CN102021208A - Method for rapidly accumulating micro-algae intracellular grease - Google Patents
Method for rapidly accumulating micro-algae intracellular grease Download PDFInfo
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Abstract
The invention relates to a method for rapidly accumulating micro-algae intracellular grease, which comprises steps of heterotrophic culturing, dilution, photo-inductive culturing, micro-algae collection and grease extraction and the like. By using the method, advantages of rapid micro-algae intracellular grease accumulation in the photo-inductive stage are brought into full play so as to provide significant technical means for solving the problem of exceeded cost caused by insufficient raw materials in process of preparing biological fuels (such as diesel oil, aviation kerosene and the like) in a large scale.
Description
Technical field
The invention belongs to bioenergy field and/or little algae biological technical field, relate to greasy method in the little gonidium of a kind of quick accumulation.
Background technology
Little algae energy development has a extensive future, the advantage uniqueness has obtained both at home and abroad and generally acknowledged.So far; the R﹠D work of countries in the world in this field also rests on starting stage (Li Yuanguang, the Tan Tianwei, yellow wise of experimental study and pilot scale demonstration; some problem in science and analysis thereof in little algae biofuel industrialization technology; China's basic science; 2009,5,64～70); cause high this bottleneck of cost but the technology that all runs into is immature, thereby little algae energy scale preparation that still is unrealized in the whole world.
The high-efficient culture pattern of grasping the little algae of the energy is the basis of realizing little algae energy scale preparation.The general fat content of algae kind that existing growth is fast is lower, and the high algae kind growth of oleaginousness is compared slowly mostly.This contradiction is the realistic obstacles of the little algae high-efficient culture of energy mode development.Little algae is cultivated and comprises autotrophy, heterotrophism and three kinds of pattern (Yanna Liang of mixotrophism, Nicolas Sarkany, Yi Cui.Biomass and lipid productivities of Chlorella vulgaris under autotrophic, heterotrophic and mixotrophic growth conditions.Biotechnology Letters, 2009,31:1043～1049).Heterotrophism training mode (Han Xu, Xiaoling Miao, Qingyu Wu.High quality biodiesel production from a microalga Chlorella protothecoides by heterotrophic growth in fermenters.Journal of Biotechnology, 2006,126:499～507) exist " can not directly utilize sun power, can not fixation of C O
2, energy consumption is big " etc. problem, thereby be difficult to use in the large scale culturing of the little algae of the energy.The U.S.'s ASP schedule work final report in 1998 is pointed out: it is the most promising training mode (Sheehan J that the open smooth autotrophy of relatively low cost is cultivated, Dunahay T, Benemann J, et al.A Look Back at the U.S.Department of Energy ' s Aquatic Species Program---Biodieselfrom Algae.National Renewable Energy Laboratory, 1998).But, open smooth autotrophy exist " culture density is low, easily contaminated, moisture evaporation, be subjected to the such environmental effects big " etc. problem, also make it be difficult to satisfy the scale demand of Energy production.
Cultivate the problem that exists at open smooth autotrophy, the light autotrophy that people attempt to adopt closed photo bioreactor to carry out little algae is cultivated.Closed photo bioreactor comprises (Ogbonna J C such as duct type, flat, pillar, Tanaka H.Industrial-size photobioreactors.Chemical technology, 1997,27 (7): 43～49), though have cell density height, growth advantage such as fast, but, be not applied to the large scale culturing of little algae at present as yet owing to reasons such as cost height, amplifying technique are immature.
In addition, people attempt to adopt closed photo bioreactor and the combination of open pond to carry out the light autotrophy cultivation of little algae in recent years: promptly utilize the closed bioreactor to realize the high-density culture of little algae earlier, utilize open pond to reduce production costs again.This pattern possesses substantially " can utilize sun power, high-density, fixation of C O
2, low-cost, efficient " feature, but how to realize the effective control and the systems engineering optimization of whole process, be still waiting further investigation.
The generally acknowledged little algae training mode of the energy of scholar should possess " can utilize sun power, high-density, fixation of C O
2, low-cost, efficient " feature, for realizing this goal, the investigator successively explores again and has released training modes such as " mixotrophism ", " first autotrophy, back heterotrophism ":
(1) mixotrophism pattern: be meant that frustule utilizes light and organism as the energy, utilize organism and inorganics training method (the Lee YK as carbon source again, Ding SY, Hoe CH, Low CS.Mixotrophic growth of Chlorella sorokiniana in outdoor enclosed photobioreactor.Journal of applied phycology, 1996,8:163～169).In the mixotrophism culturing process of little algae, photoautotrophy of little algae and chemoheterotrophy can carry out simultaneously.This pattern can reduce production costs by combining with organic wastewaters processing such as being rich in carbon, nitrogen, but its greatest problem is to grow a large amount of assorted bacterium in the large scale culturing process easily, is difficult to realize the high-density culture of little algae.
(2) " first autotrophy, back heterotrophism " pattern: be to utilize closed bioreactor autotrophy earlier with fixation of C O
2, after carry out heterotrophism and cultivate (Xiong W, Gao C, Yan D, et al.Double CO
2Fixation in photosynthesis-fermentation model enhances algal lipid synthesis for biodiesel production.Bioresource Technology, 2010,101:2287～2293).The greatest problem that this pattern exists is can't accomplish sterile culture (Scott SA after the photoinduction culturing process is amplified; Davey MP; Dennis JS; et al.Biodiesel from algae:challenges and prospects.Current Opinion inBiotechnology; 2010,21:1-10),, the heterotrophism of little algae require the algae kind must not be with any assorted bacterium because cultivating; therefore this pattern can't be amplified, and does not have the mass-producing using value.
The relative merits of comprehensive above-mentioned several little algae training modes, pattern that the present invention has designed a kind of " heterotrophism-dilution-photoinduction series connection is cultivated ", this pattern has satisfied " can utilize sun power, high-density, fixation of C O
2, low-cost, efficient " requirement.With the chlorella is example, its flow process is as follows: at first utilize the bio-reactor heterotrophism to cultivate chlorella to obtain high-density cells, after treating that organic carbon source is exhausted in the nutrient solution, with the substratum dilution algae liquid that does not contain organic carbon source, (8～24h) light induction makes a large amount of fast accumulation of grease in the frustule in the short period of time.But the heterotrophism stage in this pattern is to carry out in the bio-reactor that shakes heterotrophism cultivations such as bottle, mechanical agitation type, air lift type, bubbling style, and purpose is in order to obtain the cell of higher density at short notice; The photoinduction stage can carry out in the system that little algae light autotrophy cultivates in any can be used for, and purpose is to improve content of oil and grease in the born of the same parents by the photoinduction effect; The heterotrophism stage separates independently with the photoinduction stage and carries out, and changes the photoinduction cultivation stage again over to after the heterotrophism stage algae liquid of emitting dilutes with the light inducing culture earlier.Need guarantee in the process to enter in the algae liquid in photoinduction stage and do not contain organic carbon source, can avoid the photoinduction stage to grow too much assorted bacterium like this; And can guarantee that by diluting effect the frustule in photoinduction stage can obtain competent illumination, realize the quick raising of fat content in the born of the same parents.This pattern is compared with above-mentioned other patterns; have the production efficiency height, do not have advantages such as the flexible and production cost of the array mode of the heterotrophism culturing process microbiological contamination problem (light autotrophy culturing process has assorted bacterium not influence growth) that the combination because of different system causes, used culture systems is low; can give full play to the advantage that chlorella accumulates fast at photoinduction stage grease, provide the important techniques means for solving the too high problem of cost that causes owing to insufficient raw material in the biofuel scale preparation process.
