CN101838629B - Method for screening polycyclic aromatic hydrocarbon-degrading bacteria - Google Patents
Method for screening polycyclic aromatic hydrocarbon-degrading bacteria Download PDFInfo
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- CN101838629B CN101838629B CN200910080013A CN200910080013A CN101838629B CN 101838629 B CN101838629 B CN 101838629B CN 200910080013 A CN200910080013 A CN 200910080013A CN 200910080013 A CN200910080013 A CN 200910080013A CN 101838629 B CN101838629 B CN 101838629B
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Abstract
The invention discloses a method for screening polycyclic aromatic hydrocarbon-degrading bacteria. The polycyclic aromatic hydrocarbon-degrading bacteria are obtained by inoculating a sample to be tested to a culture medium taking polycyclic aromatic hydrocarbons as an exclusive carbon source and culturing the sample. The method has the advantages of overcoming the defects of complex operating steps and the requirement of special instruments in the conventional screening methods (such as the spraying method and the sublimed method), saving manpower, and difficultly causing environmental pollution, along with convenient operation and easy control (such as better control of the amount of sediment and uniformity of the polycyclic aromatic hydrocarbons on the culture base plate); and in addition, the method also has the advantages of avoiding the problem that organic solvents such as ethers or acetone, and the like kill some types of polycyclic aromatic hydrocarbon-degrading bacteria in the conventional screening method, more accurately estimating the amount of polycyclic aromatic hydrocarbon-degrading bacteria in the sample to be tested, and realizing a big-flux genetic operation, such as transposable mutation and the like, which cannot be implemented by the conventional screening method.
Description
Technical field
The present invention relates to a kind of method of screening polycyclic aromatic hydrocarbon-degrading.
Background technology
Polycyclic aromatic hydrocarbons (Polycyclic Aromatic Hydrocarbons; PAHs) be meant the compound that two or more phenyl ring rearrange with chain, horn shape or string shape; Mainly come from organic incomplete combustion such as timber, fossil oil, coal-tar products or food; Polycyclic aromatic hydrocarbons distributes in environment extensively, in atmosphere, water body, soil, settling and food, all detects.The polycyclic aromatic hydrocarbons great majority are toxic substance, and many have three to cause effect, promptly carcinogenic, aberration inducing and mutagenesis, and polycyclic aromatic hydrocarbons carcinogens of having found up to now and verivate thereof surpass 400 kinds, are no lack of the such strong carcinogen of benzo (a) pyrene.Characteristics such as the half volatile that polycyclic aromatic hydrocarbons has, lipotropy, high biological accumulation property and difficult degradation, make its can be in environment existence for a long time, long-distance migration and be enriched in organism, cause long-term and serious harm for human health and ecosystem safety.Therefore; 1998; The U.S., Canada and Europe in totally 32 countries " about the cross-border air pollutant pact of long distance " of concluding with polycyclic aromatic hydrocarbons clearly be decided to be must control persistence organic pollutant (Persistent Organic Pollutants, POPs).
Polycyclic aromatic hydrocarbons in the environment is except that the photochemistry decomposition can take place in few part, and most of main biodegradation pathway that relies on slowly disappears, so microbiological deterioration is the polycyclic aromatic hydrocarbons contaminated comparatively cost-effective method of control.And polycyclic aromatic hydrocarbon-degrading bacteria obtain the basis of studying just and utilizing microbiological deterioration polycyclic aromatic hydrocarbons ability, the counting of polycyclic aromatic hydrocarbon-degrading bacteria also can be assisted and understood the contaminated degree of environment in the environmental sample simultaneously.
Dull and stereotyped cultivation can be screened degradation bacteria comparatively easily, but but relatively more difficult to the screening of polycyclic aromatic hydrocarbon-degrading bacteria.Because the solvability extreme difference of polycyclic aromatic hydrocarbons in water is volatile again, can not be evenly dispersed in the solid medium, if in substratum, add other organic cosolvent, then can't obtain with the polycyclic aromatic hydrocarbons is the degradation bacteria of the sole carbon source and the energy; And these organic solvents are unfavorable for the growth of degradation bacteria to the also toxic effect of bacterium.At present, the plate culture medium that generally adopts spray method, subliming method or entrapping method to make comes screening polycyclic aromatic hydrocarbon-degrading.But there is following shortcoming in these traditional screening methods: (1) operating process is more loaded down with trivial details, be not easy control (temperature and the homogenizing of polycyclic aromatic hydrocarbons in substratum when making flat board), and the instrument more (like atomizer, heat-resisting petridish and well heater etc.) of needs; (2) environment around the operating process of spray method and subliming method possibly polluted, and easily by other microbial contamination; (3) ether in the spray method or acetone and other organic solvent can be killed some polycyclic aromatic hydrocarbon-degrading bacterias to the toxic effect of part mikrobe; (4) molecular biology that is unfavorable for big flux such as swivel base sudden change is operated.
