CN103074205B - Electric turntable system, microalgae high-throughput screening device, and microalgae high-throughput screening method - Google Patents
Electric turntable system, microalgae high-throughput screening device, and microalgae high-throughput screening method Download PDFInfo
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Abstract
The invention discloses an electric turntable system comprising a pedestal with a driving motor and a turntable on the pedestal. A plurality of plate slots are distributed on the turntable. The invention also provides a microalgae high-throughput screening device and a microalgae high-throughput screening method. The method comprises the steps that: microalgae grown to a logarithmic phase is inoculated in culture media in microwell plates; the microwell plates inoculated with the microalgae are fixed in the plate slots of the electric turntable system; light culture is carried out under a rotation condition; absorbance of each pore in the microwell plates after culturing is measured; and the absorbance is converted into corresponding microalgae cell dry weight concentration, such that needed microalgae can be selected. The screening result obtained by the invention is consistent with that obtained by using shake flasks under same culturing conditions. With the method provided by the invention, equipment and labor investments required by algae species screening are effectively reduced, and time spent by algae species screening work is shortened.
Description
Technical field
The present invention relates to the high-throughput screening method of a kind of electric rotary system, a kind of micro-algae high flux screening device and micro-algae.
Background technology
The global warming causing due to fossil oil overexploitation and burning at present has been subject to all circles and has paid close attention to widely (IPCC (Inter-governmental Panel on Climate Change) Climate Change 2001:The Scientific Basis.Contribution of Working Group I to the Third Assessment Report of the Intergovernment Panel on Climate Change.Cambridge University press, 2001a).Carbonic acid gas, as a kind of main greenhouse gases, is one of major incentive causing Greenhouse effect.Ge great energy company of the world is all attempting Renewable Energy Development technology to realize control and the reduction of discharging to carbonic acid gas.In the middle of numerous new energy technologies, biomass energy is a very promising field, and biomass energy can additionally not increase the carbonic acid gas in atmosphere, and raw material sources are extensive, cheap.In the field of biomass energy, again one of the most promising developing direction for the comprehensive development and utilization of micro-algal biomass, because micro-algae is that in numerous biomass, photosynthetic efficiency is the highest as a kind of low one-celled plants such as grade, and micro-algal biomass also has the natural product of a lot of high added values in being rich in energy, it is fully utilized, be conducive to further reduce Energy production cost (referring to Hejazi MA and Wijffels RH, Milking of microalgae.Trends Biotechnol, 2004,22).
In the research and application process of micro-algae, the seed selection work of algae kind is one of vital link.Traditional method of selection is: algal species cultivation in shaking flask, in conjunction with different screening conditions, such as sewage, high temperature, high concentration carbon dioxide etc., then measure the index such as biomass, fat content of algae kind, examine or check thus the application prospect of this algae kind (referring to Rodolfi L et al, Microalgae for oil:Strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor.Biotechnol Bioeng, 2009,102).Although this screening method is reliable, efficiency is very low, in a large amount of algae kinds of reply or a large amount of screening conditions, must drop into a large amount of manpower and materials, this process both loaded down with trivial details also economical not (referring to
r et al., The use of miniaturized algal bioassays in comparison to the standard flask assay.Environ Toxicol Water Qual, 1998,13).
In recent years, there is research that some miniature culture techniques are used to micro-algae to improve conventional efficient (referring to Eisentraeger A et al., Development of automated high-throughput ecotoxicity and genotoxicity test systems and fields of application.Water Sci Technol, 2004,50; Nowack ECM et al., The 96-well twin-layer system:a novel approach in the cultivation of microalgae.Protist, 2005,156; Jira ' skova ' and Pouli ' c ˇ kova ', High-throughput screening technology for monitoring phytohormone production in microalgae.J Phycol, 2009,45).These technology major parts are all derived from the system of early stage Lukavsk ' y structure (referring to Lukavsky ' J, The evaluation of algal growth potential (AGP) and toxicity of water by miniaturized growth bioassay.Water Res, 1992,26).Its main purpose is in conjunction with micro-algae, some specific toxin or active substance to be detected fast, and is not suitable for the screening of the micro-algae algae of biomass energy kind.And the miniature culture technique of existing report lacks whether have enough conforming abundant research and checking with the selection result of shake-flask culture for the selection result under the micro-scale culture environment of algae kind.
To the carrying out aspect high flux screening of the micro-algae of enduring high-concentration carbonic acid gas biomass energy, at present relevant patent report is very limited.Chinese patent application CN101412965A discloses the preparation method of a kind of micro-algae of stabilizing carbon dioxide, has wherein reported the seed selection under high concentration carbon dioxide condition to certain chlorella, and this patent application does not relate to the high flux screening work of algae kind.Chinese patent application CN101126712A discloses a kind of for the high-throughout 96 orifice plate screening methods of weedicide, and this technology system is the rapid screening to weedicide characteristic and consumption based on chlorella, does not relate to the quick breeding of tolerance carbonic acid gas biomass energy algae kind.