Summary of the invention
The invention provides greasy method in the little gonidium of a kind of quick accumulation, this method comprises the step that little algae heterotrophism is cultivated, heterotrophism is cultivated the step of carrying out the photoinduction cultivation after the micro algae culturing liquid dilution that is obtained, and optional frustule is gathered, the step of grease separation and Extraction.
The invention provides a kind of production method of microalgae grease, this method comprises the heterotrophic step of little algae, carry out the step that photoinduction is cultivated after the heterotrophism nutrient solution dilution with little algae, and frustule is gathered, the step of grease separation and Extraction.This method can realize greasy quick accumulation in the born of the same parents, have that floor space is few, area productive rate height, (cell density during photoinduction is higher because of the high cost of gathering that brings of cell density is low for little relatively algae light autotrophy, be generally 2～5g/L), thereby improved production efficiency greatly, reduced production cost.
In an embodiment, described little algae is selected from: the Chlorella pyrenoidesa in the Chlorophyta Chlorella (Chlorella pyrenoidosa), Chlorella vulgaris (Chlorella vulgaris), chlorella ellipsoidea (Chlorella ellipsoidea), Chlorella emersonii, Chlorella sorokiniana, Chlorella saccharophila, Chlorella regularis, Chlorella minutissima, Chlorella protothecoides, Chlorella zofingiensis, and the Brachiomonas submarina in the Chlorophyta, Chlamydobonas reinhardtii, Chlamydomonas acidophila, Haematococcus pluvialis, Haematococcus lacustris, Scenedesmus obliquus, Spongiococcum exetriccium, Tetraselmis suecica, Tetraselmis chuii, Tetraselmis tetrathele, Tetraselmis verrucosa, Micractinium pusillum;
The Cylindrotheca fusiformis of Bacillariophyta, Nitzschia laevis, Nitzschia alba, Nitzschia fonticola, Navicula incerta, Navicula pelliculosa;
The Anabaena variabilis of Cyanophyta;
The Poterioochromonas malhamensis of Chrysophyta;
The Amphidinium carterae of Pyrrophyta, Crypthecodinium cohnii;
The Euglena gricilis of Euglenophyta;
The Galdieria sulphuraria of rhodophyta.
In an embodiment, described little algae is selected from the algae of Chlorophyta Chlorella.Especially, described little algae is selected from the Chlorella pyrenoidesa (Chlorellapyrenoidosa) in the Chlorophyta Chlorella, Chlorella vulgaris (Chlorella vulgaris), chlorella ellipsoidea (Chlorella ellipsoidea), Chlorella emersonii, Chlorella sorokiniana, Chlorella saccharophila, Chlorella regularis, Chlorella minutissima, Chlorella protothecoides and Chlorella zofingiensis.
In an embodiment, described little algae is selected from Chlorella pyrenoidesa (Chlorella pyrenoidosa), Chlorella vulgaris (Chlorella vulgaris) and chlorella ellipsoidea (Chlorella ellipsoidea).
In an embodiment, the heterotrophic step of described little algae comprises: adding pH is 4.0～10.0 substratum in bio-reactor, insert little algae algae kind by 0.1～30% of working volume and carry out batch culture, fed batch cultivation, cultured continuously or semicontinuous cultivation, culture temperature is 10～50 ℃, pH is less than 10.0 in control, and the control dissolved oxygen is more than 1%.
In an embodiment, described algae liquid dilution comprises that it is 0.1～50 grams per liter that the algae liquid that heterotrophism is obtained with substratum is diluted to cell density, and described substratum does not contain organic carbon source, and its pH is 4.0～10.0.
In an embodiment, described photoinduction is cultivated and is comprised the algae liquid after the dilution changed over to and carry out photoinduction in the photoinduction device, continuous illumination or intermittent illumination, and culture temperature is 5～50 ℃, intensity of illumination is 0.1～150klx, and the photoinduction culture cycle is 1～150 hour.
In an embodiment, the heterotrophism substratum is made up of nitrogenous source, organic carbon source and small amounts of inorganic salt, trace element and water; The photoinduction substratum is made up of nitrogenous source, inorganic salt and water.
In an embodiment, but described heterotrophism step is carried out in the bio-reactor that shakes bottle, mechanical agitation type, air lift type or the cultivation of bubbling style heterotrophism, described photoinduction culturing step is shaking bottle or is being selected from the runway pond of open type or any can be used for such as circle pond, enclosed flat bioreactor or duct type bioreactor or pillar bioreactor or the upright bag of film and Pig bioreactor carries out in the device that little algae light autotrophy cultivates, and illumination condition is that natural light or artificial light shine.
In an embodiment, when chlorella was Chlorella vulgaris, the employed substratum of heterotrophism was grouped into by following one-tenth basically: KNO
35～15 grams per liters, glucose 10～60 grams per liters, KH
2PO
40.3～0.9 grams per liter, Na
2HPO
412H
2O 1.0～10.0 grams per liters, MgSO
47H
2O 0.2～1.0 grams per liter, CaCl
20.05～0.3 grams per liter, FeSO
47H
2O 0.01～0.05 grams per liter; Trace element 0.5～4ml, wherein trace element consists of H
3BO
35～15 grams per liters, ZnSO
47H
2O 5.0～10.0 grams per liters, MnCl
2H
2O 1.0～2.0 grams per liters, (NH
4)
6Mo
7O
244H
2O 0.5～1.5 grams per liter, CuSO
45H
2O 1.0～2.0 grams per liters, Co (NO
3)
26H
2O 0.1～0.9 grams per liter; Water.In an embodiment, when chlorella was Chlorella pyrenoidesa, the employed substratum of heterotrophism was grouped into by following one-tenth basically: glucose 10～60 grams per liters, urea 2～8 grams per liters, KH
2PO
41～2 grams per liter, Na
2HPO
412H
2O 1.0～10.0 grams per liters, MgSO
47H
2O 1～2 grams per liter, CaCl
20.05～0.1 grams per liter, trisodium citrate 0.1～2.0 grams per liter, Fe-EDTA solution 0.5～1mL, A5 solution 1～5mL; Wherein the Fe-EDTA solution formula is FeSO
47H
2O 20～30 grams per liters and EDTA 20～40 grams per liters; The A5 solution formula is H
3BO
32.5～4.0 grams per liters, MnCl
24H
2O 1.0～2.0 grams per liters, ZnSO
47H
2O 0.1～0.6 grams per liter, CuSO
45H
2O 5～10 grams per liters, Na
2MoO
40.01～0.05 grams per liter; Water.
