CN105087625A - Genetic conversion method of halophilic and basophilic sulfur alkali vibrio - Google Patents
Genetic conversion method of halophilic and basophilic sulfur alkali vibrio Download PDFInfo
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Abstract
The invention relates to the field of molecular biology, in particular to a genetic conversion method of halophilic and basophilic sulfur alkali vibrio. The genetic conversion method includes the following steps: 1), inoculating a strain after being separated and purified in a sulfur alkali vibrio synthetic culture medium for culturing for 12-18h, and collecting thalli when OD600 value is from 0.5 to 0.9; 2), preparing competent cell with capability of adsorbing exogenous DNA through a CaC12-NaC1 solution treatment method; 3), guiding exogenous plasmid with resistance into the competent cell in step 2) through heat shock response; 4), incubating the competent cell in which the plasmid is guided, coating on a resistant flat plate, and screening converted strains with resistance. By the genetic conversion method, a favorable tool is provided for studying molecular biology and gene engineering transformation of the sulfur alkali vibrio.
Description
Technical field
The present invention relates to biology field, particularly, the present invention relates to a kind of genetic transforming method addicted to salt basophilic sulphur alkali vibrios.
Background technology
Sulphur cycle, as integral part main in geochemical cycle, plays an important role in human being's production life.Particularly in recent years, along with the continuous deterioration of environment, take acid rain as the sulphur pollution of representative, bring great threat to the existence of the mankind.The sulfurous gas that acid rain is mainly produced by combustion of fossil fuel and volcanic eruption produces through oxidation.Sweet natural gas, as the one of fossil oil, can produce sulfurous gas in combustion.Mainly because containing a certain amount of hydrogen sulfide in Sweet natural gas, not only there is strong corrosive nature to equipment such as drilling well, sleeve pipe, gathering lines, and the sulfur dioxide pollution environment produced.The sulfide contained in Sweet natural gas has H
2s, mercaptan, thioether, dithiocarbonic anhydride and carbonyl sulfide, the process of Sweet natural gas biological desulfurization process to as if H2S, hydrogen sulfide (H2S) is a kind of hypertoxic gas, to all toxic effect of animal, microorganism, also can cause the poisoning of catalyst of petroleum refining process, reduce product yield, cause equipment and corrosion of piping, reduce work-ing life.Therefore, desulfurization is the important procedure of oil and natural gas oil-gas gathering and transportation process and petroleum chemicals processing.In the various sulfur methods of oil and natural gas, biological desulfurizing technology has the advantage such as economy, environmental protection, and correlative study at home and abroad in recent years all shows with application the impetus increased fast.H
2the removing sulfuldioxide of S mainly contains LO-CAT technique, Amine-Claus technique, membrane separation process and biological desulphurization etc.Biological desulphurization utilizes the metabolism of microorganism by H
2s is converted into elemental sulfur.H in the gas such as Sweet natural gas, biogas
2s, first by alkali liquor absorption, then, utilizes microbiological treatment absorption liquid, by H
2s is converted into elemental sulfur.Biological desulphurization has less energy-consumption, high-level efficiency, equipment simply, not produce the advantages such as secondary pollution.The scale that biological desulfurization process is applicable to is 100 ~ 10000Kg mono-Zhi Liu ∕ days; At this treatment scale, biological desulfurization process than LO-CAT technique and Amine-Claus technique advantageously.