Summary of the invention
The method that the purpose of this invention is to provide a kind of screening polycyclic aromatic hydrocarbon-degrading.
The method of screening polycyclic aromatic hydrocarbon-degrading provided by the present invention is that testing sample is seeded in the polycyclic aromatic hydrocarbons is in the substratum of sole carbon source, cultivates and obtains polycyclic aromatic hydrocarbon-degrading bacteria.
In the aforesaid method, said substratum is a solid medium, and said solid culture primary surface is covered with cellulose membrane, is added with polycyclic aromatic hydrocarbons on the said cellulose membrane.
During concrete the application, can earlier said polycyclic aromatic hydrocarbons be dissolved in the volatile organic solvent, be added on the cellulose membrane again.Said organic solvent specifically can be ether.
In the aforesaid method, said polycyclic aromatic hydrocarbons is various polycyclic aromatic hydrocarbonss such as phenanthrene, anthracene, pyrene or fluoranthene.
Said cellulose membrane is acetic acid-nitric acid cellulose mixture film, specifically can be the acetic acid-nitric acid cellulose mixture film in 0.45 μ m aperture.
In the aforesaid method, said is luxuriant and rich with fragrance with the polycyclic aromatic hydrocarbons, and said is the consisting of of substratum of sole carbon source with the phenanthrene: NaCl 79.45g/L, MgCl
26H
2O 6.9g/L, MgSO
47H
2O 10.45g/L, CaCl
27H
2O 0.75g/L, KCl 2.1g/L, NaHCO
30.1g/L, NaBr 0.25g/L, (NH
4)
2SO
44.0g/L, trace element solution 1mL/L, luxuriant and rich with fragrance 1mg/L, all the other are water;
Consisting of of said trace element solution: Ca (NO
3)
24H
2O 800mg/L, FeSO
4.7H
2O 400mg/L, CuSO
45H
2O 80mg/L, ZnSO
47H
2O 40mg/L, MnSO
44H
2O 40mg/L, NaMO
42H
2O8mg/L, H
3BO
36mg/L, K
2HPO
4500mg/L, all the other are water;
The pH value of said substratum is 7.5.
The inventive method has overcome complex operating steps in the conventional screening methods (like spray method and subliming method) and to the needs of particular instrument; Have advantages such as artificial, easy and simple to handle, the easy control (as controlling the deposition and the homogenizing of polycyclic aromatic hydrocarbons on the culture medium flat plate preferably) of saving, difficult pollution surrounding environment; Also avoided ethers in the conventional screening methods or acetone and other organic solvent killing action to some polycyclic aromatic hydrocarbon-degrading bacterias; Thereby can estimate the quantity of polycyclic aromatic hydrocarbon-degrading bacteria in the testing sample more accurately, and can carry out the genetic manipulation of the big flux that employing conventional screening methods such as swivel base sudden change can't carry out.
Description of drawings
The luxuriant and rich with fragrance degradation bacteria of Fig. 1 for adopting the inventive method from the soil sample of oil field, to filter out.
Embodiment
Experimental technique described in the following embodiment like no specified otherwise, is ordinary method; Said reagent and biomaterial like no specified otherwise, all can obtain from commercial sources.
The acquisition of embodiment 1, polycyclic aromatic hydrocarbon-degrading bacteria
The composition of related substratum and reagent is following in the experiment:
Sea salt synthetic medium (SSDM) consists of: NaCl 79.45g/L, MgCl
26H
2O 6.9g/L, MgSO
47H
2O 10.45g/L, CaCl
27H
2O 0.75g/L, KCl 2.1g/L, NaHCO
30.1g/L, NaBr0.25g/L, trace element solution 1mL/L; More than the corresponding salinity of content of each composition be 10% substratum (salinity of substratum equal in the substratum each component of inorganic salts quality sum divided by the volume of substratum), the substratum of other salinity can be prepared in this ratio; Other contains (NH
4)
2SO
44.0g/L all the other are water; Adopt 5M KOH liquid concentrator to regulate the pH to 7.5 of substratum.Wherein, consisting of of trace element solution: Ca (NO
3)
24H
2O800mg/L, FeSO
4.7H
2O 400mg/L, CuSO
45H
2O 80mg/L, ZnSO
47H
2O 40mg/L, MnSO
44H
2O 40mg/L, NaMO
42H
2O 8mg/L, H
3BO
36mg/L, K
2HPO
4500mg/L, all the other are water.
Luxuriant and rich with fragrance diethyl ether solution: phenanthrene is dissolved in the ether, is formulated as the diethyl ether solution that concentration is the phenanthrene of 1mg/mL.
