CN100478438C - Method for quick filtering out bacteria having characterization of adsorption for degrading polycyclic aromatic hydrocarbon - Google Patents

Method for quick filtering out bacteria having characterization of adsorption for degrading polycyclic aromatic hydrocarbon Download PDF

Info

Publication number
CN100478438C
CN100478438C CNB2007100108035A CN200710010803A CN100478438C CN 100478438 C CN100478438 C CN 100478438C CN B2007100108035 A CNB2007100108035 A CN B2007100108035A CN 200710010803 A CN200710010803 A CN 200710010803A CN 100478438 C CN100478438 C CN 100478438C
Authority
CN
China
Prior art keywords
polycyclic aromatic
aromatic hydrocarbons
inorganic salt
substratum
aromatic hydrocarbon
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related
Application number
CNB2007100108035A
Other languages
Chinese (zh)
Other versions
CN101050421A (en
Inventor
张颖
王荣
任瑞霞
徐慧
李慧
史荣久
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institute of Applied Ecology of CAS
Original Assignee
Institute of Applied Ecology of CAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institute of Applied Ecology of CAS filed Critical Institute of Applied Ecology of CAS
Priority to CNB2007100108035A priority Critical patent/CN100478438C/en
Publication of CN101050421A publication Critical patent/CN101050421A/en
Application granted granted Critical
Publication of CN100478438C publication Critical patent/CN100478438C/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Abstract

This invention relates to a novel method for screening polycyclic aromatic hydrocarbon degrading strains by using adsorption carrier. Polycyclic aromatic hydrocarbons (4 or higher) have low water solubility, and can be adsorbed onto solid particles whose surface microenvironment may contain polycyclic aromatic hydrocarbon degrading strains. This invention provides a rapid and highly efficient method for screening polycyclic aromatic hydrocarbon degrading strains with adsorption characteristic. The method utilizes adsorption carrier to culture and separating polycyclic aromatic hydrocarbon degrading strains, and double-layered flat plat technology to primarily screen, and screens polycyclic aromatic hydrocarbon degrading strains according to the degradation efficiency. The combination of primary and final screening can largely increase the screening efficiency.