In a concrete embodiment, described grease separating and extracting method adopts the organic solvent extraction method.
In another specific embodiment, described microalgae culture method also comprises the step of extracting microalgae grease.
The application also provides a kind of grease production method, and this method comprises that heterotrophism cultivates the step of little algae, will carry out the step that photoinduction is cultivated after the dilution of heterotrophism cultured microalgae algae liquid, and frustule is gathered, the step of grease separation and Extraction.
Little algae, substratum and the culture condition that uses in the application's the grease production method like this paper above and hereinafter the embodiment part describe in detail.
Cultivate chlorella with heterotrophism-dilution disclosed by the invention-photoinduction serial connection technology, at first cultivate chlorella at closed bio-reactor middle-high density heterotrophism, after treating that organic carbon source is exhausted in the nutrient solution, with the substratum dilution algae liquid that does not contain organic carbon source, (8～24h) light induction in the short period of time, can make a large amount of fast accumulation of fat content in the frustule, fat content (for example 8～24 hours) at short notice doubles (fat content generally is no more than 10% in the heterotrophism stage born of the same parents, can reach 20～30% after photoinduction) than the frustule in heterotrophism stage.
The accompanying drawing summary
Fig. 1 is presented at and adopts heterotrophism-dilution-photoinduction series connection to cultivate the Chlorella pyrenoidesa greasy result of accumulation (having measured other main biochemical components in the cell simultaneously) fast in the flat bioreactor series system of 50L bio-reactor/3L.
Fig. 2 shows that the 5T fermentor tank is connected serially to the change curve of outdoor 30L flat board and outdoor natural light intensity of the big basin cultured continuously of 60L Chlorella vulgaris photoinduction stage and natural temperature.
Fig. 3 shows that the 5T fermentor tank is connected serially to the dull and stereotyped Chlorella vulgaris greasy conditional curve of accumulation (having measured other main biochemical components in the cell simultaneously) fast of cultivating of outdoor 30L.
Fig. 4 shows that the 5T fermentor tank is connected serially to the big basin of outdoor 60L and cultivates the Chlorella vulgaris greasy conditional curve of accumulation (having measured other main biochemical components in the cell simultaneously) fast.
Fig. 5 shows that the 5T fermentor tank is connected serially to outdoor 20m
2Runway pond and 3.14m
2The change curve of outdoor natural light intensity of circle pond cultured continuously Chlorella vulgaris photoinduction stage and natural temperature.
Fig. 6 shows that the 5T fermentor tank is connected serially to outdoor 20m
2The Chlorella vulgaris greasy conditional curve of accumulation (having measured other main biochemical components in the cell simultaneously) is fast cultivated in the runway pond.
Fig. 7 shows that the 5T fermentor tank is connected serially to outdoor 3.14m
2The Chlorella vulgaris greasy conditional curve of accumulation (having measured other main biochemical components in the cell simultaneously) is fast cultivated in the circle pond.
Fig. 8 shows that 500mL shakes the change curve that main biochemical component in the chlorella ellipsoidea born of the same parents is cultivated in the series connection of bottle/3L column type photoreactor.
Specific embodiments
The little algae that is applicable to the application includes but not limited to the Chlorella pyrenoidesa (Chlorella pyrenoidosa) in the Chlorophyta Chlorella, Chlorella vulgaris (Chlorella vulgaris), chlorella ellipsoidea (Chlorella ellipsoidea), Chlorella emersonii, Chlorella sorokiniana, Chlorella saccharophila, Chlorella regularis, Chlorella minutissima, Chlorella protothecoides, Chlorella zofingiensis, and the Brachiomonas submarina in the Chlorophyta, Chlamydobonas reinhardtii, Chlamydomonas acidophila, Haematococcus pluvialis, Haematococcus lacustris, Scenedesmus obliquus, Spongiococcum exetriccium, Tetraselmis suecica, Tetraselmis chuii, Tetraselmis tetrathele, Tetraselmis verrucosa, Micractinium pusillum;
The Cylindrotheca fusiformis of Bacillariophyta, Nitzschia laevis, Nitzschia alba, Nitzschia fonticola, Navicula incerta, Navicula pelliculosa;
The Anabaena variabilis of Cyanophyta;
The Poterioochromonas malhamensis of Chrysophyta;
The Amphidinium carterae of Pyrrophyta, Crypthecodinium cohnii;
The Euglena gricilis of Euglenophyta;
The Galdieria sulphuraria of rhodophyta.
In preferred embodiment, the present invention adopts the algae of the Chlorophyta Chlorella fat that produces oil.In preferred embodiment, the present invention adopts Chlorella pyrenoidesa, Chlorella vulgaris or the chlorella ellipsoidea fat that produces oil.
1. the high Density Heterotrophic of little algae in bio-reactor
The purpose of this step is in order to obtain a large amount of frustules fast, for the various activeconstituentss of quick dynamic accumulation of photoinduction stage, especially to comprise grease, pigment, protein, polysaccharide etc.
Can adopt various substratum well known in the art to carry out little algae heterotrophism cultivates.Usually, the heterotrophism substratum contains nitrogenous source, organic carbon source, small amounts of inorganic salt, trace element and water.Be applicable to that nitrogenous source that little algae cultivates, organic carbon source, inorganic salt, trace element etc. are that this area is known.For example, as nitrogenous source, spendable have urea or various nitrate, as KNO
3Deng; As organic carbon source, spendable have for example glucose etc.
This class substratum comprises HA-SK substratum (Chinese patent ZL 200610024004.9), Endo substratum (Ogbonna J.C., Masui.H., Tanaka.H.Sequential heterotrophic:autotrophic cultivation-an efficient method of producing Chlorella biomass for health foodand animal feed.J.Appl.Phycol.1997,9,359～366) etc.
The used HA-SK substratum of the present invention is basically by KNO
3, glucose and small amounts of inorganic salt, trace element and water forms.In described technical scheme, described trace element should be selected from H
3BO
3, ZnSO
47H
2O, MnCl
2H
2O, (NH
4)
6Mo
7O
244H
2O, CuSO
45H
2O, Co (NO
3)
26H
2Among the O one or more or whole.
Term used herein " basically by ... form " in the expression composition of the present invention except containing main ingredient KNO
3, outside glucose and small amounts of inorganic salt, trace element and the water, also can comprise some for the fundamental characteristics of composition or new characteristic (can keep little algae and reach higher level at short culture cycle inner cell density, activity substance content is cultivated to compare with conventional heterotrophism and the had a more substantial increase simultaneously) component of influence in fact not.Term used herein " by ... form " expression composition of the present invention is made up of pointed concrete component, do not have other components, but can have the impurity of content in common scope.
