CN105505915A - Method for screening high-activity formaldehyde-tolerant formaldehyde-degrading bacterium mutant strains through atmospheric and room temperature plasma (ARTP) mutagenesis - Google Patents
Method for screening high-activity formaldehyde-tolerant formaldehyde-degrading bacterium mutant strains through atmospheric and room temperature plasma (ARTP) mutagenesis Download PDFInfo
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Abstract
The invention relates to a method for screening high-activity formaldehyde-tolerant formaldehyde-degrading bacterium mutant strains through ARTP mutagenesis. A mutagenesis breeding system for a formaldehyde-degrading bacterial strain is constructed by using a novel ARTP jet mutagenesis system and is cooperated with a high-concentration formaldehyde gradient double-layer agar medium screening means so as to obtain a series of high-activity formaldehyde-tolerant mutant strains with changed degradation efficiency. Compared with formaldehyde tolerance concentrations reported at home and abroad, the mutagenesis bacterial strains prepared in the invention has a greatly improved formaldehyde tolerance concentration and can thus degrade formaldehyde at a high concentration.
Description
Technical field
The invention belongs to the application of plasma technique in field of biology, be specifically related to one and utilize the mutagenesis of atmospheric pressure at room plasma body to screen high reactivity tolerance formaldehyde degrading bacteria mutant strain method.
Background technology
Formaldehyde is a kind of colourless organic pollutant having intense stimulus smell, its primary pollution source is from the waste gas, waste water etc. of the industry discharges such as organic synthesis, plastics, coating, art work and paint, because formaldehyde is more easily rapidly absorbed into human body in contact site, biomass cells nuclear gene mutation, infringement DNA can be caused again after respiratory tract absorbs; High-concentration formaldehyde is then all toxic to neural system, immunity system etc., and formaldehyde is risen to I class carcinogenic substance by international cancer research institution (IARC), and therefore to carry out preventing and controlling significant to human health for PARA FORMALDEHYDE PRILLS(91,95) pollutent.The method of current removal formaldehyde pollution has charcoal absorption, nano-photo catalytic, ozone anion, plant to decompose, but these methods exist reagent consumption amount greatly, costly, the shortcoming such as formaldehyde effectively can not to be removed.And biological process degradation of formaldehyde has effective, the advantages such as cost is low, non-secondary pollution, therefore the microorganism that screening and separating has a stronger degradation of formaldehyde pollutent has become recent domestic study hotspot, and degradation effect is that lower concentration arrives high density screening trend on the whole.Current Degradation Formaldehyde bacterial strain does not have higher tolerance methanal concentration, and this is a great difficult problem.
Combined novel atmospheric pressure at room plasma body (ARTP) the biological induced-mutation breeding technique of independent research with Beijing Siqingyuan Bioscience Co., Ltd. by Tsing-Hua University, because it has jet temperature low (25 ~ 35 DEG C), active particle is evenly distributed, to human body and environment without harm, operate the advantages such as simple and easy, therefore ARTP provides platform method for utilizing systems biology and synthetic biology means to build high-performance bacterial strain.But at present there are no the report about use ARTP mutagenesis screening high reactivity tolerance formaldehyde degrading bacteria mutant strain method.
Summary of the invention
In order to solve above-mentioned prior art problem, the object of the present invention is to provide a kind of use ARTP mutagenesis screening high reactivity tolerance formaldehyde degrading bacteria mutant strain method, the mutagenic strain prepared of the method is adopted to be greatly improved at formaldehyde tolerable concentration, and then can degradation of formaldehyde at higher concentrations.
