CN103642694A - High-yield cultivation method of botryococcus - Google Patents
High-yield cultivation method of botryococcus Download PDFInfo
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- CN103642694A CN103642694A CN201310683781.4A CN201310683781A CN103642694A CN 103642694 A CN103642694 A CN 103642694A CN 201310683781 A CN201310683781 A CN 201310683781A CN 103642694 A CN103642694 A CN 103642694A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
Abstract
The invention relates to a high-yield cultivation method of botryococcus and a production method of grease and/or hydrocarbon. The method comprises a heterotrophic or poly-culture (also named as mixed-culture) cultivation step of botryococcus algae and a photoautotrophic cultivation step which takes algae cells obtained by the heterotrophic or poly-culture cultivation as seeds. The method disclosed by the invention can be adopted to sufficiently bring rapid growth advantages of the algae cells of the botryococcus in the heterotrophic or poly-culture stage, so that a great deal of algae is provided for the botryococcus in the heterotrophic stage, and the botryococcus quickly grows in the heterotrophic stage and is accumulated with helpful substances (grease and hydrocarbon), and therefore, important technical means are provided for solving the problems that the botryococcus is low in cell growth rate in the heterotrophic stage and low in yield of the helpful substances (grease and hydrocarbon).
Description
Technical field
The invention belongs to bioenergy field, relate to a kind of method that quick acquisition has high grease and/or hydrocarbon productive rate grape algae.
Background technology
Grape algae (Botryococcus spp.) is the raw unicellular micro-algae of a kind of groups, is distributed widely in the freshwater of the torrid zone and Temperate Region in China, also in brackish water or seawater, sees once in a while (Huang et al.1999; Volova et al.2003).Various lipid, hydrocarbon (Brown and Knights1969 can be synthesized and accumulate to an important feature for grape algae exactly in a large number; Knights et al.1970) and composition (the Metzger and Casadevall1991 such as ether fat; Metzger1999).The hydrocarbonaceous amount of grape algae can account for the 15-80% of dry cell weight, is much higher than the hydrocarbonaceous amount (Banerjee et al.2002) of other microorganism.Because hydrocarbonaceous amount is high, and there is prolific ability (Wake and Hillen1980 under certain condition; Townsend2001), grape algae is considered to a kind of renewable resources (Casadevall et al.1985) that can be used to produce liquid fuel.
Botryococcus braunii (Botryococcus braunii) cell ovalize, wide 3～6 microns, long 6～12 microns, being embedded in the doline colloid of staggered stratification, is often that 2 or 4 cells become one group, and each colloid funnel is combined mutually in central authorities, all cells are radiation radially more or less all, cell is near around closely squeezing mutually, and therefore, a quite uniform shape appears in whole colony.Group size can reach 100 microns～0.5 millimeter.The colony of grape algae is cell string not of uniform size, and shape is like grape, and intercellular is connected with transparent refraction filament.Atrichia, containing chlorophyll a, b and α, the pigments such as β-carotene, xenthophylls.According to source, particularly the structure of the hydrocarbon that produces is different, and grape algae is divided into A, B, tri-strains of L.The optimum growth temperature of each strain of grape algae has difference, and the optimum growth temperature of A strain is 25 ℃, has surpassed 30 ℃ of speeds of growth just slack-off; The applicable growth temperature of B strain is higher, still can grow, but in the time of 25 ℃, produce hydrocarbon amount maximum at 35 ℃.A strain is produced C
23-C
33the unramified straight chain diolefine of odd number carbon and alkatrienes, B strain generates the C with polyunsaturated acid, branched-chain hydrocarbon
30-C
37terpenoid, L strain generates C
40h
78this single tetraene hydrocarbon polymer.
The research of cultivating for grape algae both at home and abroad at present mainly concentrates on light autotrophy aspect.Although the light autotrophy culturing process of grape algae can accumulate a large amount of greases and hydrocarbon, but the grease of high-energy synthetic or hydrocarbon are extremely processes for power consumption in cell, this light autophyting growth speed that also just means grape algae is lower than other micro-algaes, and the doubling time is longer than other micro-algaes.At present, for the light autotrophy of grape algae, cultivate the following problem that still exists: 1) inoculum density is low causes being vulnerable to miscellaneous bacteria algae and protozoic pollution and the impact of natural environment and climate condition etc.; 2) seed spreads cultivation and all adopts the cultivation of light autotrophy at present, its cycle very very long (generally at least needing 1～2 month if obtain a certain amount of algae kind access great Chi light autotrophy), algae kind is large at constantly the spread cultivation a large amount of culture apparatus of middle needs and equipment and floor space of some months, and it is low that productive rate is cultivated by unit; 3) if the algae kind that outdoor light autotrophy is cultivated is subject to the pollutions such as assorted algae or protozoon, planktonic organism, this batch of algae kind will be scrapped so, need again to carry out the work that spreads cultivation of algae kind, also this can cause next step light autotrophy to be cultivated does not have the problem of algae kind for cultivating, and has a strong impact on production again.
