CN102212471B - Microalgae high-throughout culture equipment - Google Patents
Microalgae high-throughout culture equipment Download PDFInfo
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Abstract
The invention provides microalgae high-throughout culture equipment comprising a frame, an oscillating mechanism, a temperature control device, a humidity control device, a ventilation control device, an objective table and a lighting device, wherein the objective table is suitable for fixing a culturing structure for containing an algae seed or algae liquid, the lighting device is arranged below the objective table and is suitable for providing even lighting for microalgae culture, the lighting device consists of a light source and a light guiding structure which comprises a light guiding layer, light reflection structures are arranged on the side surfaces and bottom surface of the light guiding layer so as to guide light from the light source to emit out from one surface of the light guide layer, therefore, light from the light source is evenly provided to the culturing structure containing the algae seed or algae liquid, and the lighting uniformity degree and lighting intensity for microalgae high-throughout culture can be improved so as to meet the demands of microalgae growth and high-throughout screening.
Description
Technical field
The present invention relates to little algae commercial application field, and particularly, relate to being suitable in the high flux screening that is used in the algae kind and carry out the equipment that little algae high-throughput is cultivated.
Background technology
Little algae be a class on land, the high holophyte of widely distributed, nutritious, the photosynthetic availability in ocean.Many little algaes can accumulate a large amount of greases in cell, fatty acid content can reach 80% of dry cell weight when some algae lacked at the nitrogen element, can extract grease from the little algae of this class by the method for biorefinery, for the preparation of edible oil and biofuel; Little algae also can be used in the industrial processes such as wastewater treatment, to reduce environmental pollution.
The environmental friendliness characteristic of little algae, to the health care characteristic of humans and animals and the applications well of bioenergy thereof; caused global little algae bioenergy research and development upsurge; and it is close towards mass-producing, industrialization gradually; but commercial development also has many technical bottlenecks to need to break through; wherein the improvement of little algae algae kind is the most important, and the screening of good little algae algae strain is the most important thing.The screening of good little algae algae strain must compare by they different performances, as the biochemical composition composition in the speed of growth, the cell, to the resistance of environment etc., and then takes all factors into consideration and selects the algae strain that is applicable to that mass-producing is cultivated.
(High throughput screening, HTS) technology is a kind of new and high technology for aspects such as new compound exploitation and purpose strain improvements that grows up the eighties in 20th century to high flux screening.Its basic characteristics are to be carrier with the microwell plate, adopt the method for high-density, micro-automatization application of sample, can fast and parallel ground test great amount of samples.High-throughput techniques has parallelization, automatization, traceization and intensive advantage concurrently.High flux screening becomes drug development and microorganism field has a lot of comparatively successful application.
Utilizing little algae to prepare in the research of biofuel and high value added product, the screening of algae kind occupies an important position, and wherein high flux screening will play an important role, and reduces in the research process consumption for human and material resources and time widely.In addition, can also provide strong technical guarantee for the bigger genetic engineering breeding of development potentiality, advance the process of little algae related industriesization.Rational and effective cultural method and condition control are cultivated for algae kind high-throughput has important effect.
At present, the system that the laboratory is used for the cultivation of algae kind high-throughput is relatively simple, the cultivation of algae strain generally all is to place growth cabinet in the triangular flask system, the ventpipe aerated culture is cultivated or is simply inserted in stuffiness, illumination and temperature are regulated and control by growth cabinet, temperature control is more even, but this mode is difficult to realize the consistent of illumination and ventilatory effect.Also have a kind of culture systems to place shaking table exactly in addition, illumination and temperature are regulated and control by shaking table, by shaking the acceleration gaseous interchange, the ventilatory effect of this mode is relative with temperature consistent, but faces the problem that uneven illumination is even, intensity of illumination can't satisfy algal grown and high flux screening needs equally.In addition, also there is the big and micro algae growth of system problem slowly in above-mentioned dual mode.
And existing illumination box or illumination shaking table are to adopt fluorescent tube to carry out illumination, not only exist outside the problems such as uneven illumination is even, the illuminance decay is obvious, also have defectives such as heat production is big, the life-span short, the illuminance adjusting is discontinuous.Such culture apparatus only is fit to adopt the triangular flask training method at present, if be used for cultivating microminiaturized laboratory sample, these problems can be more outstanding.In addition, the top light of traditional shaking table photograph and vessel fixed form have also limited the use of microwell plate, aspect solution ventilation, mixing, crossed contamination, evaporation and illumination, can't satisfy microminiaturized high-throughout experimental technique and implement especially.
Summary of the invention
In view of this, the objective of the invention is to solve at least one aspect of the above-mentioned problems in the prior art and defective.