In this substratum, each component of substratum can change within the specific limits and can very big materially affect do not arranged to microalgae cell density and quality.Therefore, the consumption of these components is not limited by the strictness of embodiment should.As known to those skilled in the art, also can add small amounts of inorganic salt in the substratum, for example sal epsom, calcium chloride, ferrous sulfate and phosphoric acid salt etc., and a small amount of trace element is as Mn, Zn, B, I, M, Cu, Co etc.
In the present invention, preferable micro-component should be selected from H
3BO
3, ZnSO
47H
2O, MnCl
2H
2O, (NH
4)
6Mo
7O
244H
2O, CuSO
45H
2O, Co (NO
3)
26H
2Among the O one or more.The consumption of inorganic salt and trace element can be determined according to conventional knowledge.
HA-SK substratum of the present invention is grouped into by following one-tenth basically: KNO
35～15 grams per liters, glucose 10～60 grams per liters, KH
2PO
40.3～0.9 grams per liter, Na
2HPO
412H
2O 1.0～10.0 grams per liters, MgSO
47H
2O 0.2～1.0 grams per liter, CaCl
20.05～0.3 grams per liter, FeSO
47H
2O 0.01～0.05 grams per liter; Trace element 0.5～4ml, wherein trace element consists of H
3BO
35～15 grams per liters, ZnSO
47H
2O 5.0～10.0 grams per liters, MnCl
2H
2O 1.0～2.0 grams per liters, (NH
4)
6Mo
7O
244H
2O0.5～1.5 grams per liters, CuSO
45H
2O 1.0～2.0 grams per liters, Co (NO
3)
26H
2O 0.1～0.9 grams per liter; Water.
In a preferable embodiment, HA-SK culture media composition of the present invention should be grouped into by following one-tenth: KNO
37 grams per liters, glucose 40 grams per liters, KH
2PO
40.6 grams per liter, Na
2HPO
412H
2O 2.0 grams per liters, MgSO
47H
2O 0.8 grams per liter, CaCl
20.2 grams per liter, FeSO
47H
2O 0.03 grams per liter; Trace element 1.5mL, wherein trace element consists of H
3BO
311～12 grams per liters, ZnSO
47H
2O 8.5～9.5 grams per liters, MnCl
2H
2O 1.4～1.5 grams per liters, (NH
4)
6Mo
7O
244H
2O 0.8～0.9 grams per liter, CuSO
45H
2O 1.5～1.6 grams per liters, Co (NO
3)
26H
2O 0.45～0.55 grams per liter; Water 1000mL.
The used Endo substratum of the present invention is grouped into by following one-tenth basically: glucose 10～60 grams per liters, urea 2～8 grams per liters, KH
2PO
41～2 grams per liter, Na
2HPO
412H
2O 1.0～10.0 grams per liters, MgSO
47H
2O1～2 grams per liters, CaCl
20.05～0.1 grams per liter, trisodium citrate 0.1～2.0 grams per liter, Fe-EDTA solution 0.5～1mL, A5 solution 1～5mL; Wherein the Fe-EDTA solution formula is FeSO
47H
2O 20～30 grams per liters and EDTA 20～40 grams per liters; The A5 solution formula is H
3BO
32.5～4.0 grams per liters, MnCl
24H
2O 1.0～2.0 grams per liters, ZnSO
47H
2O 0.1～0.6 grams per liter, CuSO
45H
2O 5～10 grams per liters, Na
2MoO
40.01～0.05 grams per liter; Water.
In a preferable embodiment, described Endo substratum is grouped into by following one-tenth: glucose 40 grams per liters, urea 6.0 grams per liters, KH
2PO
41.5 grams per liter, Na
2HPO
412H
2O 5.0 grams per liters, MgSO
47H
2The O1.8 grams per liter, CaCl
20.05 grams per liter, trisodium citrate 0.4 grams per liter, Fe-EDTA solution 0.8mL, A5 solution 2.0mL, wherein the Fe-EDTA solution formula is FeSO
47H
2O 25 grams per liters and EDTA 33.5 grams per liters, the A5 solution formula is H
3BO
32.86 grams per liter, MnCl
24H
2O 1.81 grams per liters, ZnSO
47H
2The O0.222 grams per liter, CuSO
45H
2O 0.07 grams per liter, Na
2MoO
40.021 grams per liter; Water.
After according to above-mentioned formulated substratum, available conventional means as acid or alkali with as described in the pH of substratum be adjusted to 4.0～9.0, and 115～120 ℃ of following autoclavings 15～20 minutes.Can adopt four kinds of modes such as batch culture, fed batch cultivation, semicontinuous cultivation (band is put) or cultured continuously to implement heterotrophism cultivates.
When carrying out the heterotrophism cultivation, corresponding prepared culture medium is joined in the bio-reactor, benefit adds water to working volume, usually coefficient is 0.6～0.8, steam sterilizing is (121 ℃ then, kept about 20 minutes), when temperature is reduced to 30～35 ℃, insert little algae by 1～15% of working volume and begin the heterotrophism cultivation.
No matter adopt which kind of training method, in culturing process, must make little algae normal growth by the suitable culture condition of control.Usually, controlled temperature is 20～35 ℃, and for example 28～30 ℃, dissolved oxygen is not less than 5% air saturation concentration, and pH is not higher than 9.0.In a preferred embodiment, dissolved oxygen is not less than 10% air saturation concentration, and pH is not higher than 8.5.In other preferred embodiment, dissolved oxygen is not less than 15% air saturation concentration, and pH is not higher than 8.
In culturing process, pH is unsuitable too high or too low, generally along with the carrying out of cultivating, pH can slowly rise (obvious especially) for this phenomenon of Chlorella vulgaris, growth has a negative impact the too high meeting of pH to frustule, regulate so use acid (for example 10% sulfuric acid), make pH not be higher than 9.0, preferable pH should be 6.5～7.5.
No matter adopt which kind of heterotrophism training method, change photoinduced algae liquid over to, in better scheme, should make in the algae liquid organic carbon source for example glucose content be zero or approach zero.
But heterotrophism can carry out in the bio-reactor that shakes heterotrophism cultivations such as bottle, mechanical agitation type, air lift type, bubbling style.
2. the dilution of high density algae liquid
The purpose of this step is in order to make the chlorella that changes the photoinduction cultivation over to absorb luminous energy efficiently, to improve optical energy utilization efficiency, reducing the mortality ratio of frustule simultaneously.Because light intensity is " time, sky, non-linear " decay in algae liquid, so under high-cell density, the frustule major part is in the dark space in the reactor, almost can't accept illumination, frustule is easy to death and can influences photoinduced efficient like this.
