CN104711249A - Artificial microalgae strain breeding method - Google Patents
Artificial microalgae strain breeding method Download PDFInfo
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- CN104711249A CN104711249A CN201310691127.8A CN201310691127A CN104711249A CN 104711249 A CN104711249 A CN 104711249A CN 201310691127 A CN201310691127 A CN 201310691127A CN 104711249 A CN104711249 A CN 104711249A
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Abstract
The present invention relates to an artificial microalgae strain breeding method, which comprises normal pressure and room temperature plasma mutagenesis of microalgae strains and algae strain screening based on absorbance and specific dye luminescence intensity change. The artificial microalgae strain breeding method is characterized in that the normal pressure and room temperature plasma mutagenesis technology, the microalgae growing testing based on absorbance, and the microalgae grease evaluation method based on the fluorescent dye are combined, and the artificial microalgae strain breeding method has characteristics of efficient and rapid target algae strain obtaining compared with the existing microalgae strain breeding method.
Description
Technical field
This patent belongs to microdisk electrode technical field (C12N1/12), is a kind of micro-algae algae strain method for artificially breeding.
Background technology
Micro-algae is autotrophic type microorganism, because there is photosynthesizer in its cell also referred to as micro-algae plant.The ability (i.e. photosynthetic capacity) that micro-algae has efficient absorption sun power, stabilizing carbon dioxide produces biomass; Micro-algae is as the one of microorganism simultaneously, and its rate of propagation is very fast.As unicellular organism, micro-algae mainly consist of albumen, fat, carbohydrate and nucleic acid etc.At present, micro-algae be important breeding bait by breeding enterprise widespread use, micro-algae has also become the source of the protective foodss such as carotenoid, protein, polysaccharide simultaneously.Along with the expection of fossil energy exhaustion aggravates, micro-algae have also been obtained very big concern in the potential application of energy goods and fine chemicals.
At present, many directly the separation from nature of the algae kind of production application obtains, and the proterties of offspring is single, easily occurs the problems such as and algae kind degeneration poor to the adaptive faculty of environment.Carry out rejuvenation or the excellent algae strain of seed selection again of algae kind in a conventional manner, often need the longer cycle and spend a large amount of manpowers.In order to obtain high yield and meet the algae strain that downstream refines demand, the breeding technique of micro-algae must be improved, thus the kind matter of micro-algae is improved.
Atmospheric pressure at room plasma body (ARTP) is a kind of new plasma source grown up in recent years, can produce under normal pressure (1am) temperature between 25 ~ 40 DEG C, the plasma jet with high reactivity particle (as being in the helium atom, Sauerstoffatom, nitrogen-atoms, OH free radical etc. of excited state) concentration.Scientific research shows, in plasma body, the active particle of suitable dosage acts on microorganism, can make structure and the permeability changes of microorganism wall/film, and cause gene damage, and then make microbial gene sequences and metabolism network noticeable change thereof, finally cause microorganism to produce teratogenesis sudden change.
Conventional micro-algae Strain selection is also a longer job consuming time, because detection method is all carried out based on acquisition certain biomass basis mostly.As, conventional microalgae grease assessment at least needs hundreds of milliliter micro-algae algae liquid, on average needs the incubation time of month from solid plate to required biomass.And simultaneously, mutagenesis screening may obtain up to a hundred alternative algae strains, therefore, after suitable mutagenesis, triage techniques is also that micro-algae algae is selected good strains in the field for seed an important ring of educating.
Along with the raising of various dyeing process sensitivity, utilizing staining to carry out assessment to microalgae biomass needs biomass to be greatly less than ordinary method, possibility is provided for improving screening, as, iodine liquid can dye to intracellular starch, can dye etc. with fluorescence dye Nile red to intracellular neutral fat.
Therefore, based on above-mentioned analysis, the present invention proposes a kind of novel micro-algae algae strain method for artificially breeding, plasma body mutagenesis is combined with based on the rapid screening dyeed, there is efficient, to obtain the strain of target algae fast feature.
Summary of the invention
The object of this invention is to provide a kind of micro-algae algae strain method for artificially breeding.
The domestication of wild algae strain and improvement are that microalgae industryization production realizes necessary, to select good strains in the field for seed the speed of educating to improve micro-algae algae, present invention incorporates the atmospheric pressure at room plasma body mutagenesis of green non-pollution, the Strain selection technology based on absorbancy and specific fluorescence dye fluorescence intensity change, realize target algae plant height effect, the object obtained fast.
The present invention utilizes atmospheric pressure at room plasma body to carry out mutagenesis to wild algae strain, namely uses room temperature atmospheric plasma instrument to carry out mutagenesis to micro algae culturing liquid.Wherein microalgae density to be mutagenic is in 100-1000 ten thousand cells/ml, mutagenesis micro-algae algae liquid is at 10 ~ 200 μ L, and output power of power supply is 50 ~ 200W, and working gas flow is 1 ~ 20SLM, the spacing of plasma emission source and sample is 1 ~ 10mm, 5 seconds ~ 2 minutes discharge time.
