CN102851234A - Rhodococcus JZX-01 used for degrading petroleum pollutants, and culturing method thereof - Google Patents
Rhodococcus JZX-01 used for degrading petroleum pollutants, and culturing method thereof Download PDFInfo
- Publication number
- CN102851234A CN102851234A CN2012101989483A CN201210198948A CN102851234A CN 102851234 A CN102851234 A CN 102851234A CN 2012101989483 A CN2012101989483 A CN 2012101989483A CN 201210198948 A CN201210198948 A CN 201210198948A CN 102851234 A CN102851234 A CN 102851234A
- Authority
- CN
- China
- Prior art keywords
- jzx
- rhodococcus
- cultural method
- culturing
- nutrient solution
- 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.)
- Pending
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a rhodococcus JZX-01 used for degrading petroleum pollutants, and a culturing method thereof. The identification of the rhodococcus is achieved through 16S rDNA sequencing and comparison on NCBI. An adopted 16S rDNA upstream primer is a universal primer 27F with a specific sequence of 5'-AGA GTT TGA TCC TGGCTCAG-3'. A downstream universal primer is 1492R with a specific sequence of 5'-GGT TAC CTT GTT ACG ACTT-3'. According to the invention, an inducing agent is added into a culture medium or culture solution, and soil is added into the culture solution or culture medium; culturing is carried out in a shaking table under a culturing temperature of 10-40 DED C and a culturing rotation speed of 100-300rpm; when culturing is carried out for 1-4days, a product is transferred into a fresh culture medium or culture solution, and screening is carried out. The rhodococcus strain is collected by China General Microbiological Culture Collection Center, and has a collection register number of CGMCC No. 5958. The collection date is April 9th, 2012. With a surfactant-producing capacity of rhodococcus JZX-01, petroleum degradation efficiency is further enhanced. The rhodococcus JZX-01 can be used in biological treatments of petroleum pollutants.
Description
Technical field
The invention belongs to gene engineering technology field, relate to specifically a kind of rhodococcus JZX-01, CGMCC No.5958, (Rhodococcus sp.JZX-01) cultural method and purposes; His culture condition and the effect of this bacterial strain in the petroleum pollution degradation process.
Background technology
In fields such as daily life and industrial production, oil and derived product thereof have very important effect.But the discharging of oil in the process of exploitation, smelting, storage, transportation and use caused huge infringement to environment, adds that the oil accident of frequent occurrence has aggravated this harm more all over the world.Most waters, especially ocean suffer the pollution of oil throughout the year on the earth, have caused global showing great attention to.
Compositions different in the oil have different impacts on human with animals and plants.When lower boiling stable hydrocarbon can cause paralysis, stupor, excessive concentration even can cause death.High boiling hydro carbons easily is attached to the root system surface of plant, forms the mucous membrane that one deck impeding nutritious substance and oxygen pass, and causes plant root to rot, and affects the growth of plant.Reactive group in the oil, inorganic nitrogen that can be in soil and phosphorus are combined and are limited nitrification and dephosphorylation.Thereby make effectively nitrogen and phosphorus content minimizing in the soil, affect the output of crop.Although the multiring aromatic hydrocarbon substance content in the oil is few, it is the material that has strong toxicity and can produce to human body carcinogenesis.Polycyclic aromatic hydrocarbons can enrichment in animals and plants especially ocean mussels organism, by human healthy of food chain impact.The marine site that causes after the Oil spills and the pollution on land also are the very big infringements to local natural landscape, and in the contaminated alkali flat, the infiltration of oil spilling can reach the very dark degree of depth, are difficult to remove, and produce long-term harmful effect.To the processing problem of the pollutent of oil become one serious, enjoy the key subjects of attracting attention in the world wide.
The treatment process of petroleum pollution mainly comprises three kinds, is respectively Physical, chemical method and biological process.Biological degradation method wherein is owing to having little, adaptable, the highly effective and safe of cost and not having the advantage of secondary pollution to become the focus of research.
The ability of microbiological deterioration petroleum hydrocarbon depends on the composition of various components in the oil, the complexity of carbochain and polycyclic aromatic hydrocarbons, ambient conditions, bacterial strain kind, whether the bacterium of tensio-active agent, generation tensio-active agent exists, and the combined influence of all (ratio of PH, temperature, C:N:P) factors such as physico-chemical property of environment.To sum up, affect microorganism the abiotic influence factor of oil degradation is mainly comprised two large classes: the firstth, the situation of petroleum hydrocarbon, the secondth, environmental factors.The hydro carbons situation of oil comprises: the chemical constitution of the state machine oil that dissolves each other of petroleum concentration, oil and water.Environmental factors comprises: temperature, PH, oxygen, nutritive substance and salinity etc.
