CN101245366B - Improved solid double-layer flat plate method for quick high-flux sifting of polycyclic aromatic hydrocarbon degradation bacterium - Google Patents
Improved solid double-layer flat plate method for quick high-flux sifting of polycyclic aromatic hydrocarbon degradation bacterium Download PDFInfo
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Abstract
The invention relates to an improved solid double-layer flat plate method for the rapid high -throughput screening of PAHs degrading bacteria, which pertains to the field of the bioremediation technology of contaminated soil and is applicable to the excavation of bioremediation microbial resources of the PAHs contaminated soil. The adopting solid double-layer flat plate method is that a lower layer culture medium is an ordinary inorganic slat agar culture medium which is added with a variety of vitamins and trace elements, an upper layer culture medium is an inorganic salt low-melting point agarose culture medium which takes the PAHs as a sole carbon source; the low-melting point agarose, which is selected to replace the ordinary agar to produce the upper layer culture medium, can prevent the degradation or volatilization due to the high temperature when the PAHs is added on the one hand and allow the degradation dark circle to be more clear to be identified on the other hand. The method develops the solid double-layer flat plate method against the shortcomings of blindness, random nature, heavy workload, time-consuming and effort-consuming, etc. of the traditional screening of the degrading bacterial strain, thus providing a rapid and convenient means for the high-throughput screening of PAHs degrading bacteria in a laboratory and the excavation of the new bioremediation microbial resources.
Description
Technical field
The present invention relates to a kind of improvement solid double-layer flat band method of fast high-flux screening polycyclic aromatic hydrocarbon-degrading, adopt improved solid double-layer flat band method quick, high flux screening polycyclic aromatic hydrocarbon-degrading bacteria from pedotheque, belong to contaminated soil bioremediation technology field, be applicable to from environmental sample, excavate polycyclic aromatic hydrocarbon pollution biological restoration Microbial resources.
Background technology
Polycyclic aromatic hydrocarbons (Polycyclic Aromatic Hydrocarbon, PAHs) is large class environmental pollutant that receive much attention, and is widely distributed, and all can detect the PAHs component in air, soil, surface water and groundwater.Because its bioavailable degree is poor, the transformation period is longer, and has potential teratogenesis, carcinogenic and mutagenic effect, therefore human health has been produced grave danger.16 kinds of polycyclic aromatic hydrocarbon compounds such as phenanthrene, pyrene, fluoranthene, benzopyrene are listed in the priority pollutants Black List by EPA (EPA).Industrial sewage discharging and oil spilling leakage accident in Oil extraction and the smelting process are the major causes that causes P in soil AHs to pollute, the China Petroleum development rapidly, but also bring serious problem of environmental pollution thereupon, have nearly ten million mu arable land to be subject in various degree petroleum hydrocarbon contaminated.Therefore, its ecological functions are repaired, recovered to the PAHs contaminated soil to ensure that agricultural product security is a very urgent task.
PAHs contaminating microorganisms degradation technique is a kind of novel biology in situ recovery technique.It utilizes species diversity principle and the characteristics such as microbe species is various, metabolic type is very abundant, by the efficient PAHs degradation bacteria strains of screening and tieback original position contaminated soil from indigenous microorganism group, reaches and removes the purpose that P in soil AHs pollutes.Adopt biological restoration degraded PAHs pollutent simple to operate, can overcome physics, chemical treatment and repair that difficulty is large, cost is high, also has the defective of secondary pollution, have a extensive future.Wherein, efficient PAHs degraded screening is the key problem in technology that carries out effective biological restoration.
The technological line of " enrichment culture → picking bacterial strain → liquid shaking bottle is verified degradation rate at random " is generally taked in the screening of conventional P AHs degradation function bacterial strain, this method is often with certain blindness and randomness, workload is large, waste time and energy, and adopts high performance liquid chromatography (HPLC) cost of determination higher.Developing a kind of quick, convenient, high-throughout screening method, to separate PAHs degradation function Microbial resources from environmental sample be a kind of active demand for the environmental microorganism scholar, and similarly research method also can be offered reference for the exploitation of other functional microorganism resources.