The high-density algae liquid that heterotrophism cultivate to obtain should dilute operation, and water and the substratum that does not contain organic carbon source dilute highdensity algae liquid, make cell density maintain 0.1～10 grams per liter, adjusting pH to 5.0～8.0.In other embodiments, dilution algae liquid makes cell density maintain 1～8 grams per liter.In a preferred embodiment, make cell density maintain 1.0～5.0 grams per liters.In other embodiments, make cell density maintain 2～5 grams per liters or 2～4 grams per liters.The pH of algae liquid of dilution can be adjusted to 5.5～7.5, and for example 6.0～7.5 etc.
Can adopt various known diluted mediums to dilute algae liquid.Usually, can use the photoinduction substratum to dilute.The photoinduction substratum contains nitrogenous source, inorganic salt and water usually, does not contain organic carbon source with respect to the heterotrophism substratum.In preferred embodiments, the nitrogen concentration of described substratum is 0.1～10 grams per liter, is preferably 0.3～6 grams per liter, is more preferred from 0.5～5 grams per liter.Described nitrogenous source can be identical or different with used nitrogenous source in the heterotrophism culturing step.
In a preferred concrete scheme, the high-density frustule that heterotrophism cultivate to obtain should with nitrogen concentration be 0.1～10 grams per liter and the initial medium that do not contain organic carbon source (for example, adopt the HA-SK substratum that does not contain glucose when cultivating Chlorella vulgaris, adopt the Endo substratum that does not contain glucose when cultivating Chlorella pyrenoidesa) suitably dilute.
The substratum that dilution is adopted need not autoclaving, and preparing adjusting pH to 5.0～8.0, back can use.
In embodiment, to Chlorella vulgaris, diluted medium (photoinduction substratum) is grouped into by following one-tenth: KNO
30.1～5 grams per liters, MgSO
47H
2O, 0.5～5.0 grams per liter, CaCl
20.01～0.06 grams per liter, FeSO
47H
2O 0.01～0.06 grams per liter, EDTA 0.020～0.052 grams per liter.
To Chlorella pyrenoidesa, diluted medium (photoinduction substratum) is grouped into by following one-tenth: urea 0.1～5.0 grams per liter, MgSO
47H
2O, 0.5～5.0 grams per liter, CaCl
20.01～0.06 grams per liter, FeSO
47H
2O0.01～0.06, EDTA 0.020～0.052 grams per liter, Trisodium Citrate 0.02～0.5 grams per liter.
3. photoinduction is cultivated
The purpose of this step is to allow little algae accept illumination, makes the fast a large amount of various activeconstituentss of dynamic accumulation of frustule by photoinduction.
The high-density micro algae culturing liquid is after dilution as mentioned above, the gained diluent changed over to carry out photoinduction in the photoinduction device and cultivate.The photoinduction substratum that adds is as indicated above, and temperature is controlled at 5～50 ℃, and intensity of illumination is 0.11～150klx, continuous illumination or intermittent illumination, and the photoinduction culture cycle is 1～150 hour, air flow is 0.1～2.0vvm.Wherein said bioreactor comprises all closed photo bioreactors (shake bottle, duct type, flat, pillar, the upright bag of film with Pig etc.) and all open bioreactor (runway pond, justify pond and the big basin of bubbling style etc.).
Usually, culture temperature can be controlled in 15～35 ℃ the scope, for example 18～35 ℃, 20～35 ℃, 20～30 ℃ etc.Usually, intensity of illumination is 1～70klx, for example, 1～60,1～50,1～40,1～30,1～20,1～10klx etc., decide by the concrete condition of production.Usually, the controlled 0.15～2.0vvm that is made as of air flow, for example, 0.2～1.8,0.5～1.5,0.8～1.5,1.0～1.5vvm etc.In other embodiments, culture temperature is controlled at 10～50 ℃, and intensity of illumination is 1～10klx, and air flow is 0.05～2.0vvm.
In other embodiments, the photoinduction culture cycle is 8～100 hours, and for example, according to the weather condition of reality, the photoinduction culture cycle can not wait for 8～90 hours, 8～80 hours, 8～60 hours, 8～48 hours, 8～24 hours; Perhaps, photoinduction culture cycle can not wait or not wait in 24～60 hours, 24～48 hours for 12～72 hours, 12～60 hours, 12～48 hours, 12～36 hours, 12～24 hours.
In this application, " photoinduction culture cycle " comprised overall optical inducing culture process, and for example, the outdoor inducing culture cycle in cultivation time comprises the time that does not have illumination night.
In this application, " light application time " refers to use the described intensity of illumination of the application that little algae is implemented the time that photoinduction is cultivated, and promptly should the time not comprise the time that does not have illumination night.In certain embodiments, the light application time of photoinduction culturing step is 8～48 hours, do not wait in for example 8～36 hours, 8～24 hours, 8～18 hours, 8～12 hours, 12～36 hours, 12～24 hours, and any duration in the above-mentioned scope.
Therefore, the application's photoinduction culturing step comprises that also light application time is 8～48 hours photoinduction culturing steps in the scope.Can adopt the mode of artificial lighting to carry out the photoinduction cultivation, also can utilize the mode of natural lighting to carry out the photoinduction cultivation out of doors.
In the photoinduction process, (maximum value that the different microalgae fat content reaches is different when fat content reaches maximum, can be by measuring fat content curve judgement in the born of the same parents in the photoinduction process, chlorella is generally about 20～30%) time can finish photoinduction and cultivate, and the results frustule carries out follow-up greasy separation and Extraction.
Frustule gather, the comprehensive utilization of greasy extraction and frond
Photoinduction is carried out centrifugal gathering to chlorella after cultivating and finishing, and obtains wet frond.The collecting method of frustule includes but not limited to high speed centrifugation, flocculation, technology such as air supporting or filtration; The frustule wall-breaking method includes but not limited to Wet-process wall breaking methods such as frond self-dissolving, high-pressure homogenization, enzymic hydrolysis, water pyrolysis.
Greasy extraction and determination method includes but not limited to organic solvent extractionprocess in the born of the same parents, that is: frond is dried to constant weight under 80～105 ℃, adopt chloroform methanol standard extraction solvent from dry algae powder, to extract grease after grinding the algae powder, extraction solvent extracts repeatedly until algae powder color and becomes white, and rotary evaporation is removed solvent.
Other compositions in the supernatant liquor progressively separation and Extraction obtain lipid acid, chlorophyll etc., or directly with all the components in the supernatant liquor and the dry chlorella powder that obtains of frond precipitation mixed atomizing.
Among the present invention, can little algae of cultivating gained be fully utilized, extract various activeconstituentss such as wherein pigment, protein, polysaccharide.Extraction of active ingredients there is no particular restriction in proper order, can not cause the composition of back extraction to lose this prerequisite but will satisfy the step of extracting earlier usually.
