CN103275967A - Method for obtaining botryococcus braunii single cells by combining Tween80 and ultrasonic waves - Google Patents
Method for obtaining botryococcus braunii single cells by combining Tween80 and ultrasonic waves Download PDFInfo
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- CN103275967A CN103275967A CN2013102049361A CN201310204936A CN103275967A CN 103275967 A CN103275967 A CN 103275967A CN 2013102049361 A CN2013102049361 A CN 2013102049361A CN 201310204936 A CN201310204936 A CN 201310204936A CN 103275967 A CN103275967 A CN 103275967A
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Abstract
The invention discloses a method for obtaining botryococcus braunii single cells by combining Tween80 and ultrasonic waves, which comprises the following steps of: collecting botryococcus braunii cells in a stable stage, and incubating the botryococcus braunii cells by use of Tween80; breaking the incubated botryococcus braunii cells by use of the mechanical force of ultrasonic waves; separating to obtain a single free cell; detecting the activity of the single cell and the like. The method disclosed by the invention successfully obtains the botryococcus braunii single cells, the nutrient substances and illumination are uniformly absorbed in a culture process, the preparation time is short, and the yield of the botryococcus braunii single cells is high.
Description
Technical field
The present invention relates to the single celled method of a kind of preparation clump grain algae, particularly a kind of use tween 80 and ultrasonic wave obtain the single celled method of clump grain algae.
Background technology
(1) present Research and the researching value of clump grain algae
From nineteen sixty-eight Maxwell etc. find out be full of the oily substance of being formed by unsaturated hydro carbons material in the grape phycocolloid colony after, and the The Nomenclature Composition and Structure of Complexes of grape hydrocarbon that algae produces is extremely similar to oil, and the little algae researcher in the whole world produces the hydrocarbon condition to its growth and carried out a large amount of research.Be clump grain algae (the Botryococcus braunii that string shape colony is given birth to, translate the grape algae again) be a kind of in the temperate zone, the little algae of the unicellular green of photoautotrophy type that widely distributes of the ground such as land, fresh water and brackish lake water of the torrid zone and continental climate band, its hydrocarbonaceous amount can be up to 86% of dry cell weight, be generally 25%-40%, therefore the product hydrocarbon amount that is higher than other microorganism far away utilizes grape algae production oil substitutes to be paid much attention to.Abroad just rise as far back as nineteen seventies and this algae is produced substitute energy launched a series of research, China's starting then later and rarely have research always.Analyzing its major cause is that clump grain algae poor growth, doubling time are long, becomes this algae and realizes industrialized one big obstacle.
(2) the little algae clump of the energy grain algae urgency of cultivating and dealing with problems
Still can't realize at present its large scale culturing well, because frond is the colony growth, frond blocks mutually in process of growth, influences frond and absorbs nutritive substance and illumination etc., and growth velocity is to limit this algae to produce hydrocarbon industrialized one big obstacle slowly.And a plurality of cells disunity on genetic background, be difficult to by the method for chemistry or physics it be carried out the seed selection of selection by mutation.Simultaneously a clump grain algae directly separates from nature, and proterties is generally than single stable, in the process of the little algae industrialization of the energy, is difficult to have breakthrough in the output of hydrocarbon and fat.Clump grain algae is main mode with schizogamy mainly, is difficult to carry out fine-variety breeding by the mode of sexual propagation.In the face of a kind of like this hydrocarbon fat content very high little algae of the energy all, cultivate not only that cost is low, output is high but also little to the pollution of environment, we should be devoted to solve the seed selection problem of the slow and good algae kind of its speed of growth.
At present, mainly be the related fields that concentrate on Physiology and biochemistry about the research of clump grain algae, seed selection and large-scale cultivation for good algae kind yet there are no relevant report., clump grain algae is cultivated the industrialization process that moves towards the industrialization from laboratory study, still face some problems: 1, still can't realize its large scale culturing at present well, growth velocity is to limit this algae to produce hydrocarbon industrialized one big obstacle slowly.Clump grain algae is a kind of frond that is the growth of string shape colony, frond is formed to a hundreds of cell by tens, size is about 300-500 μ m, because frond is the colony growth, frond blocks mutually in process of growth, influence frond and absorb nutritive substance and illumination etc., the frond poor growth, the doubling time is long; 2, a clump grain algae directly separates from nature, and proterties is generally than single stable, in the process of the little algae industrialization of the energy, is difficult to have breakthrough in the output of hydrocarbon and fat; 3, the mode of reproduction of clump grain algae mainly is monogony, based on schizogamy, for the seed selection of good algae kind, is difficult to carry out fine-variety breeding by the mode of sexual propagation; 4, the colony growth conditions of clump grain algae not only defines its growth velocity, and a plurality of cell disunity on genetic background, is difficult to by the method for chemistry or physics it be carried out the seed selection of selection by mutation.