Because the solvability of petroleum hydrocarbon in water is extremely low, so that the utilization ratio of petroleum hydrocarbon is very low, cause the ability of biological degradation oil to descend.Addressing this problem, proposing to utilize bio-surfactant to carry out emulsification oil phase and water, making oil be more prone to touch bacterium.Bio-surfactant is the Polyampholyte that microorganisms is arranged, and it not only can be attached on cell surface, and can directly be rejected to outside the born of the same parents.Bio-surfactant is applied in the petroleum industry; not only reduced the difficulty of exploitation and the transportation of oil; and increased the produced quantity of oil, for the environment protection aspect, bio-surfactant can increase the biodegradable ability of revealing the petroleum pollution in environment.
The at present biodegradable emphasis of the petroleum pollution screening that concentrates on high efficient petroleum degrading bacteria with separate, what the most easily be degraded in the petroleum hydrocarbon is the alkane of short chain, the C10 in discovery of oil hydrocarbon in 2002 such as Subarna the most easily is degraded to the alkane of C20,2009, Le Thi Nhi-Cong etc. find in the degradation process of petroleum hydrocarbon, what at first decompose is straight-chain paraffin, has the long chain alkane of branch can be accumulated in together in degradation process, can be degraded hardly.Yet the discoveries such as Rontani in 1986 etc. and calendar year 2001 Alvarez have some bacteriums to have the ability of these obstinate substrates of oxidation.For example, 1993, the discovery genus bacillus such as the Sorkhon hydro carbons of C15 to C17 of can only degrading; The discoveries such as calendar year 2001 Kato happiness heat bite oily bacillus can degrade alkane to C23; 2005, the fatty ground bacillus of the discoveries such as Nazina only can grown in the alkane of C16 carbon source at C6.Only have a few bacterial strain to have large-scale degradation capability, Sakai etc. separated the acinetobacter that obtains in 1994 can degrade C13 to the C44 paraffinic hydrocarbons; 2003, the Rhod of the screenings such as van Beilen can be degraded to C36 with long chain alkane.
Therefore find can degrade in extensive range, C38 in the petroleum pollution can be had very large theory and actual application value with the microorganism of interior component efficient degradation, this is in the biological restoration in petroleum pollution zone, and improvement ecotope and human safety and Health aspect have great importance.
Summary of the invention
The purpose of this invention is to provide a kind of new petroleum pollution degradation bacterial strain, i.e. rhodococcus JZX-01.
Another object of the present invention provides the culture condition of above-mentioned rhodococcus JZX-01.
Purpose of the present invention also comprises provides the application of rhodococcus JZX-01 in real life, and being used for degraded oil or former wet goods pollutent specifically is the environmental friendliness material.
The bacterial strain that the present invention is encircleed is the method for taking step-sizing, and directly screening contains required purpose bacterial classification and separation and purification and obtains from soil, and provides the culture condition of this bacterial classification and in the purposes of oil or former wet goods contaminant degradation.
The evaluation of the 16S rDNA of rhodococcus JZX-01 of the present invention is JQ648650 by carrying out in the state-run biotechnology NCBI of information center of the U.S. by the sequence number of submitting the 16S rDNA that obtains after the sequence to.
Technical scheme of the present invention is as follows:
A kind of rhodococcus JZX-01, by China Committee for Culture Collection of Microorganisms common micro-organisms center, the registration number of preservation is the rhodococcus of CGMCC No.5958; Preservation date on April 9th, 2012.
A kind of rhodococcus JZX-01, compare by 16S rDNA order-checking and at NCBI and to obtain, the upstream and downstream primer all is universal primer, upstream primer is 27F, concrete sequence is 5 '-AGA GTT TGATCC TGG CTC AG-3 ', downstream primer is 1492R, and concrete sequence is 5 '-GGT TAC CTT GTT ACGACT T-3 '.
The cultural method of rhodococcus JZX-01 of the present invention, step is as follows:
(1) rhodococcus JZX-01 thalline or spore are received on the inclined-plane, cultivated 1-4 days on the inclined-plane, described medium component is carbon source 1-20g/L, univalent metal salt or its hydrate 1-5g/L of the monovalent metallic ion of nitrogenous source 1-5g/L, NaCl 1-2g/L, micro-mixing solutions solution 1-20mL/L, agar 1-30g/L, phosphate radical or its hydrate 1-5g/L, biphosphate, adding distil water is settled to 1L.
(2) thalline is obtained from the inclined-plane after, be transferred in the nutrient solution and cultivated 1-4 days, the composition of described nutrient solution is: the univalent metal salt of the monovalent metallic ion of nitrogenous source 1-5g/L, NaCl 1-2g/L, trace element solution 1-20mL/L, phosphate radical or its hydrate 1-5g/L, biphosphate or its hydrate 1-5g/L, inductor 1-10ML/L, adding distil water is settled to 1L, the pH value is 3-9, and culture temperature is 5 ℃-45 ℃.
Described carbon source is to be Zulkovsky starch, glucose or glycerine.
