CN1869199A - A strain denitrogen paracoccus and its culturing method and application - Google Patents
A strain denitrogen paracoccus and its culturing method and application Download PDFInfo
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- CN1869199A CN1869199A CN 200610081490 CN200610081490A CN1869199A CN 1869199 A CN1869199 A CN 1869199A CN 200610081490 CN200610081490 CN 200610081490 CN 200610081490 A CN200610081490 A CN 200610081490A CN 1869199 A CN1869199 A CN 1869199A
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Abstract
The invention discloses a denitrogenation paracoccus strain and the cultivating method and the application. The strain is Paracoccus denitrificans W12 CGMCC No.1673. It has the strong degradation ability to organic pollutant, and could be used to take the biology treatment to the polluted water containing benzene, xylene, quinoline and cyanide and to the polluted soil. Especially, it has high degradation activity to benzene.
Description
Technical field
The present invention relates to a strain denitrogen paracoccus and cultural method thereof and application.
Background technology
Opened in the biological treating of the earth " self-cleaning " process and had advantages such as cost is low, secondary pollution is light, Environmental compatibility is good, the biological treating technology has now become one of preferred option in the Pollution abatement technology, as the activated sludge process that can be used for sewage/wastewater treatment that now produced good result, oxidation ditch process etc.Successful, effective, the suitable factor of decision biochemical processing process, except processing condition and operational administrative, the effect that is used for the functional microorganism colony of processes such as contaminant degradation, conversion also is crucial.Therefore, to separation, the screening of extraordinary microorganism, the separation of functional gene, extraction, and make up various environmental engineering bacterium whereby and become research focus in environmental science, the life science.
Biological reinforcing technology (the Bioaugmentation that arises at the historic moment, abbreviation BA technology), gene and strengthening technology (Gene-Enhanced-Technology, be called for short the GET technology) be the function stem/flora that in the pollution system, adds artificial culture, the import feature gene is with degraded, the conversion of target substrates in the enhancing system.Some external scientific research institutions such as the U.S., Japan, Britain now successfully develop commercial environmental organism preparation, the wherein famous EM preparation that Japan is arranged, the AM preparation of the U.S..
Pyridine is a widely used compound in the chemical industry, as the chemosynthesis monomer, is a kind of common toxic pollutent therefore, is the feature pollution factor of trade effluents such as coking chemical waste water, agricultural chemicals waste water.In recent years, pyridine is unusual important chemical synthon, the existing market investigation shows, whole world pyridine steady demand, therefore, the foreign trader falls over each other to found the factory in China, as world-renowned Nepera company and Reilly industrial, respectively in Beijing, Nanjing has been built up or the factory of pyridine under preparation.Infer that in view of the above in the near future, the organic contamination of pyridine and the relevant leakage that causes, the problems such as processing of pollution may seem more and more outstanding and serious in the production of pyridine, application, sales process.In addition, along with the increase of weedicide consumption, developing rapidly of its production compound probability, especially no-tillage/as to subtract the non-agrotechnical promotion of ploughing such as cultivated, Paraquat because of its unique chemical characteristic as 1) quick-acting; 2) only plant shoot is divided effectively, invalid to root and rhizome; 3) combine rapidly with soil and lose activity, therefore, the production of this agricultural chemicals, use with sale and all be lasting ascendant trend, increase 5-10% every year approximately in the sales volume of U.S.'s Paraquat.Paraquat is one of main derivative of pyridine, accounts for 30% of pyridine consumption.Therefore, the biodegradable systematic study to this pollutent is very necessary.
Though, biodegradable research about pyridine just has relevant report as far back as the seventies in 20th century, but, because pyridine itself belongs to the difficult degradation organic heterocyclic molecule, occurring in nature is difficult to find the microorganism that can effectively degrade to it, so so far various countries will pyridine as in addition primary study and the processing of the priority pollutants in the pollution system and biodegradable organic compounds, wish to find some effective microorganisms that it is controlled, thereby guarantee that pyridine content is unlikely to environment is polluted in the trade effluent of various dischargings.