Description

A kind of rapid screening has the method for the polycyclic aromatic hydrocarbon-degrading bacteria of characterization of adsorption
Technical field
The present invention relates to the microorganism recovery technique field of contaminated soil, be specially a kind of novel method of utilizing the adsorptive support screening polycyclic aromatic hydrocarbon-degrading.
Background technology
Polycyclic aromatic hydrocarbons is a big class environmental organic pollutant, has carcinogenic, teratogenesis, mutagenic effect, wherein has 16 kinds to be classified as priority pollutant by Environmental Protection Agency.Eliminate the pollution of polycyclic aromatic hydrocarbons, for improving environment, improving people's living standard and have very important significance.Polycyclic aromatic hydrocarbons in the environment is mainly by microbiological deterioration, so the screening efficient degrading bacteria is a key of polycyclic aromatic hydrocarbon pollution bioremediation technology.At present, traditional method is to adopt liquid shaking bottle culture method screening degradation bacteria strains.The main drawback of this method is only to screen the microbe groups of growing fast in suspension liquid, and the degradation bacteria with adsorptive power is difficult to screen, and pollutent mostly is adsorbed on the solid particulate matter in the contaminate environment.
Summary of the invention
The purpose of this invention is to provide a kind of true and reliable, method of screening polycyclic aromatic hydrocarbon-degrading rapidly and efficiently.
Technical scheme of the present invention is:
A kind of rapid screening has the method for the polycyclic aromatic hydrocarbon-degrading bacteria of characterization of adsorption, in conjunction with polycyclic aromatic hydrocarbons water-soluble very low, very easily be adsorbed on characteristic on the particulate matter, utilize adsorptive support enrichment polycyclic aromatic hydrocarbon-degrading bacteria, adopt the double-layer plate method to carry out primary dcreening operation, and measure its degradation efficiency to polycyclic aromatic hydrocarbons with high-efficient liquid phase chromatogram technology, finishing screen is chosen efficient degrading bacteria.
1, the contaminated soil sample takes and pre-treatment
Take 0-20cm deep soil sample, cross the 1mm sieve, 4 ℃ of preservations.
2, utilize adsorptive support enrichment and separation degradation bacteria
Add adsorptive support in triangular flask, its add-on is advisable (10-20g) to cover in the triangular flask bottom, bind up with gauze, and in 121 ℃, moist heat sterilization 20 minutes.The polycyclic aromatic hydrocarbons acetone soln 50uL of 1mg/mL is sprayed on the carrier, leaves standstill 2h, acetone is volatilized naturally, add 40mL minimal medium and 10mL soil supension at last, 30 ℃ of lucifuge shaking tables are cultivated.Getting 3～4g carrier in per 10 days is transferred in the freshly prepared inorganic salt liquid substratum that contains carrier.After 30 days carrier is taken out, fall the thalline of soil particle and non-absorption with aseptic water washing, placing with the polycyclic aromatic hydrocarbons is on the inorganic salt solid medium flat board that contains nystatin of sole carbon source, cultivates 4～6 days for 30 ℃.Lawn around the carrier is scraped, and line separates single bacterium colony on above-mentioned flat board, and the inclined-plane is preserved.Wherein,
Adsorptive support can be for improving soil texture perlite commonly used, rice husk etc.
Being prepared as follows of soil supension: claim soil sample 0.5g, pour in the aseptic water bottle of 49.5mL of band granulated glass sphere (consumption of granulated glass sphere to be best at the bottom of being full of bottle) rapidly into, vibrated 5～10 minutes, soil sample is fully broken up, promptly become 10 -2Soil supension.
Inorganic salt liquid substratum composed as follows: K 2HPO 41g; KH 2PO 41g; NaCl 0.5g; NH 4Cl 1g; MgSO 47H 2O 0.2g; CaCl 215mg; FeSO 47H 2O 2mg; CuSO 45H 2O0.4mg; KI 1mg; MnSO 4H 2O 4mg; H 3BO 35mg; CoCl 26H 2O 1mg; Na 2MoO 42H 2O 2mg; NiCl 26H 2O 2mg is dissolved in the 1L distilled water; PH 7.0 ± 0.2;
Inorganic salt liquid substratum specifically composed as follows that contains carrier: add adsorptive support in triangular flask earlier, its add-on is advisable (10-20g) to cover in the triangular flask bottom, binds up with gauze 121 ℃ of moist heat sterilizations 20 minutes; After treating its cooling, the polycyclic aromatic hydrocarbons acetone soln 50uL of 1mg/mL is sprayed on the carrier, leaves standstill 2h, acetone is volatilized naturally, add 50mL inorganic salt liquid substratum then.