Before this, also there is the scholar to propose to adopt " individual layer flat band method " preliminary screening PAHs degradation bacteria." the dull and stereotyped partition method of individual layer " for be coated with the acetone or alcohol solution of one deck PAHs at the solid inorganic substratum, after organic solvent fully volatilized, inoculation pedotheque suspension made it to be evenly distributed on the PAHs top layer; 1-3 is after week, and what can form a degraded circle on the next door of bacterium colony then is the PAHs degradation bacteria.But in our practice, find, adopt the coating method preparation infeasible take PAHs as sole carbon source individual layer solid plate, because the organic solvent volatile ratio is very fast, do not wait coating evenly just to separate out the PAHs crystal, therefore can not prepare uniform PAHs solid plate; It is slightly good than the coating method effect that the employing spray method prepares the individual layer solid plate, and the PAHs crystal distributes more even, but the degraded circle edge that forms is irregular, is not easy identification, and spray process needs carry out very difficult control aseptic condition in stink cupboard.
Summary of the invention
The purpose of this invention is to provide a kind of from pedotheque the solid " double-layer plate partition method " of fast high-flux screening polycyclic aromatic hydrocarbons efficient degrading bacteria.For the blindness of traditional random screening method and the shortcoming that wastes time and energy, a kind of fast in the improved basis exploitation of individual layer flat band method, screening method separates PAHs degradation function microorganism from environmental sample easily.Overcome many deficiencies of individual layer flat band method, adopted the double-layer plate method can make the PAHs crystal be uniformly distributed in the upper strata substratum, the bacterial plaque edge clear of formation is easy to identification, and can operates in super clean bench, controls easily aseptic condition.Thereby, satisfy the requirement laboratory fast high-flux preliminary screening polycyclic aromatic hydrocarbons efficient degradation function stem.
Technical scheme of the present invention is as follows:
The invention provides a kind of solid double-layer flat band method of fast high-flux screening polycyclic aromatic hydrocarbon-degrading, principal character is: lower floor's substratum is that common inorganic salt nutrient agar adds multivitamin and trace element, thus nutritional needs that can the various microorganisms of As soon as possible Promising Policy; The upper strata substratum is the inorganic salt low melting-point agarose substratum take polycyclic aromatic hydrocarbons (PAHs) as sole carbon source, select low melting-point agarose to replace plain agar to make the upper strata substratum, the degraded or the volatilization that cause owing to high temperature in the time of can preventing from adding PAHs on the one hand; Can make on the other hand that the dark circle of degraded is more clear easily to be distinguished.
Use above-mentioned " solid double-layer flat band method " main technological route of fast high-flux screening PAHs efficient degrading bacteria from pedotheque to be: the enrichment culture of collecting soil sample → polycyclic aromatic hydrocarbons tolerance flora and strains separation → application double-layer plate method preliminary screening polycyclic aromatic hydrocarbon-degrading bacteria → HPLC method are measured primary dcreening operation bacterial strain PAHs degradation rate and are sieved again → confirm high efficient strain.Implementation step is in detail:
1) collecting soil sample and processing: gather top layer pedotheque (0-20cm), cross the 1mm sieve, 4 ℃ of Refrigerator stores;
2) enrichment culture of polycyclic aromatic hydrocarbons tolerance flora: adopt the method that progressively improves concentration of substrate that polycyclic aromatic hydrocarbons tolerance flora is carried out enrichment culture.Take by weighing for examination pedotheque 5g (dry weight), in the liquid minimal medium of access 45ml take certain PAHs component as sole carbon source, the starting point concentration of PAHs is 50mg L
-1, place 180rpm enrichment culture on 30 ℃ of constant-temperature tables.Behind the enrichment culture certain hour (7～10 days), pipette enrichment culture liquid for the first time by in the fresh liquid minimal medium take PAHs as sole carbon source of 10% volume ratio access 45ml, carry out the enrichment culture second time, repeat altogether 4 times, every switching 1 time, the concentration of PAHs improves 50mg L
-1, the concentration of PAHs progressively is increased to 200mg L
-1
3) the enrichment culture liquid the separation of polycyclic aromatic hydrocarbons tolerance flora: with 2) adopts stroke-physiological saline solution (weight concentration 0.85%NaCl solution) to carry out 10 times of serial gradient dilutions under aseptic condition, gets 0.1ml, extent of dilution is 10
-5～10
-7Enrichment culture liquid coating LB solid medium, place 30 ℃ of constant incubators, cultivate 48h.Picking has each some strain of single bacterium colony of different colonial morphologies, and dibbling and is carried out serial number to bacterium colony on the LB solid medium again.Place 30 ℃ of constant incubators, cultivate 48h.