Paracoccus denitrificans is the quite common a kind of microorganism of occurring in nature, and ubiquity in soil, water body also is a kind of mode trickle biology of studying more thoroughly, and is quite extensive about the research of aspects such as Physiology and biochemistry, pathways metabolism.At present, research emphasis mainly concentrates on the nitrogen metabolism aspect, and becomes the mode trickle biology in the nitrogen metabolism research.
The pollution condition of China is obviously different with other country, and existing commercialization biotechnological formulation is difficult to play effective regulation effect to " three wastes ", and serious pollution condition needs to be resolved hurrily.The method contaminated solution of biological restoration has crucial meaning.
Summary of the invention
First purpose of the present invention provide a strain effectively the degradable organic pollutant pyrido be used for Paracoccus denitrificans (Paracoccus denitrificans) W12 of environmental pollution improvement.
Paracoccus denitrificans (Paracoccus denitrificans) W12 that is used for environmental improvement provided by the present invention was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on 04 11st, 2006, and deposit number is CGMCC No.1673.
Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCC No.1673 can add on pyridine (1.96g/L pyridine) screening culture medium at common bacteria LB substratum and inorganic salt and grows, bacteria colony white, rounded, the edge is smooth, it is the G-tyrothricin that opticmicroscope is observed it down, single or paired, do not move.Aerobic, respiratory metabolism; When nitrate, nitrite or nitrogen oxide exist, can be electron acceptor(EA) battalion anaerobic growth with them.Catalase, oxidase positive.
Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCC № 1673 can cultivate down at 30 ℃～35 ℃ with LB substratum or the screening culture medium that contains pyridine; The described screening culture medium that contains pyridine is to contain 1.96g/L pyridine, Na among every 1000mL
2HPO
42H
2O 7.0g, KH
2PO
43.0g, NaCl 0.25g, MgSO
47H
2O0.3g, CaCl
22H
2O 0.02g, FeCl
36H
2O 0.045g, MnSO
44H
2O 0.01g, ZnSO
47H
2O0.01g, CuSO
45H
2O 0.002g, CoCl
26H
2O 0.003g, NiCl
26H
2O 0.003g, Na
2MoO
42H
2O0.002g, pH 7-8.
Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCC No.1673 in the LB substratum 30 ℃～35 ℃, 150rpm cultivates 18-22h, and viable count can reach 10
9～10
10Cfu/ml; In containing the screening culture medium of pyridine 30 ℃～35 ℃, 150rpm cultivates 38h～42h, and viable count can reach 10
8～10
9Cfu/ml, pyridine degradable reaches 99%.
Second purpose of the present invention provides a kind of special cultural method of Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCC № 1673, this cultural method with coking chemical waste water as its sole carbon source, the energy, nitrogenous source.
The method of cultivation Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCC № 1673 provided by the present invention is that coking chemical waste water is cultivated as the substratum of Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCC № 1673.
The pH of described coking chemical waste water is 7.0-7.5, and COD is 1680mg/L.
Culture temperature in the described cultural method can be 30 ℃～35 ℃.
Incubation time in the described cultural method can be 3～5 days.
Paracoccus denitrificans of the present invention (Paracoccus denitrificans) W12 CGMCC No.1673 degradable pyridine, benzene, dimethylbenzene, quinoline, prussiate, particularly pyridine there is very high degrading activity, experiment shows, this bacterial strain reaches 99% to pyridine degradable, and be not subjected to the influence of microorganism in the environment, this is more rare in this quasi-microorganism of Paracoccus denitrificans, therefore, to system, the further investigation of this bacterium, can enrich the content of the biochemical aspect of this mode trickle biological physiology.Experiment shows that Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCCNo.1673 also can utilize coking chemical waste water to grow as its sole carbon source, the energy, nitrogenous source.
Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCC No.1673 can have effective degrading activity to pyridine on the one hand under aerobic conditions, on the other hand again can be under oxygen free condition metabolism nitrogenous compound effectively, and well-known, several importances of the improvement of coking chemical waste water comprise: the 1) biological degradation of organic pollutant such as phenol, pyridine, quinoline etc.; 2) ammonia nitrogen and other nitrogenous elemental substances removes; Therefore, biochemical functions from microorganism, Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCC No.1673 is the biological treating that is suitable for very much coking chemical waste water, can be used as biological reinforced dose in the coking chemical waste water biological treating technology.Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCC No.1673 has very high degradation capability to organic pollutant, can be used for containing the biological treating and the contaminated soil reparation of the waste water of organic pollutants such as pyridine, benzene, dimethylbenzene, quinoline, prussiate, and develop corresponding environment friendly biological preparation whereby, have higher research, application and marketable value.
Description of drawings
Fig. 1 is the electromicroscopic photograph of Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCC No.1673.
Fig. 2 is pyridine degradable curve and the thalli growth curve of Paracoccus denitrificans in the pollutant effluents (Paracoccus denitrificans) W12 CGMCC No.1673
Fig. 3 is for polluting the pyridine degradable curve of Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCC No.1673 in the mud
Fig. 4 is the growth curve of Paracoccus denitrificans in the coking chemical waste water (Paracoccus denitrificans) W12 CGMCC No.1673
Fig. 5 is that the plasmid of Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCC No.1673 extracts electrophorogram.
Embodiment
Following experimental technique is ordinary method if no special instructions, and the solvent in all substratum is water.
BS minimal medium: contain in every 1000mL water: Na
2HPO
42H
2O 7.0g, KH
2PO
43.0g, NaCl0.25g, MgSO
47H
2O 0.3g, CaCl
22H
2O 0.02g, FeCl
36H
2O 0.045g, MnSO
44H
2O0.01g, ZnSO
47H
2O 0.01g, CuSO
45H
2O 0.002g, CoCl
26H
2O 0.003g, NiCl
26H
2O0.003g, Na
2MoO
42H
2O 0.002g, pH 7.5, (solid medium adds 15g agar).
The detection method of pyridine adopts high performance liquid chromatography (HPLC) method:
Sample preparation: in the aerobic degradation process, sampling at regular intervals, water sample is centrifugal 10min under 8000rpm, and supernatant is used for the HPLC quantitative analysis of pyridine.
Chromatographic instrument is Tianjin, island SHIMADZU, and LC-VP series, chromatographic column are Diamosil (TM) diamond C18 post, 250mm * 4.6mm, and granularity is 5 μ m; Moving phase is 80% (v/v) methanol aqueous solution, and flow rate of mobile phase is 1.0ml/min.Measure wavelength 254nm.
Separation, the purifying of embodiment 1, Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCC No.1673
Paracoccus denitrificans (Paracoccus denitrificans) W12 be adopt the active sludge poisonous, that noxious industry waste water is tamed cultivation for a long time from Tsing-Hua University environmental engineering system separate, a strain G-bacterium that purifying obtains, concrete enrichment, separation, purge process are as follows:
Under the condition of selective pressure pyridine 2000mg/L, just with pyridine as sole carbon source and nitrogenous source, from the active sludge of raising and train for a long time, obtain Paracoccus denitrificans (Paracoccusdenitrificans) W12 CGMCC No.1673 through steps such as enrichment, separation, screening, purifying.
Concrete sepn process is as follows: the sample of gathering is inoculated into contains liquid B S pyridine and select shaking in the bottle of substratum (adding the 2000mg/L pyridine as sole carbon source and nitrogenous source in the BS inorganic salt solution), 35 ℃, one week of shaking culture under the 120rpm.The pregnant solution dilution is applied to solid BS pyridine selects on the substratum, 35 ℃, constant temperature culture is until growing obvious visible bacterium colony.Picking list bacterium colony moves and receives in the liquid B S pyridine selection substratum, and 35 ℃, shaking culture under the 120rpm reaches 10 until cell concn
7-10
8Individual cell/mL.Diluting the applying solid flat board once more, detect purity, is the purebred bacterial strain of sole carbon source and nitrogenous source growth until obtaining with the pyridine.The bacterial strain that obtains is carried out morphologic observation and Physiology and biochemistry evaluation, the result shows that this bacterial strain can add on pyridine (1.96g/L pyridine) screening culture medium at common bacteria LB substratum and inorganic salt and grows, bacteria colony white, rounded, the edge is smooth, it is the G-tyrothricin that opticmicroscope is observed it down, be about 0.9 μ m, the about 0.3 μ m of diameter.(Fig. 1), single or paired, do not move.Aerobic, respiratory metabolism; When nitrate, nitrite or nitrogen oxide exist, can be electron acceptor(EA) battalion anaerobic growth with them.Catalase, oxidase positive.Again the 16srRNA of thalline is checked order in addition, and compare with the sequence in the database, last combining form feature, physiological and biochemical property and nucleotide sequence feature are Paracoccus denitrificans (Paracoccus denitrificans) with this identification of strains.With this bacterial strain called after Paracoccus denitrificans (Paracoccusdenitrificans) W12.This bacterial strain has been preserved in Chinese common micro-organisms culture presevation management committee common micro-organisms center on 04 11st, 2006, deposit number is CGMCC No.1673.