With the polycyclic aromatic hydrocarbons inorganic salt solid medium flat board that contains nystatin composed as follows of sole carbon source: K 2HPO 41g; KH 2PO 41g; NaCl 0.5g; NH 4Cl 1g; MgSO 47H 2O 0.2g; CaCl 215mg; FeSO 47H 2O 2mg; CuSO 45H 2O 0.4mg; KI 1mg; MnSO 4H 2O4mg; H 3BO 35mg; CoCl 26H 2O 1mg; Na 2MoO 42H 2O 2mg; NiCl 26H 2O 2mg is dissolved in the 1L distilled water, adds 20g agar; PH 7.0 ± 0.2.Be divided in the triangular flask of 4 500mL, the bottled 250mL of each triangle cultivates based on 121 ℃ of moist heat sterilizations 20 minutes.When falling flat board, add the nystatin of 12.5mg in each triangular flask, the 0.25mg polycyclic aromatic hydrocarbons also shakes up.
Used polycyclic aromatic hydrocarbons can be phenanthrene, pyrene, fluoranthene, benzopyrene etc.
3, the primary dcreening operation of degradation bacteria
Adopt the double-layer plate method to carry out the primary dcreening operation of degradation bacteria.Doubling plate lower floor is the inorganic salt solid medium, and the upper strata is polycyclic aromatic hydrocarbons sole carbon source inorganic salt solid mediums.Connect bacterium on doubling plate, cultivated 6-10 days for 30 ℃, observe under ultraviolet lamp, if polycyclic aromatic hydrocarbons is degraded, then this periphery of bacterial colonies substratum absorption under UV-light weakens, and showing as thalline generation on every side has the non-bright dark circle that exposes to the sun.Wherein,
Consisting of of inorganic salt solid medium: K 2HPO 41g; KH 2PO 41g; NaCl 0.5g; NH 4Cl 1g; MgSO 47H 2O 0.2g; CaCl 215mg; FeSO 47H 2O 2mg; CuSO 45H 2O0.4mg; KI 1mg; MnSO 4H 2O 4mg; H 3BO 35mg; CoCl 26H 2O 1mg; Na 2MoO 42H 2O 2mg; NiCl 26H 2O 2mg is dissolved in the 1L distilled water, adds 20g agar; PH 7.0 ± 0.2.
Polycyclic aromatic hydrocarbons sole carbon source inorganic salt solid medium composed as follows: K 2HPO 41g; KH 2PO 41g; NaCl 0.5g; NH 4Cl 1g; MgSO 47H 2O 0.2g; CaCl 215mg; FeSO 47H 2O 2mg; CuSO 45H 2O 0.4mg; KI 1mg; MnSO 4H 2O 4mg; H 3BO 35mg; CoCl 26H 2O 1mg; Na 2MoO 42H 2O 2mg; NiCl 26H 2O 2mg is dissolved in the 1L distilled water, adds 20g agar; PH 7.0 ± 0.2.Be divided in the triangular flask of 4 500mL, the bottled 250mL of each triangle cultivates based on 121 ℃ of moist heat sterilizations 20 minutes.When falling flat board, add the nystatin of 12.5mg in each triangular flask, the 0.25mg polycyclic aromatic hydrocarbons also shakes up.
4, the multiple sieve of degradation bacteria
It is to cultivate 10 days in the inorganic salt liquid substratum of sole carbon source that the primary dcreening operation degradation bacteria strains is inserted with the polycyclic aromatic hydrocarbons, filter and collect thalline, and with sterilized water washing thalline 2 times, be OD with freshly prepared inorganic salt liquid substratum adjusting cell concentration 660=0.3, to get the access of 1mL bacteria suspension and be equipped with in the culturing bottle of 9mL inorganic salt liquid substratum, culturing bottle adds 1mgmL in advance -1Polycyclic aromatic hydrocarbons acetone soln 10uL, as phenanthrene, pyrene, benzopyrene etc., and acetone vapored away, the ultimate density of polycyclic aromatic hydrocarbons is 1mgL in the substratum -130 ℃ of lucifuge shaking tables were cultivated 10 days, and remaining polycyclic aromatic hydrocarbons adopts high performance liquid chromatography (HPLC) method to measure in the substratum.Finally choose the high bacterial strain of degradation rate as efficient degrading bacteria.Wherein,
Consisting of of the liquid minimal medium of polycyclic aromatic hydrocarbons sole carbon source: K 2HPO 41g; KH 2PO 41g; NaCl 0.5g; NH 4Cl 1g; MgSO 47H 2O 0.2g; CaCl 215mg; FeSO 47H 2O 2mg; CuSO 45H 2O 0.4mg; KI 1mg; MnSO 4H 2O 4mg; H 3BO 35mg; CoCl 26H 2O 1mg; Na 2MoO 42H 2O 2mg; NiCl 26H 2O 2mg is dissolved in the 1L distilled water, adds 20g agar; PH 7.0 ± 0.2.Be divided in the triangular flask of 4 500mL, the bottled 250mL of each triangle cultivates based on 121 ℃ of moist heat sterilizations 20 minutes.When falling flat board, add the nystatin of 12.5mg in each triangular flask, the 0.25mg polycyclic aromatic hydrocarbons also shakes up.
Consisting of of inorganic salt liquid substratum: K 2HPO 41g; KH 2PO 41g; NaCl 0.5g; NH 4Cl 1g; MgSO 47H 2O 0.2g; CaCl 215mg; FeSO 47H 2O 2mg; CuSO 45H 2O0.4mg; KI 1mg; MnSO 4H 2O 4mg; H 3BO 35mg; CoCl 26H 2O 1mg; Na 2MoO 42H 2O 2mg; NiCl 26H 2O 2mg is dissolved in the 1L distilled water.