Summary of the invention
The objective of the invention is to solve above-mentioned the deficiencies in the prior art, provide the free clump grain algae of a kind of preparation single celled method, it is unicellular that this method successfully obtains clump grain algae, and nutritive substance and illumination homogenization absorb in the culturing process, the preparation time cycle is short, the unicellular pick-up rate height of clump grain algae.
For achieving the above object, the present invention by the following technical solutions.
A kind of use tensio-active agent obtains the single celled method of clump grain algae in conjunction with ultrasonic wave, comprises following concrete steps:
1. collect the frustule that is in stationary phase, hatch frustule with tensio-active agent:
A clump grain algae frustule enters the stationary phase frustule because the accumulation of carotenoid becomes orange from green, also can in metabolic process, produce simultaneously and beneficiation of hydrocarbons and lipid, have only hydrocarbon and the lipid of few part can be secreted in the water surrounding, microscopically just can see that with tweezers applying light cover glass oil droplet constantly discharges from frond, with the observation of dyeing of Nile red fluorochrome, can find the accumulation of a large amount of hydrocarbon and lipid material, according to the accumulation of hydrocarbon fat, the color of dyeing becomes orange to arrive golden yellow not wait.
Use bolting silk to get rid of clump grain algae in process of growth behind own discharge unicellular, the algae liquid of collection 100ml is dissolved in the tensio-active agent of 10ml; The preferred 500 mesh sieve thin,tough silk that use, preferred surfactant is that volume fraction is 5% tween 80; Because clump grain algae frustule has the phenomenon of natural subsidence, and the centrifuge tube that frustule is housed is placed on the vibration instrument, makes hydro carbons and the complete emulsification of lipid material between frustule; Preferred vibration instrument is ultrasonic wave, vortex instrument and shaking culture instrument etc., and preferred rotating speed is 150rpm/min, hatches 30min;
(2) utilize hyperacoustic mechanical force that the frustule of having hatched is carried out fragmentation:
Frustule after hatching in the tensio-active agent in (1) is carried out ultrasonication; Preferred ultrasonic power is 200w, and ultrasonic time is 5s, and be 5s off time, and overall treatment time is 1min;
Supersound process is finished later frustule, with vacuum filtration pump and filter membrane, separates tween and frustule; The filter membrane of preferred 8 μ m.
Because the concentrated surfactant long time treatment can have stronger toxic action to frustule, can remove most tensio-active agent by suction filtration, after suction filtration is intact the frustule of staying on the filter membrane is resuspended in the substratum, preferred BG-11 substratum all washes the frustule on the filter membrane with pipettor.
Carry out the ultrasonication of a short period of time again for resuspended frustule, collect frustule with 8 μ m filter membranes through method of pumping filtration again; Preferred ultrasonic power 200w, ultrasonic 10s.Can find still to have the existence of tensio-active agent this moment, damages for fear of the late growing stage of residual surfactant to frustule, uses the resuspended flushing of aseptic substratum again.At last the frustule of handling is resuspended in the substratum, preferred culture medium is BG-11.Carry out microscopic examination, find to have occurred unicellular, but unicellular be with not fully the algae colony of cracking mix.
(3) separation obtains single free cell
The mixed algae liquid of the unicellular and algae colony that obtains with silk cover filtering is stayed bigger algae colony on the bolting silk, preferably uses 500 purpose bolting silks; Most single celled algae and bicelluar algae are filtered in the liquid, and the nutrient solution that filters is collected by filter membrane, are resuspended in the aseptic BG-11 substratum, preferably use the filter membrane of 8 μ m; Optimum regime can obtain 50.2% unicellular.
(4) detection of single cell active
Owing to consider in experimentation, tensio-active agent and ultrasonication be to the damaging action of frustule, so unicellularly carried out active detection for what obtain.In continuous training sample and observation process, find greater than the existing state of 90% frustule better: the cell proterties is stable, does not have breaking and the stripping of pigment of cytolemma, and part obtains unicellularly can also further divide after cultivating 3-4 days.