Concrete screening process is following:
, salinity adds agarose in being 5% sea salt synthetic medium; Making its final concentration in substratum is 1.5%～1.8%; To cultivate based on the 20min that sterilizes under the 0.06MPa condition; Culture medium after sterilization evenly is poured in the glass culture dish while hot, treat culture medium solidifying after, be positioned over 37 ℃ the moisture of solid culture primary surface evaporated fully.
Under aseptic condition; With tweezers clamp through the sterilization acetic acid-nitric acid cellulose mixture film (millipore filtration, aperture 0.45 μ m, diameter 90mm, available from Beijing through biochemical reagents biotech company of HTC of section) the edge; Be placed on the above-mentioned solid culture primary surface for preparing gently; Push and push away flat then with the coated glass rod gently, cellulose membrane and substratum are fitted tightly, process the cellulose membrane flat board.
The diethyl ether solution of getting 1mL concentration and be the phenanthrene of 1mg/mL joins on the above-mentioned cellulose membrane flat board, is coated with spreading rod rapidly, makes luxuriant and rich with fragrancely on the cellulose membrane flat board, to be evenly distributed; Reserve 5% space when petridish is covered, make diethyl ether solution can evaporate effusion, approximately build the petridish lid fully behind the 5mim, this moment, phenanthrene evenly was fixed on the cellulose membrane.To not contain luxuriant and rich with fragrance ether simultaneously is coated on the above-mentioned cellulose membrane flat board as blank.
It is in 5% the sea salt synthetic medium that the pedotheque that picks up from Dongying city, Shandong Province Xian He town Shengli Oil Field is joined the 100mL salinity, 200r/min vibration 30min under the normal temperature condition; Draw 1mL suspension-s in centrifuge tube, the centrifugal 10min of 5000r/min; Abandoning supernatant, adding 1mL again, to be added with salinity be that 5% above-mentioned sea salt synthetic medium washs.Above washing process repeats 3 times; It is last that to be resuspended in an amount of salinity be in 5% synthetic cultivation of sea salt and carry out a series of gradient dilutions with bacterial sediment after centrifugal; With the bacterium liquid after the dilution be coated on above-mentioned contain on the luxuriant and rich with fragrance cellulose membrane flat board with the blank flat board on, the flat board after the coating placed to leave standstill under 30 ℃ of conditions cultivated 15 days.Three repetitions are established in experiment.
The result shows to have no colony growth on the blank flat board, explains not add on the luxuriant and rich with fragrance flat board as carbon source, and the mikrobe in the soil sample can't grow; Accordingly, there is bacterial strain to grow being added with on the luxuriant and rich with fragrance flat board, explains that this bacterial strain phenanthro-of can degrading is the sole carbon source growth with the phenanthrene; The average quantity of the Sino-Philippines degradation bacteria of unit mass soil sample is 5.6 * 10
6CFU/g, wherein CFU is that bacterium colony forms number (colony-forming units), soil quality calculates with dry weight.Bacterial strain is as shown in Figure 1 at the upgrowth situation that contains on the luxuriant and rich with fragrance cellulose membrane flat board, picking single bacterium colony wherein, and called after P-4 and A-1 carry out enrichment culture respectively to P-4 and A-1 respectively.
The evaluation of embodiment 2, bacterial strain
One, the morphological specificity of bacterial strain
The colony diameter of the bacterial strain P-4 that the foregoing description 1 screens is 2～4mm, rounded, smooth, protuberance, cream-colored or little yellow, glossy, neat in edge; Under electron microscope, observe, somatic cells is knee shape or spirrillum, atrichia, does not have mobility, and size is (1.03～1.65) μ m * (0.33～0.56) μ m.
The colony diameter of the strains A that the foregoing description 1 screens-1 is 2～4mm, rounded, smooth, the protuberance, yellow, glossy, neat in edge; Under electron microscope, observe, somatic cells is shaft-like, atrichia does not have mobility, and size is (1.15～1.40) μ m * (0.50～0.63) μ m.
Two, the growth characteristics of bacterial strain
Bacterial strain P-4 and A-1 that the foregoing description 1 screens all belong to Gram-negative, aerobic bacteria.The righttest growth pH value is 7.2, and the pH scope of can growing is 5.5～9.0; Optimum growth temperature is 30 ℃, and the TR that can grow is 10～42 ℃.The growth of bacterial strain P-4 be unable to do without inorganic salt, belongs to halophile, can in 0.1～17% salinity range, carry out growth and breeding, and suitable growth salt rangeability is 3～6%.Strains A-1 belongs to the moderate halophilic bacteria, can in 0.05～27.5% salinity range, carry out growth and breeding, and suitable growth salt rangeability is 4～10%.