Basis of the present invention is the characteristic of polycyclic aromatic hydrocarbons: the macromole polycyclic aromatic hydrocarbons more than Fourth Ring or the Fourth Ring is water-soluble very low, very easily is adsorbed on the solid particulate matter, and may contains the degradation bacteria of utilizing polycyclic aromatic hydrocarbons in the microenvironment on these particulate matter surfaces.Influence that the major cause of degrading polycyclic aromatic hydrocarbons is that these compound water solubles are very low in the edatope, very easily be adsorbed on the soil particle, cause the bioavailability of these compounds very low.But since the bacterium of adsorbing on these compounds and the carrier be in together one with the very different microenvironment of liquid environment in, so than the bacterium utilization that is easier to adsorb on the suppressed by vector.Adsorptive support sieve bacterium method is a kind of screening method of high efficiency polycyclic aromatic hydrocarbon-degrading bacteria.The main points of the method for the invention are: in triangular flask, add adsorptive support and polycyclic aromatic hydrocarbons, and make its absorption, add the inorganic salt nutrient solution then, and the inoculation contaminated soil, shaking table is cultivated; Bacterial strain after the concentration and separation utilizes double-layer plate method primary dcreening operation and the actual degradation efficiency of its polycyclic aromatic hydrocarbons of mensuration to sieve again again and determines whether it is the bacterial strain that we need.
The invention has the beneficial effects as follows:
1, the present invention utilizes adsorptive support culture of isolated degradation bacteria, utilizes the double-layer plate technology to carry out primary dcreening operation, finally according to degradation efficiency screening degradation bacteria strains.Primary dcreening operation and multiple sieve combine, and have improved screening efficiency greatly.
2, the present invention combines the characterization of adsorption of polycyclic aromatic hydrocarbon-degrading bacteria and the adsorptivity of polycyclic aromatic hydrocarbons, and the actual utility value of screening degradation bacteria is improved.
3, the adsorptive support sieve bacterium method selected for use of the present invention for the preparation of the later microbial inoculum condition of providing convenience, is fit to drop in the practical application very much.
Embodiment
The present invention is research object with the oil-polluted soils, and the pyrene of Fourth Ring structure is as representative polycyclic aromatic hydrocarbons, and the concrete operations step is as follows:
1, the collection of pedotheque and processing
Take the 0-20cm soil layer of oil-polluted soils, 1mm sieve, 4 ℃ of preservations.
2, utilize adsorptive support enrichment and separation degradation bacteria
15g binds up with gauze with adsorptive support, place triangular flask sterilization (121 ℃ of moist heat sterilization 20min), then 1mg/mL pyrene acetone soln 50uL is sprayed on the carrier, leave standstill 2h, acetone is volatilized naturally, add 40mL inorganic salt liquid substratum and 10mL soil supension at last, 30 ℃ of lucifuge shaking tables are cultivated.Getting the 3-4g carrier in per 10 days is transferred in the freshly prepared inorganic salt liquid substratum that contains carrier.After 30 days carrier is taken out, with the thalline of aseptic water washing soil particle and non-absorption, placing with the pyrene is on the inorganic salt solid medium flat board that contains nystatin of sole carbon source, cultivates 4～6 days for 30 ℃.Lawn around the carrier is scraped, and line separates single bacterium colony on above-mentioned flat board, and the inclined-plane is preserved.Wherein,
Adsorptive support can be for improving soil texture perlite commonly used, rice husk etc.
Being prepared as follows of soil supension: claim soil sample 0.5g, pour in the aseptic water bottle of 49.5mL of band granulated glass sphere (consumption of granulated glass sphere to be best at the bottom of being full of bottle) rapidly into, vibrated 5～10 minutes, soil sample is fully broken up, promptly become 10 -2Soil supension.
Inorganic salt liquid substratum composed as follows: K 2HPO 41g; KH 2PO 41g; NaCl 0.5g; NH 4Cl 1g; MgSO 47H 2O 0.2g; CaCl 215mg; FeSO 47H 2O 2mg; CuSO 45H 2O0.4mg; KI 1mg; MnSO 4H 2O 4mg; H 3BO 35mg; CoCl 26H 2O 1mg; Na 2MoO 42H 2O 2mg; NiCl 26H 2O 2mg is dissolved in the 1L distilled water; PH 7.0 ± 0.2;