Technical scheme of the present invention is as follows: a kind of use ARTP mutagenesis screening high reactivity tolerance formaldehyde degrading bacteria mutant strain method, comprises the steps:
1) ARTP mutagenesis device is utilized to carry out mutagenesis to the Degradation Formaldehyde bacterial strain filtered out, using 99.99% helium as working gas, power be 115W, working gas flow is 10L/min, distance between plasma emission source and bacterial strain is 2mm, service temperature 23.0 ~ 35.0 ° of C, mutagenic treatment time 0.5min ~ 3min;
2) Agar Plating of concentration of formaldehyde in 1000 ~ 15000mg/L linearly gradient is prepared, one end concentration is high, the other end concentration is low, double-deck gradient plate is coated with the bacterial strain after ARTP mutagenesis, 30 DEG C of constant temperature culture a couple of days, select well-grown bacterium colony access slant medium in area with high mercury, obtain high reactivity tolerance methanal mutant strain.The described Degradation Formaldehyde bacterial strain filtered out is pseudomonas, and bacterial strain is negative bacterium, and bacterial strain is rod-short.
Further improve as the present invention, the described mutagenic treatment time is 2min, its lethality rate controls more than 99%, may be conducive to improving the tolerance degree of bacterial strain PARA FORMALDEHYDE PRILLS(91,95) and improving the degradation efficiency of PARA FORMALDEHYDE PRILLS(91,95), consider the factors such as energy conservation, choosing ARTP treatment time 2min is best Induced dosage
As the further improvement of such scheme, the inhibition concentration of described mutant strain is 10000mg/L≤MIC≤15000mg/L, this for ARTP treatment time 2min be the minimum inhibitory concentration of mutant strain, the upgrowth situation that relative wild strain is good in the formaldehyde flat board of 500mg/L, the tolerance of 2min mutant strain PARA FORMALDEHYDE PRILLS(91,95) improves nearly 20 times more than.
The described Degradation Formaldehyde bacterial strain filtered out obtains through enrichment culture and strains separation, described 1) enrichment culture: each for the active sludge of long period of soaking waste water 5g is added stirring in the Erlenmeyer flask of 100mLLB substratum and leave standstill, LB substratum (g/L): peptone 10 weight part, yeast extract paste 5 weight part, Nacl10 weight part, distilled water 1l, adjust pH to 7.0, 1 × 105Pa sterilizing 20min, getting 2mL supernatant liquor, to join concentration 50mg/L formaldehyde be in the LB substratum of carbon source, be placed in 30 ° of C shaking culture on 150r/min shaking table, 3d transfers 1 bacterium liquid, each by 2%(m/v) inoculum size is transferred to shaking culture in fresh culture, repetition like this 10 times, concentration of formaldehyde is increased to 100mg/L from 50mg/L simultaneously, 2) strains separation: on the solid plate utilizing coubling dilution to coat containing formaldehyde (50mg/L), purifying of single bacterium colony numbering that flat board grows also repeatedly being rule, to guarantee the purity of bacterial strain and the stable of Degradation Formaldehyde performance.
Accompanying drawing explanation
The bacterium colony of Fig. 1 strain isolated and the thalli morphology examined under a microscope;
The growth curve of Fig. 2 bacterial strain W1 and Degradation Formaldehyde rate curve figure;
The ARTP mutagenesis lethality rate curve of Fig. 3 bacterial strain W1;
The part bacterium colony enlarged diagram of Fig. 4 mutant bacteria;
Fig. 5 high reactivity mutant strain is to different mass concentration Degradation Formaldehyde process.
Embodiment
Below in conjunction with accompanying drawing, introduce embodiments of the invention in detail.
The screening and separating of 1.1 bacterial strains
Active sludge (being soaked by dyeing waste water for a long time) is gathered as bacterial screening sample from Wuhu Zhong Tian printing and dyeing mill.
1.2 reagent and instrument
Extractum carnis, peptone, formaldehyde solution massfraction is 37%, Nacl, MgSO
4, FeSO
47H
2o, MnSO
4h
2o, CaCl
22H
2o, (NH
4)
2sO
4, KH
2pO
4and K
2hPO
4thering is provided etc. fastening extra large Chemical Reagent Co., Ltd., Sinopharm Group, being analytical pure.