Three, the biochemical character of bacterial strain
Bacterial strain P-4 and the A-1 that the foregoing description 1 is screened carries out utilization of carbon source respectively, carbohydrate produces acid, starch, gelatin and tween 80 hydrolysising experiment; Measure its oxydase, katalase, DNA enzyme and urease activity; Measure its nitrate reduction ability, the result is following:
The bacterial strain P-4 that the foregoing description 1 screens can utilize other carbohydrate except that utilizing D-pectinose and alpha-lactose to produce the acid, produces acid like sucrose, SANMALT-S, D-fructose, D-N.F,USP MANNITOL, D-seminose, D-wood sugar, D-semi-lactosi and D-glucose; This bacterial strain can hydrolysis tween 80 and gelatin, but can not the hydrolysis Zulkovsky starch; The oxydase of this bacterial strain, katalase and urease are positive, and the DNA enzyme is negative; It is nitrogen that this bacterial strain can make nitrate reduction; This bacterial strain can utilize pentitol, ribitol, L-arabinose, D-arabitol, D-cellobiose, D-fructose, gentiobiose, alpha-D-glucose, D-seminose, D-sorbyl alcohol, turanose, D-galactosonic acid lactones, glucono-, D-glucose amino acid, α-Tong Wuersuan, D, and L-lactic acid, quininic acid/quinic acid, D-glucaric acid, D-L-Ala, L-L-Ala, L-alanyl-glycine, L-L-glutamic acid, glycyl-L-aspartic acid, glycyl-L-glutamic acid, L-proline(Pro) and L-Pyrrolidonecarboxylic acid are as the sole carbon source and the energy.
The strains A that the foregoing description 1 screens-1 can not utilize sucrose, SANMALT-S, alpha-lactose and D-wood sugar to produce acid, and D-capable of using can draw uncle's sugar, D-fructose, D-N.F,USP MANNITOL, D-seminose, D-semi-lactosi and D-glucose to produce acid; This bacterial strain can not the hydrolysis tween 80, gelatin and Zulkovsky starch; This bacterial strain has the activity of oxydase, katalase, urase and nitrate reduction, but does not have DNA enzyme, nitrous acid reductive activity; This bacterial strain can utilize D-pectinose, cellobiose, lactose, tyrosine, altheine as the sole carbon source and the energy.
Four, antibiotics resistance
Bacterial strain P-4 and A-1 that the foregoing description 1 is screened carry out the antibiotics resistance experiment respectively.
The result shows that the bacterial strain P-4 that the foregoing description 1 screens is responsive to the penbritin in the microbiotic, Cephalothin sodium, clarithromycin, Clindamicin, Oxacyclotetradecane,erythromycin deriv, Synnematin B and vancomyein; And ceftriaxone, CEFOTAXIME SODIUM STERILE, CIPROFLOXACIN USP 24, qingfengmeisu qiong, Streptomycin sulphate and tsiklomitsin are had resistance.
Sulfamethoxazole in the strains A that the foregoing description 1 screens-1 pair microbiotic, Clindamicin and vancomyein are responsive; And ceftriaxone, CEFOTAXIME SODIUM STERILE, Cephalothin sodium, CIPROFLOXACIN USP 24, qingfengmeisu qiong, Streptomycin sulphate, penbritin, clarithromycin, Oxacyclotetradecane,erythromycin deriv, Synnematin B and tsiklomitsin are had resistance.
Five, cytochemistry composition analysis
The detection that bacterial strain P-4 that the foregoing description 1 is screened and A-1 breathe quinone and lipid acid respectively.
The result shows, bacterial strain P-4 and A-1 all mainly with Q9 (ubiquinone 9) as the main quinone of breathing; Mainly 18 carbon ω 7c monounsaturated fatty acids (35.00%), 16 carbon sfass (25.01%), 16 carbon ω 7c monounsaturated fatty acids (17.89%), 14 carbon sfass (6.16%) and ring type 17 carbon sfass (5.22%) in the cell walls lipid acid of bacterial strain P-4; Also contain a spot of 3-hydroxy-16 carbon sfas (3.43%), 18 carbon sfass (1.66%) and 3-hydroxyl 14 carbon sfass; And mainly comprise 16 carbon sfass (27.1%), 16 carbon ω 7c monounsaturated fatty acids (24.0%), 18 carbon ω 7c monounsaturated fatty acids (21.27%), ω 8c-ring type 19 carbon sfass (8.0%) and 3-hydroxyl 12 carbon sfass in the cell walls lipid acid of strains A-1, also contain a spot of 12 carbon sfass (3.84%) and ten carbon sfass (2.79%).
Six, the 16S rRNA gene sequencing and the Phylogenetic Analysis of bacterial strain
Bacterial strain P-4 and A-1 that the foregoing description 1 is screened carry out 16S rRNA gene sequencing and Phylogenetic Analysis respectively.