Bacterial strain at present for biological desulphurization mainly contains sulphate reducing bacteria and oxide sulfate bacterium.Wherein sulphate reducing bacteria (SRB) is first found by Beijerinck for 1895, so far the history of existing over one hundred year.SRB is a kind of anerobe carrying out sulfate reduction metabolism, and in Gram-negative, take organism as electron donor, vitriol is electron acceptor(EA).According to different organism utilities, SRB is divided into 8 genus: Desulfovibrio (Desulfovibrio); Desulfotomaculum (Desulfotomaculum); Desulfurization monospore Pseudomonas (Desulfomonas); Desulfurization onion shape Pseudomonas (Desulfobulbus); Desulfobacter (Desulfobacter); Desulfococcus (Desulfococcus); Desulfurization Sarcina (Desulfosarcina); Desulfurization Spirochaetes (Desulfonema).Wherein front 4 genus utilize lactic acid salt, propionic salt, ethanol etc. as growth matrix, are only oxidized to acetate level, therefore are also called incomplete oxidation bacterium.Rear 4 genus are oxidized some lipid acid, particularly acetic acid in specific manner, and lactic acid, succsinic acid, phenylformic acid etc., are finally thoroughly degraded to CO
2, therefore be also called complete oxidation bacterium.SRB is quite active in the anaerobic waste water microbiological treatment being rich in vitriol, and it utilizes organism as electron donor, with SO
4 2-as final electron acceptor(EA), by dissimilation organic in waste water, obtain the material of synthetic cell and the required energy that sustains life.SO
4 2-the sulfide that reduction is formed is toxic and restraining effect, particularly H to system flora
2the toxicity of S is maximum.Sulfur oxidizing bacterium SOB is mainly divided three classes: thread thiobacterium, photosynthetic sulfur bacteria and colorless sulfur bacteria, major part belongs to chemosynthetic autotroph.Thread thiobacterium mainly comprises two genus, i.e. Beggiatoa (Beggiatoa) and Thiothrix (Thiothix).Because the elemental sulfur of generation is stored in cell paste by this bacterioid, bring difficulty to isolation andpurification, apply less in actual production.Colorless sulfur bacteria (Colourlesssulfurbacteria, CSB), wherein Thiobacillus (Thibacillus) is modal a kind of colorless sulfur bacteria in soil and natural water, is widely used in sulfate wastewater treatment research.But thiobacillus life condition is limited, can not survives in high salt high alkalinity environment, be subject to certain restrictions in the application.Sulphur alkali vibrios (Thioalkalivibrio) is a class photosynthetic sulfur bacteria, using sulfide or thiosulphate as electron donor, can obtain energy from light source, rely on special photosynthetic pigments in body, assimilation CO
2carry out photosynthesis.This bacterial strain in pH6.5 ~ 10.5, can grow under the condition of salinity 0.2 ~ 4.0M, and the sulfide-oxidation in environment can be become elemental sulfur, sulphite, vitriol etc.In addition, such biological sulfur oxidizing bacterium has the feature of high-sulfur rate of oxidation and high elemental sulfur productive rate, and does not need additionally to add organism, considerably reduces processing cost, is thus with a wide range of applications in the removing of hydrogen sulfide in natural gas.Sulphur alkali vibrios is typical autotrophic bacteria, and its growth velocity is slow, and if the sulphur simple substance produced reclaim not in time, be easy to be further oxided into inferior sulfate radical and enter liquid, bringing difficulty to industrial cycle process.Therefore, need the energy regeneration mechanism to this bacterium self, and associated metabolic mechanism is studied, this just needs us in gene aspect to its research and transformation.But at present more early stage separation and purification qualification and reactor application stage are still rested on to the research major part of this genus bacterium, about the correlated inheritance working method of this genus bacterium yet there are no bibliographical information.
At present, directed toward bacteria genetic transforming method has physical transformations, conjugal transfer, chemical conversion.Physical, mainly utilizes the method for electric shock to make cell surface occur aperture, DNA is entered in cell.This laboratory attempt electric shocking method transforms, and does not succeed.Conjugal transfer is applicable to most bacteriums, but needs to build transfer system, operates more loaded down with trivial details.The permeability that chemical method mainly changes film by the interpolation of some chemical reagent makes exogenous plasmid enter in cell to go, and the method that now conventional chemical method prepares competent cell has Hanahan method, Inoue method and CaCl
2method.These two kinds of methods are mainly for colibacillary, and operational requirement is high, particularly Hanahan method, and require very strict, agents useful for same purity requirement is very high, and cost is high.The method for transformation of present most of bacterium all by Mandel and Higa based on the experimental result of 1970, they proposed the CaCl with precooling at that time
2solution-treated bacterium, after of short duration heating, K phage DNA can infect bacteria.But, sulphur alkali vibrios as a kind of extreme microorganism, its cellularstructure and physiological habit special, by traditional CaCl
2method directly applies to this bacterium, is difficult to prepare competent cell, and reason is that sulphur alkali vibrios must have certain Na
+survive under the environment existed, receive ion in maintenance Premeabilisation of cells pressure, proton pump, flagellar movement aspect plays an important role.In addition, relatively deficient for the research prepared addicted to salt halophile competence, can the research data of reference relatively less, therefore there is certain initiative.