On the other hand; heterotrophism or raise together with cultivation and can obtain high-cell density and high Growth of Cells speed; thereby heterotrophism or raise together with culture density and the cell yield that cultivation can improve grape algae greatly; but the content of grease and hydrocarbon is lower in the frustule obtaining, therefore also cannot be for take high Lipid-producing and hydrocarbon as the cultivation of object grape algae large-scale.In addition, heterotrophism or raise together with process, conventionally the substratum of selecting is common micro-algae culture medium, do not add and promote growing plants growth hormones material, culturing process is not is not conventionally regulated and controled or is added without the supplemented medium of optimizing by intermittent flow, this method is not considered grape algae heterotrophism and raise together with the difference existing with ordinary light autotrophy on nutritional needs to cause substratum not good to the culture effect of cell, and Growth of Cells is relatively slow.
Therefore, this area is needed a kind of efficient grape algae culture process and method badly.
Summary of the invention
For the problems referred to above, the invention provides a kind of effective solution, first adopt heterotrophism or raise together with (claim again hold concurrently support) training method and cultivate grape algae, using subsequently heterotrophism or raise together with the frustule that cultivation obtained and carry out the cultivation of light autotrophy as seed.Adopt the inventive method can give full play to grape algae at heterotrophism or raise together with the advantage of stage frustule Fast Growth, for micro-algae provided a large amount of algae kinds and micro-algae at light autotrophy stage Fast Growth and accumulates useful matter (grease and hydrocarbon) and obtain high cell yield and metabolic substd productive rate in the light autotrophy stage, for cell growth rate and useful matter (grease and the hydrocarbon) problem that productive rate is low in solution grape algae light autotrophy culturing process provide important technique means.
Therefore, first aspect present invention provides a kind of grape algae cultural method, and the method comprises the heterotrophism of grape frustule or raises together with culturing step and using heterotrophism or raise together with the light autotrophy culturing step that frustule that cultivation obtained is implemented as seed.
Second aspect present invention provides the production method of a kind of grease and/or hydrocarbon, and described method comprises the heterotrophism of grape frustule or raises together with culturing step, usings heterotrophism or raise together with the light autotrophy culturing step that frustule that cultivation obtained implements as seed and the step that frustule is gathered and grease/total hydrocarbon extracts.
In an embodiment, aforesaid method of the present invention comprises:
(1) heterotrophism or raise together with and cultivate grape frustule, wherein, culturing process, by control of additive raw material pH, the element such as constant and carbon, nitrogen and/or phosphorus is stabilized within the scope of finite concentration, and heterotrophism or raise together with cultivates that while finishing, to control the concentration of the nutritive ingredients such as carbon, nitrogen and/or phosphorus lower be even zero;
(2) using heterotrophism or raise together with gained frustule and implement light autotrophy as seed and cultivate, and
(3) gather frustule and separation and Extraction grease and/or hydrocarbon.
In an embodiment, grape algae algae kind used includes but not limited to Botryococcus braunii, Botryococcus sudeticus, Botryococcus sp., Botryococcus spp..
In an embodiment, for heterotrophism or raise together with gained frustule, dilutable water then carries out the cultivation of light autotrophy after adding light autotrophy substratum in dilution algae liquid; Or directly to heterotrophism or raise together with and cultivate the grape frustule obtaining and carry out the cultivation of light autotrophy.
In an embodiment, described grape algae adopts any in Botryococcus braunii A, B and tri-strains of L.
In an embodiment, described grape algae algae kind heterotrophism or the step of raising together with comprise: in bio-reactor, add that pH is 4.0～11.0, the preferred substratum of 7.5-8.5,0.1～30% access algae kind by working volume is carried out batch culture, fed batch cultivation, repeated fed-batch culture, semicontinuous cultivation or cultured continuously, culture temperature is 10～40 ℃, control pH and be less than 10.0, be preferably less than 9.0, control dissolved oxygen more than 1%.
In an embodiment, when described grape algae algae kind heterotrophism is cultivated, do not need to carry out illumination.Described grape algae is raised together with while cultivating, and need to carry out illumination, range of light intensity 1-2500 μ molm
-2s
-1.
In an embodiment, the step of described grape algae light autotrophy comprises: by heterotrophism or raise together with (hold concurrently support) grape algae algae kind and be inoculated in light autotrophy culture apparatus and carry out light autotrophy, culture temperature is 5～50 ℃, continuous illumination or intermittent illumination, and intensity of illumination is 1～2500 μ molm
-2s
-1, light autotrophy culture cycle is 1～180 day, preferably 5～40 days, and Initial seeding density is 0.01～5.0 grams per liter, and described substratum is not containing organic carbon source, and its pH is 4.0～9.0.