The result shows; The 16S rRNA gene order of the bacterial strain P-4 that the foregoing description 1 screens is shown in sequence in the sequence table 1; Revolve the homology that bacterium (Thalassospira xiamenensis), deep sea thalassospira (Thalassospira profundimaris) and Lu Sentanhai revolve the 16SrRNA gene order of bacterium (Thalassospira lucentensis) with the type strain Sha Menhai that has delivered and be respectively 99.2%, 98.9% and 98.0%; The 16S rRNA gene order of the strains A that the foregoing description 1 screens-1 is shown in GenBank Accession No.EF421176, with the type strain expectation salt Zymomonas mobilis DSM 9502 of salt zygosaccharomyces
T(Halomonas desiderata DSM 9502
T), the living salt Zymomonas mobilis of wall DSM 14789
T(Halomonas muralis DSM 14789
T), Pan Tailailiya salt Zymomonas mobilis DSM14789
T(Halomonas pantelleriensis DSM 9661
T) and Campania salt Zymomonas mobilis DSM 14789
T(Halomonas campaniensis DSM 15293
T) the homology of 16S rRNA gene order be respectively 96.0%, 95.6%, 95.5% and 95.3%, be lower than 95% with the similarity of the 16S rRNA gene order of other bacterial strain of salt zygosaccharomyces.
Seven, DNA-DNA hybridization analysis
The bacterial strain P-4 that the foregoing description 1 is screened carries out the DNA-DNA hybridization analysis with the type strain that Pseudomonas is revolved in the sea, and the strains A-1 that the foregoing description 1 is screened is carried out the DNA-DNA hybridization analysis with the type strain of salt zygosaccharomyces.
The result shows; The DNA of the bacterial strain P-4 that the foregoing description 1 screens and the type strain Sha Menhai that has delivered revolve bacterium (T.xiamenensis), deep sea thalassospira (T.profundimaris) and Lu Sentanhai to revolve the dna homology property of bacterium (T.lucentensis) all lower, are respectively 39%, 12% and 3%.And the G+C molar content of this bacterial strain is 61.6%, is respectively 52.6%, 47.0% and 54.7% and Sha Menhai revolves the G+C molar content that bacterium, deep sea thalassospira and Lu Sentanhai revolve bacterium.
Based on above mensuration result; The bacterial strain P-4 that the foregoing description 1 is screened is accredited as Xian Hehai and revolves bacterium (Thalassospira xianhensis) P-4; And be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on March 6th, 2009 and (be called for short CGMCC; The address is: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), preservation registration number is CGMCC № 2942.
The type strain Pan Tailailiya salt Zymomonas mobilis DSM 9661 of the strains A that the foregoing description 1 screens-1 and salt zygosaccharomyces that delivered
T(Halomonas pantelleriensis DSM 9661
T) and the living salt Zymomonas mobilis of wall DSM 14789
T(Halomonas muralis DSM 14789
T) dna homology property all lower, be respectively 11% and 18%.And the G+C molar content of this bacterial strain is 67.1%, and Pan Tailailiya salt Zymomonas mobilis DSM9661
TWith the living salt Zymomonas mobilis of wall DSM 14789
TThe G+C molar content be respectively 65.0% and 62.4%.
Based on above mensuration result; The strains A-1 that the foregoing description 1 is screened is accredited as celestial river salt Zymomonas mobilis (Halomonas xianhensis) A-1; And be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on March 6th, 2009 and (be called for short CGMCC; The address is: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), preservation registration number is CGMCC № 2941.
The mensuration of embodiment 3, degrading polycyclic aromatic hydrocarbons ability
1, Xian Hehai revolves the mensuration of the luxuriant and rich with fragrance ability of bacterium (Thalassospira xianhensis) P-4 CGMCC № 2942 degradeds
Phenanthrene is dissolved in the N filtration sterilization; In salinity is the sea salt synthetic medium of the foregoing description 1 of 5%, add yeast powder, making its final concentration in substratum is 0.1%, in substratum, adds the luxuriant and rich with fragrance solution of above-mentioned filtration sterilization simultaneously, and making luxuriant and rich with fragrance final concentration in substratum is 100mg/L.
Bacterium (Thalassospira xianhensis) P-4 CGMCC № 2942 being revolved in sea, celestial river insert in the above-mentioned substratum according to 5% inoculum size, is to cultivate 10d under the aerobic lucifuge condition of 200r/min at 30 ℃, rotating speed.Simultaneously, with Jia Fei not but the substratum that adds N as contrast, be used to check that sea, celestial river revolves bacterium (Thalassospira xianhensis) P-4 CGMCC № 2942 and whether can utilize N; With inoculated bacteria not as blank, be used to deduct luxuriant and rich with fragrance evaporable influence; Revolve contrasting of bacterium (Thalassospiraxianhensis) P-4 CGMCC № 2942 dead thalline to inoculate sea, celestial river, be used to deduct the influence of thalline absorption as dead bacterium.