4) the dull and stereotyped preparation of polycyclic aromatic hydrocarbons solid double-layer: first impouring 20ml lower floor substratum in aseptic plate, both the solid inorganic salt culture medium adds 0.1ml/L VITAMIN mother liquor.After lower floor's culture medium solidifying, add again 10ml upper strata substratum, both the solid inorganic salt low melting-point agarose substratum take PAHs as sole carbon source.Upper strata substratum compound method: measure weight concentration 1%PAHs sterile alcohol solution 100 μ l, add 10ml and be cooled in 35 ℃ the inorganic salt low melting-point agarose substratum, making the PAHs final concentration is 100mg L
-1
5) use double-layer plate method high flux screening polycyclic aromatic hydrocarbon-degrading bacteria: with sterilizing toothpick with 3) in cultured bacterium colony dibbling in 4) on the polycyclic aromatic hydrocarbons solid double-layer flat board for preparing, in 30 ℃ of constant incubators, cultivated for 2～4 weeks (incubation time according to different PAHs and difference).Observe under ultraviolet lamp, periphery of bacterial colonies the dark circle of degraded occurs and shows that then substrate is degraded, and this bacterial strain can be confirmed as polycyclic aromatic hydrocarbon-degrading bacteria.
6) carry out multiple sieve according to degradation rate: choose 5) the larger part bacterial strain of the dark circle of middle degraded, be inoculated in the LB liquid nutrient medium, be cultured to OD
600=0.6～0.8, in the liquid minimal medium of 10% inoculum size access take PAHs as sole carbon source, place 5-10 days (incubation time is decided according to different PAHs components) of 180rpm cultivation on 30 ℃ of constant-temperature tables.Extract residue PAHs component in the substratum, adopt the HPLC method to measure, and compare, calculate degradation rate.
Among the present invention, liquid minimal medium prescription is: K
2HPO
42g, KH
2PO
40.5g, NaCl 0.5g, NH
4Cl 0.5g, MgSO
40.2g, CaCl
210mg, trace element solution 1ml are settled to 1000ml with distilled water; Wherein, the trace element solution prescription is: FeSO
27H
2O 2g, MnSO
4H
2O 2g, Na
2MoSO
42H
2O 0.5g, H
3BO
40.2g, CuSO
45H
2O 0.4g, ZnSO
40.5g, NH
4VO
30.2g, CoCl
26H
2O 0.5g, NiCl
26H
2O 0.2g is settled to 1000ml with distilled water.
Among the present invention, liquid minimal medium compound method take PAHs as sole carbon source: the PAHs mother liquor of getting the acetone preparation, concentration is 5g/L, 0.22 μ m filter membrane degerming, measure 50～200 μ l PAHs acetone mother liquors, add in the sterilized 250ml triangular flask, treat that the acetone volatilization is complete, add the liquid minimal medium of 50ml sterilization, the final concentration that makes PAHs is 50～200mg L
-1
Among the present invention, solid inorganic salt nutrient agar prescription is: the liquid minimal medium adds the plain agar powder of 2% weight.
Among the present invention, solid inorganic salt low melting-point agarose culture medium prescription is: the liquid minimal medium adds the low melting-point agarose (fusing point is 30 ℃) of 1% weight.
Among the present invention, VITAMIN mother liquor prescription is: VitB1 10mg, riboflavin 5mg, vitamin B6 5mg, calcium pantothenate 20mg, Para-Aminobenzoic 5mg, nicotine 5mg, inositol 100mg are settled to 1000ml with sterilized water, with 0.4 μ m membrane filtration degerming.