One of purpose of the present invention is to provide a kind of equipment that little algae high-throughput is cultivated that is suitable for carrying out, and it can improve illumination uniformity coefficient and the intensity of illumination of cultivating for little algae high-throughput, satisfies the needs of micro algae growth and high flux screening.
According to an aspect of the present invention, a kind of equipment that little algae high-throughput is cultivated that is suitable for carrying out is provided, comprise framework, oscillating mechanism, temperature-control device, humidity conditioner, ventilation controller, Stage microscope and means of illumination, wherein, Stage microscope is suitable for being fixed for holding the cultivation structure of algae kind or algae liquid, means of illumination is arranged on the below of Stage microscope, be suitable for little algae cultivation uniform illumination is provided, and means of illumination comprises light source and light guide structure, and light guide structure is configured to be suitable for the light from light source is provided equably to the cultivation structure that accommodates algae kind or algae liquid.
Be suitable for carrying out in the equipment of little algae high-throughput cultivation above-mentioned, this light guide structure can be multilayered structure, comprise: optical waveguide layer, be suitable for the light from light source is reflected equably to all directions, incide the optical waveguide layer from a pair of side of optical waveguide layer from the light of light source; The bottom reflection film covers the bottom surface of optical waveguide layer, is suitable for returning optical waveguide layer to the luminous reflectance of described bottom reflection by optical waveguide layer; With the offside reflection film, another of covering optical waveguide layer is suitable for and will returns optical waveguide layer to described another luminous reflectance to offside reflection by optical waveguide layer the side.
Be suitable for carrying out the equipment that little algae high-throughput is cultivated above-mentioned, the ratio of the area of each side in the area of the end face of optical waveguide layer and the described a pair of side can be greater than 10: 1.
Be suitable for carrying out on the submarginal summit portion of optical waveguide layer, can also being provided with reflectance coating in the equipment of little algae high-throughput cultivation above-mentioned, last reflectance coating is suitable for the light reflected back optical waveguide layer from end face periphery inclination outgoing.
Carry out in the equipment of little algae high-throughput cultivation above-mentioned being suitable for, at least one in described bottom reflection film and the offside reflection film can be total reflection film.
Be suitable for carrying out in the equipment of little algae high-throughput cultivation above-mentioned, optical waveguide layer can be made of base material and the leaded light powder or the leaded light particle that are evenly distributed in the base material, the light that enters optical waveguide layer reflects when meeting with leaded light powder or leaded light particle, at least a in reflection and the transmission, and base material can comprise polymethyl methacrylate materials, and leaded light powder or leaded light particle can comprise nanostructure.
Be suitable for carrying out in the equipment that little algae high-throughput cultivates above-mentioned, this multilayered structure can also comprise all light film that is positioned on the optical waveguide layer end face, is suitable for the upwards emission equably of light from the end face outgoing of optical waveguide layer.
Be suitable for carrying out in the equipment that little algae high-throughput cultivates above-mentioned, this multilayered structure can also comprise equal tabula rasa, is arranged on the top of optical waveguide layer, is suitable for the light from the end face outgoing of optical waveguide layer upwards is transmitted into the cultivation structure that accommodates algae kind or algae liquid equably.
Be suitable for carrying out going up equal tabula rasa and all can separating predetermined distance between the light film in the equipment that little algae high-throughput cultivates above-mentioned, with at last equal tabula rasa with all form the space of being scheduled between the light film.
Be suitable for carrying out in the equipment that little algae high-throughput cultivates above-mentioned, at last equal tabula rasa with all can be provided with bracing or strutting arrangement between the light film, this bracing or strutting arrangement is suitable for adjusting the described predetermined distance that separates, to adjust the volume in described predetermined space.
Carry out in the equipment of little algae high-throughput cultivation above-mentioned being suitable for, described light source can comprise led array, and can be provided with lens between led array and light guide structure, and these lens project in the optical waveguide layer after being suitable for assembling from the light of led array.
Above-mentionedly be suitable for carrying out the equipment that little algae high-throughput cultivates and can also comprise light intensity controller, it provides light intensity to the current control of led array from the led array outgoing by control.
Be suitable for carrying out in the equipment of little algae high-throughput cultivation above-mentioned, framework is suitable for forming and carries out little algae cultured microalgae culture environment, wherein Stage microscope and means of illumination are arranged in this framework, temperature-control device is suitable for controlling the temperature in little algae culture environment, humidity conditioner is suitable for controlling the humidity in little algae culture environment, ventilation controller is suitable for controlling the ventilation in little algae culture environment, and oscillating mechanism is suitable for vibrating in little algae culturing process, and wherein means of illumination is arranged on the oscillating mechanism.