Described nitrogenous source is extractum carnis, beef leaching thing, peptone, yeast extract, analysis for soybean powder immersion liquid, Pidolidone ammonia, ammonium sulfate, ammonium chloride, SODIUMNITRATE or saltpetre.
Described trace element is manganese, zinc, cobalt, nickel, magnesium, iron, calcium, copper, chromium, selenium, molybdenum, cobalt or fluorine.
Described inductor adds in substratum or the nutrient solution sole carbon source as JZX-01 to be cultivated, and guarantees that bacterial classification can only utilize this kind carbon source to carry out growth metabolism; Described inductor is alkane, crude oil, diesel oil, gasoline, liquid paraffin or polycyclic aromatic hydrocarbons.
Cultivate nutrient solution after 1-4 days under the rotating speed of 1000-10000rpm, carried out under 0-20 ℃ the temperature centrifugal 1-10 minute.
The thalline JZX-01 that arrives usefulness after the centrifugal not minimal medium of carbonaceous sources is washed 1-3 time, removes the nutritive substance in the substratum, pure JZX-01 is joined in the substratum cultivate.
Rhodococcus provided by the invention on April 9th, 2012 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, preservation be numbered CGMCC No.5958, Classification And Nomenclature is rhodococcus (Rhodococcus sp).
Effect of the present invention is that rhodococcus JZX-01 can be with very high efficient degraded inductor, and degradation rate is higher in the short period of time.
Effect of the present invention also has rhodococcus JZX-01 can produce efficiently bio-surfactant, and reduces the surface tension of nutrient solution, increases the mutual emulsifying capacity of inductor and inorganic salt solution.
Rhodococcus JZX-01 of the present invention has the ability that produces bio-surfactant, and this ability is significant in the degradation process take inductors such as petroleum hydrocarbons as substrate.
The physiological and biochemical property of rhodococcus JZX-01 provided by the invention, specifically such as following table:
| Experimental project | The result | Experimental project | The result | Experimental project | The result | Experimental project | The result |
| Arginine | - | Nitrate reduction | + | ONPG | + | Hydrogen sulfide | + |
| Ornithine | - | Urea | + | Sucrose | + | Glucose | + |
| Methionin | - | Vitamin C2 | - | Maltose | + | Oxydase | + |
| Citrate salt | + | The indole test | - | Wood sugar | - | Gramstaining | + |
Annotate: in the table+expression is positive, and-expression is negative.
Be described as follows:
The evaluation of rhodococcus of the present invention is compared by 16S rDNA order-checking and at NCBI and is obtained, the upstream primer of used 16S rDNA is universal primer 27F, concrete sequence is 5'-AGA GTTTGATCC TGG CTC AG-3', the universal primer in downstream is 1492R, and concrete sequence is 5'-GGT TAC CTT GTTACG ACT T-3'.
The screening method of rhodococcus JZX-01 of the present invention, specifically inductor is joined in substratum or the nutrient solution, and soil is joined nutrient solution or substratum, in shaking table, cultivate, the temperature of cultivating is 10-40 ℃, and the rotating speed of cultivation is 100-300rpm.Cultivate after 1-4 days, be transferred in fresh substratum or the nutrient solution and screen.
The cultural method of rhodococcus JZX-01 of the present invention, specifically with the inoculating needle picking rhodococcus jZX-01 thalline that the separation screening purifying obtains from soil or spore inoculating to the inclined-plane, slant culture basal growth 1-4 days, described medium component is carbon source (Zulkovsky starch, glucose or glycerine) 1-20g/L, nitrogenous source 1-5g/L, NaCl 1-2g/L, trace element solution 1-20mL/L, agar 1-30g/L, the monovalent metallic ion of phosphate radical or its hydrate 1-5g/L, the univalent metal salt of biphosphate or its hydrate 1-5g/L adding distil water are settled to 1L.In well-grown slant medium, add the liquid minimal medium that does not have inductor, the washing hypothallus is directly poured thalline in the nutrient solution into afterwards and is cultivated, culture temperature is 5 ℃-45 ℃, the composition of substratum or nutrient solution is nitrogenous source 1-5g/L, NaCl 0-2g/L, trace element mixing solutions 1-20mL/L, the monovalent metallic ion of phosphate radical or its hydrate 1-5g/L, the univalent metal salt of biphosphate or its hydrate 0-5g/L, inductor 1-50ML/L, the adding distil water constant volume, pH value is 3-9, culture temperature is 5 ℃-45 ℃, and described inductor is alkane, crude oil, diesel oil, gasoline, liquid paraffin or polycyclic aromatic hydrocarbons.
In the cultural method of the present invention, through the thalline that arrives that separates after the nutrient solution cultivation, can wash thalline with the substratum of removing carbon source or nutrient solution, again centrifugation, resulting flora can directly join the DeR of carrying out inductor in the minimal medium that contains inductor.