Among the present invention, LB solid culture based formulas is: Tryptones 10g, yeast soak powder 5g, NaCl 10g, and agar powder 20g is settled to 1000ml with distilled water, and pH 7.2～7.4.
Among the present invention, polycyclic aromatic hydrocarbons is phenanthrene, pyrene or fluoranthene etc.
Beneficial effect of the present invention is as follows:
1, the invention provides a kind of improved " solid double-layer flat band method ", simple and efficient to handle, can from pedotheque, separate the bacterial strain with degraded PAHs function by high-throughput, greatly improved first screen capacity.The shortcomings such as blindness, randomness and the workload that the present invention is directed to traditional degradation bacteria strains screening is large, waste time and energy, develop a kind of improved solid double-layer flat band method, be high flux screening polycyclic aromatic hydrocarbon-degrading bacteria in the laboratory, excavate new biological restoration Microbial resources a kind of quickly and easily means are provided.
2, solid double-layer flat band method of the present invention, lower floor's substratum are that common inorganic salt nutrient agar adds multivitamin and trace element; The upper strata substratum is the inorganic salt low melting-point agarose substratum take polycyclic aromatic hydrocarbons (PAHs) as sole carbon source, select low melting-point agarose to replace plain agar to make the upper strata substratum, the degraded or the volatilization that cause owing to high temperature in the time of can preventing from adding PAHs on the one hand; Can make on the other hand that the dark circle of degraded is more clear easily to be distinguished.
3, compare with " individual layer flat band method ", adopt " double-layer plate method " can make the PAHs crystal be uniformly distributed in the upper strata substratum, the bacterial plaque edge clear of formation is easy to identification.The lower floor substratum adds multivitamin and trace element, can the various microorganisms of As soon as possible Promising Policy to the demand of VITAMIN and trace element, thereby accomplish not leak sieve as far as possible; When preparing the upper strata substratum, select low melting-point agarose to replace plain agar, PAHs can be down to when hanging down in the substratum temperature add, can avoid high temperature to add the fashionable volatilization that causes, thereby the health that reduces experiment operator endangers.
4, laboratory experiment shows, adopts " solid double-layer flat band method " provided by the invention, can rapid, high volume separate from oil-polluted soils that degraded is luxuriant and rich with fragrance, the bacterial strain of pyrene and fluoranthene, thereby further adopt the liquid shaking bottle method to sieve again.
Description of drawings
Fig. 1 is the dark circle that luxuriant and rich with fragrance degradation bacteria forms at double-layer plate.
Fig. 2 is the dark circle that the pyrene degradation bacteria forms at double-layer plate.
Fig. 3 is the dark circle that the fluoranthene degradation bacteria forms at double-layer plate.
Embodiment
Embodiment 1
Gather Shen and comfort top layer, rice field (0-20cm) soil that the irrigated area petroleum waste water was irrigated more than 50 years, cross the 1mm sieve, 4 ℃ of Refrigerator stores.Take by weighing for examination pedotheque 5g (dry weight), (starting point concentration of PAHs is 50mg L in the liquid minimal medium of access 45ml take PAHs as sole carbon source
-1), can select to add granulated glass sphere, place 180rpm enrichment culture on 30 ℃ of constant-temperature tables.Behind the enrichment culture certain hour (7～10 days), pipette enrichment culture liquid for the first time by in the fresh liquid minimal medium take PAHs as sole carbon source of 10% volume ratio access 45ml, carry out the enrichment culture second time, repeat altogether 4 times, every switching 1 time, the concentration of PAHs improves 50mg L
-1, the concentration of PAHs progressively is increased to 200mg L
-1Above-mentioned enrichment culture liquid stroke-physiological saline solution (weight concentration 0.85%NaCl solution) is carried out 10 times of serial gradient dilutions under aseptic condition, get 0.1ml, extent of dilution is 10
-5～10
-7Enrichment culture liquid coating LB solid medium, place 30 ℃ of constant incubators, cultivate 48h.Picking has each some strain of single bacterium colony of different colonial morphologies, and dibbling and is carried out serial number to bacterium colony on the LB solid medium again.Place 30 ℃ of constant incubators, cultivate 48h.