The factor that influences the inventive method implementation result is mainly the power of the time of ultrasonication, surfactant concentrations and ultrasonication; The optimum combination that the present invention draws is: ultrasonication 1min, and the concentration of tween 80 is 5%, the power of ultrasonication is 200w.
Advantage of the present invention is: at present, for solving clump grain algae speed of growth problem slowly, most researchers concentrates on the Physiology and biochemistry aspect to main energy, in the hope of strengthening clump cultivation of grain algae from improving the nutritional condition aspect, cycle is long like this, infusion of financial resources also can increase, can be from not solving the poky problem of clump grain algae in essence.Carry out genetic modification from the genetically engineered aspect to clump grain algae, the stretch that also still needs to grow very much will be walked.Utilize tween and ultrasonic wave to combine and obtain the unicellular of clump grain algae, namely solved clump grain algae because of the poky problem in settlement, also provide material foundation for utilizing induced-mutation technique to change its genetic material.The present invention has also successfully solved: 1, the problem that the homogenization of nutritive substance and illumination absorbs in the culturing process; The 2 clumps of grain algae poor growths, long problem of doubling time; 3, can strengthen the problem of the selection of germplasm to a certain extent; 4, carry out the problem that selection by mutation provides genetic background to unify material by chemical process and physical method, accelerate the seed selection speed of good algae kind.The present invention can conveniently obtain unicellular, and can repeatedly repeat in order to obtain a large amount of unicellular
Description of drawings
Fig. 1 is the unicellular figure of clump grain algae.
Fig. 2 is the detection figure of single cell active.
Embodiment
Embodiment 1:
1. collect the frustule that is in stationary phase, hatch frustule with tween 80
A clump grain algae frustule enters the stationary phase frustule because the accumulation of carotenoid becomes orange from green, also can in metabolic process, produce simultaneously and beneficiation of hydrocarbons and lipid, have only hydrocarbon and the lipid of few part can be secreted in the water surrounding, microscopically just can see that with tweezers applying light cover glass oil droplet constantly discharges from frond, with the observation of dyeing of Nile red fluorochrome, can find the accumulation of a large amount of hydrocarbon and lipid material, according to the accumulation of hydrocarbon fat, the color of dyeing becomes orange to arrive golden yellow not wait.
Being dissolved in the 10ml volume fraction with the algae liquid of 500 mesh sieve thin,tough silk (getting rid of clump grain algae own discharge unicellular in process of growth) collection 100ml is in 5% the tween 80, because clump grain algae frustule has the phenomenon of natural subsidence, the centrifuge tube that frustule is housed is placed on the vibration shaking table, rotating speed is 150rpm/min, hatch 30min, make hydro carbons and the complete emulsification of lipid material between frustule;
(2) utilize hyperacoustic mechanical force that the frustule of having hatched is carried out fragmentation;
Hatch 30min in tween 80 after, frustule is carried out ultrasonication by following condition: power is 200w, and ultrasonic time is 5s, and be 5s off time, and overall treatment time is 1min.
Supersound process is finished later frustule, and the filter membrane (the long 9 μ m-15 μ m of frustule, wide 6 μ m-9 μ m) with vacuum filtration pump and 8 μ m separates tween and frustule.Because high density tween 80 long time treatment can have stronger toxic action to frustule, can remove most tween 80 by suction filtration.After suction filtration is intact the frustule of staying on the filter membrane is resuspended in the 10mlBG-11 substratum, the pipettor of the frustule on the filter membrane with 1ml all washed.
Carry out the ultrasonication of a short period of time (power 200w, ultrasonic 10s) again for resuspended frustule, collect frustule with 8 μ m filter membranes through method of pumping filtration again.Can find still to have the existence of tween 80 this moment, damages for fear of the late growing stage of residual tween 80 to frustule, uses the aseptic resuspended flushing of BG-11 substratum again.The frustule of handling is resuspended in the BG-11 substratum of 10ml at last.Carry out microscopic examination, find to have occurred unicellular, but unicellular be with not fully the algae colony of cracking mix.
(3) separation obtains single free cell
The mixed algae liquid of the unicellular and algae colony that obtains with 500 purpose silk cover filterings, bigger algae colony is stayed on the 500 mesh sieve thin,tough silk, most single celled algae and bicelluar algae are filtered in the liquid, the nutrient solution that filters is collected by 8 μ m filter membranes, be resuspended in the aseptic BG-11 substratum, optimum regime can obtain 50.2% unicellular, it is as shown in Figure 1 unicellular that microscopy obtains.