Summary of the invention
The object of the invention is the demand that can study and apply for micro-algal biomass at present for adapting to, the high-throughput screening method of a kind of micro-algae is provided, the method can be cultivated under multiple condition multiple micro-algae simultaneously, has greatly improved screening efficiency.In addition, the present invention also provides a kind of electric rotary system and a kind of micro-algae high flux screening device that can be used for the method.
The invention provides a kind of electric rotary system, this electric rotary system comprises with the base of drive-motor and is arranged on the rotating disk on base, it is characterized in that, is distributed with multiple tabular draw-in grooves on described rotating disk.
The present invention also provides a kind of micro-algae high flux screening device, it is characterized in that, this device comprises above-mentioned electric rotary system and microwell plate, in the dismountable tabular draw-in groove that is fixed on described electric rotary system of described microwell plate.
In addition, the present invention also provides the high-throughput screening method of a kind of micro-algae, it is characterized in that, the method comprises, the micro-algae that grows to logarithmic phase is inoculated in the substratum of microwell plate, then this microwell plate of the micro-algae of inoculation is fixed in the tabular draw-in groove of above-mentioned electric rotary system, and under rotation condition, carry out illumination cultivation, measure the absorbancy of cultivating every hole in rear microwell plate, or further absorbancy is converted into corresponding microalgae cell dry weight concentrations, and select required micro-algae according to the absorbancy obtaining or microalgae cell dry weight concentrations.
Technique effect of the present invention is as follows:
Adopt the inventive method can utilize microwell plate under multiple culture condition, multiple micro-algae algae kinds to be cultivated and screened, assessment algae kind is as the potentiality of biological energy raw material simultaneously.The selection result that experimental result shows to utilize method of the present invention with utilize shaking flask consistent in the selection result under same culture conditions, illustrate that method of the present invention has reliability.
In addition, method of the present invention has reduced required equipment and the human input of Strain selection effectively, has shortened the required time of Strain selection work simultaneously.
Brief description of the drawings
Accompanying drawing is used to provide a further understanding of the present invention, and forms a part for specification sheets, is used from explanation the present invention, but is not construed as limiting the invention with embodiment one below.In the accompanying drawings:
Fig. 1 a is the schematic diagram of electric rotary system in one embodiment of the present invention; Fig. 1 b is the schema of the high-throughput screening method of micro-algae in one embodiment of the present invention.
Fig. 2 shows in one embodiment of the present invention Chlorella pyrenoidesa under different gas concentration lwevels, the comparative result of growing state in 96 orifice plate rotating table systems and shake-flask culture.
Fig. 3 shows in one embodiment of the present invention Chlorella vulgaris under different gas concentration lwevels, the comparative result of growing state in 96 orifice plate rotating table systems and shake-flask culture.
Fig. 4 shows in one embodiment of the present invention scenedesmus obliquus under different gas concentration lwevels, the comparative result of growing state in 96 orifice plate rotating table systems and shake-flask culture.
Fig. 5 shows Chlorella vulgaris in one embodiment of the present invention, Chlorella pyrenoidesa, scenedesmus obliquus under the suitableeest gas concentration lwevel separately, in 96 orifice plate rotating table systems and shake-flask culture, and the comparative result of remaining nitrate concentration in substratum.
Fig. 6 shows Chlorella vulgaris in one embodiment of the present invention, Chlorella pyrenoidesa, scenedesmus obliquus under the suitableeest gas concentration lwevel separately, in 96 orifice plate rotating table systems and shake-flask culture, and the comparative result that the pH value of substratum changes.
Fig. 7 shows in one embodiment of the present invention, the result that the relevant parameter in 96 orifice plate rotating table systems and shake-flask culture is carried out to linear regression analysis.
Description of reference numerals
The tabular draw-in groove of 1 base 2 rotating disk 3
Embodiment
Term:
Unless otherwise, all technology and scientific terminology used and the routine that the present invention relates to the those of ordinary skill of technical field are understood otherwise in the present invention identical meanings.
" biomass energy ", refers to that sun power is stored in the form of energy in biomass with chemical energy form, i.e. the energy taking biomass as carrier.It derives from the photosynthesis of green plants directly or indirectly, can be converted into conventional solid-state, liquid state and gaseous fuel, inexhaustible, nexhaustible, is a kind of renewable energy source, is also the reproducible carbon source of unique one simultaneously.
" greenhouse gases ", refer to lay equal stress on some gases of new emitted radiation of the solar radiation that can absorb land return in atmosphere, as water vapour, carbonic acid gas, most of refrigeration agent etc.Their effect is to make earth surface become warmer, the effect that is similar to greenhouse and holds back air in solar radiation heating greenhouse.This greenhouse gases make the earth become warmer to be called " Greenhouse effect ".Steam (H
2o), carbonic acid gas (CO
2), Nitrous Oxide (N
2o), methane (CH
4) and ozone (O
3) be greenhouse gases main in earth atmosphere.