The present invention, on the basis of the method, transforms it and optimizes, and develops a set of genetic transforming method being applicable to sulphur alkali vibrios.The present invention provides molecular tool for this bacterium genetic manipulation, has very profound significance.Meanwhile, this invention is also for the foundation of the genetic operating system of sulfide linkage vibrios provides certain reference.
Summary of the invention
The object of this invention is to provide the high-efficiency genetic transforming method of a kind of sulphur alkali vibrios.
The genetic transforming method of sulphur alkali vibrios of the present invention, comprises the steps:
1) inoculation after separation and purification cultivated in sulphur alkali vibrios synthetic medium, incubation time is 12 ~ 18h, OD
600thalline is collected when value is between 0.5 ~ 0.9;
2) CaCl is passed through
2the method of-NaCl solution process, preparation has the competent cell of absorption foreign DNA ability;
3) by heat shock response, the plasmid steps for importing 2 with resistance by external source) competent cell;
4) hatched by the competent cell importing plasmid, be coated in resistant panel, screening has the conversion bacterial strain of resistance.
According to method of the present invention, described sulphur alkali vibrios, specifically can be ThioalkalivibrioversutusD301, its culture presevation is numbered CGMCCNo.8497.This sulphur alkali vibrios ThioalkalivibrioversutusD301 is preserved in (No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center on November 25th, 2013, Institute of Microorganism, Academia Sinica, 100101)
Above-mentioned addicted to salt basophilia sulphur alkali vibrios, there is (pH11.0,4.0MNa under the high-alkali condition of high salt
+) ability of oxidation of sulfureted produce elemental sulfur, elemental sulfur productive rate >90%, sulfur purity >99%.(pH9.0 ~ 12.0,0.5 ~ 4.0MNa in wider pH and salinity range
+) keep this stable ability, can process <3, the sulfide of 000mg/L, this bacterial strain has good application in gaseous bio desulfurization-sulfur recovery.
According to method of the present invention, described sulphur alkali vibrios liquid nutrient medium consists of:
NaHCO
30.7moL/L, NaOH0.125moL/L, Na
2s
2o
35H
2o0.08moL/L, K
2hPO
43H
2o0.009moL/L, NH
4cl0.005moL/L, KNO
30.005moL/L, MgCl
26H
2the trace element solution of O0.005moL/L, 2mL/L.
Trace element solution forms: EDTA0.02 × 10
-3moL/L, FeSO
47H
2o0.007 × 10
-3moL/L, ZnSO
47H
2o0.3 × 10
-3moL/L, MnCl
24H
2o0.15 × 10
-3moL/L, CoCl6H
2o0.84 × 10
-3moL/L, NiCl
26H
2o0.08 × 10
-3moL/L, Na
2moO
42H
2o0.12 × 10
-3moL/L, CaCl
22H
2o0.07 × 10
-3moL/L, H
3bO
34.84 × 10
-3moL/L.
According to method of the present invention, described sulphur alkali vibrios solid medium consists of in aforesaid liquid substratum the agar powder adding 1.5% ~ 1.7%, after sterilizing, is poured on solid plate, namely obtains solid medium.
According to method of the present invention, the CaCl adopted
2-NaCl solution total concn is 0.1 ~ 1.0mol/L, wherein, and Ca
2+with Na
+mol ratio is 5:1 ~ 1:1.
According to method of the present invention, step 3) in the temperature of heat shock response be 40 ~ 50 DEG C, the time is 1 ~ 3min.
According to method of the present invention, step 3) described in plasmid be preferably pKT230 or pJAK12.
The genetic transforming method of sulphur alkali vibrios of the present invention, concrete operation steps is as follows:
1) with 5% inoculum size inoculation, at 30 DEG C, cultivate 15 ~ 18 hours under 180rpm, measure its OD
600between 0.5 ~ 0.9; It is placed on rapidly in 0 DEG C of frozen water and freeze 1 ~ 2 hour, be divided in sterilized centrifuge tube by the bacterium liquid of gained, at 4 DEG C, rotating speed 8000rpm, centrifugal 10min, collect thalline;
2) be the CaCl of 0.1 ~ 1.0mol/L by total concn
2-NaCl solution (Ca
2+with Na
+mol ratio is 5:1 ~ 1:1) clean bacterial strain, carry out repeatedly successively, obtain competent cell; Thalline through cleaning is freezed 1 ~ 2 hour in 0 DEG C of frozen water, packing composition.