Contaminant degradation spectrum and the resistance spectrum of embodiment 2, Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCC No.1673
1) contaminant degradation of Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCC No.1673 spectrum
The BS minimal medium is as minimum medium, with the energy, carbon source, the nitrogenous source of the organic pollutant shown in the table 1 as unique interpolation, adopts the mode of solid culture, 30 ℃, leave standstill and cultivate a week, observe the situation that bacterium colony forms.Test result to compounds such as benzene, phenol, toluene, dimethylbenzene, pyridine, prussiate, quinoline, imidazoles is as shown in table 1.The result shows that Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCC No.1673 has the degraded spectrum of broad, can utilize multiple organic pollutant growth, especially pyridine is had higher degrading activity.
Table 1 W12 utilizes situation to the degraded of organic pollutant
| Pyridine | Dimethylbenzene | Benzene | Phenol | Toluene | Quinoline | CN - | Imidazoles | |
| Concentration | 1.96g/L | 1.72g/L | 4.39g/L | 50mg/L | 1.72g/L | 0.5g/L | 200mg/L | 50g/L |
| Degradation characteristic | √ | ++ | + | - | - | + | +++ | - |
Annotate: "+" expression thalline is grown on corresponding pollutent flat board in the table, and "+" multilist more shows that bacterium looks good more; "-" expression thalline is not long on corresponding pollutent flat board, and " √ " expression thalline is grown vigorous on corresponding pollutent flat board, pollutent to bacterium without any effect.
2) resistance of Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCC No.1673 detects
This bacterium is inoculated in respectively in the antibiotic LB solid medium of carboxylic benzyl mycin, kantlex, spectinomycin, paraxin, Totomycin, tsiklomitsin, cephamycin, ammonia benzyl mycin or Rifampin that contains different concns as shown in table 2 (50mg/L-100mg/L), 30 ℃, leave standstill and cultivate a week, observe thalline forms bacterium colony on media surface situation.The result is as shown in table 2.The result shows that Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCCNo.1673 is except spectinomycin and Totomycin are had the resistance, and is all responsive to all the other microbiotic.
Table 2 W12 is to antibiotic tolerated
| Cephalo | Paraxin | Grand | Block that | The carboxylic benzyl | Rifampin | Totomycin | Tsiklomitsin | The ammonia benzyl | ||||||||||
| Concentration (mg/L) | 50 | 100 | 50 | 100 | 150 | 200 | 50 | 100 | 50 | 100 | 50 | 100 | 50 | 100 | 50 | 100 | 50 | 100 |
| Resistance | - | - | + | + | - | - | - | ++ | + | - | - | |||||||
Annotate: "+" expression thalline is grown on corresponding resistant panel in the table, and "+" multilist more shows that bacterium looks good more; "-" expression thalline is not long on corresponding resistant panel.
Having investigated the resistance of Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCC No.1673 to heavy metal in addition, is example with Pb, this bacterium is inoculated in contain different concns Pb (NO
3)
2The LB solid medium in, cultivate down at 35 ℃, the result shows that thalline can reach 50mg/L to Pb tolerance concentration, so have higher heavy metal resistance.
1) Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCC No.1673 is to the biological degradation of pyridine pollutant effluents
Be inoculated in the 300ml triangular flask that contains 50ml LB liquid nutrient medium from inclined-plane picking 2 ring Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCC No.1673 bacterium, 35 ℃, 180rpm shaking culture 36h, the centrifugal 10min of 8000rpm collects thalline, wash this bacterium liquid with stroke-physiological saline solution again, centrifugal again, twice so repeatedly, centrifugal condition also is 8000rpm, 10min.At last that the gained thalline is resuspended with being added with the BS nutrient solution of pyridine, and insert and contain in the 300ml triangular flask of waste water that 50ml adds pyridine, pyridine concentration is about 50mg/l, 100mg/l, 200mg/l, 400mg/l, 900mg/l, 1400mg/l respectively; 35 ℃, 150rpm, carry out aerobic degradation, with the corresponding waste water that does not connect bacterium is blank, after having cultivated 0h, 10h, 20h, 30h, 40h, 45h, 47h, 60h, detect the pyridine content in each bottle respectively, and mensuration thalline biomass, draw the degradation kinetics curve, the result shows that Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCC No.1673 all has degradation effect preferably to the pyridine in the waste water that contains each concentration pyridine, and the degradation rate to examination concentration pyridine in 25h～45h all reaches more than 99%.Wherein, Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCC No.1673 in the waste water that contains 1400mg/l concentration pyridine to the degradation kinetics curve of pyridine as shown in Figure 2, Fig. 2 shows, preceding 20h is because thalline is in lag phase, so pyridine is not degraded almost, along with the growth of thalline, pyridine begins rapid degraded behind the 20h, behind 45h, pyridine is almost degraded fully.Lag phase is got rid of the back Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCC No.1673 degradation curve is fitted, found that at quick degradation period pyridine degradable curve to meet the zeroth order reaction dynamic law substantially.Its kinetic equation is: y=-55.894x+2735R
2=0.9132; Y is a pyridine concentration, the mg/l of unit; X is the reaction times, the h of unit.
The result shows, Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCC No.1673 has good effect for the pyridine degradable in the pyridine pollutant effluents, substantially can in a short period of time pyridine be dropped to can emission standards, shows that Paracoccus denitrificans W12 has the biodegradable great potential of heterogeneous ring compound such as the pollution of being used for system pyridine.
2) Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCC No.1673 pollutes the biological restoration of mud to pyridine
2 ring Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCC No.1673 are inoculated in the 300ml triangular flask that contains 50ml LB liquid nutrient medium from the inclined-plane picking, 35 ℃, 180rpm shaking culture 36h, bacterium liquid is inserted respectively in the soil and the natural soils of 5g without sterilization that 5g dries behind autoclave sterilization, and making final soil throw the bacterium amount is 5.6 * 10
10CFU/g soil, adding concentration then respectively is that each 1ml of 8266mg/l pyridine solution makes pollution level be about 1.5mg/g soil, adds the 9ml sterilized water more respectively, the last mixing and the 150ml triangular flask of packing into are made the mud contamination treatment reactor.Then triangular flask is placed 190rpm, carry out the aerobic degradation experiment under 35 ℃.Sampling analysis pyridine concentration when 0h, 10h, 20h, 22h, 25h, 30h, 45h, each sampling back replenishes corresponding sterilized water, and water content is constant in the maintenance triangular flask.Draw out at last Paracoccus denitrificans (Paracoccusdenitrificans) W12 CGMCC No.1673 sterilization with natural mud system in to the degradation curve (Fig. 3) of pyridine.Fig. 3 shows that Paracoccus denitrificans under aerobic condition (Paracoccus denitrificans) W12 CGMCCNo.1673 can effectively utilize the pyridine in the mud, and is basically that the pyridine degradable in the mud is complete in 20 hours.And the microorganism in the natural mud is to the not significantly influence of effect of Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCCNo.1673 degraded pyridine.
This test shows that Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCC No.1673 has good effect for the pyridine degradable that pyridine pollutes in the mud, can in a short period of time pyridine be dropped to on-contaminated standard, this shows that Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCC No.1673 can be used for the biological restoration that pyridine pollutes mud or soil.