In the culturing process, timing sampling utilizes HPLC (HPLC) to measure luxuriant and rich with fragrance degradation rate; Waters600E liquid chromatograph, chromatographic column are Supelcosil LC
18-DB, moving phase (first alcohol and water, volume ratio are 80: 20), flow velocity 1mL/min detects wavelength 254nm.
Three repetitions are established in experiment.The result shows that Xian Hehai revolves bacterium (Thalassospira xianhensis) P-4 CGMCC № 2942 average degradation rate to phenanthrene in 9 days can reach 10.8mg/ (Ld).
2, Xian Hehai revolves the performance of bacterium (Thalassospira xianhensis) P-4 CGMCC № 2942 degraded naphthalenes
Naphthalene is dissolved in the N filtration sterilization; In salinity is the sea salt synthetic medium of the foregoing description 1 of 5%, add yeast powder, making its final concentration in substratum is 0.1%, in substratum, adds the naphthalene solution of above-mentioned filtration sterilization simultaneously, and making the final concentration of naphthalene in substratum is 100mg/L.
Bacterium (Thalassospira xianhensis) P-4 CGMCC № 2942 is revolved in sea, celestial river to be inserted in the above-mentioned substratum according to 5% inoculum size; Be to cultivate 10d under aerobic lucifuge sealing (supply with for guaranteeing oxygen, add the 30mL substratum in the 300mL culturing bottle) condition of 200r/min at 30 ℃, rotating speed.Simultaneously, not add naphthalene but the substratum that adds N as contrast, be used to check that sea, celestial river revolves bacterium (Thalassospira xianhensis) P-4CGMCC № 2942 and whether can utilize N; With inoculated bacteria not as blank, be used to deduct the influence of naphthalene evaporable; Revolve contrasting of bacterium (Thalassospira xianhensis) P-4 CGMCC № 2942 dead thalline to inoculate sea, celestial river, be used to deduct the influence of thalline absorption as dead bacterium.
Take a sample after cultivating 10d, utilize HPLC (HPLC) to measure the degradation rate of naphthalene; Waters 600E liquid chromatograph, chromatographic column are Supelcosil LC
18-DB, moving phase (first alcohol and water, volume ratio are 80: 20), flow velocity 1mL/min detects wavelength 276nm.
Three repetitions are established in experiment.The result shows, behind the cultivation 10d, is added with and does not detect naphthalene containing in the viable bacteria sample of naphthalene, all detects tangible naphthalene and be added with the blank of naphthalene and contain in the dead bacterium contrast; Be added with containing viable bacteria contrast, being added with the blank of naphthalene and containing in the dead bacterium contrast and do not have colour-change all the time of N; And be added with the containing in the viable bacteria sample of naphthalene gradually by the colourless yellow that becomes, explain that sea, celestial river revolves bacterium (Thalassospira xianhensis) P-4 CGMCC № 2942 naphthalene of can degrading.
3, Xian Hehai revolves the performance of bacterium (Thalassospira xianhensis) P-4 CGMCC № 2942 degraded pyrenes
Pyrene is dissolved in the ether, and preparation becomes the solution that concentration is 1mg/mL.
After salinity is that to add the quality percentage composition in 5% the above-mentioned sea salt synthetic medium be 1.5～1.8% agarose; The 20min 0.06MPa sterilize under the condition; Evenly be poured into substratum in the glass culture dish while hot subsequently; After culture medium solidifying, be positioned in 37 ℃ of environment, the moisture of solid culture primary surface is evaporated fully.Be covered with sterilized acetic acid-nitric acid cellulose mixture film (millipore filtration subsequently on the surface of plate culture medium; Aperture 0.45 μ m, diameter 90mm; Available from Beijing through biochemical reagents biotech company of HTC of section); The concentration that on film, adds the above-mentioned preparation of 1mL be 1mg/mL pyrene diethyl ether solution and coating evenly, treat that ether volatilizees fully after, build dull and stereotyped for use.The ether that will not contain pyrene simultaneously is coated on the above-mentioned cellulose membrane flat board as blank.
Bacterium (Thalassospira xianhensis) P-4 CGMCC № 2942 is revolved in sea, celestial river be inoculated in the SSDMY liquid nutrient medium, the 140r/min shaking culture is 4 days under the normal temperature condition; Draw the 1mL bacteria suspension in centrifuge tube, the centrifugal 10min of 5000r/min; Abandoning supernatant is coated on bacterial sediment on the above-mentioned acetic acid-nitric acid cellulose mixture film flat board that contains pyrene, the flat board after the coating is placed to leave standstill under 30 ℃ of conditions cultivated 15 days.