Description of drawings
Consult follow-up diagram and can understand above-mentioned and further feature and advantage of the present invention better with description.Not detailed row and non-restrictive example then please refer to the description of this subsequent figures in the literary composition.Element in the diagram might not meet ratio, and is that the mode of emphasizing is depicted principle of the present invention.In each diagram, components identical is represented with identical Reference numeral in the difference diagram.In the accompanying drawings:
Fig. 1 illustrates the synoptic diagram that is suitable for carrying out the component part of the equipment that little algae high-throughput cultivates according to of the present invention;
Fig. 2 shows and wherein is provided with the shaker platform that is suitable for carrying out the means of illumination of the equipment that little algae high-throughput cultivates according to of the present invention;
Fig. 3 A is the enlarged diagram of the A part among Fig. 2;
Fig. 3 B is the enlarged diagram of the B part among Fig. 2;
Fig. 3 C is the enlarged diagram of the C part among Fig. 2;
Fig. 3 D is the enlarged diagram of the D part among Fig. 2;
Fig. 4 is the synoptic diagram that illustrates from the direct of travel of light in light guide structure of light source; And
Fig. 5 illustrate the frustule that utilizes high-throughput culture device of the present invention microminiaturized cultivate and conventional training method under dependency.
Embodiment
The present invention is described further below in conjunction with the drawings and the specific embodiments.
Fig. 1 shows the equipment that is suitable for carrying out little algae high-throughput cultivation according to of the present invention.As shown in Figure 1, be suitable for carrying out the equipment 100 that little algae high-throughput cultivates and comprise casing or skeleton construction 1.Body structure 1 is divided into two portions, and in the left part in Fig. 1, the transmission rig 3 that is provided with shaker platform 2 and supports shaker platform 2 and be suitable for shaker platform 2 is vibrated can be realized the shaking culture of little algae thus.Wherein, shaker platform 2 is provided with means of illumination 5, and it is used for having the microwell plate of little algae or porous plate 10 that illumination is provided to the inoculation that is placed on shaker platform 2 tops.Porous plate 10 can comprise 96 orifice plates, 48 orifice plates, 24 orifice plates and 12 orifice plates etc.According to one embodiment of the present invention, porous plate 10 can be cultivated bearing structure by other little algae and replace, as shakes bottle or triangular flask etc.
In one embodiment, porous plate 10 is fixed in the body structure 1 by Stage microscope (not shown), means of illumination 5 is positioned at the below of porous plate 10, supply with light source from its bottom, thereby be convenient to the porous plate top seal, can prevent crossed contamination, guarantee ventilation, evaporate consistence, increase rotating speed, increase ventilatory effect etc.
In body structure 1, can also be provided with temperature sensor 6, add wet hole 7, heating blower 8, refrigeration fan 9, vaporizer and compressor (not shown) etc., be provided with control device 4 in the left part in Fig. 1, thus to cultivating the environment of little algae in the body structure 1, comprise that temperature, humidity, ventilation etc. control, realize being suitable for the good environment that little algae is cultivated.
Fig. 2 A shows and wherein is provided with the shaker platform that is suitable for carrying out the means of illumination in the equipment that little algae high-throughput cultivates according to of the present invention.Wherein in Fig. 2, the lower left is the vertical view of shaker platform, and the upper left side is the sectional view of edge I-I line intercepting wherein, and the lower right is the sectional view of edge II-II line intercepting wherein.According to one embodiment of the present invention, means of illumination 5 is arranged in the shaker platform 2, can shake with shaker platform 2, and have the porous plate 10 of little algae that illumination is provided to the inoculation of shaker platform 2 tops, can carry out wave and culture to little algae thus.
As shown in Figure 2, shaker platform 2 surfaces are provided with a plurality of perforates 201, as square opening, make and can pass perforate 201 projections to porous plate from the light that is arranged on shaker platform 2 subsurface means of illumination 5.Can fit together by screw 202 etc. between shaker platform 2 and the means of illumination under it 5.According to the embodiment of the present invention, means of illumination 5 is between upper cover plate 203 and base plate 204.Upper cover plate 203 only covers the bight part of means of illumination 5, so that beam projecting.Base plate 204 covers means of illumination 5 fully and provides support to it.
As shown in Figure 2, means of illumination 5 comprises light source 501 and light guide structure, and it is arranged on the both sides of light guide structure, preferably is arranged on the relative both sides of light guide structure, incident beam in the light guide structure.According to one embodiment of the present invention, light source 501 adopts led array, this can overcome, and heat radiation such as conventional light sources such as fluorescent tubes is big, uneven illumination even, the illuminance decay obviously, short, illuminance of life-span regulates defectives such as discontinuous.Be supplied to the electric current of led array by adjusting, can regulate its intensity of illumination.
In the present invention, light guide structure is configured to be suitable for providing the porous plate 10 that little algae is arranged to inoculation equably with the light from light source.Embodiments of the present invention adopt the bottom for the light mode, and means of illumination 5 is arranged on the below of porous plate 10, and the light guide structure guiding is upwards throwed towards porous plate 10 equably from the light of light source 501.