MG96G
tMpCR amplification instrument Lang Ji scientific instrument company limited; TGL-16G high speed freezing centrifuge Anting Scientific Instrument Factory, Shanghai; UV-2100 spectrophotometer UNICO(Shanghai) Instruments Co., Ltd.; OlympusCX41 biomicroscope Olympus Corp; DYCP-31D Horizontal electrophoresis tank Beijing Liuyi Instrument Factory; The automatic triple pure water distiller Shanghai Ben Bo Instrument Ltd. of SZ-97; Scanning electron microscope (ScanningElectronicMicroscope, SEM) FEIQuanta200 is the public instrument in Anhui Polytechnic University's Bioexperiment center.
1.3 substratum and culture condition
LB substratum (g/L): peptone 10, yeast extract paste 5, Nacl10, distilled water 1l, adjusts pH to 7.0,1 × 10
5pa sterilizing 20min.
Basic inorganic salt substratum (g/L): (NH
4)
2sO
41.0, K
2hPO
42.16, MgSO
40.1, KH
2pO
40.85, MnSO
4h
2o0.05, CaCl
2h
2o0.03, FeSO
47H
2o0.01, adding distil water is settled to 1L, adjusts pH to 7.0,0.68 × 10
5pa sterilizing 30min.
Methyl ethyl diketone solution: 6mL glacial acetic acid, 50g ammonium acetate, 0.5mL methyl ethyl diketone and 100mL distilled water.
Culture condition: following experiment is as without particular case, and all 30 ° of C constant incubator shaking culture, shaking speed 150r/min, cultivates 24h.
1.4 experimental technique
1.4.1 the screening and separating of Formaldehyde degradation bacterium:
Enrichment culture: each 5g of active sludge collecting long period of soaking dyeing waste water adds stirring in the Erlenmeyer flask of 100mLLB substratum and leaves standstill, getting 2mL supernatant liquor, to join concentration 50mg/L formaldehyde be in the LB substratum of carbon source, is placed in 30 ° of C shaking culture on 150r/min shaking table.3d transfers 1 bacterium liquid, presses 2%(at every turn
m/v) inoculum size is transferred to shaking culture in fresh culture, so repeat 10 times, concentration of formaldehyde is increased to 100mg/L from 50mg/L simultaneously.
Strains separation: on the solid plate utilizing coubling dilution to coat containing formaldehyde (50mg/L), purifying of single bacterium colony numbering that flat board grows also repeatedly being rule, to guarantee the purity of bacterial strain and the stable of Degradation Formaldehyde performance.
1.4.2 the drafting of bacterial strain W1 growth curve and the mensuration of degradation of formaldehyde
By the bacterial strain that separates by 2% inoculum size (
m/v) to be seeded in concentration of formaldehyde be in the LB nutrient solution of 100mg/L, under 30 ° of C conditions, shaking culture on rotating speed 150r/min shaking table, with LB nutrient solution for blank, every 2h pipettes rapidly 1mL in the centrifuge tube of sterilizing at sterilizing super clean bench, gets 1mL bacterium liquid in 414nm wavelength place densitometric
oDvalue, makes
oD-t curve, synchronized sampling measures the formaldehyde content of nutrient solution at 414nm place.
With reference to GB/T13197-1991.Get appropriate testing sample and add 25mL tool plug scale test tube, after distilled water diluting to scale marks, draw 2.5mL methyl ethyl diketone solution and add this test tube, and put upside down and for several times make it mix, 60 ° of C water-bath 15min, detecting the absorbancy at wavelength 414nm place after taking out room temperature cooling 1h, take water as reference.
1.4.3 the qualification of bacterial strain
Bacterial strain dyeing and physiological and biochemical analysis, adopt transmission electron microscope observing strain morphology.
Extract this bacterial strain complete genome DNA, adopt universal primer amplification 16SrDNA, amplified production submits Shanghai Sangon Biological Engineering Technology And Service Co., Ltd's order-checking to, is compared by this sequence BLAST on ncbi database.