(4) detection of single cell active
Owing to consider in experimentation, tween 80 and ultrasonication be to the damaging action of frustule, so unicellularly carried out active detection for what obtain.In continuous training sample and observation process, find greater than the existing state of 90% frustule better: the cell proterties is stable, does not have breaking and the stripping of pigment of cytolemma, and part obtains unicellularly can also further divide after cultivating 3-4 days.As Fig. 2: observe at 10 * 100 times microscopicallies: A figure is the single cell that expands; B figure is the frustule that is carrying out longitudinal division, and this type of cell fission becomes the cell among the E figure; C figure is the frustule that is tiltedly dividing, and the cell fission among this figure becomes the cell among the F figure; D figure is the cell that preliminary division is finished; G-H figure is the settlement of three cells and four cells.
Claims (7)
1. one kind is used tensio-active agent to obtain the single celled method of clump grain algae in conjunction with ultrasonic wave, comprises following concrete steps:
(1). collect the frustule that is in stationary phase, hatch frustule with tensio-active agent:
Use bolting silk to get rid of clump grain algae in process of growth behind own discharge unicellular, collection algae liquid was dissolved in the tensio-active agent in 10: 1 by volume; And place vibration to shake on the instrument, make hydro carbons and the complete emulsification of lipid material between frustule;
(2) utilize hyperacoustic mechanical force that the frustule of having hatched is carried out fragmentation:
Frustule after hatching in the tensio-active agent in (1) is carried out ultrasonication;
Supersound process is finished later frustule, with vacuum filtration pump and filter membrane, carries out separate surfactants and frustule; After suction filtration is intact the frustule of staying on the filter membrane is resuspended in the substratum, carries out the ultrasonication of a short period of time again for resuspended frustule, collect frustule with filter membrane through method of pumping filtration again; Use the resuspended flushing of aseptic substratum again; At last the frustule of handling is resuspended in the substratum, carries out microscopic examination, find to have occurred unicellular, but unicellular be with not fully the algae colony of cracking mix;
(3) separation obtains single free cell
The mixed algae liquid of the unicellular and algae colony that obtains with silk cover filtering, bigger algae colony is stayed on the bolting silk, most single celled algae and bicelluar algae are filtered in the liquid, and the nutrient solution that filters is collected by filter membrane, are resuspended in the aseptic substratum;
(4) detection of single cell active
In continuous training sample and observation process, find greater than the existing state of 90% frustule better: the cell proterties is stable, does not have breaking and the stripping of pigment of cytolemma, and part obtains unicellularly can also further divide after cultivating 3-4 days.
2. the method for claim 1, wherein bolting silk is 500 orders.
3. the method for claim 1, wherein described concussion instrument is ultrasonic wave, vortex instrument or shaking culture instrument.
4. the method for claim 1, wherein ultrasonic power is 200w in (2), and ultrasonic time is 5s, and be 5s off time, and overall treatment time is 1min.
5. the method for claim 1, wherein the filter membrane aperture is 8 μ m.
6. the method for claim 1, wherein substratum is the BG-11 substratum.
7. the method for claim 1, wherein described tensio-active agent is that percent by volume is 5% tween 80.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1654629A (en) * | 2005-01-17 | 2005-08-17 | 山东大学 | Process for preparing intact phycobilisome |
| CN1654627A (en) * | 2005-01-17 | 2005-08-17 | 山东大学 | Stabilization technology for intact phycobilisomes of red algae and blue algae |
| CN102321541A (en) * | 2011-08-05 | 2012-01-18 | 中国科学院过程工程研究所 | Botryococcus braunii magnetic separation method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1654629A (en) * | 2005-01-17 | 2005-08-17 | 山东大学 | Process for preparing intact phycobilisome |
| CN1654627A (en) * | 2005-01-17 | 2005-08-17 | 山东大学 | Stabilization technology for intact phycobilisomes of red algae and blue algae |
| CN102321541A (en) * | 2011-08-05 | 2012-01-18 | 中国科学院过程工程研究所 | Botryococcus braunii magnetic separation method |
Non-Patent Citations (1)
| Title |
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| 孙晓璐，等: "对产油脂酵母的细胞破碎方法及油脂提取效果的比较", 《河南工业大学学报(自然科学版)》 * |
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