Fig. 3 A-3D shows in detail the structure according to means of illumination of the present invention, wherein Fig. 3 A is the enlarged diagram of the A part among Fig. 2, Fig. 3 B is the enlarged diagram of the B part among Fig. 2, and Fig. 3 C is the enlarged diagram of the C part among Fig. 2, and Fig. 3 D is the enlarged diagram of the D part among Fig. 2.
According to the embodiment of the present invention, shown in Fig. 3 A and 3B, light guide structure is multilayered structure, the bottom reflection film 504 that comprises optical waveguide layer or light guiding plate 503 and covering optical waveguide layer 503 bottom surfaces, optical waveguide layer 503 be suitable for from the light of light source 501 incidents equably to all directions reflection, refraction or transmission, bottom reflection film 504 is suitable in the light reflected back optical waveguide layer 503 with directive optical waveguide layer 503 bottom surfaces.Below bottom reflection film 504, reflector 506 can also be set, its can be further with the light of directive optical waveguide layer 503 bottom surfaces with penetrate in the light reflected back optical waveguide layer 503 of bottom reflection film 503.
According to one embodiment of the present invention, on the end face of optical waveguide layer 503, can also be provided with all light films 507, all light film 507 can carry out homogenizing to the light from optical waveguide layer 503 outgoing.In another embodiment of the invention, on the submarginal summit portion of optical waveguide layer 503, reflectance coating 509 can also be set, last reflectance coating 509 is suitable for and will attempts from the light reflected back optical waveguide layer 503 of end face periphery inclination outgoing.
In the present invention, reflector 506, bottom reflection film 504, optical waveguide layer 503, all light film 507, go up reflectance coating 509 and be arranged in turn between base plate 204 and the upper cover plate 203, and be formed with in the upper cover plate 203 and be suitable for the opening that passes through from the light of optical waveguide layer 503 outgoing.According to one embodiment of the present invention, can also be provided with all tabula rasas 508 above the optical waveguide layer 503, it is positioned at the following of upper cover plate 203 and supports upper cover plate 203, and is suitable for further homogenizing from the light of optical waveguide layer 503 outgoing.
According to one embodiment of the present invention, shown in Fig. 3 A-3C, going up can be spaced a predetermined distance between equal tabula rasa 508 and the optical waveguide layer 503, in order to form certain space 510 between last equal tabula rasa 508 and optical waveguide layer 503.From the light of optical waveguide layer 503 outgoing in this space 510 with the air collision of for example air and so on, diffuse-reflectance takes place, make that the light of outgoing is more even.In one embodiment, between last equal tabula rasa 508 and optical waveguide layer 503, be provided with adjustable for height bracing or strutting arrangement, support copper post 511 shown in Fig. 3 B, it can adjust the size of above-mentioned predetermined distance, in order to adjust the volume in described space 510, irreflexive degree takes place thereby can adjust from the light of optical waveguide layer 503 outgoing, namely described space is more big, it is more abundant that diffuse-reflectance is carried out, and the light of outgoing is more even.
According to the embodiment of the present invention, shown in Fig. 3 A and 3B, led array 501 is arranged on the substrate 502, as aluminium base.Substrate 502 is fixed on the side group plate 205, and led array 501 is to two light incident side face throw lights of optical waveguide layer 503.Side group plate 205 can be L shaped shape, covers the side of light guide structure fully, and extends certain-length below light guide structure.The side group plate 205 that light guide structure extends below is fixing by the coupling device such as screw 206 with base plate 204, to form skeleton construction.Between optical waveguide layer 503 and the side group plate 205 and on bracing or strutting arrangement 208 and 209 such as packing ring all can be set between tabula rasa 508 and the optical waveguide layer 503.
In the present invention, shown in Fig. 3 C, two other place, side at light guide structure namely in non-two sides (for example, perpendicular to two sides of light incident side face) that are subjected to light of optical waveguide layer 503, also is provided with offside reflection film 505.Light from led array 501 wherein incides in the optical waveguide layer 503 after lens 511 (shown in Fig. 3 D) are assembled, reflect the back from the end face outgoing of optical waveguide layer 503 through bottom reflection film 504 and offside reflection film 505, projecting inoculation equably has on the porous plate 10 of little algae.
Fig. 3 D shows the detailed construction of light source.Wherein, led array 501 is electrically connected to substrate 502, is provided with lens 511 above the LED unit at each, and lens 511 are used for converging to optical waveguide layer 503 from the light that led array penetrates.
Fig. 4 illustrates light path or the direct of travel of incident light in light guide structure from led array in more detail.As shown in Figure 4, optical waveguide layer 503 is made of base material 5030 and the leaded light powder or the leaded light particle 5031 that are evenly distributed in the base material 5030.Base material 5030 for example can be polymethylmethacrylate (PMMA) material, and it has high refractive index usually.Leaded light powder or leaded light particle 5031 can be nanometer leaded light powder or particle.