3) competent cell is placed on ice, adds a certain amount of plasmid pKT230 or pJAK12, mix fully, after leaving standstill 30 ~ 40min on ice, cell mixture is placed on rapidly in the water-bath of 40 ~ 50 DEG C, leave standstill 1 ~ 3min;
4) cell mixture is transferred to rapidly in 0 DEG C of frozen water the 1 ~ 2min that freezes, then add the substratum of 890uL, at 30 DEG C, rotating speed 120rpm, vibrate in shaking table 1 ~ 2h; From centrifuge tube, draw the cell suspension of 100 μ L, be coated on and have on antibiotic flat board, then cultivate 2 ~ 3 days in the incubator of 30 DEG C, observe colony growth situation, the bacterium colony picking grown out is done PCR qualification.
The invention provides a kind of CaCl
2-NaCl prepares competent cell jointly, the method for heat shock High-efficient Genetic Transformation bacterial strain.In method of the present invention, first bacterial strain is cultivated in synthetic medium OD
600between 0.5 ~ 1.0, then collect thalline, then with the CaCl containing NaCl
2solution cleans it, cleans multipass successively, finally places it in 0 DEG C of frozen water the 1 ~ 2h that freezes, packing composition.Efficient competent cell can being obtained by this step, the DNA of competent cell with 10 ~ 20 μ g is mixed, by leaving standstill 30min on ice, making the Ca combining DNA
2+can be adsorbed on the surface of cell, at a certain temperature, there is aperture in cytolemma, thus the DNA combined can be entered in the middle of cell, and then complete the conversion of foreign DNA in cell.In this process, Ca
2+very weak with the bonding force of cell.Therefore, should avoid rocking in operating process as far as possible, the transformation efficiency of bacterium can be improved so significantly.
What method provided by the invention can solve long-standing problem people to a certain extent is easy to dead addicted to competent cell in salt halophile genetic transformation process, the problem that transformation efficiency is lower, the conversion addicted to salt halophile not easily transformed not being only other provides reference, also for sulphur alkali vibrios molecular biology and genetic engineering modified wait study provide favourable instrument.The present invention also has that working method is easy, source chemicals is easy to get, preparation time is short, frozen service efficiency advantages of higher.
Accompanying drawing explanation
Fig. 1 is the strain culturing figure of the embodiment of the present invention.
Fig. 2 is the preparation figure of the competent cell of the embodiment of the present invention.
Fig. 3 is that the competent cell of the embodiment of the present invention hatches figure on ice.
Fig. 4 is the heat-shock transformed figure of plasmid of the embodiment of the present invention.
Fig. 5 is bacterial strain plate screening figure after the conversion of the embodiment of the present invention.
Fig. 6 is that the conversion bacterial strain of the embodiment of the present invention extracts plasmid gel electrophoresis qualification.
Fig. 7 is the pKT230 plasmid/pJAK12 Plastid transformation bacterial strain streptomycin resistance gene PCR gel electrophoresis qualification of the embodiment of the present invention.
ThioalkalivibrioversutusD301 is preserved in (No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center on November 25th, 2013, Institute of Microorganism, Academia Sinica, 100101).
Embodiment
The experimental technique used in following embodiment if no special instructions, be ordinary method, specifically can refer to concrete grammar listed in " Molecular Cloning: A Laboratory guide " (third edition) J. Pehanorm Brooker one book to carry out, or carry out according to test kit and product description; Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
The screening and separating purifying of embodiment 1 sulphur alkali vibrios Thioalkalivibrioversutus
Gather the saline and alkaline lakebed mud in Ordos, inner Mongolia area, get 1g sample and be placed in 100ml10g/L Sulfothiorine substratum, other compositions of substratum are as follows: 0.5 ~ 4.0MNaCl, 10g/LNaHCO
3, 5g/LKNO
3, 2g/LKH
2pO
4, 0.1g/LFeCl
3, 0.01g/LMnCl
2, 1MNaOH adjust pH to 10.30 DEG C, 180rpm shaking table shaking culture 48h, get 10ml suspension liquid and be inoculated in the above-mentioned Sulfothiorine substratum of 100mL, continue 30 DEG C, 48h cultivated by 180rpm shaking table.Above-mentioned enrichment culture liquid is carried out plate isolation, more multiple sieve in picking individual colonies access Sulfothiorine substratum.