The measuring method that relates to biochemical component in frustule dry weight, fat content and the born of the same parents herein is as follows:
The frustule dry weight is measured: get nutrient solution V milliliter in little algae (as chlorella) culturing process, centrifugal 10 minutes of 8000rpm with deionized water wash 3 times of the frond after centrifugal, is transferred to weighing bottle (W
1(gram)) in, in 105 ℃ of baking ovens, dry to constant weight W
2(gram).Frond dry weight Cx can calculate according to following formula: Cx (grams per liter)=(W
2-W
1)/V/1000.
Grease is measured: get a certain amount of each cultivation stage and dry frustule to constant weight, in mortar, be ground to Powdered, (0.2～0.5g) carefully is transferred in the centrifuge tube to take by weighing an amount of algae powder, add an amount of extraction solvent (chloroform: sonic oscillation 30min in ultrasonator methyl alcohol=2: 1), the centrifugal 10min of 8000rpm, supernatant is transferred in the dry rotary evaporation bottle of known weight, it is colourless until supernatant to repeat above-mentioned steps.Rotate evaporate to dryness after merging supernatant, weigh and calculate fat content.
Fat content (%) is calculated as follows:
Grease (%)=(W
2-W
0)/W
1* 100
In the formula: W
1---be algae grain weight amount, g; W
0---for drying the rotary evaporation bottle weight to constant weight, g; W
2---be the weight of evaporative flask behind the oil extraction liquid evaporate to dryness, g.
The mensuration of carbohydrate: with reference to pharmacopeia 2005 editions, select anthrone-sulfuric acid color reaction, adopt dextrose anhydrous product in contrast, measure at the 625nm place with colorimetry.
Protein content determination: the mensuration of total protein content employing Kjeldahl determination in little algae (as chlorella) cell (Ning Zhengxiang. the food composition analysis handbook. Beijing: China Light Industry Press, 1998,76-78).
The chloroplast pigment assay: little algae (as chlorella) chloroplast pigment assay employing Lichtenthaler etc. has carried out revised method (Lichtenthaler HK to the Arnon method, Wellburn AR.Determinations of total carotenoids and chlorophylls a and b of leaf extracts indifferent sovents.Biochemical Society Transactions, 1983,11:591～592).
Determination of ash: little algae (as chlorella) ash content adopts 550 ℃ of calcination to constant weights to measure, i.e. GB GB6438-86 method.
Cultivate the greasy method of accumulation with the little algae of the existing energy and compare, method of the present invention has the following advantages: can obtain highdensity microalgae cell at short notice, the fat content in the microalgae cell is improved fast.The present invention cultivates Synthetic Oil with little algae and is divided into frustule growth and two stages of grease dynamic accumulation, and promptly heterotrophism is cultivated and the photoinduction cultivation stage.The purpose that heterotrophism is cultivated little algae is in order to obtain a large amount of frustules fast; and the purpose that photoinduction is cultivated is in order to induce a large amount of grease of the quick dynamic accumulation of microalgae cell; adopt heterotrophism-dilution-photoinduction series connection training method to cultivate little algae and improved greasy productive rate; solved the synthetic contradiction of not carrying out simultaneously of growth of energy microalgae cell and grease to a certain extent; greatly reduce cost, provide new technique means for solving the too high problem of cost that causes owing to insufficient raw material in the biofuel scale preparation process.
In addition, before the application, the generally understanding of this area is to have only by the pattern of light autotrophy to utilize little algae to come the scale operation grease.Yet the light autotrophy is cultivated and is cultivated different fully with photoinduction of the present invention.During the light autotrophy was cultivated, substratum did not contain organic carbon source, and little algae utilizes the CO in the atmosphere
2, water, light and small amounts of inorganic nutritive salt carries out photoautotrophy, keeps the growth of self, general inoculum density lower (about 0.1g/L), growth is slow (sometimes in order to promote growth, also need manually to feed CO in culturing process
2), culture cycle is long (generally to be wanted more than the week, outdoor light autotrophy culturing cell density refractory is to reach higher level), especially in large scale culturing, in order to provide large scale culturing required inoculation algae liquid, chronic (generally the needing 1-2 month) that seed spreads cultivation and needs, production efficiency is very low.And the substratum of used substratum of the application's photoinduction and light autotrophy is different, owing to contain nutritive ingredients such as abundant inorganic salt and trace element in the heterotrophism stage algae liquid, so after dilution, required photoinduction substratum is simply more a lot of than light autotrophy substratum, for example compare with common little algae light autotrophy substratum BG-11 on the document, do not need to add phosphoric acid salt, yellow soda ash, ferric ammonium citrate and trace element in the photoinduction substratum of dilution usefulness, the nitrate consumption of Tian Jiaing also lacks a lot than light autotrophy substratum consumption simultaneously.Cultivation by the heterotrophism stage; can accumulate a large amount of frustules at short notice; when mass-producing is cultivated; after dilution; (with the chlorella is example to enter photoinduced algae cell density; density range is about 2～5g/L) higher more than 20 times than the inoculum density of light autotrophy training method; it is low well to have solved light autotrophy training method inoculum density; the seed shortcomings such as the cycle is long that spread cultivation, simultaneously at the light induction of short period in the cycle (general 8～24 hours), algae cell density almost remain unchanged (being its not regrowth); fat content but can accumulate fast in the born of the same parents; content is doubled and redoubled (being increased to about 20～30% by 10%), light autotrophy so relatively, and floor space is dwindled greatly; the area productive rate significantly improves; whole culture cycle obviously shortens, and the cost of gathering reduces, and production efficiency improves greatly.
Below will be further described related content of the present invention by embodiment.Unless have describedly in addition, each component concentration is all used grams per liter (g/L) expression in the substratum that the present invention adopts.Should be understood that among the application " contain ", " comprising " also comprise " by ... form ", " by ... constitute " implication.
Following heterotrophism substratum of adding and tap water are sterilized to 25L in the 50L bio-reactor, insert Chlorella pyrenoidesa by 12% of working volume when temperature is reduced to 30 ℃, and the beginning heterotrophism is cultivated.
The heterotrophism culture condition: temperature is 30 ℃, and initial speed is 150r/min, and air flow quantity is 1vvm, and pH controls dissolved oxygen 15% or more by regulating rotating speed less than 8.0 in the culturing process.
The high-density algae liquid that glucose in the heterotrophism culturing process has been consumed is diluted to about 2.70g/L, and adds following photoinduction substratum, change over to carry out the photoinduction cultivation in the flat photoreactor of 3L, and 30 ℃ of temperature, light intensity 8000lx, air flow quantity is 1vvm.12h is cultivated in photoinduction, and cell density is reduced to 2.65g/L from 2.70g/L, and fat content rises to 19.70% (see figure 1) from 9.70%.