According to the embodiment of the present invention, the light beam that led array 501 sends throws after lens 511 are assembled and enters in the optical waveguide layer 503, because leaded light particle 5031 distributes in base material 5030 equably, therefore every bundle light must have two kinds of possibilities after entering optical waveguide layer, the one, move ahead along original route, another kind then be meet with leaded light particle 5031 reflect, reflection or transmission.The light that enters optical waveguide layer 503 experienced repeatedly reflect, reflect after, six of the directive optical waveguide layer faces equably.Owing to be subjected to two sides of light and place, bottom surface to be provided with reflectance coating or reflector respectively optical waveguide layer non-, as 504,506 and 505, the bottom reflection film 504 that covers optical waveguide layer 503 bottom surfaces will be attempted the light that penetrates from optical waveguide layer 503 bottom surfaces reflected back optical waveguide layer 503 again, offside reflection film 505 will be attempted to be subjected to light that two sides of light penetrate reflected back optical waveguide layer 503 again from optical waveguide layer 503 non-, and final light can only be from end face and the outgoing of two light incident side faces of optical waveguide layer 503.According to the present invention, the area of the end face of optical waveguide layer 503 is set to much larger than the area of light incident side face, and then all light is almost all from the top surface side outgoing equably of optical waveguide layer 503.According to one embodiment of the present invention, the end face of optical waveguide layer is set to greater than about 10: 1 with described another ratio to the area of each side in the side, make the light that enters light guide structure from light source that the porous plate 10 of little algae almost all be arranged from the even outgoing of top surface side to the inoculation of optical waveguide layer, thus the raising light efficiency.
From the light of the top surface side outgoing of optical waveguide layer 505 through equal light films 507 and on all tabula rasa 508 and project behind the gas homogenization the space therebetween above inoculation the porous plate 10 of little algae is arranged, little algae algae kind is carried out illumination cultivation.According to one embodiment of the present invention, for fear of the loss of incident beam in light guide structure, bottom reflection film 504 and offside reflection film 505 can be made of total reflection layer or total reflection film.Below will come verification setting that the performance of the shaking table of the means of illumination of cultivating for little algae high-throughput of the present invention is arranged by specific embodiment.
In the present embodiment, whether the light distribution of estimating designed shaking table by the light intensity of measuring the different positions place reaches the requirement of little algae high flux screening.
Experimental procedure is, the temperature of setting shaking table is 25 ℃, and light intensity is 4000lux, measures the intensity of illumination of designed the shaking table upper left corner, the upper right corner, the lower left corner, the lower right corner and center position with luxmeter.During mensuration with 96 microwell plates as measuring basis.
Data processing adopts SPSS Statistics 17.0 softwares.By to the illumination homogeneity between the calculating of the variation coefficient (CV) of place, each hole in each microwell plate intensity of illumination and each hole that one-way analysis of variance (ANOVA) is estimated each microwell plate.By the illumination homogeneity that calculating and the one-way analysis of variance (ANOVA) of the variation coefficient (CV) of light intensity mean value in 5 microwell plates of different positions are estimated the different positions place.In the above data processing with the measurement of error P＜0.05 as conspicuous level.
Be respectively by the CV that calculates between each hole of each microwell plate: the upper left corner 9.32%, the upper right corner 9.85%, the lower left corner 9.43%, the lower right corner 10.22%, center 8.34%.Obtain degree of freedom F＞F0.05 between each hole of each microwell plate by one-way analysis of variance, error P＜0.05, therefore illustrate that the growth differences of the algae strain between each hole does not reach conspicuous level, illustrate that namely light distribution between each hole of microwell plate of position than homogeneous, can satisfy the high flux screening of little algae.
Be 9.58% by the CV that calculates between the microwell plate of position, variance analysis obtains F＞F0.05, P＜0.05, therefore illustrate that the difference between light intensity does not reach conspicuous level between each microwell plate, the light distribution that is whole Stage microscope different positions place is homogeneous, can satisfy the high flux screening of little algae.
In the present embodiment, select 96 orifice plates, 48 orifice plates, 24 orifice plates, 12 orifice plates respectively, adopt the means of illumination among the present invention that illumination is provided, algae strain Chlorella vulgaris (is purchased in hydrobiont institute of Chinese Academy of Sciences algae kind storehouse, be numbered 1068) cultivate, adopt BG11 substratum (1-3 sees the following form).