" high flux screening ", refer to taking the experimental technique of molecular level and cell levels as basis, using microplate format as experimental tool carrier, gather experimental result data with sensitive detecting instrument fast, process experimental data with Computer Analysis, detect at one time the technical system of number with ten million sample, it has trace, the feature such as quick, sensitive and accurate.
" micro-algae ", refers to unicellular planktonic algae, is preferably the unicellular planktonic algae of any clarified liq substratum pure culture.
Below at length introduce concrete steps of the present invention.
The invention provides a kind of electric rotary system, as shown in Figure 1a, this electric rotary system comprises with the base 1 of drive-motor and is arranged on the rotating disk 2 on base 1, it is characterized in that, is distributed with multiple tabular draw-in grooves 3 on described rotating disk 2.
According to the present invention, described rotating disk 2 can be vertical dial or horizontal rotating disc, and preferably, in order to reach well-mixed object, described rotating disk 2 is preferably vertical dial.
Described drive-motor is preferably alternating current motor.
To the size of described tabular draw-in groove 3, there is no particular limitation with size in the present invention, preferably, described tabular draw-in groove 3 is for can be fixed on microwell plate the tabular draw-in groove on rotating disk 2, further preferably, each tabular draw-in groove 3 is applicable to fix a microwell plate, the size of described tabular draw-in groove 3 preferably adapts with microwell plate used, concrete size those skilled in the art can select as required, for example, in the present invention, microwell plate used is conventional aseptic polypropylene 96 orifice plates, therefore, the length and width of the tabular draw-in groove 3 being suitable for are identical with aseptic polypropylene 96 orifice plates of this routine, the degree of depth is preferably 0.7 centimetre.
For making full use of the space of described rotating disk 2 and reaching abundant mixing and make the object of condition homogeneous, described multiple tabular draw-in groove 3 is center radiation symmetric offset spread, as shown in Figure 1a, the number of described tabular draw-in groove 3 can be selected as required, in a kind of preferred implementation of the present invention, in electric rotary system used, the number of tabular draw-in groove 3 is 8.
To the shape and size of described rotating disk 2, there is no particular limitation in the present invention, preferably, in the applicable electric rotary system of conventional 96 orifice plates, the cross section of described rotating disk 2 is that radius is the circle of 30-50 centimetre, in a kind of preferred implementation of the present invention, the cross section of described rotating disk 2 is that radius is the circle of 30 centimetres, and the geometric centre of each tabular draw-in groove 3 is 20 centimetres apart from the center of rotating disk 2.
The invention provides a kind of micro-algae high flux screening device, it is characterized in that, this device comprises above-mentioned electric rotary system and microwell plate, in the dismountable tabular draw-in groove 3 that is fixed on described electric rotary system of described microwell plate.
Wherein, the concept of described microwell plate is known to the skilled person, conventional at least one as in 24 orifice plates, 96 orifice plates and 384 orifice plates.
The present invention also provides the high-throughput screening method of a kind of micro-algae, it is characterized in that, the method comprises, the micro-algae that grows to logarithmic phase is inoculated in the substratum of microwell plate, then this microwell plate of the micro-algae of inoculation is fixed in the tabular draw-in groove 3 of above-mentioned electric rotary system, and under rotation condition, carry out illumination cultivation, measure the absorbancy of cultivating every hole in rear microwell plate, or further absorbancy is converted into corresponding microalgae cell dry weight concentrations, and select required micro-algae according to the absorbancy obtaining or microalgae cell dry weight concentrations.
Method of the present invention also can be carried out in above-mentioned micro-algae high flux screening device.
In the time using vertical dial, preferably microwell plate is sealed to prevent that nutrient solution is excessive, the mode of described sealing is known to the skilled person, as sealed with aseptic Parafilm.
The preparation method of the described micro-algae that grows to logarithmic phase is known to the skilled person, and for example, micro-algae is carried out in shaking flask to preculture.Described pre-incubated condition is known to the skilled person, and does not repeat them here.
A kind of method that essence of the present invention is to provide high-throughput cultivates micro-algae, therefore, those skilled in the art can set screening conditions as required, to select needed micro-algae.
For example method of the present invention is applicable to the screening of micro-algae of enduring high-concentration carbonic acid gas.Particularly, method of the present invention also can comprise, in micro-algae is inoculated in to microwell plate after and this microwell plate of the micro-algae of inoculation is fixed in tabular draw-in groove 3 before, in microwell plate, enclose the gas of high carbon dioxide content.Wherein, the concentration of the carbon dioxide in gas of described high carbon dioxide content can be 2-50 volume %.
The method of the described gas of enclosing high carbon dioxide content in microwell plate can be the method for this area routine, for example, under aseptic technique, in the super-clean environment of gas that is full of high carbon dioxide content, seals this microwell plate with aseptic Parafilm.Described super-clean environment is for example closed Bechtop.Particularly, can in closed Bechtop, be filled with the gas of high carbon dioxide content, after gas concentration lwevel in worktable is stable, across operation gloves, off-the-shelf aseptic Parafilm be sealed microwell plate.