After described mutagenesis operation terminates, the micro-algae after mutagenic treatment is coated on solid medium, cultivates under selective pressure (temperature, light intensity, defect substratum) or regular culture conditions, in contrast with the algae liquid of non-mutagenic treatment simultaneously.The successful criterion of mutagenesis: under regular culture conditions, after being born growing to algae, carry out algae to mutagenesis and control group respectively to fall to counting, be more than or equal to 90% index that is successful mutagenesis with lethality rate=(1-mutagenesis algae fall number/contrast algae to fall number) × 100%, drop into row 96 orifice plate cultivate screening to growing mutagenesis algae; For under selective pressure, the algae grown falls and namely cultivates screening for 96 orifice plates.
Described algae is dropped in 96 orifice plates and carries out liquid culture, and the spectrophotometer utilizing microplate reader or have an orifice plate scanning function measures micro-algae OD value that orifice plate is cultivated, and obtains frustule growing state.Can select different determined wavelength according to the strain of different microalgae algae, as chrysophyceae uses 650 ~ 680nm to detect usually, green alga uses 750nm to detect usually.
The micro-algae of described liquid culture is dyeed by specific stain agent, and to develop the color, power detects the target metabolite condition of production.Usually utilize iodine liquid to dye to starch in cell, with fluorescence dye Nile red or BODIPY, neutral fat in cell or grease are dyeed.
Finally, according to screening object, in conjunction with being grown on production of metabolites situation, corresponding mutagenesis algae strain is determined, for follow-up study or production practice.
The present invention uses plasma body to carry out mutagenesis to micro-algae under room temperature, normal atmosphere, low voltage environment, and avoid the toxicity of Typical physical chemomorphosis method or strong cell injury, the treatment time is short simultaneously, mutagenic treatment process only clock in a measure; 96 orifice plates are utilized to cultivate as screening basis, OD is detected and specific chromogenic reaction bonded, avoid in conventional sense process and need longer incubation time to obtain the drawback of enough biomass, can measure a large amount of algae strain simultaneously, fall from algae time shorten to the week left and right that preliminary screening goes out the strain of target algae.Therefore the relatively conventional micro-algae algae of the present invention select good strains in the field for seed educate be a kind of green, efficiently, novel method fast.
Accompanying drawing explanation
Fig. 1 lsochrysis zhanjiangensis mutagenic treatment time and lethality rate curve.
The relative growth rate of Fig. 2 wild strain and 23 strain mutagenesis algae strains.
The relative growth rate of the relative wild strain of Fig. 3 mutagenesis algae strain and relative unit OD Nile red fluorescence intensity.
The mutagenesis Strain selection that Fig. 4 is target with high temperature resistant and high grease.
Embodiment
Embodiment 1:
With lsochrysis zhanjiangensis (Isochrysis zhangjiangensis FACHB-1750, be preserved in Chinese Academy of Sciences's Wuhan hydrobiont institute algae kind preservation center) be object, utilize the atmospheric pressure at room plasma body breeding machine (ARTP) of Tsing-Hua University's chemical engineering and Department of Engineering Physics's joint research and development to carry out the mutagenesis of chrysophyceae.
1, lsochrysis zhanjiangensis nutrient solution concentration is adjusted to 100 ~ 1,000 ten thousand cells/ml, gets 10 ~ 200 microlitre algae liquid and be put on slide glass.
2, be placed on microscope carrier by slide glass, the distance of adjustment jet exit and slide glass is less than or equal to 10mm.
3, output power of power supply is 50 ~ 200W, and working gas flow is 1 ~ 20SLM, 5 seconds ~ 2 minutes discharge time.
4, slide glass 800 microlitre golden alga culture solutions (natural sea-water+F/2 substratum) are rinsed, and as far as possible all reclaim nutrient solution, be coated with for follow-up solid medium.
5, the mutagenesis algae liquid under the different treatment condition being coated with recovery on solid F/2 substratum, is positioned over 25 degree, periodicity of illumination 14h:10h, light intensity 50 μ Em
-2s
-1cultivate in incubator.Cultivate on solid medium in contrast with the algae of the non-mutagenic treatment of same volume.
6, through three to four days, visible solid substratum grows single algae and fall.Be more than or equal to 90% for standard with lethality rate, fall to being transferred in 96 orifice plates by the micro-algae algae on solid medium under this condition, each algae falls to taking 1 hole, has added 100 microlitre golden alga culture solutions in hole in advance.96 orifice plates are positioned over 25 degree, periodicity of illumination 14h:10h, light intensity 50 μ Em
-2s
-1cultivate in incubator.
7, microplate reader is utilized every day to carry out OD mensuration at 655nm place.
8, according to the Nile red dyeing condition of standard, the chrysophyceae that orifice plate is cultivated being dyeed, using the spectrophotofluorometer with orifice plate scanning function to detect, for detecting neutral fat content in cell.