The heterotrophism substratum:
Glucose 60.0 gram urea 8.0 gram MgSO
47H
2O 2.0 grams
KH
2PO
41.1 gram Na
2HPO
412H
2O 9.0 gram CaCl
20.02 gram
Trisodium citrate 1.8 grams
Fe-EDTA solution 1.0ml trace element solution 4.5ml water 1000ml
Wherein the Fe-EDTA solution formula is FeSO
47H
2O 15 grams per liters and EDTA1.4 grams per liter, trace element solution prescription are H
3BO
32.11 grams per liter, MnCl
24H
2O 0.81 grams per liter, ZnSO
47H
2O 0.11 grams per liter, CuSO
45H
2O 10.0 grams per liters, Na
2MoO
40.05 grams per liter.
The photoinduction substratum:
Urea 0.5 gram MgSO
47H
2O 1.0 gram trisodium citrates 0.05 gram
CaCl
20.01 gram Fe-EDTA solution 0.4ml water 1000ml
Wherein the Fe-EDTA solution formula is 15 grams per liters and EDTA1.4 grams per liter.
Adding heterotrophism substratum and tap water are sterilized to 2.5T in the 5T fermentor tank, when temperature is reduced to 30 ℃, with the Chlorella vulgaris of 500L fermentor tank as seed tank culture, regulate tank pressure, air flow and mixing speed according to dissolved oxygen level in cell growing state, the fermented liquid in the culturing process, keep competent oxygen level in the nutrient solution, guarantee the quick growth of frustule.
The high-density algae liquid that glucose in the early stage fed-batch heterotrophism culturing process has been consumed goes to and carries out photoinduction in flat photoreactor of 30L and the big basin of 60L bubbling style out of doors and cultivate after the dilution of photoinduction substratum.The photoinduction culture condition: natural temperature, temperature be at 13～30 ℃, natural lighting, and light intensity is in 0～36klx (see figure 2), and air flow quantity is 1vvm.After 8h is cultivated in photoinduction, algae cell density is reduced to 1.46g/L from 1.60g/L in the 30L flat-plate reactor, fat content rises to 13.27% from 7.87%, laggardly go into night, whole evening, algae cell density was reduced to 1.16g/L from 1.46g/L, the range of decrease is obvious than daytime, fat content also has small size decline, drop to 12.93% from 13.27%, after inducing through the evanescent light of 4h second day daytime, algae cell density slightly rises, rise to 1.18g/L from 1.16g/L, fat content rises rapidly simultaneously, is increased to 18.58% (see figure 3) from 12.93%, Fig. 4 is a photoinduction situation in the big basin of 60L bubbling style, and the rule of data presentation Changing Pattern and the flat photoreactor of 30L is similar.
High-density Chlorella vulgaris algae liquid with after the semicontinuous heterotrophism cultivation end after the dilution of photoinduction substratum, changes 3T runway pond and 3.14m over to
2Tiltedly the photoinduction cultivation is carried out in leaf stirring-type circle pond out of doors.The photoinduction culture condition: low natural temperature, temperature be at 5～18 ℃, natural lighting, and light intensity is in 0～34klx (see figure 5), and air flow quantity is 1vvm.Because of this batch 5T fermentor tank semicontinuous cultivation concluding time between the lights, so frustule promptly enters night after changing photoinduction over to, the photoinduction stage has been carried out 90h altogether, weather was fine in preceding 2 days, but temperature is lower, put into that night of algae liquid, the lowest temperature has only 5 ℃, and the two days highest temperatures in back are 14 ℃, the 3rd day beginning weather light rain of having turned out cloudy, temperature begins slight rebound, the 3rd day begins sultry humidity at dusk, and whole evening, the 4th day morning, the moon had light rain with thundershower, afternoon rain stops, and finishes to cultivate and the frustule of gathering.
By Fig. 6,7 as can be seen, because temperature is lower, be unfavorable for the growth metabolism of frustule on the one hand; On the other hand because the 3rd it beginning is rained successively, and rainwater has diluted algae liquid, has caused density loss in the Da Chi inner cell, so overall optical induces the process algae cell density to descend obviously.
Because postvaccinal preceding 12 hours, it just in time is evening, photosynthesis can not carry out, so pigment and carbohydrate all decline to a great extent, fat content but significantly raises, and rises to about 18% from 12%, and that night, outdoor temperature was lower, low temperature stress may be induced greasy dynamic accumulation in the frustule, and this also is frustule fat metabolic a kind of response mechanism of environmental change to external world.Temperature was all lower in next two days, the highest temperature is no more than 14 ℃, composition does not have considerable change in the frustule born of the same parents, beginning in the 3rd day, though rainwater is arranged, temperature significantly gos up, and sultry humidity, from experimental data, the actual climatic condition in this open air helps the lifting of photoinduction process frond quality on the contrary, but lift velocity is slower.In the outdoor photoinduction process of whole 90h, although low temperature, wet weather and thunderstorm weather occur, by prolonging the photoinduction time, the quality of frond also can be increased dramatically, and final fat content all significantly is promoted to about 20%.
The heterotrophism substratum:
Glucose 60.0 gram saltpetre 10.0 gram MgSO
47H
2O 0.2 gram
KH
2PO
40.3 gram Na
2HPO
412H
2O 8.8 gram CaCl2 0.02 gram
Fe-EDTA solution 1.0ml trace element solution 3.5ml water 1000ml
Wherein the Fe-EDTA solution formula is FeSO
47H
2O 15 grams per liters and EDTA 1.4 grams per liters, the trace element solution prescription is H
3BO
32.86 grams per liter, MnCl
24H
2O 0.11 grams per liter, ZnSO
47H
2The O9.22 grams per liter, CuSO
45H
2O 1.00 grams per liters, (NH
4)
6Mo
7O
244H
2O 0.1 grams per liter, Co (NO
3)
26H
2O 0.9 grams per liter.
The photoinduction substratum:
Saltpetre 0.5 gram MgSO
47H
2O 0.6 gram
CaCl
20.03 gram Fe-EDTA solution 1.5ml water 1000ml
Wherein the Fe-EDTA solution formula is 8 grams per liters and EDTA10.4 grams per liter.
Embodiment 3: the main biochemical component Study of variation law of chlorella ellipsoidea in heterotrophism-dilution-photoinduction series connection culturing process
The variation that present embodiment shakes main biochemical component in bottle/3L column type bioreactor level determination chlorella ellipsoidea heterotrophism-dilution-photoinduction series connection culturing process at 500mL.
Adopt with embodiment 1 and 2 similar modes with heterotrophism-dilution-photoinduction culture technique cultivation chlorella ellipsoidea of connecting, 500mL shakes bottle, liquid amount 200mL, 28 ℃, 150rpm is when treating that algae cell density reaches the above and glucose of 10g/L and exhausts, change 3L column type bioreactor over to and carry out the photoinduction cultivation, the about 2g/L of algae cell density, 30 ℃ of temperature, light intensity 10Klx.The substratum that heterotrophism and photoinduction are adopted and the corresponding substratum of Chlorella pyrenoidesa are consistent.When heterotrophism finishes in the frustule fat content be 13.94%, change 12h after the photoinduction over to, the frustule fat content is increased to 23.91% (see figure 8) fast.