Composition and the content thereof of table 1 BG11 substratum
| Sequence number | Composition | Amount (/L) | |
| 1 | NaNO 3 | 1.5 |
|
| 2 | MgSO 4·7H 2O | 0.075 |
|
| 3 | CaCl 2·2H 2O | 0.036g | |
| 4 | Citric acid | 0.006 |
|
| 5 | The Fe-EDTA | 1mL | |
| 6 | Na 2CO 3 | 0.02g | |
| 7 | A5 Trace element | 1.0ml |
Table 2 A
5Trace element forms and content
| Sequence number | Composition | Amount (g/L) |
| 1 | H 3BO 3 | 2.86 |
| 2 | MnCl 2·4H 2O | 1.81 |
| 3 | ZnSO 4·7H 2O | 0.222 |
| 4 | Na 2MoO 4·2H 2O | 0.39 |
| 5 | CuSO 4·5H 2O | 0.079 |
| 6 | Co(NO 3) 2·6H 2O | 0.0494 |
Composition and the content thereof of table 3 Fe-EDTA liquid storage
| Sequence number | Composition | Amount (g/L) |
| 1 | FeCl 3·6H 2O | 3.15 |
| 2 | Na 2EDTA·2H 2O | 4.36 |
The algae strain of exponential phase of growth of some amount is inoculated in the sterilized substratum of 300mL by 20% inoculum size, with 8 passage pipettors, measure corresponding algae liquid by following liquid feeding and join in each microwell plate: the liquid amount of 96 microwell plates, 48 microwell plates, 24 microwell plates, 12 microwell plates is respectively 200 μ L, 500 μ L, 1000 μ l, 2000 μ l after mixing.Every type plate do 3 parallel, cover plate lid sealing (above experiment is all carried out) at last under aseptic condition.Other experiment condition is: 25 ℃ of temperature, illumination 30 μ molm
-2S
-1, photoperiod 12hL/12hD, shaking speed 180rpm.Cultivate the OD that measures respectively after 14 days in each hole
750(expression algae cell density).
Analyze the algae strain biomass in each hole of same type microwell plate, obtain that the CV data see the following form between the hole of each orifice plate.Data as seen from following table 4, no matter adopt the microwell plate (96 orifice plates, 48 orifice plates, 24 orifice plates, 12 orifice plates) of which kind of type, the collimation of frustule biomass is all very good between each hole, this show the means of illumination that is provided with among the present invention the high-throughput culture device be applicable to various types of microwell plates on the market.
Table 4
| The orifice plate type | CV between the hole | The crossed |
| 96 holes | 10.2％ | 0 |
| 48 holes | 7.6％ | 0 |
| 24 holes | 6.4％ | 0 |
| 12 holes | 6.3％ | 0 |
In an embodiment, will to the frustule that utilizes high-throughput culture device of the present invention microminiaturized cultivate and conventional training method under dependency analyze.Under certain test conditions, relatively estimating with high-throughput culture device of the present invention of the energy for growth in shaking bottle and microwell plate cultivated little algae and whether had higher dependency with shake-flask culture between little algae by microwell plate by three kinds of different algae kinds.
Testing used algae kind is respectively: Chlorella vulgaris, Scenedesmus quadricauda, Oscillatoria sp (above-mentioned algae kind is all purchased in hydrobiont institute of Chinese Academy of Sciences algae kind storehouse), all adopt BG11 substratum (seeing Table 1), the microwell plate type of selecting for use is 48 holes, by silica-gel plate lid sealing microwell plate.
Experimental procedure is: the algae liquid that some amount is in logarithmic phase is inoculated in the sterilized substratum by 20% inoculum size, the liquid amount that shakes bottle is 100mL/250mL, the liquid amount of 48 microwell plates is 500 μ L, shake bottle and seal with 8 layers of gauze, microwell plate seals (above experiment is all carried out) with the silica-gel plate lid under aseptic condition.The culture condition that experiment is set is: 25 ℃ of temperature, illumination 30 μ molm
-2S
-1, photoperiod 12hL/12hD, shaking speed 180rpm, culture cycle is 14 days.Regularly get the algae liquid of 2mL every day in the cultivation from shake bottle, and the algae liquid of getting 5 holes from 48 microwell plates carries out the mensuration of OD750 (expression algae cell density).Be X-coordinate at last with time, OD
750For ordinate zou is drawn out the growth curve of each algae kind, as shown in Figure 5, again to shaking the OD at each time point place in bottle and the microwell plate
750Carrying out curve estimates and linear regression processing.Wherein, in Fig. 5, Cv, Sq, Os represent algae kind Chlorella vulgaris respectively, Scenedesmus quadricauda, and Oscillatoria sp, MTP represent the growth curve in the microwell plate.