Method of the present invention is also applicable to the screening of the micro-algae that tolerates sewage.Particularly, described substratum can be sewage.Described sewage can be for example anaerobic pond sewage and/or Aerobic Pond sewage.
It should be noted that, the technical scheme that above-mentioned substratum is sewage is only one embodiment of the present invention, and in fact, in method of the present invention, substratum used can be the substratum of the various routines in this area, as BG-11 substratum, f/2 substratum etc.Those skilled in the art can be according to algae need to select suitable substratum.
Method of the present invention is also suitable for the screening of micro-algae with other character, does not repeat one by one at this, and these all should be considered as content disclosed by the invention.
According to the present invention, the inoculum density of described micro-algae can be conventional selection, is preferably every liter of substratum 0.01-0.03g dry cell weight.Described dry cell weight can obtain by centrifugal direct measurement dry cell weight, also can obtain by the absorbancy (OD value) of test micro algae culturing liquid.For 96 orifice plates of standard specifications, its single hole volume is 300 microlitres, preferred every hole inoculation 50-200 microlitre, more preferably 100 microlitres.
For every kind under single screening conditions micro-algae, can set up 3-6 parallel test to guarantee the accuracy of result.
According to the present invention, the condition of described illumination cultivation can be conventional selection, and illumination methods can be for making one-sided directional light irradiate perpendicular to described microwell plate; Illumination kind can be white light, and intensity of illumination can be 1000-12000 Lux (depending on the intensity of illumination at the card place of nearly rotating disk 2), and the illumination cultivation cycle can be 7-14 days.Temperature and pressure can be selected as required, and for example temperature can be 20-30 DEG C, and pressure can be normal pressure.For different micro-algaes, those skilled in the art can select different conditions to screen.
Those skilled in the art can recognize, except for screening micro-algae, method of the present invention also can be for determining the optimal culture condition of target microalgae, or only for cultivating in multiple micro-algae is under multiple condition.These all should be considered as content disclosed by the invention.
According to the present invention, for the algae liquid in microwell plate is mixed, avoid algae liquid excessive simultaneously, the rotating speed of described rotation is preferably 10-80 rev/min, more preferably 30-50 rev/min.
According to the present invention, in described measurement microwell plate, the method for the absorbancy in every hole can be the ordinary method of this area: measure the absorbancy of every hole at 570nm by microplate reader.Described microplate reader can be for example Thermo Multiskan MK3 type microplate reader.
According to the present invention, for the experiment that only can judge micro algae growth situation by measuring absorbancy, without absorbancy being converted into microalgae cell dry weight, and maybe must judge by microalgae cell dry weight and the situation of micro algae growth situation absorbancy further need to be converted into microalgae cell dry weight for measuring different microalgae.
The described method that absorbancy is converted into corresponding microalgae cell dry weight concentrations preferably includes: the standard working curve of making described absorbancy and described microalgae cell dry weight concentrations, record after cultivating in microwell plate standard working curve described in the absorbancy in every hole substitution, ask and calculate microalgae cell dry weight concentrations.
Particularly, the method for the standard working curve of the described absorbancy of described making and described microalgae cell dry weight concentrations comprises, by fask oscillating method, micro-algae is cultivated, and the algae liquid measuring with constant duration is at the absorbancy X at 570nm place
1-X
m, get the algae liquid of quantitative volume simultaneously, record the dry cell weight Y of this algae liquid
1-Y
m, set up the one-variable linear regression equation of absorbancy and dry cell weight:
y＝ax-b，
Wherein: a is that Monomial coefficient, b are that constant term, m represent number of times and m>=3 measured, by absorbancy X
1-X
mand Y
1-Y
mone-variable linear regression equation described in substitution, asks and calculates a value and b value.
In illumination cultivation process, as need are measured the growth curve of micro-algae, every other day, in Bechtop, open Parafilm, utilize microplate reader to measure the OD value in every hole in microwell plate, then again with aseptic Parafilm, microwell plate is sealed by above-mentioned steps, continue illumination cultivation.Measure growth curve if do not needed, only need in cultivating end, measure OD value one time, between incubation period, every 2-3 days, the carbonic acid gas in microwell plate is once upgraded.
The method of the standard working curve of the described absorbancy of described making and described microalgae cell dry weight concentrations for example, corresponding algae kind is first carried out to the preculture of 7 days by a definite date in the shaking flask of 400 milliliters, from the 3rd day, measure the absorbancy of 570nm wavelength every day, collect 40 milliliters of centrifugal supernatants that go of algae liquid simultaneously, the lyophilize 24 hours at-40 DEG C of residue frustule, weigh dry weight, set up the one-variable linear regression equation of dry weight and absorbancy.The OD value of measurement is converted into dry weight concentrations, thereby algae kind or screening conditions are compared and assessed.