Above-mentioned carbon source is Zulkovsky starch, glycerine, glucose.Wherein the growth of thalline in glucose, glycerine and Zulkovsky starch is vigorous.
Above-mentioned nitrogenous source is extractum carnis, beef leaching thing, peptone, yeast extract, analysis for soybean powder immersion liquid, Pidolidone ammonia, ammonium sulfate, ammonium chloride, SODIUMNITRATE, saltpetre.Wherein under the existence of extractum carnis, beef leaching thing, SODIUMNITRATE or ammonium sulfate, thalli growth is good, in the situation that the nitrate ion Individual existence, thalline also can well be grown.Show that JZX-01 possesses the effect of oxidation high price nitrogen element.Peptone is convenient to bacterium and is absorbed because the character of its pair property electrolysis possesses good shock absorption, but Growth of Cells is slow in use.
Above-mentioned inductor is alkane, crude oil, diesel oil, gasoline, liquid paraffin or polycyclic aromatic hydrocarbons.During as inductor, JZX-01 shows good degradation property with alkane, crude oil, diesel oil and gasoline.Wherein with alkane or diesel oil during as inductor, JZX-01 can also produce bio-surfactant, reduces the surface tension of bacterium liquid, promotes emulsifying effect and the Degradation of these water-insoluble substance.Inductor is to be that the thalline initial OD values of 400-700 is to add under the condition of 0.1-2 at wavelength.The amount of inductor has the growth of thalline and promotes first the effect that suppresses afterwards, and the suitable inductor of every liter of solution is 1-20g.
Above-mentioned trace element is that trace element is manganese, zinc, cobalt, nickel, magnesium, iron, calcium, copper, chromium, selenium, molybdenum, cobalt or fluorine.Trace element solution is such as, MnSO
44H
2O, ZnSO
47H
2O, Na
2MoO
42H
2O, CuSO
45H
2O, FeSO
47H
2O, CaCl
2, NaSO
4Or H
3BO
3Deng.
The preferred medium component of the present invention is Zulkovsky starch 1-5g/L, beef leaching thing 1-5g/L, SODIUMNITRATE 1-5g/L, K2HPO4 0.5-5g/L, KH2PO4 0.5-5g/L, NaCl 0.1-2g/L, trace element solution 1-20ML/L.
The rhodococcus JZX-01 that the present invention screening obtains can the degraded oil pollutent, degrades in extensive range, the C38 in the petroleum pollution can be decomposed efficiently with interior component.Moreover, the branched-chain component of hard degradation, for example pristane and phytane can be by most degradeds.The ability of rhodococcus JZX-01 generation tensio-active agent has further increased again the efficient of oil degradation.Through investigating, the degraded that rhodococcus JZX-01 can be efficient, safe with petroleum pollution can be used for the biological treatment to petroleum pollution.
Description of drawings
Fig. 1 is rhodococcus JZX-01 and the evolutionary tree of relevant bacterial strain.The 16S rDNA of JZX-01 is by being JQ648650 at the U.S.'s state-run biotechnology NCBI of information center sequence number that identify and that obtain 16S rDNA.Adopt the N-J method, the value of bootstrapping is the systematic evolution tree that obtains JZX-01 under 1000 the condition.
Embodiment
By will helping further to understand the present invention below in conjunction with specific examples, but protection scope of the present invention is not restricted to this:
Embodiment 1:
The screening of rhodococcus JZX-01
Contaminated soil is fetched in petroleum-polluted serious zone from ring area, the Bohai Sea, in the process of collecting pedotheque, carries out the sampling of soil in the different degree of depth and site.The pedotheque of fetching is used inductor is joined in substratum or the nutrient solution, and soil is joined nutrient solution or substratum, cultivates in shaking table, and the temperature of cultivation is 10-40 ℃, and the rotating speed of cultivation is 100-300rpm.Cultivate after 1-4 days, be transferred in fresh substratum or the nutrient solution and screen.Wherein, described medium component is that ammonium sulfate 5g/L, NaCl 0.5g/L, micro-mixing solutions 10mL/L, dipotassium hydrogen phosphate 3g/L, potassium primary phosphate 3g/L adding distil water are settled to 1L.The inductor 5mL/L that adds, culture temperature is 30 ℃, and pH value is 7.0, and described inductor is diesel oil.Above-mentioned substratum or nutrient solution are all 121 ℃ of sterilizations 30 minutes.Cultivate after 3 days, bacterium liquid is transferred in the fresh substratum or nutrient solution, add the inductor of equivalent, repetitive operation 3 times.