According to the pseudomonas putida of announcing in NCBI (
pseudomonasputida) gene order, devise formaldehyde dehydrogenase (FDH) gene-specific primer.Fdh-F:5 '-CCATATGCTGGCAATCGTGGAGGT-3 ', fdh-R sequence is: 5 '-CTACTCGAGCGCCGCACCCCACATCTTGTGCG-3 ', is synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
1.4.4 the ARTP mutafacient system of bacterial strain
ARTP mutagenesis device is utilized to carry out mutagenesis to the Degradation Formaldehyde bacterial strain filtered out, using 99.99% helium as working gas, power be 115W, working gas flow is 10L/min, distance between plasma emission source and sample is 2mm, service temperature 23.0 ~ 35.0 ° of C.In order to find the best mutagenic treatment time, investigate bacteria lethal rate when irradiation time is 0.5min, 1min, 1.5min, 2min, 2.5min, 3min first respectively.To the sample processed coated plate under suitable extent of dilution, calculate its lethality rate by CFU, and obtain destruction curve
[9].
Lethality rate is calculated as follows:
LethalityUT(%)=（
U-T）/
U×100%
Note: wherein U is the total colony number without mutagenic treatment contrast bacterium, and T is total colony number corresponding after mutagenic treatment.
1.4.5 E-test screening high reactivity tolerance methanal bacterial strain
The Agar Plating of preparation concentration of formaldehyde distribution gradient, concentration of formaldehyde in substratum is gradient (1000 ~ 15000mg/L) linearly, one end concentration is high, the other end concentration is low, double-deck gradient plate is coated with the bacterial strain after ARTP mutagenesis, 30 DEG C of constant temperature culture a couple of days, selects well-grown bacterium colony in area with high mercury and access on slant medium, namely obtain high reactivity tolerance methanal mutant strain, cultivate the mensuration of ripe laggard row Degradation Formaldehyde rate.
2.1 Formaldehyde degradation bacterium screening and separating and qualifications
2.1.1 the identification of morphology of bacterial strain
Through enrichment repeatedly with add formaldehyde with minimal medium for sole carbon source purifying and cultivate, from the active sludge of dye house effluents process, screen a strain Formaldehyde degradation bacterium, called after W1.Bacterial strain W1 cultivates 24h under 30 ° of C conditions, and bacterium colony is spheroidal, regular edges, and smooth surface is moistening, has stickiness, has projection, is creamy white; Bacterial Gram dyeing display, this bacterial strain is negative bacterium.Be rod-short (Figure 1A: bacterium colony by scanning electron microscopic observation bacterial strain W1,1B: gramstaining (100 × oily mirror), 1C: scanning electron microscope image (5kV, 8.7mm × 10.0k)), measure the multinomial physiological and biochemical index of bacterial strain W1, result is as shown in table 1.With reference to " uncle Jie Shi Bacteria Identification handbook " and " common bacteria system identification handbook " etc., can preliminary evaluation bacterial strain W1 be pseudomonas.
Table 1 bacterial strain W1 bio-chemical characteristics result
2.1.2 the 16SrDNA sequential analysis of bacterial strain
The complete genome DNA extracting bacterial strain W1 does template, 16SrDNA universal primer 27F/1492R is utilized to carry out pcr amplification, result display amplified production size is about about 1.5kb, product is submitted to Shanghai Sangon Biological Engineering Technology And Service Co., Ltd's cloning and sequencing, the 16SrDNA nucleotide sequence reported in sequence and GenBank database is carried out BLAST compare of analysis, with pseudomonas putida (
pseudomonasputida) similarity reaches 96%, sequence length is 1485bp (AccessionNo.NC_002947.3).