The result shows to have no colony growth on the blank flat board, explains not add on the flat board of pyrene as carbon source, and Xian Hehai revolves bacterium (Thalassospira xianhensis) P-4 CGMCC № 2942 and can't grow; Accordingly; Revolve bacterium (Thalassospira xianhensis) P-4 CGMCC № 2942 and can grow being added with on the flat board of pyrene sea, celestial river, explain that sea, celestial river revolves can degrade pyrene and be the sole carbon source growth with the pyrene of bacterium (Thalassospira xianhensis) P-4 CGMCC № 2942.
4, the mensuration of the luxuriant and rich with fragrance ability of celestial river salt Zymomonas mobilis (Halomonas xianhensis) A-1 CGMCC № 2941 degradeds
Phenanthrene is dissolved in the N filtration sterilization; In salinity is the sea salt synthetic medium of the foregoing description 1 of 5%, add yeast powder, making its final concentration in substratum is 0.5%, in substratum, adds the luxuriant and rich with fragrance solution of above-mentioned filtration sterilization simultaneously, and making luxuriant and rich with fragrance final concentration in substratum is 100mg/L.
Celestial river salt Zymomonas mobilis (Halomonas xianhensis) A-1 CGMCC № 2941 is inserted in the above-mentioned substratum according to 5% inoculum size, is to cultivate 10d under the aerobic lucifuge condition of 200r/min at 30 ℃, rotating speed.Simultaneously, with Jia Fei not but the substratum that adds N as contrast, be used to check whether celestial river salt Zymomonas mobilis (Halomonas xianhensis) A-1 CGMCC № 2941 can utilize N; With inoculated bacteria not as blank, be used to deduct luxuriant and rich with fragrance evaporable influence; To inoculate contrasting of celestial river salt Zymomonas mobilis (Halomonasxianhensis) A-1 CGMCC № 2941 dead thalline, be used to deduct the influence of thalline absorption as dead bacterium.
In the culturing process, timing sampling utilizes HPLC (HPLC) to measure luxuriant and rich with fragrance degradation rate; Waters600E liquid chromatograph, chromatographic column are Supelcosil LC
18-DB, moving phase (first alcohol and water, volume ratio are 80: 20), flow velocity 1mL/min detects wavelength 254nm.
Three repetitions are established in experiment.The result shows, celestial river salt Zymomonas mobilis (Halomonas xianhensis) A-1 CGMCC № 2941 can be with luxuriant and rich with fragrance all degradeds of 100mg/L in 10 days, can reach 10mg/ (Ld) to the average degradation rate of phenanthrene.
5, the performance of celestial river salt Zymomonas mobilis (Halomonas xianhensis) A-1 CGMCC № 2941 degraded anthracenes is dissolved in anthracene in the ether, and preparation becomes the solution that concentration is 1mg/mL.
After salinity is that to add the quality percentage composition in 5% the above-mentioned sea salt synthetic medium be 1.5～1.8% agarose; The 20min 0.06MPa sterilize under the condition; Evenly be poured into substratum in the glass culture dish while hot subsequently; After culture medium solidifying, be positioned in 37 ℃ of environment, the moisture of solid culture primary surface is evaporated fully.Be covered with sterilized acetic acid-nitric acid cellulose mixture film (millipore filtration subsequently on the surface of plate culture medium; Aperture 0.45 μ m, diameter 90mm; Available from Beijing through biochemical reagents biotech company of HTC of section); The concentration that on film, adds the above-mentioned preparation of 1mL be 1mg/mL anthracene diethyl ether solution and coating evenly, treat that ether volatilizees fully after, build dull and stereotyped for use.The ether that will not contain anthracene simultaneously is coated on the above-mentioned cellulose membrane flat board as blank.
Celestial river salt Zymomonas mobilis (Halomonas xianhensis) A-1 CGMCC № 2941 is inoculated in the SSDMY liquid nutrient medium, and the 140r/min shaking culture is 4 days under the normal temperature condition; Draw the 1mL bacteria suspension in centrifuge tube, the centrifugal 10min of 5000r/min; Abandoning supernatant is coated on bacterial sediment on the above-mentioned acetic acid-nitric acid cellulose mixture film flat board that contains anthracene, the flat board after the coating is placed to leave standstill under 30 ℃ of conditions cultivated 15 days.
The result shows to have no colony growth on the blank flat board, explains not add on the flat board of anthracene as carbon source, and celestial river salt Zymomonas mobilis (Halomonas xianhensis) A-1 CGMCC № 2941 can't grow; Accordingly; Celestial river salt Zymomonas mobilis (Halomonas xianhensis) A-1 CGMCC № 2941 can grow on the flat board of anthracene being added with, and explains that celestial river salt Zymomonas mobilis (Halomonas xianhensis) A-1 CGMCC № 2941 anthra of can degrading is the sole carbon source growth with the anthracene.
6, the performance of celestial river salt Zymomonas mobilis (Halomonas xianhensis) A-1 CGMCC № 2941 degraded fluoranthene
Fluoranthene is dissolved in the ether, and preparation becomes the solution that concentration is 1mg/mL.