For different algae kinds, dry weight and OD
570the one-variable linear regression equation of value has different parameters, and table 1 has been listed the parameter of part algae kind:
Table 1
| Algae kind | Equation of linear regression | R 2 |
| Chlorella vulgaris | y＝1.7083x-0.1254 | 0.997 |
| Pyrenoids bead | y＝2.1024x-0.0985 | 0.989 |
| Scenedesmus obliquus | y＝2.3207x-0.1104 | 0.998 |
| Husky angle chlamydomonas | y＝2.8956x-0.0948 | 0.998 |
| Goat's horn crescent moon algae | y＝2.1067x-0.1107 | 0.981 |
| Primary election Dunaliella salina | y＝2.6777x-0.0657 | 0.996 |
Wherein, x represents OD
570; Y represents dry cell weight (g/L); R
2for slope coefficient
At present to the assessment of biomass energy algae kind be mainly measure that the indexs such as biomass, fat content are examined or check and the application prospect of more different algae kinds (referring to Rodolfi L et al, Microalgae for oil:Strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor.Biotechnol Bioeng, 2009,102).The concentration of algae kind concentration ratio that in general small-scale cultivation and screening obtain industry amplification culture all wants high, and in cultivation on a small scale, can to reach more than 1.2g/L be moderate-yield level to biomass.
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is elaborated.Should be understood that, embodiment described herein only, for description and interpretation the present invention, is not limited to the present invention.Fig. 1 b shows the schema of the high-throughput screening method of micro-algae in one embodiment of the present invention.As shown in Figure 1 b, sealing, high carbon dioxide concentration culture condition Parafilm 96 orifice plates are packed in the tabular draw-in groove 3 in electric rotary system, one-sided directional light lateral vertical and described 96 orifice plates irradiate, in culturing process, 96 orifice plates can be unloaded, and measure the absorbancy in every hole in 96 orifice plates by microplate reader.
Below describe the just mode description of a preferred embodiment thereof with embodiment, but not intention limits the invention.In following examples, electric rotary system used is 96 orifice plate rotating table systems, as shown in Figure 1a, wherein, the vertical dial that rotating disk 2 is colourless synthetic glass, its cross section is the circle of 30 centimetres of radiuses, on rotating disk 2, there are 8 radiosymmetric tabular draw-in grooves 3, in each tabular draw-in groove 3, can fix 96 orifice plates of a standard specifications (purchased from Jiangsu glasswork company limited, aseptic polypropylene material, single hole volume is 300 microlitres), particularly, the length of each tabular draw-in groove 3 is 127 millimeters, wide is 85 millimeters, height is 14 millimeters, the geometric centre of each tabular draw-in groove 3 is 20 centimetres apart from the center of rotating disk 2.Wherein, absorbancy is measured by Thermo Multiskan MK3 type microplate reader.
Embodiment 1
(1) preparation of substratum: the BG-11 substratum after improving with distilled water preparation, every liter contains 1.5g NaNO
3, 0.04g K
2hPO
4, 0.075g MgSO
47H
2o, 0.036g CaCl
22H
2the A5 trace element of O, 0.006g citric acid, 0.006g ferric ammonium citrate, 0.001g EDTA and 1.0ml, every liter of A5 trace element contains: 2.86g H
3bO
3, 1.81g MnCl
24H
2o, 0.05g CoCl
26H
2o, 0.79g CuSO
45H
2o, 0.22g ZnSO
47H
2o and 0.39g Na
2moO
4.
(2) preculture of Chlorella pyrenoidesa: Chlorella pyrenoidesa (Chlorellapyrenoidosa) (domestication through 3-5 for high concentration carbon dioxide) is inoculated in 400 milliliters of BG-11 substratum, and illumination is 8000lux (Lux); Temperature is 25 DEG C; Initial inoculation OD
570=0.050 (0.02g dry cell weight/L substratum).From the 3rd day, measured OD every day
570, collect 40 milliliters of centrifugal supernatants that go of algae liquid simultaneously, the lyophilize 24 hours at-40 DEG C of residue frustule, weighs dry weight, and dry weight and absorbancy are set up to one-variable linear regression equation, as shown in table 1.
(3) utilize shake-flask culture to investigate the biomass that Chlorella pyrenoidesa is grown under high concentration carbon dioxide: in step (2) preculture process, in the 4th day (logarithmic phase) sampling, utilize through the BG-11 substratum of sterilizing and be diluted to OD
570=0.050.This algae liquid 85ml is placed in to 250ml Erlenmeyer flask, in Erlenmeyer flask, pass into the air (volumetric concentration of carbonic acid gas is followed successively by 5%, 10%, 20%, 30%) that contains different concns carbonic acid gas, each concentration is set up three Duplicate Samples, utilize transparent sealed membrane to seal Erlenmeyer flask, above operation all completes in Bechtop.Finally Erlenmeyer flask is placed in to 36 hole shaking tables and cultivates, rotating speed is 110 revs/min; Illumination is 8000lux; Temperature is 25 DEG C.In culturing process every day sampling and measuring OD
570, supplement the carbon dioxide of respective concentration simultaneously.Growth of Cells stops after entering stationary phase cultivating.