2.2 bacterial strain W1 growth curves and Degradation Formaldehyde rate curve
As can be seen from Figure 3, the Degradation Formaldehyde curve of bacterial strain W1 and growth curve are substantially identical, and add substratum 10h early stage at bacterium liquid, be in lag phase because bacterial strain grows under new environment, thalli growth is slow, and therefore PARA FORMALDEHYDE PRILLS(91,95) degraded is also slightly slow; During entering logarithmic growth subsequently (10 40h), nutrient solution
oDvalue rises to 0.680 by 0.105, and Degradation Formaldehyde rate rises to 92.0% from 21%, illustrates that bacterial strain W1 also increases the degradation amount of PARA FORMALDEHYDE PRILLS(91,95) while of mushroom after media environment adaptation; After 40h
oDvalue lift velocity slows down until be tending towards constant, starts to enter stationary phase, and therefore Degradation Formaldehyde rate remains unchanged substantially; Because cell proliferation is more and more slower after stationary phase, thalline self-dissolving, death toll increases, and after 50h, cell enters decline phase, and Degradation Formaldehyde rate starts to be tending towards declining, and drops to 60% after 56h.Can determine that bacterial strain W1 is in incubation time 12 40h scope needed for logarithmic phase thus.
The mensuration of 2.3ARTP induced mutation rate lethality rate curve
Measure according to the ARTP lethality rate curve of method 1.4.4 PARA FORMALDEHYDE PRILLS(91,95) degradation bacteria strains W1, as shown in Figure 3.
As shown in Figure 4, ARTP has very strong lethal effect to bacterial strain W1, calculates by the lethality rate of formula to thalline, and during ARTP process 2min, its lethality rate reaches 99.25%, and during process 150s and 180s, lethality rate reaches 100%.Due to what suddenly change, there is randomness, relation between lethality rate and gain mutant is not fully aware of, lethality rate is controlled more than 99%, may be conducive to improving the tolerance degree of bacterial strain PARA FORMALDEHYDE PRILLS(91,95) and improving the degradation efficiency of PARA FORMALDEHYDE PRILLS(91,95), consider the factors such as energy conservation, ARTP treatment time 2min is chosen in this research is thus best Induced dosage.
The colonial morphology research of 2.4 mutant bacterias
According to the size of colony growth, rounding, abundance and growth characteristic, the bacterial strain W1 mutant strain obtained is observed.The mutant strain that mutagenesis 1.5min and 2min screens belongs to colony growthing slow type mutant, when through cultivating 3d, almost not long any bacterium colony, incubation time grows to 5d again, its colony diameter can not reach 2mm, but now naked eyes can be distinguished, again through the slat chain conveyor of 14d, at this moment mutant colonies becomes growth vigor bacterium, the colony diameter that 1.5min and 2min mutant is formed comparatively wild strain W1 large 100% and 200% respectively, after t test, difference reaches pole conspicuous level (P<0.01), and part bacterium colony enlarged view as shown in Figure 4.
The concentration gradient flat band method rapid screening of 2.5 high reactivity tolerance mutant strains
Adopt in the double-deck nutrient agar of different concns formaldehyde gradient and directly screen, test wild strain W1, the final tolerant formaldehyde maximum concentration of 1.5min and 2min mutant strain, different concns gradient plate statistics is as shown in table 2.
The impact that the different concentration of formaldehyde of table 2 grows wild mushroom W1 and mutant strain
Note: +++ represent and look; ++ represent and look general; + represent and look poor;-represent that single colony counts is rare
As shown in Table 2, along with the increase of concentration of formaldehyde, the single colony number of wild strain W1 on screening flat board reduces gradually, when concentration of formaldehyde reaches 1500mg/L, on flat board, colonial morphology is poor, and concentration of formaldehyde to repeat dull and stereotyped single bacterium colony when reaching 3000mg/L not long, to continue to increase when concentration of formaldehyde reaches 4000mg/L single bacterium colony on flat board not long yet, therefore can determine that the minimum inhibitory concentration (MIC, MinimumInhibitoryConcentration) of concentration of formaldehyde to wild strain W1 is 3000mg/L.And relative wild strain W1, the tolerable concentration of mutant strain 1.5min and 2min PARA FORMALDEHYDE PRILLS(91,95) has obvious degree to improve.Wherein 1.5min mutant strain starts to occur the rare phenomenon of bacterium colony at formaldehyde 7000mg/L, and 2min mutant strain growing way on the concentration of formaldehyde flat board of 10000mg/L is still better at present, and without any growth sign in the formaldehyde gradient plate of 15000mg/L, therefore concentration of formaldehyde is 10000mg/L≤MIC≤15000mg/L to the minimum inhibitory concentration of 2min mutant strain.The upgrowth situation that relative wild strain W1 is good in the formaldehyde flat board of 500mg/L, the tolerance of 2min mutant strain PARA FORMALDEHYDE PRILLS(91,95) improves nearly 20 times more than.