After salinity is that to add the quality percentage composition in 5% the above-mentioned sea salt synthetic medium be 1.5～1.8% agarose; The 20min 0.06MPa sterilize under the condition; Evenly be poured into substratum in the glass culture dish while hot subsequently; After culture medium solidifying, be positioned in 37 ℃ of environment, the moisture of solid culture primary surface is evaporated fully.Be covered with sterilized acetic acid-nitric acid cellulose mixture film (millipore filtration subsequently on the surface of plate culture medium; Aperture 0.45 μ m, diameter 90mm; Available from Beijing through biochemical reagents biotech company of HTC of section); The concentration that on film, adds the above-mentioned preparation of 1mL be 1mg/mL fluoranthene diethyl ether solution and coating evenly, treat that ether volatilizees fully after, build dull and stereotyped for use.The ether that will not contain fluoranthene simultaneously is coated on the above-mentioned cellulose membrane flat board as blank.
Celestial river salt Zymomonas mobilis (Halomonas xianhensis) A-1 CGMCC № 2941 is inoculated in the SSDMY liquid nutrient medium, and the 140r/min shaking culture is 4 days under the normal temperature condition; Draw the 1mL bacteria suspension in centrifuge tube, the centrifugal 10min of 5000r/min; Abandoning supernatant is coated on bacterial sediment on the above-mentioned acetic acid-nitric acid cellulose mixture film flat board that contains fluoranthene, the flat board after the coating is placed to leave standstill under 30 ℃ of conditions cultivated 15 days.
The result shows to have no colony growth on the blank flat board, explains not add on the flat board of fluoranthene as carbon source, and celestial river salt Zymomonas mobilis (Halomonas xianhensis) A-1 CGMCC № 2941 can't grow; Accordingly; Celestial river salt Zymomonas mobilis (Halomonas xianhensis) A-1 CGMCC № 2941 can grow on the flat board of fluoranthene being added with, and can degrade fluoranthene and be the sole carbon source growth with the fluoranthene of celestial river salt Zymomonas mobilis (Halomonas xianhensis) A-1 CGMCC № 2941 is described.
Claims (3)
1. the method for a screening polycyclic aromatic hydrocarbon-degrading is that testing sample is seeded in the polycyclic aromatic hydrocarbons is in the substratum of sole carbon source, cultivates and obtains polycyclic aromatic hydrocarbon-degrading bacteria; Said substratum is a solid medium, and said solid culture primary surface is covered with cellulose membrane, is added with polycyclic aromatic hydrocarbons on the said cellulose membrane;
Said polycyclic aromatic hydrocarbons is dissolved in earlier in the volatile organic solvent, is added on the cellulose membrane again;
Said polycyclic aromatic hydrocarbons is phenanthrene, anthracene, pyrene or fluoranthene;
Consisting of of said solid medium: NaCl 79.45g/L, MgCl
26H
2O 6.9g/L, MgSO
47H
2O10.45g/L, CaCl
27H
2O 0.75g/L, KCl 2.1g/L, NaHCO
30.1g/L, NaBr 0.25g/L, (NH
4)
2SO
44.0g/L, trace element solution 1mL/L, luxuriant and rich with fragrance 1mg/L, all the other are water;
Consisting of of said trace element solution: Ca (NO
3)
24H
2O 800mg/L, FeSO
4.7H
2O 400mg/L, CuSO
45H
2O 80mg/L, ZnSO
47H
2O 40mg/L, MnSO
44H
2O 40mg/L, NaMO
42H
2O8mg/L, H
3BO
36mg/L, K
2HPO
4500mg/L, all the other are water;
The pH value of said substratum is 7.5.
2. method according to claim 1 is characterized in that: said organic solvent is an ether.
3. method according to claim 1 and 2 is characterized in that: said cellulose membrane is acetic acid-nitric acid cellulose mixture film.
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| CN101050421A (en) * | 2007-03-30 | 2007-10-10 | 中国科学院沈阳应用生态研究所 | Method for quick filtering out bacteria having characterization of adsorption for degrading polycyclic aromatic hydrocarbon |
| CN101195810A (en) * | 2007-11-30 | 2008-06-11 | 华南理工大学 | High-efficiency degradation bacterium series of polycyclic aromatic hydrocarbon and uses thereof |
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| CN101050421A (en) * | 2007-03-30 | 2007-10-10 | 中国科学院沈阳应用生态研究所 | Method for quick filtering out bacteria having characterization of adsorption for degrading polycyclic aromatic hydrocarbon |
| CN101195810A (en) * | 2007-11-30 | 2008-06-11 | 华南理工大学 | High-efficiency degradation bacterium series of polycyclic aromatic hydrocarbon and uses thereof |
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