Embodiment 2
The purifying of rhodococcus JZX-01
To be coated with in solid nutrient medium after the bacterium liquid dilution that obtain, the composition of solid nutrient medium is: the monovalent metallic ion of Zulkovsky starch 2g/L, ammonium sulfate 5g/L, NaCl 0.5g/L, trace element solution 10mL/L, agar 20g/L, phosphate radical or its hydrate 3g/L, dipotassium hydrogen phosphate 3g/L, potassium primary phosphate 3g/L adding distil water are settled to 1L.Picking list bacterium colony carries out preservation.After obtaining single bacterium colony, verify respectively each bacterial strain to the degradation capability of diesel oil, wherein the best bacterial strain called after JZX-01 of a strain degradation capability.
Embodiment 3
Carbon source is to rhodococcus JZX-01 affects on the growth
The rhodococcus JZX-01 that obtains carries out enlarged culturing in liquid nutrient media, the carbon source of liquid nutrient media is respectively Zulkovsky starch 2g/L, glycerine 2g/L, glucose 2g/L, nitrogenous source is ammonium sulfate 5g/L, NaCl0.5g/L, trace element solution 10mL/L, dipotassium hydrogen phosphate 3g/L, potassium primary phosphate 3g/L adding distil water is settled to 1L.Investigate respectively above-mentioned three kinds of carbon sources to the impact of thalli growth, found that the upgrowth situation of JZX-01 is best in the substratum that adds Zulkovsky starch 2g/L.
Nitrogenous source is to rhodococcus JZX-01 affects on the growth
The rhodococcus JZX-01 that obtains carries out enlarged culturing in liquid nutrient media, the nitrogenous source of liquid nutrient media is respectively extractum carnis 5g/L, beef leaching thing 5g/L, peptone 5g/L, yeast extract 5g/L, analysis for soybean powder immersion liquid 5g/L, Pidolidone ammonia 5g/L, ammonium sulfate 5g/L, ammonium chloride 5g/L, SODIUMNITRATE 5g/L, saltpetre 5g/L, carbon source is Zulkovsky starch 2g/L, NaCl 0.5g/L, trace element solution 10mL/L, dipotassium hydrogen phosphate 3g/L, potassium primary phosphate 3g/L adding distil water is settled to 1L.Investigate respectively above-mentioned ten kinds of carbon sources to the impact of thalli growth, found that the upgrowth situation of JZX-01 is best in the substratum that adds ammonium sulfate 5g/L.
Embodiment 5
Be used for cultivating the micro-mixed solution of JZX-01
Trace element of the present invention is the mixing solutions that must add in solid nutrient medium, liquid nutrient media and the minimal medium, for rhodococcus JZX-01 growth provides essential trace element.The composition of trace element mixed solution is: Na
2EDTAH
2O 1.2g/L, solid NaOH 0.6g/L, MnSO
44H
2O 0.4g/L, ZnSO
47H
2O 0.08g/L, 98% vitriol oil 0.06g/L, Na
2M
oO
42H
2O 0.2g/L, FeSO
47H
2O 0.03g/L, CuSO
45H
2O 0.01g/L, CaCl
20.3g/L and solid NaSO
40.5g/L.
Embodiment 6
Different inductors are to rhodococcus JZX-01 affects on the growth
Rhodococcus JZX-01 is in minimal medium, use different inductors to grow, the inductor of minimal medium is respectively that volume ratio is 1% alkane, crude oil, diesel oil, gasoline, liquid paraffin and polycyclic aromatic hydrocarbons, nitrogenous source is ammonium sulfate 5g/L, NaCl 0.5g/L, trace element solution 10mL/L, dipotassium hydrogen phosphate 3g/L, potassium primary phosphate 3g/L adding distil water is settled to 1L.Investigate respectively above-mentioned six kinds of inductors to the impact of thalli growth, found that the upgrowth situation of JZX-01 is best in the diesel oil substratum that adds 1% volume ratio.
Embodiment 7
The acquisition of the pure thalline of rhodococcus JZX-01 after the enlarged culturing
Rhodococcus JZX-01 is in liquid nutrient media, 30 ℃, growth is after 3 days in the shaking table of 200rpm, the nutrient solution of drawing 10mL exists, and after the abandoning supernatant, carries out shaking washing 10 minutes with aseptic inorganic salt solution in the whirlpool oscillator of 1000rpm, with the solution that mixes in the whizzer of 8000rpm centrifugal 10 minutes again, repeat said process 3 times, abandon clean supernatant liquor, obtain a large amount of rhodococcus JZX-01.