Can obtain each algae kind from Fig. 5 by above-mentioned data processing and all show separately characteristics shaking the variation tendency of growth curve on bottle and the microwell plate, illustrate that used algae kind high flux screening device can be not influential to the feature of each algae kind self.And by to shaking the OD at each time point place in bottle and the microwell plate
750Linear regression processing can obtain the dependency R value of each algae kind under two kinds of training methods and be respectively R
Cv=0.986, R
Sq=0.991, R
Os=0.985, namely these two kinds of training methods show higher dependency in used algae kind high flux screening device.
According to above description as can be seen, in the present invention, by offside reflection film and the bottom reflection film at optical waveguide layer, so that guiding is mainly penetrated from the end face of optical waveguide layer from the light of light source, improve the illumination uniformity coefficient and the intensity of illumination that are used for little algae high-throughput cultivation, satisfy the needs of micro algae growth and high flux screening.And, adopt led array as light source, can overcome that heat radiation such as conventional light sources such as fluorescent tubes is big, uneven illumination even, the illuminance decay obviously, short, illuminance of life-span regulates defectives such as discontinuous, be supplied to the electric current of led array by adjusting, can easily regulate its intensity of illumination, thereby realization algae kind is cultivated under the homogeneous illumination condition.In addition, have by adjusting different optical bands the LED lamp quantity and arrange, adjust the illumination condition that is fit to micro algae growth.According to means of illumination of the present invention, can be set to uniform twinkler by whole shaking table table top, can satisfy the use of various forms microwell plate or porous plate simultaneously, be not subjected to the restriction of orifice plate form structure.And the present invention adopts the bottom for the light mode, is convenient to the top seal with porous plate, can prevent crossed contamination, guarantees ventilation, evaporates consistence, increases rotating speed, increase ventilatory effect etc.
At present, the device that does not have little algae high-throughput to cultivate as yet, those skilled in the art cultivate for the high-throughput of realizing little algae, once attempted light source is installed in the microbiological incubator, for not influencing experimental implementation, can only be installed in top or the side of microbiological incubator, still, top or side cause incubator inside for photoconduction and are subjected to light inhomogeneous, influence The selection result, and light path is longer, the illuminance decay is high, at the bottom of the light utilization efficiency, illumination is regulated insensitive.The trial that designs multiple scheme by the inventor is implemented in the Stage microscope bottom for light, has realized that illumination is even in little algae high-throughput cultivation, reduces the illuminance decay, improves light utilization efficiency, and sensitivity is regulated in illumination, can satisfy the demand that little algae high-throughput is cultivated.The led array that means of illumination of the present invention adopts does not have the many shortcomings of common fluorescent tube heat radiation.
Can combine by conventional shaking table according to means of illumination of the present invention, can realize that little algae controls illumination, temperature control, control high-throughput wet and control gas and cultivate shaking under the state, in suitable algae plant height flux culture condition scope, temperature, humidity, speed, illuminance are adjustable continuously.And high-throughput culture device of the present invention can adopt the module stacked system, stackable many groups, and every pack module can independent temperature control, control is wet, control speed, control light, further improves the handiness that it is used for screening comparative study.Further, the means of illumination among the present invention adopts means such as sidelight irradiation, diffuse-reflectance, even light can realize more uniform illuminance.Thus, be suitable for carrying out the breeding techniques such as screening, selection by mutation and genetic engineering breeding that equipment that little algae high-throughput cultivates can be applied to a large amount of algae kinds of nature according to of the present invention.
Though below described the present invention with reference to preferred embodiment, it will be recognized by those skilled in the art, under the prerequisite that does not depart from marrow of the present invention or scope, can change in form and details.
Claims (12)
1. one kind is suitable for carrying out the equipment that little algae high-throughput is cultivated, and comprises framework, oscillating mechanism, temperature-control device, humidity conditioner, ventilation controller, Stage microscope and means of illumination, wherein,
Stage microscope is suitable for being fixed for holding the cultivation structure of algae kind or algae liquid,
Means of illumination is arranged on the below of Stage microscope, and be suitable for little algae cultivation uniform illumination is provided, and
Means of illumination comprises light source and light guide structure, and light guide structure is configured to be suitable for the light from light source is provided equably to the cultivation structure that accommodates algae kind or algae liquid,
Wherein this light guide structure is multilayered structure, comprising:
Optical waveguide layer is suitable for the light from light source is reflected equably to all directions, incides the optical waveguide layer from a pair of side of optical waveguide layer from the light of light source;
The bottom reflection film covers the bottom surface of optical waveguide layer, is suitable for returning optical waveguide layer to the luminous reflectance of described bottom reflection by optical waveguide layer; With
The offside reflection film, another of covering optical waveguide layer is suitable for and will returns optical waveguide layer to described another luminous reflectance to offside reflection by optical waveguide layer the side.
2. according to claim 1ly be suitable for carrying out the equipment that little algae high-throughput is cultivated, wherein
The ratio of the area of each side in the area of the end face of optical waveguide layer and the described a pair of side was greater than 10: 1.