Obtain a strain sulfur oxidizing bacterium through separation and purification repeatedly, bacterium colony is light yellow, and circular edge is neat, surface drying.Gramstaining is negative, and thalli morphology is vibrios.It is 99% that 16SrDNA sequential analysis shows with ThioalkalivibrioversutusDSM13738 homology, this sulfur oxidizing bacterium is accredited as a strain Thioalkalivibrioversutus, called after D301, this bacterium is preserved in (No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center on November 25th, 2013, Institute of Microorganism, Academia Sinica, 100101).
Sulfothiorine in substratum is replaced with sodium sulphite, adds sodium sulphite with the speed of 1g/ (Lh) to stream in substratum, this sulfur oxidizing bacterium can utilize sodium sulphite grow and produce elemental sulfur.Through X-ray diffraction spectroscopic identification, the crystalline form producing elemental sulfur is S
8.
The saline and alkaline lake mud of the Ordos, inner Mongolia taken a morsel further containing bacterium liquid, put in sulphur alkali vibrios liquid nutrient medium, at 30 DEG C, rotating speed 180rpm, shaking culture about 5 days in shaking table, from accessing in liquid nutrient medium containing drawing 5mL bacteria liquid, enrichment culture 3 generation successively, getting 100 μ L is coated on sulphur alkali vibrios solid medium, picking forms the bacterium colony of obvious sulphur, cultivate 2 days in access liquid nutrient medium, getting 100 μ L is again coated on solid medium, cultivate according to liquid-solid, purifying 3 generation, carry out the qualification of bacterial classification 16SrRNA, qualification result carries out Blast sequential analysis, similarity is higher than 98%, can tentatively predicate pure bacterium, further alternative way is that repurity cultivated for two generations, carry out identification and analysis, if result is stablized, namely can think that gained list bacterium colony is the pure bacterium of the sulphur alkali vibrios needed for us, to its preservation that spreads cultivation.
The preparation of embodiment 2 sulphur alkali vibrios bacterium liquid
Draw the seed liquor spread cultivation, with the inoculum size of 5%, at 30 DEG C, rotating speed 120rpm, shaking culture 15 ~ 18h in shaking table, measure OD
600scope is between 0.5 ~ 0.7, and in this stage, bacterial strain just enters logarithmic phase, produces sulphur less.
The preparation of embodiment 3 sulphur alkali vibrios competent cell
Be placed on rapidly by cultured bacterial strain in the mixture of ice and water of 0 DEG C, freeze at least 30min, is divided in sterilized centrifuge tube by the bacterium liquid of gained, and at 4 DEG C, rotating speed 8000rpm, centrifugal 10min, collect thalline.With 0.1 ~ 1.0mol/L, Ca
2+with Na
+mol ratio is the CaCl of 5:1 ~ 1:1
2-NaCl solution cleaning bacterial strain, at 4 DEG C, rotating speed 8000rpm, centrifugal 10min, collect thalline.With 0.1 ~ 1.0mol/L, Ca
2+with Na
+mol ratio is the CaCl2-NaCl solution of 5:1 ~ 1:1, and at static 30min on ice, at 4 DEG C, rotating speed 8000rpm, centrifugal 10min, collect thalline.Repeat previous step, the above-mentioned CaCl being less than 1mL will be added in gained bacterium liquid
2the solution of-NaCl solution 1/2 concentration, and add the glycerine of concentration 20% ~ 50%, phage solution is placed on and leaves standstill more than 2h on ice, namely competent cell is obtained, by obtained competent cell packing composition, take out small part and be used for transformation experiment, remaining leaves in the refrigerator of-80 DEG C, for future use.
The extraction of embodiment 4 plasmid pKT230, pJAK12
Be seeded in by intestinal bacteria containing plasmid pKT230, pJAK12 in LB substratum, cultivate after 12 ~ 18 hours, extract plasmid, the extraction of plasmid adopts AxyPrep plasmid extraction kit.
Embodiment 5 plasmid is heat-shock transformed
Competent cell is placed on ice, adds the DNA of 10 ~ 20 μ g, mix fully, leaving standstill 30min on ice.Next the cell mixture of gained is placed on rapidly in the water-bath of 40 ~ 50 DEG C, leaves standstill 1 ~ 3min.