Embodiment 8:
The extraction of above-mentioned rhodococcus (Rhodococcus.Sp JZX-01) CGMCC No.5958 bacterial strain 16SrDNA gene
JZX-01 was cultivated 3 days in substratum or nutrient solution, obtain the cell culture fluid of high density.Get the nutrient solution of 1.5mL, carry out centrifugally under the rotating speed of 8000rpm, add the sterilization minimal medium in the throw out and wash, recentrifuge is abandoned supernatant, and the precipitation that obtains adds the broken bacterium damping fluid of 100uL, whirlpool concussion mixing.Then add successively 0.4g quartz sand, 300uL phenol: chloroform: the mixed solution of primary isoamyl alcohol (ratio is 25: 24: 1), the whirlpool concussion is three minutes again, carries out the bacterium broken wall treatment.After adding the slow damping fluid of TE of 100uL, whirlpool concussion mixing.Under the rotating speed of 12000rpm centrifugal 10 minutes afterwards, get the supernatant liquor of about 300uL.In supernatant liquor, add the 3M NaAC of 30uL and the dehydrated alcohol of 600uL, leave standstill more than 30 minutes under the room temperature behind the mixing.Under the rotating speed of 12000rpm centrifugal 8 minutes afterwards, abandon clean supernatant.The ethanol that adds such as 70% in the throw out washs, and centrifugal 5 minutes of 12000rpm abandons supernatant, natural air drying.In the sample after the oven dry, the EB damping fluid that adds 50uL carries out wash-out, is positioned over preservation in-20 ℃ the refrigerator behind the wash-out.So far, obtain the JZX-01 genomic dna.Take the genomic dna that extracts as template, to buy in the universal primer 27f of Shanghai bio-engineering corporation and 1492r as primer, add upstream primer 27f 2uL, downstream primer 1492r 2uL, adding template is the genomic dna 3uL of rhodococcus (Rhodococcus.Sp JZX-01) CGMCC No.5958 bacterial strain, plus master mix taq DNA synthetic enzyme 25uL behind the additional distilled water 18uL, joins among the pcr amplification instrument.The heating schedule of pcr amplification instrument is as follows: 94 ℃ were heated 4 minutes, 94 ℃ were carried out sex change 1 minute, 53 ℃ of temperature were annealed 30 seconds, 72 ℃ were extended 2 minutes, will from 53 ℃ to 72 ℃ loop 30 times, keep 10 minutes at 72 ℃ at last, reaction mixture is fully increased, after forward 4 ℃ to and stop amplified reactions, from the pcr amplification instrument, take out sample after for some time, be positioned over and carry out in 4 ℃ of refrigerators carrying out prolonged preservation under short-term preservation or-20 ℃.The 16S rDNA product that obtains of amplification takes out 5uL, verifies in containing 0.8% agargel electrophoresis, uses the gene marker of 100bp to carry out mark.The PCR product that contains purpose fragment (1500bp) mixes, and 0.8% agarose gel electrophoresis reclaims, and the electrophoresis liquid that more renews during recovery is cut glue purification, and the qualified PCR sample of electrophoresis detection checking is checked order.
Embodiment 9
The systematic evolution tree of rhodococcus JZX-01
Sequencing result according to rhodococcus compares, and 16 SrDNA gene order length of rhodococcus (Rhodococcus.sp) CGMCC No.5958 bacterial strain are 1376bp, and nucleotide sequence is as follows:
1 tcgaacgatg aagcccagct tgctgggtgg attagtggcg aacgggtgag taacacgtgg
61 gtgatctgcc ctgcacttcg ggataagcct gggaaactgg gtctaatacc ggataggacc
121 tcgggatgca tgttccgggg tggaaaggtt ttccggtgca ggatgggccc gcggcctatc
181 agcttgttgg tggggtaacg gcccaccaag gcgacgacgg gtagccggcc tgagagggcg
241 accggccaca ctgggactga gacacggccc agactcctac gggaggcagc agtggggaat
301 attgcacaat gggcgcaagc ctgatgcagc gacgccgcgt gagggatgac ggccttcggg
361 ttgtaaacct ctttcagtac cgacgaagcg caagtgacgg taggtacaga agaagcaccg
421 gccaactacg tgccagcagc cgcggtaata cgtagggtgc gagcgttgtc cggaattact
481 gggcgtaaag agctcgtagg cggtttgtcg cgtcgtctgt gaaaacccgc agctcaactg
541 cgggcttgca ggcgatacgg gcagacttga gtactgcagg ggagactgga attcctggtg
601 tagcggtgaa atgcgcagat atcaggagga acaccggtgg cgaaggcggg tctctgggca
661 gtaactgacg ctgaggagcg aaagcgtggg tagcgaacag gattagatac cctggtagtc
721 cacgccgtaa acggtgggcg ctaggtgtgg gtttccttcc acgggatccg tgccgtagct
781 aacgcattaa gcgccccgcc tggggagtac ggccgcaagg ctaaaactca aaggaattga
841 cgggggcccg cacaagcggc ggagcatgtg gattaattcg atgcaacgcg aagaacctta
901 cctgggtttg acatacaccg gaccgcccca gagatggggt ttcccttgtg gtcggtgtac
961 aggtggtgca tggctgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga
1021 gcgcaaccct tgtcctgtgt tgccagcacg taatggtggg gactcgcagg agactgccgg
1081 ggtcaactcg gaggaaggtg gggacgacgt caagtcatca tgccccttat gtccagggct
1141 tcacacatgc tacaatggcc ggtacagagg gctgcgatac cgcgaggtgg agcgaatccc
1201 ttaaagccgg tctcagttcg gatcggggtc tgcaactcga ccccgtgaag tcggagtcgc
1261 tagtaatcgc agatcagcaa cgctgcggtg aatacgttcc cgggccttgt acacaccgcc
1321 cgtcacgtca tgaaagtcgg taacacccga agccggtggc ctaacccctc gtggga
The 16S rDNA of JZX-01 is JQ 648650 by identifying at the state-run biotechnology NCBI of information center of the U.S. by the sequence number of submitting the 16S rDNA that obtains after the sequence to.Adopt the N-J method, the value of bootstrapping is the systematic evolution tree that obtains JZX-01 under 1000 the condition, and is shown in Figure 1 such as Figure of description.Find through comparison and systematic evolution tree, JZX-01 belongs to a strain of Rhod.