(4) utilize 96 orifice plate rotating table systems to cultivate and investigate the biomass that Chlorella pyrenoidesa is grown under high concentration carbon dioxide: in step (2) preculture process, in the 4th day (logarithmic phase) sampling algae liquid, utilize through the BG-11 substratum of sterilizing algae liquid is diluted to OD
570=0.050.Algae liquid 100 μ l after dilution are inoculated in 96 orifice plates, in 96 orifice plates, pass into the carbon dioxide (concentration be followed successively by carbonic acid gas with volume of air than 5%, 10%, 20%, 30%) of different concns, each concentration is inoculated 6 holes, utilize Parafilm to seal 96 orifice plates, above operation all completes in Bechtop.
Finally 96 orifice plates are placed in to 96 orifice plate rotating table systems shown in Fig. 1 b and cultivate, rotating speed is 40 revs/min; Intensity of illumination is 8000lux; Temperature is 25 DEG C.In culturing process, directly measure OD by microplate reader every day
570, supplement carbon dioxide to maintain constant gas concentration lwevel simultaneously.Growth of Cells stops after entering stationary phase cultivating.
(5) data analysis: in conjunction with the OD value recording in cultivating, one-variable linear regression Equation for Calculating dry cell weight between the dry weight obtaining in preculture process in utilization (2) and absorbancy, draw Chlorella pyrenoidesa cell growth curve under different gas concentration lwevels in shaking flask screening and 96 orifice plate rotating table system screening methods, as shown in Figure 2.In Fig. 2, dotted line and hollow pattern (◇,, zero and △) are illustrated in the Growth of Cells in 96 orifice plate rotating table systems, solid line and solid pattern (◆, ■, ● and ▲) be illustrated in the Growth of Cells in shaking flask.Wherein diamond sign (◆ and ◇) represents 5%CO
2growth of Cells under concentration; Square mark (■ and) represents 10%CO
2growth of Cells under concentration; Trilateral mark (▲ and △) expression 20%CO
2growth of Cells under concentration; Circle marker (● and zero) expression 30%CO
2growth of Cells under concentration.Data are the mean value of parallel laboratory test.
As seen from Figure 2, the growth of Chlorella pyrenoidesa in 96 orifice plate rotating table systems revealed good consistence with the growth table in shaking flask, and 20% carbonic acid gas is its suitableeest gas concentration lwevel, and under this gas concentration lwevel, biomass reaches as high as 1.256 ± 0.032g/L.
Embodiment 2-3
Test with the method identical with embodiment 1, algae kind difference used, changes Chlorella vulgaris (Chlorella sp.) and scenedesmus obliquus (Scenedesmus obliquus) into.The growth in 96 orifice plate rotating table systems of two kinds of algaes also shows good consistence with the growth in shaking flask, the suitableeest gas concentration lwevel of Chlorella vulgaris and scenedesmus obliquus is respectively 20% and 10%, and biomass reaches as high as 1.168 ± 0.053g/L and 1.184 ± 0.036g/L.Detailed data is shown in Fig. 3 and Fig. 4 successively.
Fig. 3 is the growth curve of Chlorella vulgaris, and wherein, dotted line is illustrated in the Growth of Cells in 96 orifice plate rotating table systems, and solid line is illustrated in the Growth of Cells in shaking flask.Wherein diamond sign represents 5%CO
2growth of Cells under concentration; Square mark represents 10%CO
2growth of Cells under concentration; Trilateral mark represents 20%CO
2growth of Cells under concentration; Circle marker represents 30%CO
2growth of Cells under concentration.Data are the mean value of parallel laboratory test.
Fig. 4 is the growth curve of scenedesmus obliquus, and wherein, dotted line is illustrated in the Growth of Cells in 96 orifice plate rotating table systems, and solid line is illustrated in the Growth of Cells in shaking flask.Wherein diamond sign represents 5%CO
2growth of Cells under concentration; Square mark represents 10%CO
2growth of Cells under concentration; Trilateral mark represents 20%CO
2growth of Cells under concentration; Circle marker represents 30%CO
2growth of Cells under concentration.Data are the mean value of parallel laboratory test.
Embodiment 4-6
(1) with the identical method of step (1) to (4) in embodiment 1, by Chlorella pyrenoidesa, Chlorella vulgaris, scenedesmus obliquus respectively at cultivate in shaking flask and 96 orifice plate rotating table systems in the carbonic acid gas of 20%, 20%, 10% concentration simultaneously.Difference is the Duplicate Samples in 96 holes of each algae inoculation in 96 orifice plate rotating table systems.
(2) in culturing process, respectively get 1 milliliter of algae liquid sample (being the summation of 10 hole samples in 96 orifice plates) every day from shaking flask and 96 orifice plates, and 8000 rpms of centrifuging and taking supernatants, by 100 times of supernatant liquor dilutions, are measured OD
220value, records this absorbancy numerical value.