Although object lesson of the present invention described above, having a bit is significantly to those skilled in the art, promptly can do various variations and change to the present invention under the premise without departing from the spirit and scope of the present invention.Therefore, claims have covered all these changes within the scope of the present invention.
Claims (10)
1. one kind accumulates greasy method in little gonidium fast, it is characterized in that, this method comprises the heterotrophism culturing step of little algae, will carry out the step that photoinduction is cultivated after the dilution of heterotrophism cultured microalgae algae liquid, and optional frustule is gathered, the step of grease separation and Extraction.
2. method according to claim 1 is characterized in that described little algae is selected from:
Chlorella pyrenoidesa in the Chlorophyta Chlorella (Chlorella pyrenoidosa), Chlorella vulgaris (Chlorella vulgaris), chlorella ellipsoidea (Chlorella ellipsoidea), Chlorella emersonii, Chlorella sorokiniana, Chlorella saccharophila, Chlorella regularis, Chlorella minutissima, Chlorella protothecoides, Chlorella zofingiensis, and the Brachiomonas submarina in the Chlorophyta, Chlamydobonas reinhardtii, Chlamydomonas acidophila, Haematococcus pluvialis, Haematococcus lacustris, Scenedesmus obliquus, Spongiococcum exetriccium, Tetraselmis suecica, Tetraselmis chuii, Tetraselmis tetrathele, Tetraselmis verrucosa, Micractinium pusillum;
The Cylindrotheca fusiformis of Bacillariophyta, Nitzschia laevis, Nitzschia alba, Nitzschia fonticola, Navicula incerta, Navicula pelliculosa;
The Anabaena variabilis of Cyanophyta;
The Poterioochromonas malhamensis of Chrysophyta;
The Amphidinium carterae of Pyrrophyta, Crypthecodinium cohnii;
The Euglena gricilis of Euglenophyta; With
The Galdieria sulphuraria of rhodophyta.
3. as each described method among the claim 1-2, it is characterized in that, the step of the little algae of described heterotrophism is included in carries out the heterotrophism cultivation in the bio-reactor: adding pH is 4.0～10.0 substratum in bio-reactor, insert little algae algae kind by 0.1～30% of working volume and carry out batch culture, fed batch cultivation, semicontinuous cultivation or cultured continuously, culture temperature is 10～50 ℃, pH is less than 10.0 in control, and the control dissolved oxygen is more than 1%.
4. as each described method among the claim 1-3, it is characterized in that described algae liquid dilution comprises that it is 0.1～50 grams per liter that the algae liquid that the heterotrophism cultivation is obtained with substratum is diluted to cell density, described substratum does not contain organic carbon source, and its pH is 4.0～10.0.
5. as each described method among the claim 1-4, it is characterized in that, described photoinduction is cultivated and is comprised the algae liquid after the dilution changed over to and carry out photoinduction in the photoinduction device, culture temperature is 5～50 ℃, continuous illumination or intermittent illumination, intensity of illumination is 0.1～150klx, and the photoinduction culture cycle is 1～150 hour.
6. as each described method among the claim 1-5, it is characterized in that the heterotrophism substratum is made up of nitrogenous source, organic carbon source and small amounts of inorganic salt, trace element and water; The photoinduction substratum is made up of nitrogenous source, inorganic salt and water.
7. as each described method among the claim 1-6, it is characterized in that, but described heterotrophism step is carried out in the bio-reactor that shakes bottle, mechanical agitation type, air lift type or the cultivation of bubbling style heterotrophism, described photoinduction culturing step is shaking bottle or is being selected from the runway pond of open type or circle pond, enclosed flat bioreactor or duct type bioreactor or pillar bioreactor or the upright bag of film carry out with the device that Pig is used for little algae light autotrophy cultivation, and light source is natural light or various artificial light.
8. method as claimed in claim 2 is characterized in that, when chlorella was Chlorella vulgaris, the employed substratum of heterotrophism was grouped into by following one-tenth basically: KNO
35～15 grams per liters, glucose 10～60 grams per liters, KH
2PO
40.3～0.9 grams per liter, Na
2HPO
412H
2O 1.0～10.0 grams per liters, MgSO
47H
2O0.2～1.0 grams per liters, CaCl
20.05～0.3 grams per liter, FeSO
47H
2O 0.01～0.05 grams per liter; Trace element 0.5～4ml, wherein trace element consists of H
3BO
35～15 grams per liters, ZnSO
47H
2O 5.0～10.0 grams per liters, MnCl
2H
2O 1.0～2.0 grams per liters, (NH
4)
6Mo
7O
244H
2O 0.5～1.5 grams per liter, CuSO
45H
2O1.0～2.0 grams per liters, Co (NO
3)
26H
2O 0.1～0.9 grams per liter; Water.
9. method as claimed in claim 2 is characterized in that, when chlorella was Chlorella pyrenoidesa, the employed substratum of heterotrophism was grouped into by following one-tenth basically: glucose 10～60 grams per liters, urea 2～8 grams per liters, KH
2PO
41～2 grams per liter, Na
2HPO
412H
2O 1.0～10.0 grams per liters, MgSO
47H
2O 1～2 grams per liter, CaCl
20.05～0.1 grams per liter, trisodium citrate 0.1～2.0 grams per liter, Fe-EDTA solution 0.5～1mL, A5 solution 1～5mL; Wherein the Fe-EDTA solution formula is FeSO
47H
2O 20～30 grams per liters and EDTA20～40 grams per liters; The A5 solution formula is H
3BO
32.5～4.0 grams per liters, MnCl
24H
2O 1.0～2.0 grams per liters, ZnSO
47H
2O 0.1～0.6 grams per liter, CuSO
45H
2O 5～10 grams per liters, Na
2MoO
40.01～0.05 grams per liter; Water.
10. a grease production method is characterized in that, described method comprises that heterotrophism cultivates the step of little algae, will carry out the step that photoinduction is cultivated after the dilution of heterotrophism cultured microalgae algae liquid, and frustule is gathered, the step of grease separation and Extraction.
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| PCT/CN2011/082261 WO2012065545A1 (en) | 2010-11-16 | 2011-11-16 | Microalgae culturing method for oil and lutein rapid accumulation |
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Cited By (21)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101979498A (en) * | 2010-12-01 | 2011-02-23 | 华东理工大学 | High-yield heterotrophic culture method for microalgae |
| WO2012065545A1 (en) * | 2010-11-16 | 2012-05-24 | 华东理工大学 | Microalgae culturing method for oil and lutein rapid accumulation |
| WO2012100583A1 (en) * | 2011-01-27 | 2012-08-02 | 华东理工大学 | Culturing method for microalgae with high yields |
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