Claims (9)
1. a rhodococcus JZX-01 is characterized in that the common micro-organisms center by China Committee for Culture Collection of Microorganisms, and the registration number of preservation is the rhodococcus of CGMCC No.5958; Preservation date on April 9th, 2012.
2. the cultural method of a rhodococcus JZX-01 as claimed in claim 1 is characterized in that step is as follows:
(1) rhodococcus JZX-01 thalline or spore are received on the inclined-plane, cultivated 1-4 days on the inclined-plane, described medium component is carbon source 1-20g/L, univalent metal salt or its hydrate 1-5g/L of the monovalent metallic ion of nitrogenous source 1-5g/L, NaCl 1-2g/L, micro-mixing solutions solution 1-20mL/L, agar 1-30g/L, phosphate radical or its hydrate 1-5g/L, biphosphate, adding distil water is settled to 1L.
(2) thalline is obtained from the inclined-plane after, be transferred in the nutrient solution and cultivated 1-4 days, the composition of described nutrient solution is: the univalent metal salt of the monovalent metallic ion of nitrogenous source 1-5g/L, NaCl 1-2g/L, trace element solution 1-20mL/L, phosphate radical or its hydrate 1-5g/L, biphosphate or its hydrate 1-5g/L, inductor 1-10ML/L, adding distil water is settled to 1L, the pH value is 3-9, and culture temperature is 5 ℃-45 ℃.
3. rhodococcus JZX-01, it is characterized in that comparing by 16S rDNA order-checking and at NCBI and obtain, the upstream and downstream primer all is universal primer, upstream primer is 27F, concrete sequence is 5 '-AGAGTT TGA TCC TGG CTC AG-3 ', downstream primer is 1492R, and concrete sequence is 5 '-GGT TAC CTTGTT ACG ACT T-3 '.
4. cultural method as claimed in claim 3 is characterized in that described carbon source is to be Zulkovsky starch, glucose or glycerine.
5. cultural method as claimed in claim 3 is characterized in that described nitrogenous source is extractum carnis, beef leaching thing, peptone, yeast extract, analysis for soybean powder immersion liquid, Pidolidone ammonia, ammonium sulfate, ammonium chloride, SODIUMNITRATE or saltpetre.
6. cultural method as claimed in claim 3 is characterized in that described trace element is manganese, zinc, cobalt, nickel, magnesium, iron, calcium, copper, chromium, selenium, molybdenum, cobalt or fluorine.
7. cultural method as claimed in claim 3 is characterized in that described inductor adds in substratum or the nutrient solution sole carbon source as JZX-01 to and cultivates, and guarantees that bacterial classification can only utilize this kind carbon source to carry out growth metabolism; Described inductor is alkane, crude oil, diesel oil, gasoline, liquid paraffin or polycyclic aromatic hydrocarbons.
8. cultural method as claimed in claim 3 is characterized in that cultivating nutrient solution after 14 days at rotating speed 1000-10000rpm, carries out centrifugal 1-10 minute under temperature 0-20 ℃.