2.6 high reactivity tolerance mutant strains are to the calculating of different concns Degradation Formaldehyde rate
Sole carbon source using formaldehyde as bacterial strain metabolism, by the 2min mutant strain of the mutagenic obtained high reactivity tolerance formaldehyde degrading bacteria of ARTP, being respectively 500 containing concentration of formaldehyde, 1000,2000,3000,4000,5000, in the liquid nutrient medium of 6000mg/L, after shaking culture 24h, 48h, 72h, 96h, 120h, measure the degradation rate of this mutant strain and former wild strain W1 PARA FORMALDEHYDE PRILLS(91,95), result as shown in Figure 5.
Analyzed from the Degradation Formaldehyde rate of Fig. 5, after 24h cultivates, mutant strain 2min is when concentration of formaldehyde is 500mg/L, Degradation Formaldehyde rate is maximum, reach 70.7%, and wild strain W1 is 48.8% in this density loss solution rate, and the degradation speed of 2min mutant strain PARA FORMALDEHYDE PRILLS(91,95) is obviously accelerated than wild strain W1; As concentration of formaldehyde > 500mg/L, Degradation Formaldehyde rate tends to be steady with the increase of concentration of formaldehyde.The increase reflecting concentration of formaldehyde can in the degraded to a certain degree promoting bacterial strain to it, may be that formaldehyde is that the growth of bacterial strain provides good carbon source and the energy, along with the increase of concentration of formaldehyde, bacterium raised growth is bred, facilitate the biological degradation of PARA FORMALDEHYDE PRILLS(91,95), but when concentration of formaldehyde is excessive, likely intercept thalline and O
2contact, cause thalline can not growth and breeding well, amount of bacteria change is no longer obvious, and affect the ability of bacterial metabolism, the toxic action of formaldehyde to bacterial strain also increases, at this moment the tolerance of bacterial strain PARA FORMALDEHYDE PRILLS(91,95) reaches capacity, thus causes Degradation Formaldehyde rate larger change can not occur along with the increase of concentration of formaldehyde, shows as Degradation Formaldehyde rate < 20% cultivate 120h in the liquid nutrient medium of high-concentration formaldehyde after.But on the whole, 2min mutant strain, after the shaking culture of 120h, wherein up to 98%, also can reach 86.3% to the Degradation Formaldehyde rate of 1000mg/L to the degradation rate of concentration 500mg/L formaldehyde.
Above embodiment only in order to technical scheme of the present invention to be described, is not intended to limit; Although with reference to previous embodiment to invention has been detailed description, those of ordinary skill in the art is to be understood that; It still can be modified to the technical scheme described in foregoing embodiments, or carries out equivalent replacement to wherein portion of techniques feature; And these amendments or replacement, do not make the essence of appropriate technical solution depart from the spirit and scope of various embodiments of the present invention technical scheme.
Claims (5)
1. use an ARTP mutagenesis screening high reactivity tolerance formaldehyde degrading bacteria mutant strain method, comprise the steps:
1) ARTP mutagenesis device is utilized to carry out mutagenesis to the Degradation Formaldehyde bacterial strain filtered out, using 99.99% helium as working gas, power be 115W, working gas flow is 10L/min, distance between plasma emission source and bacterial strain is 2mm, service temperature 23.0 ~ 35.0 ° of C, mutagenic treatment time 0.5min ~ 3min;
2) Agar Plating of concentration of formaldehyde in 1000 ~ 15000mg/L linearly gradient is prepared, one end concentration is high, the other end concentration is low, double-deck gradient plate is coated with the bacterial strain after ARTP mutagenesis, 30 DEG C of constant temperature culture a couple of days, select well-grown bacterium colony access slant medium in area with high mercury, obtain high reactivity tolerance methanal mutant strain.