9, maximum specific growth rate calculating is carried out according to OD change, meanwhile, the Nile red fluorescence under Units of Account OD, and be index according to Nile red fluorescence under maximum specific growth rate and unit OD, determine the strain of candidate's mutagenesis algae, carry out subsequent production or research work.Wherein, treatment time and lethality rate relation, relative growth rate and relative unit OD Nile red fluorescence intensity level are shown in accompanying drawing 1 ~ 3.
Embodiment 2:
The operation such as mutagenesis, with embodiment 1, is that target is screened with high temperature resistant, the plate be painted with is placed in 36 DEG C of cultivations after mutagenesis; Light intensity is 30 ~ 50 μm of olm
-2s
-1, Light To Dark Ratio is 14:10.
Final acquisition 9 strain is high temperature resistant and the strain of high fat content algae, and wherein contrast is the wild strain under regular culture conditions.
Embodiment 3:
Sub-heart-shaped four slit bamboo or chopped wood algaes (Tetraselmis subcordiformisFACHB-1751 the is preserved in Chinese Academy of Sciences's Wuhan hydrobiont institute algae kind preservation center) algae of high yield starch marine green algae is selected good strains in the field for seed and educates.Carry out mutagenesis with 1,500,000 cells/ml four slit bamboo or chopped wood algaes, the spacing of plasma emission source and sample is 2mm, and nutrient solution is natural sea-water+Kang Wei side nutritive salt, mutation time 30s.After mutagenesis, four slit bamboo or chopped wood algae algae liquid are diluted 10,000 times, Tu Kangwei side's solid plate.Other operation is with embodiment 1.
Through mutagenesis, lethality rate is 99.6%.The starch content of mutagenesis algae strain with iodine dye colour developing value for index is investigated.
Claims (5)
1. a micro-algae algae strain method for artificially breeding, it is characterized in that: comprise micro-algae algae strain atmospheric pressure at room plasma body mutagenesis and the Strain selection flow process based on absorbancy and specific fluorescence dye fluorescence intensity change, concrete operations comprise:
(1) atmospheric pressure at room plasma body is utilized to carry out mutagenesis to natural algae strain: to use room temperature atmospheric plasma instrument to carry out mutagenesis to micro algae culturing liquid;
(2) the micro-algae after mutagenic treatment is coated on solid medium, cultivate under selection pressure condition or under regular culture conditions, in contrast with the algae liquid of non-mutagenic treatment simultaneously, for under regular culture conditions, after being born growing to algae, carry out algae to mutagenesis and control group respectively to fall counting, be more than or equal to 90% index that is successful mutagenesis with lethality rate=(1-mutagenesis algae fall number/contrast algae to fall number) × 100%, drop into row 96 orifice plate cultivate screening to growing mutagenesis algae; For under selective pressure, the algae grown falls and namely cultivates screening for 96 orifice plates;
(3) utilize microplate reader or spectrophotometer to measure micro-algae OD value that orifice plate is cultivated, obtain frustule growing state;
(4) utilize dyestuff to dye to the microalgae cell of liquid culture in orifice plate, measure the accumulation of endocellular metabolism thing;
(5) with the speed of growth change or metabolite content changing conditions for index, finally determine the strain of target algae.
2., according to algae algae strain method for artificially breeding micro-described in claim 1, it is characterized in that: natural algae strain comprises green alga, chrysophyceae, diatom unicellular algae or spirulina.
3. according to algae algae strain method for artificially breeding micro-described in claim 1, it is characterized in that: microalgae density to be mutagenic is in 100-1000 ten thousand cells/ml, mutagenesis micro-algae algae liquid is at 10 ~ 200 μ L, output power of power supply is 50 ~ 200W, working gas flow is 1 ~ 20SLM, the spacing of plasma emission source and sample is 1 ~ 10mm, 5 seconds ~ 2 minutes discharge time.
4., according to algae algae strain method for artificially breeding micro-described in claim 1, it is characterized in that: regular culture conditions is target microalgae conventional culture conditions; The feature of selective pressure condition for having according to the strain of expectation mutagenesis algae, under regular culture conditions, raise or reduce temperature, strengthen or weaken illumination, use auxotroph substratum or contain heavy metal ion, halogen-containing organic compound, hydrocarbon compound or antibiotic substratum.
5. according to algae algae strain method for artificially breeding micro-described in claim 1, it is characterized in that: according to the mutagenesis algae strain proterties expecting to obtain, with the speed of growth improve or reduce, can survive under selection pressure condition and cell internal object metabolite content height for index, determine final target algae strain.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110885816A (en) * | 2019-12-04 | 2020-03-17 | 西安建筑科技大学 | Method for mutagenizing and screening microalgae with high oil yield by ARTP |
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| CN101597571A (en) * | 2009-05-11 | 2009-12-09 | 新奥科技发展有限公司 | Sudden change algae strain of tool high growth rates Dunaliella salina and complex mutation breeding method thereof |
| CN102220307A (en) * | 2011-05-26 | 2011-10-19 | 武汉中科光谷绿色生物技术有限公司 | Method for mutagenizing micro algae to generate docosahexaenoic acid by use of ion beam bioengineering |
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Application publication date: 20150617 |