The composition that contains the inorganic salt liquid substratum of carrier: add adsorptive support in triangular flask earlier, its add-on is advisable (15g) to cover in the triangular flask bottom, binds up with gauze 121 ℃ of moist heat sterilization 20min; After treating its cooling, the pyrene acetone soln 50uL of 1mg/mL is sprayed on the carrier, leaves standstill 2h, acetone is volatilized naturally, add 50mL inorganic salt liquid substratum then.
With the pyrene the consisting of of the inorganic salt solid medium that contains nystatin of sole carbon source: K 2HPO 41g; KH 2PO 41g; NaCl 0.5g; NH 4Cl 1g; MgSO 47H 2O 0.2g; CaCl 215mg; FeSO 47H 2O 2mg; CuSO 45H 2O 0.4mg; KI 1mg; MnSO 4H 2O 4mg; H 3BO 35mg; CoCl 26H 2O 1mg; Na 2MoO 42H 2O 2mg; NiCl 26H 2O 2mg is dissolved in the 1L distilled water, adds 20g agar; PH 7.0 ± 0.2.Be divided in the triangular flask of 4 500mL, the bottled 250mL of each triangle cultivates based on 121 ℃ of moist heat sterilization 20min.When falling flat board, add the nystatin of 12.5mg in each triangular flask, the 0.25mg pyrene also shakes up.
3, the primary dcreening operation of degradation bacteria strains
Adopt conventional double-layer plate method to carry out the primary dcreening operation of degradation bacteria.Doubling plate lower floor is the inorganic salt solid medium, and the upper strata is pyrene sole carbon source inorganic salt solid mediums.Connect bacterium on doubling plate, cultivated 6-10 days for 30 ℃, observe under ultraviolet lamp, if pyrene is degraded, then this periphery of bacterial colonies substratum absorption under UV-light weakens, and showing as thalline generation on every side has the non-bright dark circle that exposes to the sun.Wherein,
The concrete composition of inorganic salt solid medium: K 2HPO 41g; KH 2PO 41g; NaCl 0.5g; NH 4Cl 1g; MgSO 47H 2O 0.2g; CaCl 215mg; FeSO 47H 2O 2mg; CuSO 45H 2O0.4mg; KI 1mg; MnSO 4H 2O 4mg; H 3BO 35mg; CoCl 26H 2O 1mg; Na 2MoO 42H 2O 2mg; NiCl 26H 2O 2mg is dissolved in the 1L distilled water, adds 20g agar; PH 7.0 ± 0.2.
Specifically consisting of of pyrene sole carbon source inorganic salt solid medium: K 2HPO 41g; KH 2PO 41g; NaCl 0.5g; NH 4Cl 1g; MgSO 47H 2O 0.2g; CaCl 215mg; FeSO 47H 2O 2mg; CuSO 45H 2O 0.4mg; KI 1mg; MnSO 4H 2O 4mg; H 3BO 35mg; CoCl 26H 2O1mg; Na 2MoO 42H 2O 2mg; NiCl 26H 2O 2mg is dissolved in the 1L distilled water, adds 20g agar; PH 7.0 ± 0.2.Be divided in the triangular flask of 4 500mL, the bottled 250mL of each triangle cultivates based on 121 ℃ of moist heat sterilization 20min.When falling flat board, add the nystatin of 12.5mg in each triangular flask, the 0.25mg pyrene also shakes up.
4, carry out multiple sieve according to degradation rate
The bacterial strain of the non-bright dark circle that exposes to the sun is arranged as entering multiple sieve foundation with producing around the thalline in the primary dcreening operation.It is to cultivate 10 days in the inorganic salt liquid substratum of sole carbon source that the primary dcreening operation degradation bacteria strains is inserted with the pyrene, filter and collect thalline, and with sterilized water washing thalline 2 times, be OD with freshly prepared inorganic salt liquid substratum adjusting cell concentration 660=0.3, to get the access of 1mL bacteria suspension and be equipped with in the culturing bottle of 9mL inorganic salt nutrient solution, culturing bottle adds 1mgmL in advance -1Pyrene acetone soln 10uL, and acetone vapored away, the ultimate density of pyrene is 1mgL in the substratum -130 ℃ of lucifuge shaking tables were cultivated 10 days, and remaining pyrene adopts the HPLC method to measure the degradation rate of bacterial strain to pyrene.Finally obtain the high bacterial strain of 4 strain degradation rates, degradation rate is not for being: 56%, 51%, 30%, 29%.Wherein,
Specifically consisting of of pyrene sole carbon source inorganic salt liquid substratum: K 2HPO 41g; KH 2PO 41g; NaCl 0.5g; NH 4Cl 1g; MgSO 47H 2O 0.2g; CaCl 215mg; FeSO 47H 2O 2mg; CuSO 45H 2O 0.4mg; KI 1mg; MnSO 4H 2O 4mg; H 3BO 35mg; CoCl 26H 2O1mg; Na 2MoO 42H 2O 2mg; NiCl 26H 2O 2mg is dissolved in the 1L distilled water, adds 20g agar; PH 7.0 ± 0.2.Be divided in the triangular flask of 4 500mL, the bottled 250mL of each triangle cultivates based on 121 ℃ of moist heat sterilization 20min.When falling flat board, add the nystatin of 12.5mg in each triangular flask, 0.25mg pyrene and mixing.
Specifically consisting of of inorganic salt liquid substratum: K 2HPO 41g; KH 2PO 41g; NaCl 0.5g; NH 4Cl 1g; MgSO 47H 2O 0.2g; CaCl 215mg; FeSO 47H 2O 2mg; CuSO 45H 2O0.4mg; KI 1mg; MnSO 4H 2O 4mg; H 3BO 35mg; CoCl 26H 2O 1mg; Na 2MoO 42H 2O 2mg; NiCl 26H 2O 2mg is dissolved in the 1L distilled water, and pH 7.0 ± 0.2.

