degradation bacteria strains of sole carbon source with the polycyclic aromatic hydrocarbons that the bacterial strain that can grow is.
2. described by claim 1 is the screening method of the degradation bacteria of substrate with the polycyclic aromatic hydrocarbons; It is characterized in that: said step 1) enrichment culture: bacterium appearance is inoculated in the cultivation of transferring in the liquid minimal medium that contains polycyclic aromatic hydrocarbons, the culture condition of at every turn transferring is for placing 180rmin on the 28-32 ℃ of constant temperature shaking table -1Cultivated 30 days, corotation connects 4-5 time, and switching is measured the each nutrient solution of per-cent meter 10% by volume and is inoculated in and contains in the 50ml liquid minimal medium that polycyclic aromatic hydrocarbons increases gradually, and is for use; Wherein contain in the liquid minimal medium of polycyclic aromatic hydrocarbons polycyclic aromatic hydrocarbons add-on 100 ㎎ L first -1, then with each increase by 100 ㎎ L -1Speed increase gradually.
By claim 1 or 2 described be the screening method of the degradation bacteria of substrate with the polycyclic aromatic hydrocarbons, it is characterized in that: said polycyclic aromatic hydrocarbons is at first to be dissolved in acetone, presses mass volume ratio (gml -1) for 1:1000 polycyclic aromatic hydrocarbons is dissolved in the acetone, process 1000 ㎎ L -1Mother liquor.
4. described by claim 1 is the screening method of the degradation bacteria of substrate with the polycyclic aromatic hydrocarbons, it is characterized in that: the polycyclic aromatic hydrocarbons in the said beef-protein medium that contains polycyclic aromatic hydrocarbons is dissolved in DMSO 99.8MIN., presses mass volume ratio (gml -1) for 1:500 polycyclic aromatic hydrocarbons is dissolved in the DMSO 99.8MIN., process 2000 ㎎ L -1Mother liquor.
5. described by claim 1 is the screening method of the degradation bacteria of substrate with the polycyclic aromatic hydrocarbons, it is characterized in that: said sorbyl alcohol solid medium is: add 1ml VITAMINs mother liquor in every liter of sorbyl alcohol minimal medium.
6. described by claim 5 is the screening method of the degradation bacteria of substrate with the polycyclic aromatic hydrocarbons; It is characterized in that: said sorbyl alcohol minimal medium is to add the 9g sorbyl alcohol in every liter of liquid minimal medium, 0.5g yeast powder and press 2% agar strip of substratum gross weight; Said VITAMINs mother liquor prescription is: para-amino benzoic acid 200mg, vitamin H 200mg, folic acid 200mg; Nicotinic acid 200mg, VA 100mg, Y factor 100mg; Vitamin G 100mg, VitB1 100mg, cobalamin 1mg; Be settled to 1000ml with sterilized water, with 0.4 μ m membrane filtration degerming.
By claim 2 or 6 described be the screening method of the degradation bacteria of substrate with the polycyclic aromatic hydrocarbons, it is characterized in that: said liquid minimal medium is: NaNO 30.5 g, (NH 4) 2SO 41.0g, Na 2HPO 42.5g, KH 2PO 41.0g, trace element solution 1ml, calcium magnesium solution 1ml is settled to 1000ml with zero(ppm) water; Wherein, the trace element solution prescription is: Al 2(SO 4) 318H 2O 108 mg, CoSO 47H 2O 56 mg, CuSO 45H 2O 56 mg, FeSO 47H 2O 3.0g, H 3BO 3611mg, KBr 28 mg, KI 56 mg, LiCl 28mg, MnCl 24H 2O 389mg, Na 2MoO 42H 2O 28mg, Na 2WO 42H 2O 28mg, NiCl 26H 2O 58mg, SnCl 22H 2O 28mg, ZnSO 4H 2O 34mg is settled to 1000ml with zero(ppm) water; Calcium magnesium solution prescription is: CaCl 230g, MgCl 220g is settled to 1000ml with zero(ppm) water.
8. said by claim 1 is the screening method of the degradation bacteria of substrate with the polycyclic aromatic hydrocarbons, it is characterized in that: said polycyclic aromatic hydrocarbons film: make acetone volatilization for the polycyclic aromatic hydrocarbons acetone soln with configuration places the ventilation, the polycyclic aromatic hydrocarbons film.
9. said by claim 8 is the screening method of the degradation bacteria of substrate with the polycyclic aromatic hydrocarbons, it is characterized in that: said polycyclic aromatic hydrocarbons acetone soln: press mass volume ratio (gml -1) for 1:100 polycyclic aromatic hydrocarbons is dissolved in the acetone, process 10000 ㎎ L -1Mother liquor.
By claim 8 or 9 said be the screening method of the degradation bacteria of substrate with the polycyclic aromatic hydrocarbons; It is characterized in that: the said polycyclic aromatic hydrocarbons acetone soln of getting places the ventilation to make the acetone volatilization clean; Integral body to be filmed is put into more than the fusing point that is heated to corresponding polycyclic aromatic hydrocarbons in the sand-bath; Above flat board, put ice bag, make polycyclic aromatic hydrocarbons distillation back meet dull and stereotyped cooling and attached on the beef extract-peptone solid medium.
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WO2016122333A1 (en) * 2015-01-28 2016-08-04 Statoil Petroleum As An automated method for selecting microbial strains which can degrade or emulsify oil
NO20171255A1 (en) * 2015-01-28 2017-07-27 Statoil Petroleum As An automated method for selecting microbial strains which can degrade or emulsify oil
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CN108284125A (en) * 2018-03-30 2018-07-17 昆明理工大学 A kind of method of continuous eluent solvent renovation of organic pollution soil
CN110643536A (en) * 2019-10-10 2020-01-03 中国科学院南京土壤研究所 Method for separating soil functional microorganisms by using soil matrix-membrane system
CN113403232A (en) * 2021-07-13 2021-09-17 重庆邮电大学 Method for screening phenanthrene polycyclic aromatic hydrocarbon degrading bacteria
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