Get the PAHs mother liquor of acetone preparation, concentration is 5g/L, 0.22 μ m filter membrane degerming, measure 50～200 μ lPAHs acetone solns, add in the sterilized 250ml triangular flask, treat that the acetone volatilization is complete, add the liquid minimal medium of 50ml sterilization, the final concentration that makes PAHs is 50～200mg L
-1Wherein:
Liquid minimal medium prescription is: K
2HPO
42g, KH
2PO
40.5g, NaCl 0.5g, NH
4Cl 0.5g, MgSO
40.2g, CaCl
210mg, trace element solution 1ml are settled to 1000ml with distilled water.Wherein, the trace element solution prescription is: FeSO
27H
2O 2g, MnSO
4H
2O 2g, Na
2MoSO
42H
2O 0.5g, H
3BO
40.2g, CuSO
45H
2O 0.4g, ZnSO
40.5g, NH
4VO
30.2g, CoCl
26H
2O 0.5g, NiCl
26H
2O 0.2g is settled to 1000ml with distilled water.
LB solid culture based formulas is: Tryptones 10g, yeast soak powder 5g, NaCl 10g, and agar powder 20g is settled to 1000ml with distilled water, and pH 7.2～7.4.
Embodiment 2
With sterilizing toothpick with cultured bacterial strain dibbling among the embodiment 1 on polycyclic aromatic hydrocarbons solid double-layer flat board, place 30 ℃ of constant incubators, cultivated for 2～4 weeks (incubation time according to different PAHs and difference).Observe under ultraviolet lamp, periphery of bacterial colonies the dark circle of degraded occurs and shows that then substrate is degraded, and this bacterial strain can be confirmed as alternative PAHs degradation bacteria.The dull and stereotyped preparation method of polycyclic aromatic hydrocarbons solid double-layer: first impouring 20ml lower floor substratum in aseptic plate, both the solid inorganic salt culture medium adds 0.1ml/L VITAMIN mother liquor.After lower floor's culture medium solidifying, add again 10ml upper strata substratum, both the solid inorganic salt low melting-point agarose substratum take PAHs as sole carbon source.Upper strata substratum compound method: upper strata substratum compound method: measure weight concentration 1%PAHs sterile alcohol solution 100 μ l, add 10ml and be cooled in 35 ℃ the inorganic salt low melting-point agarose substratum, making the PAHs final concentration is 100mg L
-1Wherein:
Solid inorganic salt nutrient agar prescription is: liquid minimal medium (the same) adds the agar powder of 2% weight.
Solid inorganic salt low melting-point agarose culture medium prescription is: liquid minimal medium (the same) adds the low melting-point agarose of 1% weight.
VITAMIN mother liquor prescription is: VitB1 10mg, riboflavin 5mg, vitamin B6 5mg, calcium pantothenate 20mg, Para-Aminobenzoic 5mg, nicotine 5mg, inositol 100mg are settled to 1000ml with sterilized water, with 0.4 μ m membrane filtration degerming.
Embodiment 3
Choose the larger primary dcreening operation bacterial strain of the dark circle of degraded among the embodiment 2, be inoculated in the LB liquid nutrient medium, be cultured to OD
600=0.6～0.8, in the liquid minimal medium (the same) of 10% inoculum size access take PAHs as sole carbon source, place 5-10 days (incubation time is decided according to different PAHs components) of 180rpm cultivation on 30 ℃ of constant-temperature tables.Extract residue PAHs component in the substratum, adopt the HPLC method to measure, and compare, calculate degradation rate.
LB liquid culture based formulas is: Tryptones 10g, yeast soak powder 5g, and NaCl 10g is settled to 1000ml with distilled water, and pH 7.2～7.4.