(3) the SODIUMNITRATE standardized solution of 1.5g/L is diluted to 100 times, 200 times, 500 times, 750 times, 1000 times successively, measure the OD of sample under each extension rate
220value, sets up one-variable linear regression equation, according to this equation by the OD measuring in the step of embodiment 4-6 (2)
220value is converted into the concentration of remaining nitrate radical.Draw the consumption curve of nitrate radical in substratum, see Fig. 5.
In Fig. 5, dotted line is illustrated in the cultivation in 96 orifice plate rotating table systems, and solid line is illustrated in the cultivation in shaking flask.Square mark represents that Chlorella vulgaris is at 20%CO
2substratum residue nitrate concentration under concentration; Trilateral mark represents that Chlorella pyrenoidesa is at 20%CO
2substratum residue nitrate concentration under concentration; Circle marker represents that grid algae is at 10%CO
2substratum residue nitrate concentration under concentration.As seen from Figure 5, above-mentioned three kinds of algaes in the methods of the invention with traditional shaking flask screening method in the situation of utilizing of right nitrate radical also have good consistence.
Embodiment 7-9
(1) with the method that in embodiment 4-6, step (1) is identical, by Chlorella pyrenoidesa, Chlorella vulgaris, scenedesmus obliquus respectively at cultivate in shaking flask and 96 orifice plate rotating table systems in the carbonic acid gas of 20%, 20%, 10% concentration simultaneously.
(2) in culturing process, respectively get 1 milliliter of algae liquid sample (being the summation of 10 hole samples in 96 orifice plates) every day from shaking flask and 96 orifice plates, and pH value determination is drawn the change curve of pH value in culturing process.See Fig. 6, wherein, dotted line is illustrated in the cultivation in 96 orifice plate rotating table systems, and solid line is illustrated in the cultivation in shaking flask.Square mark represents that Chlorella vulgaris is at 20%CO
2under concentration, Medium's PH Value changes; Trilateral mark represents that Chlorella pyrenoidesa is at 20%CO
2under concentration, Medium's PH Value changes; Circle marker represents that grid algae is at 10%CO
2under concentration, Medium's PH Value changes.As seen from Figure 6, above-mentioned three kinds of algaes in the methods of the invention with traditional shaking flask screening method in the changing conditions of pH value also have good consistence.
Embodiment 10
(1) with the identical method of step (1) to (4) in embodiment 1, Chlorella pyrenoidesa, Chlorella vulgaris, scenedesmus obliquus, goat's horn crescent moon algae (Solenastrum capricornutum), husky angle chlamydomonas (Chlamydomonas sajao), primary election Dunaliella salina (Dunaliella primolecta) in the carbonic acid gas of 5%, 10%, 20%, 30% concentration, are cultivated simultaneously in shaking flask and 96 orifice plate rotating table systems.The first five substratum of planting fresh water algae is BG-11, anaerobic pond sewage, Aerobic Pond sewage; The substratum of Dunaliella salina is f/2, anaerobic pond sewage, Aerobic Pond sewage.6 algaes, 12 culture condition of each algae kind altogether, amount to 72 experiment conditions, and in 96 orifice plates, each experiment condition is inoculated 6 hole Duplicate Samples, 3 Duplicate Samples of each experiment condition in shaking flask.
(2) in culturing process, measure OD every day
570value stops experiment after entering stationary phase, calculates the frustule dry weight output under each experiment condition, and experimental result is in table 2 and table 3.Result by table 2 and table 3 can find out, 6 algae kinds altogether in the screening process of 72 experiment conditions, method of the present invention (table 2) has good consistence compared with traditional shaking flask screening (table 3).
Embodiment 11
To what relate in all embodiment, biomass, remaining nitrate radical, the pH value of utilizing method of the present invention and traditional shaking flask method to measure have been carried out statistical analysis, the result of its one-variable linear regression can be referring to Fig. 7, wherein, transverse axis X-Flask represents shaking flask method, longitudinal axis Y-M96SS represents method of the present invention, and Fig. 7 a represents the linear regression analysis of cell growth dry weight in two kinds of methods; Fig. 7 b represents the linear regression of substratum residue nitrate concentration in two kinds of methods; Fig. 7 c represents that Medium's PH Value changes the linear regression analysis in two kinds of methods.Linear regression coefficient R
2scope between 0.974～0.991, p < 0.0017, illustrates that regression equation is remarkable, and the scope of linear regression coefficient is between 1.011～1.051, approaches very much 1.
This result shows, the important parameter that biomass, remaining nitrate radical, these three of pH values relate to micro-algae output has good consistence in the method compared with the shaking flask screening method of standard, utilizes method of the present invention can replace shake-flask culture algae kind to be carried out to the screening of different growth conditionss.
Method of the present invention can be saved to a certain extent experimental resources in the time answering single algae kind and less screening conditions.From goods and materials and the manpower of screening operation consumption, utilize shaking flask single algae kind to be cultivated under 4 kinds of gas concentration lwevels to (three groups of parallel laboratory tests), need to take 12 taper shaking flasks and a shaking table, every day sampling and measuring OD value and again shaking flask is enclosed again to the required experimental implementation time of respective concentration carbonic acid gas and be about 30 minutes; And utilize 96 orifice plate rotating table systems to cultivate under 4 kinds of gas concentration lwevels single algae kind Chlorella pyrenoidesa, need utilize 4 96 orifice plates and an electric rotary system, utilize every day microplate reader directly to measure OD value and saved the operating time, the operating time of experiment every day is about 20 minutes.