3) growing state of Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCC No.1673 in coking chemical waste water
Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCC No.1673 utilizes certain selective pressure (adding pyridine as unique substrate in the BS component of inorganic salts as utilizing) from the multi-functional degradation bacteria that occurring in nature separates, purifying comes out, it to be carried out following experiment:
Choose two ring bacterium accesses from the LB substratum of preservation strain and be equipped with the 300ml triangular flask of 30ml LB liquid nutrient medium, 35 ℃, 180rpm, shaking culture is spent the night.After treating that thalline enters logarithmic phase, draw 1ml bacterium liquid and insert and 30ml is housed after filtration in the 300ml triangular flask of the Shoudu Iron and Steel Co coking chemical waste water of degerming, 35 ℃, 150rpm, shaking culture 5d surveys OD every day one time
600Draw out growth curve at last, the result shows, Paracoccus denitrificans (Paracoccusdenitrificans) W12 CGMCC No.1673 can utilize the coking chemical waste water of Shoudu Iron and Steel Co to grow preferably, breed, utilize coking chemical waste water that Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCC No.1673 is carried out shaking culture, (4d～5d), the cell concn of Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCC No.1673 in nutrient solution can reach 10 after cultivating certain hour
9Cfu/mL, OD
600=1.2.Its growth curve result as shown in Figure 4, the result shows that the preceding 2d that Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCCNo.1673 grows is in lag phase substantially in coking chemical waste water, cell concentration is lower; 2d～3d enters logarithmic phase, the very fast increase of cell concentration; Begin to enter stationary phase to 4d, cell concentration is constant substantially.Paracoccus denitrificans (Paracoccus denitrificans) is though W12 CGMCC No.1673 grows slower in coking chemical waste water, but thalline can reach very high concentration in 3d, illustrate that Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCC No.1673 can well grow, and is expected to be used for the biochemical treatment of coking chemical waste water in coking chemical waste water.
Plasmid is composed the preliminary study of depositing situation and pyridine degradable gene in the bacterium
Adopt improved alkaline denaturation, extract plasmid from the nutrient solution of Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCCNo.1673, concrete plasmid leaching process is as follows:
1, material therefor:
1) substratum: LB
2) plasmid extracts used solution:
TE：50mM Tris，20mM Na
2EDTA，pH8.0；
Lysate: 3% sodium lauryl sulphate (SDS) and 0.2mol/L NaOH;
TS：1M Tris，4M NaCl，pH7.0；
TES：50mM Tris，5mM Na
2EDTA，50mM NaCl，pH8.0。
2, plasmid extracts: adopt improved alkaline denaturation, concrete steps are as follows:
1) thalli growth is to OD
600Be 1.6～1.7 o'clock, get the centrifugal 10min of 4ml bacterium liquid chamber temperature 6000rpm, outwell supernatant;
2) thalline is suspended in the 1mlTE solution, mixing does not have caked thalline as far as possible;
3) add the 2ml lysate, slowly put upside down, lysing cell 7min in room temperature or ice bath treats that lysate is limpid;
4) add 1mlTS liquid, place 1h, the centrifugal 20min of 8000rpm in ice bath;
5) collect supernatant liquor, add the cold dehydrated alcohol of equal-volume ,-20 ℃ leave standstill 30min
6) supernatant liquor that inclines, 35 ℃ of oven dry ethanol;
7) precipitation is dissolved in the 0.1mlTES solution, adds equal-volume chloroform mixing again;
8) the centrifugal 3min of 7000rpm draws the supernatant plasmid solution, adds RNase, to remove RNA;
9) electrophoresis detection
The detected through gel electrophoresis of its extraction product as shown in Figure 5, show that by figure Paracoccus denitrificans (Paracoccusdenitrificans) W12 CGMCC No.1673 carries a plasmid that fragment is bigger, size is about 100kb, the arrow of swimming lane 1 is represented the big plasmid that this bacterium is contained among Fig. 5, size is about 100Kb, and swimming lane M is molecular weight standard (1Kbplus DNA Ladder).Plasmid is eliminated Preliminary experiment results and is shown, does not have corresponding relation between the degraded of pyridine and the plasmid, infers that the gene of coding pyridine degradable enzyme may be on plasmid, and might be on genome.Find that by conjugation test Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCC No.1673 does not have engagement function.