Claims (4)

1, a kind of rapid screening has the method for the polycyclic aromatic hydrocarbon-degrading bacteria of characterization of adsorption, it is characterized in that: in conjunction with polycyclic aromatic hydrocarbons water-soluble very low, very easily be adsorbed on characteristic on the particulate matter, utilize the adsorptive support enrichment and separate polycyclic aromatic hydrocarbon-degrading bacteria, adopt the double-layer plate method to carry out primary dcreening operation, and measure its degradation efficiency to polycyclic aromatic hydrocarbons with liquid chromatography technology, finally from contaminated soil, screen degradation bacteria;
The enrichment of polycyclic aromatic hydrocarbon-degrading bacteria and separation comprise:
Add adsorptive support in triangular flask, carrier is to improve the soil texture perlite, rice husk commonly used, covers in the triangular flask bottom with carrier and is advisable, bind up with gauze, and in 121 ℃ of moist heat sterilization 20min; The polycyclic aromatic hydrocarbons acetone soln 50uL of 1mg/mL is sprayed on the carrier, leaves standstill 2h, acetone is volatilized naturally, add 40mL inorganic salt liquid substratum and 10mL soil supension at last, 30 ℃ of lucifuge shaking tables are cultivated; Get the 3g-4g carrier in per 10 days and be transferred to freshly prepared containing in the carrier inorganic salt liquid substratum; After 30 days carrier is taken out, with the thalline of aseptic water washing soil particle and non-absorption, placing with the polycyclic aromatic hydrocarbons is on the inorganic salt solid medium flat board that contains nystatin of sole carbon source, cultivates 4～6 days for 30 ℃; Lawn around the carrier is scraped, and line separates single bacterium colony on above-mentioned flat board, and the inclined-plane is preserved; Wherein,
Inorganic salt liquid substratum composed as follows: K 2HPO 41g; KH 2PO 41g; NaCl 0.5g; NH 4Cl 1g; MgSO 47H 2O 0.2g; CaCl 215mg; FeSO 47H 2O 2mg; CuSO 45H 2O0.4mg; KI 1mg; MnSO 4H 2O 4mg; H 3BO 35mg; CoCl 26H 2O 1mg; Na 2MoO 42H 2O 2mg; NiCl 26H 2O 2mg is dissolved in the 1L distilled water; PH7.0 ± 0.2;
Inorganic salt liquid substratum composed as follows that contains carrier: add adsorptive support in triangular flask earlier, add-on covers in the triangular flask bottom with carrier and is advisable, and binds up with gauze 121 ℃ of moist heat sterilization 20min; After treating its cooling, the polycyclic aromatic hydrocarbons acetone soln 50uL of 1mg/mL is sprayed on the carrier, leaves standstill 2h, acetone is volatilized naturally, add 50mL inorganic salt liquid substratum then;
With the polycyclic aromatic hydrocarbons inorganic salt solid medium that contains nystatin composed as follows of sole carbon source: K 2HPO 41g; KH 2PO 41g; NaCl 0.5g; NH 4Cl 1g; MgSO 47H 2O 0.2g; CaCl 215mg; FeSO 47H 2O 2mg; CuSO 45H 2O 0.4mg; KI 1mg; MnSO 4H 2O4mg; H 3BO 35mg; CoCl 26H 2O 1mg; Na 2MoO 42H 2O 2mg; NiCl 26H 2O 2mg is dissolved in the 1L distilled water, adds 20g agar, pH7.0 ± 0.2; Be divided in the triangular flask of 4 500mL the bottled 250mL substratum of each triangle, 121 ℃ of moist heat sterilizations 20 minutes; When falling flat board, add nystatin, 0.25mg polycyclic aromatic hydrocarbons and the mixing of 12.5mg in each triangular flask.
2, have the method for the polycyclic aromatic hydrocarbon-degrading bacteria of characterization of adsorption according to the described rapid screening of claim 1, it is characterized in that, adopt the double-layer plate method to carry out the primary dcreening operation of polycyclic aromatic hydrocarbon-degrading bacteria, comprising:
Doubling plate lower floor is the inorganic salt solid medium, and the upper strata is polycyclic aromatic hydrocarbons sole carbon source inorganic salt solid mediums; Connect bacterium on doubling plate, cultivated 6-10 days for 30 ℃, observe under ultraviolet lamp, if polycyclic aromatic hydrocarbons is degraded, then this periphery of bacterial colonies substratum absorption under UV-light weakens, and the non-bright dark circle that exposes to the sun is arranged around the thalline; Wherein,