Application examples 1
Gather Shen and comfort irrigated area top layer, rice field (0-20cm) soil in more than 50 years of petroleum waste water irrigation as for examination soil, adopt improved " solid double-layer flat band method ", take three ring PAHs phenanthrene as the medelling compound, the luxuriant and rich with fragrance degradation bacteria of screening from this pedotheque.The concentration of enrichment culture phenanthrene is from 50mg L
-1, be increased to gradually 200mg L
-1It is, every that to take turns the enrichment culture time be 7 days; The final concentration of double-layer plate upper strata substratum phenanthrene is 100mg L
-1, incubation time was 2 weeks behind the dibbling double-layer plate, had 7 strain bacterium and the dark circle of degraded (seeing Fig. 1) occurred at luxuriant and rich with fragrance double-layer plate; Choose the larger bacterial strain of the dark circle of wherein 3 strains degraded through the luxuriant and rich with fragrance degradation rate of liquid shaking bottle measuring, concentration of substrate is 200mg L
-1, incubation time is 5 days, degradation rate is respectively 87%, 95% and 92%.
Application examples 2
Gather Shen and comfort irrigated area top layer, rice field (0-20cm) soil in more than 50 years of petroleum waste water irrigation as for examination soil, adopt improved " solid double-layer flat band method ", take Fourth Ring PAHs pyrene as the medelling compound, screening pyrene degradation bacteria from this pedotheque.The concentration of enrichment culture pyrene is from 50mg L
-1, be increased to gradually 200mg L
-1It is, every that to take turns the enrichment culture time be 10 days; The final concentration of double-layer plate upper strata substratum pyrene is 100mg L
-1, incubation time was 4 weeks behind the dibbling double-layer plate, had 8 strain bacterium and the dark circle of degraded (seeing Fig. 2) occurred at the pyrene double-layer plate; Choose the larger bacterial strain of the dark circle of wherein 5 strains degraded through liquid shaking bottle measuring pyrene degradation rate, concentration of substrate is 100mg L
-1, incubation time is 10 days, degradation rate is respectively 54%, 52%, and 68%, 73% and 61%.
Application examples 3
Gather Shen and comfort irrigated area top layer, rice field (0-20cm) soil in more than 50 years of petroleum waste water irrigation as for examination soil, adopt improved " solid double-layer flat band method ", take Fourth Ring PAHs fluoranthene as the medelling compound, screening fluoranthene degradation bacteria from this pedotheque.The concentration of enrichment culture fluoranthene is from 50mg L
-1, be increased to gradually 200mgL
-1It is, every that to take turns the enrichment culture time be 10 days; The final concentration of double-layer plate upper strata substratum fluoranthene is 100mg L
-1, incubation time was 4 weeks behind the dibbling double-layer plate, only had 1 strain bacterium the dark circle of degraded (seeing Fig. 3) to occur at the fluoranthene double-layer plate; This bacterial strain is through liquid shaking bottle measuring fluoranthene degradation rate, and concentration of substrate is 100mg L
-1, incubation time is 7 days, degradation rate difference 88%.
Claims (7)
1. the improvement solid double-layer flat band method of a fast high-flux screening polycyclic aromatic hydrocarbon-degrading is characterized in that, lower floor's substratum adopts common inorganic salt nutrient agar to add multivitamin and trace element; The upper strata substratum is the inorganic salt solid medium take polycyclic aromatic hydrocarbons as sole carbon source, but selects low melting-point agarose to replace plain agar; Concrete steps are as follows:
1) enrichment culture of polycyclic aromatic hydrocarbons tolerance flora: the earth sample that fetches earth, the liquid minimal medium of inoculation take polycyclic aromatic hydrocarbons as sole carbon source adopts the method that progressively improves concentration of substrate that polycyclic aromatic hydrocarbons tolerance flora is carried out enrichment culture;
2) the enrichment culture liquid the separation of polycyclic aromatic hydrocarbons tolerance flora: with 1) adopts stroke-physiological saline solution to carry out 10 times of serial gradient dilutions under aseptic condition, gets 0.1ml, extent of dilution is 10
-5～10