Claims (10)
1, Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCC No.1673.
2, the method for a kind of cultivation Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCC No.1673 is that coking chemical waste water is cultivated as the substratum of Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCC № 1673.
3, cultural method according to claim 2 is characterized in that: the pH of described coking chemical waste water is 7.0-7.5, and COD is 1680mg/L.
4, cultural method according to claim 2 is characterized in that: the culture temperature in the described cultural method is 30 ℃～35 ℃.
5, cultural method according to claim 2 is characterized in that: the incubation time in the described cultural method is 3～5 days.
6, the application of Paracoccus denitrificans (Paracoccus denitrificans) W12 CGMCC No.1673 in degradable organic pollutant.
7, application according to claim 6 is characterized in that: described organic pollutant is pyridine, benzene, dimethylbenzene, quinoline or prussiate.
8, application according to claim 6 is characterized in that: described organic pollutant is a pyridine.
9, according to claim 6,7 or 8 described application, it is characterized in that: described organic pollutant is wastewater organic pollutant or soil organic pollutant.
10, application according to claim 9 is characterized in that: described waste water is coking chemical waste water.
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Cited By (9)
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| RU2041944C1 (en) * | 1992-12-28 | 1995-08-20 | Акционерное общество закрытого типа "Биотехинвест" | Strain of bacteria paracoccusdenitrificans being destructor of aniline, 3-chloraniline,4-chloraniline and 3,4-dichloraniline |
| CN1047155C (en) * | 1993-06-11 | 1999-12-08 | 中国科学院成都生物研究所 | New technology for treating electroplating waste water with micro-organism |
| KR100298297B1 (en) * | 1998-10-30 | 2001-12-01 | 이흥규 | Novel microbes for disposing swage of livestock farming and biological use of disposed swage |
| CN100424167C (en) * | 2002-12-04 | 2008-10-08 | 上海一本生命科技工程有限公司 | YB microbe function fungus and domestic organic garbage treating machine |
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| CN101255403B (en) * | 2007-12-26 | 2010-06-16 | 北京大学 | Pyridine degradation bacterium strain and uses thereof |
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| CN102911897B (en) * | 2012-10-11 | 2014-03-26 | 中国水产科学研究院淡水渔业研究中心 | Cultural method for paracoccus denitrificans and application of same to purifying aquaculture water |
| CN102911897A (en) * | 2012-10-11 | 2013-02-06 | 中国水产科学研究院淡水渔业研究中心 | Cultural method for paracoccus denitrificans and application of same to purifying aquaculture water |
| CN102976541B (en) * | 2012-11-13 | 2014-06-11 | 安徽国星生物化学有限公司 | New technology for separating waste water containing zinc and nickel |
| CN102976541A (en) * | 2012-11-13 | 2013-03-20 | 安徽国星生物化学有限公司 | New technology for separating waste water containing zinc and nickel |
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| CN106282069A (en) * | 2016-09-30 | 2017-01-04 | 福建省微生物研究所 | A kind of secondary coccus and the application in sewage purification |
| CN106282069B (en) * | 2016-09-30 | 2019-02-05 | 福建省微生物研究所 | A kind of pair coccus and the application in sewage purification |
| CN106520617A (en) * | 2016-11-10 | 2017-03-22 | 中国科学院成都生物研究所 | Desulfurization and denitrification paracoccus denitrificans and application thereof |
| CN106520617B (en) * | 2016-11-10 | 2019-08-20 | 广州清源生物科技有限公司 | One plant of desulfurization removing nitric pair coccus and its application |
| CN111040965A (en) * | 2019-12-16 | 2020-04-21 | 南京理工大学 | Bacterium-algae symbiotic system for reinforcing pyridine biodegradation under micro-aerobic condition |
| CN111040965B (en) * | 2019-12-16 | 2022-06-28 | 南京理工大学 | Bacterial-algae symbiotic system for strengthening pyridine biodegradation under micro-aerobic condition |
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