Inorganic salt solid medium composed as follows: K 2HPO 41g; KH 2PO 41g; NaCl 0.5g; NH 4Cl 1g; MgSO 47H 2O 0.2g; CaCl 215mg; FeSO 47H 2O 2mg; CuSO 45H 2O0.4mg; KI 1mg; MnSO 4H 2O 4mg; H 3BO 35mg; CoCl 26H 2O 1mg; Na 2MoO 42H 2O 2mg; NiCl 26H 2O 2mg is dissolved in the 1L distilled water, adds 20g agar; PH7.0 ± 0.2;
Polycyclic aromatic hydrocarbons sole carbon source inorganic salt solid medium composed as follows: K 2HPO 41g; KH 2PO 41g; NaCl 0.5g; NH 4Cl 1g; MgSO 47H 2O 0.2g; CaCl 215mg; FeSO 47H 2O 2mg; CuSO 45H 2O 0.4mg; KI 1mg; MnSO 4H 2O 4mg; H 3BO 35mg; CoCl 26H 2O 1mg; Na 2MoO 42H 2O 2mg; NiCl 26H 2O 2mg is dissolved in the 1L distilled water, adds 20g agar, pH7.0 ± 0.2; Be divided in the triangular flask of 4 500mL the bottled 250mL substratum of each triangle, 121 ℃ of moist heat sterilizations 20 minutes; Fall before the flat board, add polycyclic aromatic hydrocarbons 0.25mg, and shake up.
3, have the method for the polycyclic aromatic hydrocarbon-degrading bacteria of characterization of adsorption according to the described rapid screening of claim 1, it is characterized in that,, comprising according to the foundation of the degradation rate of bacterial strain in the inorganic salt liquid substratum that with the polycyclic aromatic hydrocarbons is sole carbon source as multiple sieve:
It is to cultivate 10 days in the inorganic salt liquid substratum of sole carbon source that the primary dcreening operation degradation bacteria strains is inserted with the polycyclic aromatic hydrocarbons, filter and collect thalline, and with sterilized water washing thalline 2 times, be OD with freshly prepared inorganic salt liquid substratum adjusting cell concentration 660=0.3, to get the access of 1mL bacteria suspension and be equipped with in the culturing bottle of 9mL inorganic salt liquid substratum, culturing bottle adds 1mgmL in advance -1Polycyclic aromatic hydrocarbons acetone soln 10uL, and acetone vapored away, the ultimate density of polycyclic aromatic hydrocarbons is 1mgL in the substratum -1, 30 ℃ of lucifuge shaking tables were cultivated 10 days, and remaining polycyclic aromatic hydrocarbons adopts liquid-phase chromatography method HPLC to measure, and finally chooses the high bacterial strain of degradation rate as degradation bacteria; Wherein,
With the polycyclic aromatic hydrocarbons inorganic salt liquid substratum composed as follows of sole carbon source: K 2HPO 41g; KH 2PO 41g; NaCl 0.5g; NH 4Cl 1g; MgSO 47H 2O 0.2g; CaCl 215mg; FeSO 47H 2O 2mg; CuSO 45H 2O 0.4mg; KI 1mg; MnSO 4H 2O 4mg; H 3BO 35mg; CoCl 26H 2O 1mg; Na 2MoO 42H 2O 2mg; NiCl 26H 2O 2mg is dissolved in the 1L distilled water, adds 20g agar, pH7.0 ± 0.2; Be divided in the triangular flask of 4 500mL, the bottled 250mL substratum of each triangle was in 121 ℃ of moist heat sterilizations 20 minutes; Fall before the flat board, add polycyclic aromatic hydrocarbons 0.25mg, and shake up;
Inorganic salt liquid substratum composed as follows: K 2HPO 41g; KH 2PO 41g; NaCl 0.5g; NH 4Cl 1g; MgSO 47H 2O 0.2g; CaCl 215mg; FeSO 47H 2O 2mg; CuSO 45H 2O0.4mg; KI 1mg; MnSO 4H 2O 4mg; H 3BO 35mg; CoCl 26H 2O 1mg; Na 2MoO 42H 2O 2mg; NiCl 26H 2O 2mg is dissolved in the 1L distilled water; PH7.0 ± 0.2.
4, have the method for the polycyclic aromatic hydrocarbon-degrading bacteria of characterization of adsorption according to the described rapid screening of claim 1, it is characterized in that, the collection of pedotheque and processing comprise:
Take the pedotheque of the 0-20cm degree of depth, cross the 1mm sieve, 4 ℃ of preservations.
CNB2007100108035A 2007-03-30 2007-03-30 Method for quick filtering out bacteria having characterization of adsorption for degrading polycyclic aromatic hydrocarbon Expired - Fee Related CN100478438C (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CNB2007100108035A CN100478438C (en) 2007-03-30 2007-03-30 Method for quick filtering out bacteria having characterization of adsorption for degrading polycyclic aromatic hydrocarbon