9. cultural method as claimed in claim 3 is characterized in that the described thalline JZX-01 that arrives after centrifugal with the minimal medium washing of carbonaceous sources not 1-3 time, removes the nutritive substance in the substratum, pure JZX-01 is joined in the substratum cultivate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012101989483A CN102851234A (en) | 2012-06-15 | 2012-06-15 | Rhodococcus JZX-01 used for degrading petroleum pollutants, and culturing method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012101989483A CN102851234A (en) | 2012-06-15 | 2012-06-15 | Rhodococcus JZX-01 used for degrading petroleum pollutants, and culturing method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102851234A true CN102851234A (en) | 2013-01-02 |
Family
ID=47398214
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012101989483A Pending CN102851234A (en) | 2012-06-15 | 2012-06-15 | Rhodococcus JZX-01 used for degrading petroleum pollutants, and culturing method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102851234A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104830721A (en) * | 2015-04-30 | 2015-08-12 | 大连民族学院 | Bacterium AJ07 having petroleum degradation function, use thereof, and seabed settlement petroleum degradation bacterium agent |
| CN105907675A (en) * | 2016-05-04 | 2016-08-31 | 大连海事大学 | Rhodococcus sp.QY-2 having low-temperature petroleum degrading function and application thereof |
| CN109182205A (en) * | 2018-10-09 | 2019-01-11 | 北京林业大学 | Rhodococcus sp and its application with carbon sequestration capacity |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974446A (en) * | 2010-07-29 | 2011-02-16 | 滨州学院 | Salt-tolerant Rhodococcus sp. JH for generating biological emulsifier and degrading alkane and application thereof in bioremediation of petroleum polluted saline-alkali soil |
-
2012
- 2012-06-15 CN CN2012101989483A patent/CN102851234A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974446A (en) * | 2010-07-29 | 2011-02-16 | 滨州学院 | Salt-tolerant Rhodococcus sp. JH for generating biological emulsifier and degrading alkane and application thereof in bioremediation of petroleum polluted saline-alkali soil |
Non-Patent Citations (1)
| Title |
|---|
| 张璐等: "石油烃降解菌Rhodococcus sp.15- 3 的分离鉴定及特性研究", 《农业环境科学学报》, 31 December 2008 (2008-12-31) * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104830721A (en) * | 2015-04-30 | 2015-08-12 | 大连民族学院 | Bacterium AJ07 having petroleum degradation function, use thereof, and seabed settlement petroleum degradation bacterium agent |
| CN105907675A (en) * | 2016-05-04 | 2016-08-31 | 大连海事大学 | Rhodococcus sp.QY-2 having low-temperature petroleum degrading function and application thereof |
| CN105907675B (en) * | 2016-05-04 | 2019-07-30 | 大连海事大学 | One plant of Rhodococcus sp and its application with Thermal degradation petroleum function |
| CN109182205A (en) * | 2018-10-09 | 2019-01-11 | 北京林业大学 | Rhodococcus sp and its application with carbon sequestration capacity |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Li et al. | Responses of ammonia-oxidizing microorganisms to biochar and compost amendments of heavy metals-polluted soil | |
| Maitra et al. | Ecological significance and phosphorus release potential of phosphate solubilizing bacteria in freshwater ecosystems | |
| CN104531585B (en) | A kind of Dell's Ford bacterium and its application | |
| CN103013859B (en) | Contaminated soil phenanthrene and application thereof in contaminated soil restoration | |
| CN109576187B (en) | Cyanide degradation strain and method for degrading cyanide by using same | |
| CN102242071B (en) | Application of Pichia guilliermondii 510-6jm in treatment of petroleum hydrocarbon contaminations | |
| CN109825449A (en) | It is a kind of degrade LAS and/or N bacterium and its application | |
| CN103215204A (en) | Arthrobacter strain highly effectively degrading phenanthrene, and application thereof | |
| CN105647838B (en) | Skin spy's acinetobacter calcoaceticus and application thereof | |
| CN100475950C (en) | Bacillus megaterium and use thereof | |
| CN107177530A (en) | A kind of new efficient domestic sewage denitrifier and its application | |
| Feng et al. | Characterization of Pseudomonas mendocina LR capable of removing nitrogen from various nitrogen-contaminated water samples when cultivated with Cyperus alternifolius L. | |
| CN101972774A (en) | Microbial repair method of oil-polluted wetland | |
| CN114854626A (en) | Pseudomonas strain for degrading polycyclic aromatic hydrocarbon pollutants and application thereof | |
| Briški et al. | Environmental microbiology | |
| Nyoyoko | Proteobacteria response to heavy metal pollution stress and their bioremediation potential | |
| KR20000034035A (en) | Bioaugmentation of oil contaminated soil by microbial composition which can decompose hydrocarbons derived from petroleum | |
| CN102851234A (en) | Rhodococcus JZX-01 used for degrading petroleum pollutants, and culturing method thereof | |
| CN108114978B (en) | A kind of method of the efficient rehabilitating soil of chemistry-microorganism | |
| Solomon et al. | Influence of biostimulation treatment using composted plant biomass on bacterial diversity of an aged petroleum contaminated soil as determined by culture-dependent and 16S rRNA gene PCR-DGGE based identification methods | |
| CN103381418B (en) | Method for processing tobacco waste or organic fluorine wastewater | |
| Sharma et al. | Potential of Citrobacter freundii for bioaccumulation of heavy metal–copper | |
| Dehnavi et al. | Biostimulation of petroleum-contaminated soils with synthetic and natural sources of NPK fertilizer | |
| CN114107101A (en) | Cadmium and chromium-fixed cupronickel bacterium and application thereof in co-remediation of cadmium and chromium composite polluted soil | |
| Uba et al. | Kinetics of biodegradation of total petroleum hydrocarbon in diesel contaminated soil as mediated by organic and inorganic nutrients |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20130102 |