What embodiment 6 transformed rear bacterial strain hatches cultivation, coated plate
Add the substratum of 890 μ L, at 30 DEG C, rotating speed 120rpm, vibrate in shaking table 1 ~ 2h, draws the conversion strain solution of 100uL, be coated on sulphur alkali vibrios streptomycin resistance solid medium, be placed in the incubator of 30 DEG C and cultivate 2h, the single bacterium colony transforming bacterial strain can be obtained.
The screening of embodiment 7 positive strain, qualification
Single bacterium colony on picking microbiotic flat board, receives in the liquid nutrient medium of resistance by its part, and a part carries out PCR clone, detects.The primer that PCR detects designs primer according to streptomycin resistance gene:
Upstream sequence 5 '-TTGAATCGAACTAATATTTTTTTGGT-3 '
Downstream sequence 5 '-CATACTGCAGACAGCGTGGACGAAC-3 '
Qualification result is as accompanying drawing 6.
In addition, the bacterial strain be transferred in liquid nutrient medium is extracted plasmid identification, and acquired results is shown in accompanying drawing 7.
Claims (7)
1. a genetic transforming method for sulphur alkali vibrios, comprises the steps:
1) inoculation after separation and purification cultivated in sulphur alkali vibrios synthetic medium, incubation time is 12 ~ 18h, OD
600thalline is collected when value is between 0.5 ~ 0.9;
2) CaCl is passed through
2the method of-NaCl solution process, preparation has the competent cell of absorption foreign DNA ability;
3) by heat shock response, the plasmid steps for importing 2 with resistance by external source) competent cell in.
2. method according to claim 1, is characterized in that, described sulphur alkali vibrios is ThioalkalivibrioversutusD301, and its culture presevation is numbered CGMCCNo.8497.
3. method according to claim 1, is characterized in that, described sulphur alkali vibrios synthetic medium comprises liquid nutrient medium, and described liquid nutrient medium consists of:
NaHCO
30.7moL/L, NaOH0.125moL/L, Na
2s
2o
35H
2o0.08moL/L, K
2hPO
43H
2o0.009moL/L, NH
4cl0.005moL/L, KNO
30.005moL/L, MgCl
26H
2the trace element solution of O0.005moL/L, 2mL/L;
Described trace element solution consists of: EDTA0.02 × 10
-3moL/L, FeSO
47H
2o0.007 × 10
-3moL/L, ZnSO
47H
2o0.3 × 10
-3moL/L, MnCl
24H
2o0.15 × 10
-3moL/L, CoCl6H
2o0.84 × 10
-3moL/L, NiCl
26H
2o0.08 × 10
-3moL/L, Na
2moO
42H
2o0.12 × 10
-3moL/L, CaCl
22H
2o0.07 × 10
-3moL/L, H
3bO
34.84 × 10
-3moL/L.
4. method according to claim 1, is characterized in that: the strain growth state preparing competent cell should be in the prometaphase of exponential growth, nutrient solution OD
600value is between 0.5 ~ 0.9.
5. method according to claim 1, is characterized in that, the CaCl adopted
2-NaCl solution total concn is 0.1 ~ 1.0mol/L, wherein, and Ca
2+with Na
+mol ratio is 5:1 ~ 1:1.
6. method according to claim 1, is characterized in that, step 3) in the temperature of heat shock response be 40 ~ 50 DEG C.
7. method according to claim 1, is characterized in that, step 3) in time of heat shock response be 1 ~ 3min.
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| CN108823128A (en) * | 2018-06-29 | 2018-11-16 | 中国科学院过程工程研究所 | A kind of sulfur oxidizing bacterium culture and active intensifying method and application |
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| CN103160535A (en) * | 2011-12-16 | 2013-06-19 | 江南大学 | Electrotransformation method for introducing shuttle plasmid into corynebacterium acetoacidophilum |
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| CN108823128A (en) * | 2018-06-29 | 2018-11-16 | 中国科学院过程工程研究所 | A kind of sulfur oxidizing bacterium culture and active intensifying method and application |
| CN108823128B (en) * | 2018-06-29 | 2021-11-02 | 中国科学院过程工程研究所 | Sulfur oxidizing bacterium culture and activity enhancement method and application |
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Application publication date: 20151125 Assignee: Yabang green process and new materials research institute Nanjing Co., Ltd Assignor: Institute of Process Engineering, Chinese Academy of Sciences Contract record no.: X2019110000013 Denomination of invention: Genetic conversion method of sulfur alkali vibrio Granted publication date: 20190315 License type: Common License Record date: 20191227 |