-7Enrichment culture liquid coating LB solid medium, place 30 ℃ of constant incubators, cultivate 48h; Picking has each some strain of single bacterium colony of different colonial morphologies, and dibbling is in the LB solid medium again, and bacterium colony is carried out serial number, places 30 ℃ of constant incubators, cultivates 48h;
3) the dull and stereotyped preparation of polycyclic aromatic hydrocarbons solid double-layer: first impouring 20ml lower floor substratum in aseptic plate, namely the solid inorganic salt culture medium adds 0.1ml/L VITAMIN mother liquor;
After lower floor's culture medium solidifying, add again 10ml upper strata substratum, i.e. solid inorganic salt low melting-point agarose substratum take PAHs as sole carbon source; Upper strata substratum compound method: measure weight concentration 1% PAHs sterile alcohol solution 100 μ l, add 10ml and be cooled in 35 ℃ the inorganic salt low melting-point agarose substratum, making the PAHs final concentration is 100mgL
-1
4) use double-layer plate method high flux screening polycyclic aromatic hydrocarbon-degrading bacteria: with sterilizing toothpick with 2) in cultured bacterium colony dibbling in 3) on the polycyclic aromatic hydrocarbons solid double-layer flat board of preparation, in 30 ℃ of constant incubators, 2～4 weeks of cultivation;
Observe under ultraviolet lamp, periphery of bacterial colonies the dark circle of degraded occurs and shows that then substrate is degraded, and this bacterial strain can be confirmed as polycyclic aromatic hydrocarbon-degrading bacteria;
The method that progressively improves concentration of substrate is: take by weighing for examination pedotheque 5g, in the liquid minimal medium of access 45ml take certain PAHs component as sole carbon source, first round enrichment culture PAHs concentration is 50mg L
-1Place 18rpm enrichment culture on 30 ℃ of constant-temperature tables, the enrichment culture time decides according to different PAHs components, be 7～10 days, after the enrichment culture, pipette for the first time enrichment culture liquid by in 10% the liquid minimal medium of volume ratio access 45ml take PAHs as sole carbon source, carry out the enrichment culture second time, repeat altogether 4 times, every switching 1 time, the concentration of PAHs improves 50mg L
-1, namely the concentration of last PAHs is increased to 200mgL
-1
2. according to solid double-layer flat band method claimed in claim 1, it is characterized in that described step 1) in, pedotheque is chosen at 0-20cm place, distance ground.
3. according to solid double-layer flat band method claimed in claim 1, it is characterized in that, liquid minimal medium preparation method take PAHs as sole carbon source: the PAHs mother liquor of getting the configuration of 5g/L acetone, 0.22 μ m filter membrane degerming, measure 50～200 μ l PAHs acetone mother liquors, add in the sterilized 250ml triangular flask, treat that the acetone volatilization is complete, add the liquid minimal medium of 50ml sterilization, the final concentration that makes PAHs is 50～200mgL
-1
4. according to solid double-layer flat band method claimed in claim 1, it is characterized in that described step 2) in, LB solid culture based formulas is: Tryptones 10g, yeast soak powder 5g, NaCl10g, and agar powder 20g is settled to 1000mL with distilled water, pH7.2～7.4.
5. according to solid double-layer flat band method claimed in claim 1, it is characterized in that described step 3) in, the solid inorganic salt culture medium prescription is: the liquid minimal medium adds the agar powder of 2% weight; VITAMIN mother liquor prescription is: VitB1 10mg, riboflavin 5mg, vitamin B6 5mg, calcium pantothenate 20mg, Para-Aminobenzoic 5mg, nicotine 5mg, inositol 100mg are settled to 1000ml with sterilized water, with 0.4 μ m membrane filtration degerming.
6. according to solid double-layer flat band method claimed in claim 1, it is characterized in that described step 3) in, inorganic salt low melting-point agarose culture medium prescription: the liquid minimal medium adds the low melting-point agarose of 1% weight.
7. according to claim 3,5 or 6 described solid double-layer flat band methods, it is characterized in that liquid minimal medium prescription is: K
2HPO
42g, KH
2PO
40.5g, NaCl 0.5g, NH
4Cl 0.5g, MgSO
40.2g, CaCl
210mg, trace element solution 1ml are settled to 1000ml with distilled water; Wherein, the trace element solution prescription is: FeSO
27H
2O 2g, MnSO
4H
2O 2g, Na
2MoSO
42H
2O 0.5g, H
3BO
40.2g, CuSO
45H
2O 0.4g, ZnSO
40.5g, NH
4VO
30.2g, CoCl
26H
2O0.5g, NiCl
26H
2O0.2g is settled to 1000ml with distilled water.
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