3. according to claim 1 being suitable for carried out the equipment that little algae high-throughput is cultivated, and wherein on the submarginal summit portion of optical waveguide layer, also is provided with reflectance coating, and last reflectance coating is suitable for the light reflected back optical waveguide layer from end face periphery inclination outgoing.
4. according to claim 1ly be suitable for carrying out the equipment that little algae high-throughput is cultivated, wherein
In described bottom reflection film and the offside reflection film at least one is total reflection film.
5. according to claim 1ly be suitable for carrying out the equipment that little algae high-throughput is cultivated, wherein
Optical waveguide layer is made of base material and the leaded light powder or the leaded light particle that are evenly distributed in the base material, and the light that enters optical waveguide layer reflects when meeting with leaded light powder or leaded light particle, at least a in reflection and the transmission, and
Base material comprises polymethyl methacrylate materials, and leaded light powder or leaded light particle comprise nanostructure.
6. according to claim 1 being suitable for carried out the equipment that little algae high-throughput is cultivated, and wherein this multilayered structure also comprises:
Be positioned at all light film on the optical waveguide layer end face, be suitable for the upwards emission equably of light from the end face outgoing of optical waveguide layer.
7. according to claim 1 being suitable for carried out the equipment that little algae high-throughput is cultivated, and wherein this multilayered structure also comprises:
Go up equal tabula rasa, be arranged on the top of optical waveguide layer, be suitable for the light from the end face outgoing of optical waveguide layer upwards is transmitted into the cultivation structure that accommodates algae kind or algae liquid equably.
8. according to claim 7ly be suitable for carrying out the equipment that little algae high-throughput is cultivated, wherein go up equal tabula rasa and all separate predetermined distance between the light film, with in last equal tabula rasa and space that all formation is scheduled between the light film.
9. according to claim 8ly be suitable for carrying out the equipment that little algae high-throughput is cultivated, wherein at last equal tabula rasa with all be provided with bracing or strutting arrangement between the light film, this bracing or strutting arrangement is suitable for adjusting the described predetermined distance that separates, to adjust the volume in described predetermined space.
10. according to claim 1ly be suitable for carrying out the equipment that little algae high-throughput is cultivated, wherein
Described light source comprises led array, and
Be provided with lens between led array and light guide structure, these lens are suitable for projecting in the optical waveguide layer after assembling from the light of led array.
11. according to claim 10 being suitable for carried out the equipment that little algae high-throughput is cultivated, wherein this equipment also comprises:
Light intensity controller provides light intensity to the current control of led array from the led array outgoing by control.
12. the equipment that is suitable for carrying out little algae high-throughput cultivation according to claim 1, wherein
Framework is suitable for forming and carries out little algae cultured microalgae culture environment, and wherein Stage microscope and means of illumination are arranged in this framework,
Temperature-control device is suitable for controlling the temperature in little algae culture environment,
Humidity conditioner is suitable for controlling the humidity in little algae culture environment,
Ventilation controller is suitable for controlling the ventilation in little algae culture environment, and
Oscillating mechanism is suitable for vibrating in little algae culturing process, and wherein means of illumination is arranged on the oscillating mechanism.
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| CN103131638A (en) * | 2011-11-30 | 2013-06-05 | 中国科学院大连化学物理研究所 | Organism cultural method and photobioreactor system used for organism cultural method |
| KR101304599B1 (en) * | 2012-02-23 | 2013-10-07 | 박승민 | Algae cultivator with maximized co2 reduction rate and algae manufacturing method using the same |
| CN102618445A (en) * | 2012-04-13 | 2012-08-01 | 暨南大学 | Method for producing microalgae grease by using eustigmatoa cf. polyphem and culture method of eustigmatoa cf. polyphem |
| CN104017726A (en) * | 2014-06-30 | 2014-09-03 | 张玟籍 | Built-in light source bioreactor and microalgae culture method |
| CN106771029A (en) * | 2016-12-16 | 2017-05-31 | 常州市环境监测中心 | One kind is used for Daphnia magna acute toxicity detection kit |
| CN111108185A (en) * | 2017-08-16 | 2020-05-05 | 日本曹达株式会社 | Microorganism culture system |
| CN110106064B (en) * | 2019-06-03 | 2020-06-16 | 中国矿业大学 | Quick microalgae screening device and quick microalgae screening method |
| CN111437716B (en) * | 2020-04-03 | 2021-11-26 | 北京航空航天大学 | Microalgae carbon sequestration method based on natural environment regulation |
| CN112920952A (en) * | 2021-03-25 | 2021-06-08 | 广东轻工职业技术学院 | Photosynthetic organism culture apparatus |
| CN113214961A (en) * | 2021-05-10 | 2021-08-06 | 东台市赐百年生物工程有限公司 | Microalgae breeding device based on LED light source |
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