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CNB2007100108035A CN100478438C (en) 2007-03-30 2007-03-30 Method for quick filtering out bacteria having characterization of adsorption for degrading polycyclic aromatic hydrocarbon

Publications (2)

Publication Number Publication Date
CN101050421A CN101050421A (en) 2007-10-10
CN100478438C true CN100478438C (en) 2009-04-15

Family

ID=38782021

Family Applications (1)

Application Number Title Priority Date Filing Date
CNB2007100108035A Expired - Fee Related CN100478438C (en) 2007-03-30 2007-03-30 Method for quick filtering out bacteria having characterization of adsorption for degrading polycyclic aromatic hydrocarbon

Country Status (1)

Country Link
CN (1) CN100478438C (en)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101245366B (en) * 2008-03-07 2013-03-20 中国科学院沈阳应用生态研究所 Improved solid double-layer flat plate method for quick high-flux sifting of polycyclic aromatic hydrocarbon degradation bacterium
CN101838629B (en) * 2009-03-17 2012-09-05 清华大学 Method for screening polycyclic aromatic hydrocarbon-degrading bacteria
CN102776135A (en) * 2011-05-12 2012-11-14 中国科学院沈阳应用生态研究所 Mixed inoculant for degrading polycyclic aromatic hydrocarbon pollutants
CN103232943B (en) * 2013-04-23 2014-12-03 中国海洋石油总公司 Instant premixed dry powder for separating polycyclic aromatic hydrocarbons and degrading fungi
CN105462838A (en) * 2015-12-29 2016-04-06 中林山水（北京）生态科技股份有限公司 Method for screening root-system symbiotic bacteria for promoting heavy metal absorption of plants
CN105543153A (en) * 2016-03-17 2016-05-04 中创宏远（北京）环保科技有限公司 Method for screening rhizosphere bacteria promoting heavy metal phytoremediation
CN109652568A (en) * 2018-12-25 2019-04-19 华南理工大学 A method of fast enriching Nitrosocosmicus belongs to ammoxidation archaeal from environment

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1223673C (en) * 2003-01-23 2005-10-19 中国科学院微生物研究所 Erythro micrococcus Em and usage for generating biologic emulsifier as well as degrading polycyclic aromatic hydrocarbon

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1223673C (en) * 2003-01-23 2005-10-19 中国科学院微生物研究所 Erythro micrococcus Em and usage for generating biologic emulsifier as well as degrading polycyclic aromatic hydrocarbon

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
《工业微生物育种学》. 施七琴、吴构刚主编，正文第100-124页，科学出版社. 1991 *
Isolation of Soil Bacteria Adapted To Degrade Humic.D.J.Vacca等.APPLIED AND ENVIRONMENTAL MICROBIOLOGY,Vol.71 No.7. 2005 *

Also Published As

Publication number Publication date
CN101050421A (en) 2007-10-10

Similar Documents

Publication Publication Date Title
CN100478438C (en) Method for quick filtering out bacteria having characterization of adsorption for degrading polycyclic aromatic hydrocarbon
CN107376940B (en) Composite catalyst material for quickly and efficiently repairing organic contaminated soil and preparation method thereof
Dean et al. Phenanthrene degradation in soils co‐inoculated with phenanthrene‐degrading and biosurfactant‐producing bacteria
CN102899271B (en) Mycobacterium 16F for efficiently degrading polycyclic aromatic hydrocarbons and benzene organic matters and application thereof
CN104946620A (en) Immobilized microbial inoculant for restoring petroleum-hydrocarbon-polluted alkaline-saline soil and preparation method thereof
GB2431926A (en) Charred biological material carrying microbes
CN111748483B (en) Bacillus for degrading petroleum hydrocarbon and application thereof
Ke et al. Negative effects of humic acid addition on phytoremediation of pyrene-contaminated sediments by mangrove seedlings
CN111014281B (en) Multi-biochar combined microorganism composite material for petroleum-polluted soil remediation and preparation method and application thereof
CN110508613B (en) Remediation method for polycyclic aromatic hydrocarbon contaminated soil
CN111088187B (en) Charcoal-based immobilized polycyclic aromatic hydrocarbon efficient degradation microbial inoculum and preparation method and application thereof
CN113293157A (en) Ferroferric oxide modified charcoal immobilized polycyclic aromatic hydrocarbon degrading microbial inoculum and preparation and application thereof
CN103451105B (en) A kind of filamentous fungus Penicllium chrysogenum J-5 of high absorption cadmium and preparation method and application
Li et al. Effects of tall fescue biochar on the adsorption and desorption of atrazine in different types of soil
Cahan et al. Innovative utilization of coal bottom ash for bioremediation of toxic organic pollutants
CN109095728B (en) Rainwater runoff purification device and purification method
CN114540337A (en) Preparation method and application of microorganism immobilized composite particles
CN101935631A (en) Ralstoniasp. and application thereof in bioremediation of petroleum-contaminated saline-alkali soil
CN101654656B (en) Separation and purification method and application of aerobic degradation pure culture of polycyclic aromatic hydrocarbon
CN101597590A (en) The double-layer plate method of fast simple screening polycyclic aromatic hydrocarbon-degrading and screening thereof are dull and stereotyped
CN116948882A (en) Achromobacter madepuralensis and application thereof
CN104974955B (en) Greedy copper bacterium YNS-85 and its application in soil remediation
CN108467841B (en) Vermiculite immobilized low-temperature degradation-resistant polycyclic aromatic hydrocarbon mixed bacteria particle and preparation method and application thereof
Wang et al. Removal of pyrene in simulated wetland by joint application of Kyllinga brevifolia Rottb. and immobilized microbes
CN108570325A (en) Microorganism formulation and preparation method thereof for soil remediation with high salt

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C14 Grant of patent or utility model
GR01 Patent grant
C17 Cessation of patent right
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20090415

Termination date: 20100330