2. use ARTP mutagenesis screening high reactivity tolerance formaldehyde degrading bacteria mutant strain method according to claim 1, is characterized in that: described in the Degradation Formaldehyde bacterial strain that filters out be pseudomonas.
3. use ARTP mutagenesis screening high reactivity tolerance formaldehyde degrading bacteria mutant strain method according to claim 1, is characterized in that: the described mutagenic treatment time is 2min.
4. use ARTP mutagenesis screening high reactivity tolerance formaldehyde degrading bacteria mutant strain method according to claim 3, is characterized in that: the inhibition concentration of described mutant strain is 10000mg/L≤MIC≤15000mg/L.
5. use ARTP mutagenesis screening high reactivity tolerance formaldehyde degrading bacteria mutant strain method as claimed in any of claims 1 to 4, is characterized in that: described in the Degradation Formaldehyde bacterial strain that filters out obtain through enrichment culture and strains separation, described in
1) enrichment culture: each for the active sludge of long period of soaking waste water 5g is added stirring in the Erlenmeyer flask of 100mLLB substratum and leave standstill, LB substratum (g/L): peptone 10 weight part, yeast extract paste 5 weight part, Nacl10 weight part, distilled water 1l, adjusts pH to 7.0,1 × 10
5pa sterilizing 20min, getting 2mL supernatant liquor, to join concentration 50mg/L formaldehyde be in the LB substratum of carbon source, is placed in 30 ° of C shaking culture on 150r/min shaking table, and 3d transfers 1 bacterium liquid, presses 2%(at every turn
m/v) inoculum size is transferred to shaking culture in fresh culture, so repeat 10 times, concentration of formaldehyde is increased to 100mg/L from 50mg/L simultaneously;
2) strains separation: on the solid plate utilizing coubling dilution to coat containing formaldehyde (50mg/L), purifying that single bacterium colony numbering that flat board grows also repeatedly is rule.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109182183A (en) * | 2018-09-17 | 2019-01-11 | 天津科技大学 | A kind of method of high throughput breeding oil degradation mutant strain |
| CN114774403A (en) * | 2022-04-27 | 2022-07-22 | 湖州师范学院 | Targeting breeding method of agricultural lactic acid bacteria |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101993837A (en) * | 2010-06-21 | 2011-03-30 | 浙江工业大学 | High-activity high-tolerance formaldehyde degrading bacteria and application thereof |
| CN103031264A (en) * | 2012-12-31 | 2013-04-10 | 浙江升华拜克生物股份有限公司 | Salinomycin high-yield strain |
| CN104017756A (en) * | 2012-12-31 | 2014-09-03 | 浙江升华拜克生物股份有限公司 | Salinomycin strain |
-
2014
- 2014-09-26 CN CN201410502482.0A patent/CN105505915A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101993837A (en) * | 2010-06-21 | 2011-03-30 | 浙江工业大学 | High-activity high-tolerance formaldehyde degrading bacteria and application thereof |
| CN103031264A (en) * | 2012-12-31 | 2013-04-10 | 浙江升华拜克生物股份有限公司 | Salinomycin high-yield strain |
| CN104017756A (en) * | 2012-12-31 | 2014-09-03 | 浙江升华拜克生物股份有限公司 | Salinomycin strain |
Non-Patent Citations (1)
| Title |
|---|
| 孔芳 等: "一株甲醛降解菌的筛选及降解特性的研究", 《微生物学通报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109182183A (en) * | 2018-09-17 | 2019-01-11 | 天津科技大学 | A kind of method of high throughput breeding oil degradation mutant strain |
| CN114774403A (en) * | 2022-04-27 | 2022-07-22 | 湖州师范学院 | Targeting breeding method of agricultural lactic acid bacteria |
| CN114774403B (en) * | 2022-04-27 | 2023-08-08 | 至农科技发展(浙江)有限公司 | Targeted breeding method for agricultural lactic acid bacteria |
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