And for multiple algae kinds the screening situation under multiple condition, present method has demonstrated larger advantage saving on goods and materials and manpower.Taking embodiment 10 as example, now there are 72 experiment conditions, in the time carrying out the screening and culturing of shaking flask, the in the situation that of only having a 36 hole shaking table in laboratory, experiment is had to be divided into 6 batches and is completed, if will complete 72 screening conditions under traditional method simultaneously, must purchase 6 shaking tables or larger shaking table, meanwhile, taking every day, the experimental implementation time of 4 experiment conditions is about 30 minutes as example, the experimental implementation time of 72 experiment conditions every day is about 9 hours, so huge workload, and an obvious laboratory technician cannot complete.And adopt present method to carry out such Strain selection experiment, and only the algae kind under all experiment conditions need be inoculated on 8 96 orifice plates and cultivate, to utilize every day microplate reader directly to measure OD value and saved the operating time, the operating time of experiment every day is only 40 minutes.
The patent of related reference herein and open by reference to being all incorporated to herein, comprises all figure and table.
In sum, the present invention can be by carrying out micro-scale cultivation to algae kind, obtains rapidly some with the relevant useful information of algae kind, and the selection result is reliable, has effectively saved the required manpower and materials of Strain selection work simultaneously compared with traditional shaking flask method.The present invention designs specially for adapting to the current growing Research Requirements to the micro-algae of biomass, can effectively improve the efficiency of Strain selection research work.
In addition, also can carry out arbitrary combination between various embodiment of the present invention, as long as it is without prejudice to thought of the present invention, it should be considered as content disclosed in this invention equally.
Claims (12)
1. the high-throughput screening method of a micro-algae, it is characterized in that, the method comprises, the micro-algae that grows to logarithmic phase is inoculated in the substratum of microwell plate, then this microwell plate of the micro-algae of inoculation is fixed in the tabular draw-in groove (3) of electric rotary system, and under rotation condition, carry out illumination cultivation, measure the absorbancy of cultivating every hole in rear microwell plate, or further absorbancy is converted into corresponding microalgae cell dry weight concentrations, and select required micro-algae according to the absorbancy obtaining or microalgae cell dry weight concentrations;
Wherein, the method also comprises, after in micro-algae is inoculated in to microwell plate and before this microwell plate of the micro-algae of inoculation is fixed in tabular draw-in groove (3), in microwell plate, enclose the gas of high carbon dioxide content, the concentration of the carbon dioxide in gas of described high carbon dioxide content is 2-50 volume %;
Wherein, described electric rotary system comprises the base (1) with drive-motor and is arranged on the rotating disk (2) on base (1), is distributed with multiple tabular draw-in grooves (3) on described rotating disk (2).
2. method according to claim 1, wherein, described rotating disk (2) is vertical dial or horizontal rotating disc.
3. method according to claim 1, wherein, described multiple tabular draw-in grooves (3) are center radiation symmetric offset spread.
4. method according to claim 1, wherein, the cross section of described rotating disk (2) is that radius is the circle of 30-50 centimetre, described tabular draw-in groove (3) is for can be fixed on microwell plate the tabular draw-in groove on rotating disk (2).
5. method according to claim 1, wherein, the method for the described gas of enclosing high carbon dioxide content in microwell plate comprises, under aseptic technique, in the super-clean environment of gas that is full of high carbon dioxide content, seal this microwell plate with aseptic Parafilm.
6. method according to claim 1, wherein, described substratum is sewage.
7. method according to claim 6, wherein, described sewage is anaerobic pond sewage and/or Aerobic Pond sewage.
8. according to the method described in any one in claim 1-7, wherein, the inoculum density of described micro-algae is every liter of substratum 0.01-0.03g dry cell weight.
9. according to the method described in any one in claim 1-7, wherein, the condition of described illumination cultivation comprises: illumination methods is for making one-sided directional light irradiate perpendicular to described microwell plate; Illumination kind is white light, and intensity of illumination is 1000-12000 Lux, and the illumination cultivation cycle is 7-14 days.
10. according to the method described in any one in claim 1-7, wherein, the rotating speed of described rotation is 10-80 rev/min.
11. according to the method described in any one in claim 1-7, and wherein, the method for measuring the absorbancy in every hole in microwell plate is: measure the absorbancy of every hole at 570nm by microplate reader.
12. according to the method described in any one in claim 1-7, wherein, the described method that absorbancy is converted into corresponding microalgae cell dry weight concentrations comprises: the standard working curve of making described absorbancy and described microalgae cell dry weight concentrations, record after cultivating in microwell plate standard working curve described in the absorbancy in every hole substitution, ask and calculate microalgae cell dry weight concentrations.
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