In an embodiment, heterotrophism or raise together with substratum and formed by nitrogenous source, organic carbon source, plant growth hormones, inorganic salt, trace element and water; Light autotrophy substratum is comprised of nitrogenous source, plant growth hormones, inorganic salt, trace element and water.
In an embodiment, described heterotrophism step can be carried out in the bio-reactor of heterotrophism cultivation at shaking flask, mechanical agitation type, air lift type or bubbling style; Described algae kind raise together with step shaking flask, can steam sterilizing closed photo bioreactor (transparent), and carry out in the bio-reactor (containing external light source and/or internal light source) containing illumination system such as mechanical agitation type; Described smooth autotrophy culturing step is in shaking flask or be selected from the raceway pond of open type or any can be used for such as the light autotrophy culture systems of circle pond, enclosed flat bioreactor or duct type bioreactor or pillar bioreactor or film vertical bag bioreactor or open type and closed hybridization or adherent culture system carries out in device that micro-algae light autotrophy cultivates, and illumination condition is natural light or artificial light irradiation.
In an embodiment, when algae kind is Botryococcus braunii, heterotrophism or raise together with used substratum and substantially consist of the following composition: KNO
30.1～15 grams per liter, organic carbon source 0.1～50 grams per liter, K
2hPO
40.1～10.0 grams per liter, MgSO
47H
2o0.1～10 grams per liter, CaCl
22H
2o0.01～5 grams per liter, plant growth hormones 2,4-dichlorphenoxyacetic acid 0.001-5 mg/litre, benzyladenine 0.001-5 mg/litre, dormin 0.001-5 mg/litre, Plant hormones regulators,gibberellins 0.001-5 mg/litre, 3-indolebutyric acid 0.001-5 mg/litre, naphthylacetic acid 0.001-5 mg/litre, rapin 0.001-5 mg/litre, ironic citrate 0.01～5 grams per liter, citric acid 0.1～15 grams per liter, trace element 0.5～20ml and water, wherein trace element consists of H
3bO
30.1～10 grams per liter, ZnSO
47H
2o0.1～10.0 grams per liter, MnCl
24H
2o0.5～10.0 grams per liter, (NH
4)
6mo
7o
244H
2o0.01～5 grams per liter, CuSO
45H
2o0.01～5.0 grams per liter, Co (NO
3)
26H
2o0.01～5 grams per liter.
In an embodiment, organic carbon source comprises any carbon source that can be used as microorganism culturing, includes but not limited to glucose, maltose, sucrose, lactose, starch, the hydrolyzate of Mierocrystalline cellulose hemicellulose, glycerine, sodium-acetate, acetic acid.Ordinary priority is considered glucose.
In a specific embodiment, when heterotrophism or raise together with after organic carbon source in nutrient solution runs out of, finish heterotrophism or raise together with cultivation, and using heterotrophism or raise together with and cultivate gained frustule and implement light autotrophy culturing step as seed.
In other embodiment of the application, can use substratum, culture condition described in the application to implement above-mentioned grape algae cultural method.
With the heterotrophism of grape algae algae kind or raise together with and cultivate and take heterotrophism or raise together with frustule that cultivation obtained and cultivate and produce grease and/or hydrocarbon as example as the light autotrophy of seed, describe advantage of the present invention in detail:
(1) can greatly improve algal species cultivation speed.The Initial seeding density that the general outdoor great Chi of take cultivates micro-algae is 0.05～0.1g/l, and it is example that the volume of great Chi is generally 5000L, if need meet this requirement, needs micro-algae of 250～500g.If adopt traditional light autotrophy algae kind that spreads cultivation, need for 1～February (algae cell density that grape algae kind light autotrophy spreads cultivation 10 days is 0.2g/l, and volume of culture is 500L); And employing heterotrophism/raise together with and cultivate algae kind, only need a couple of days to complete.
(2) cultivate and compare with traditional algae kind light autotrophy, can reduce device and number of devices in algal species cultivation process, and floor space is little, unit culture area productive rate is high.Algae kind light autotrophy spreads cultivation needs the culture apparatus of 500L and holding time to grow (1～2 month), and algae kind heterotrophism/raise together with cultivation only to need the culture apparatus of 50L and holding time shorter (10-20 days).
(3) cultivate and to compare with algae kind light autotrophy, algae kind heterotrophism or raise together with the impact that cultivation is not subject to outdoor weather and environment.When algae kind light autotrophy is cultivated, in the time of cannot continuing to cultivate if run into the rainy weather of short-term, need to move into indoor and add artificial light and continue light autotrophy cultivation algae kind, thus unwanted artificial lighting expense while having increased the cultivation of algae kind heterotrophism.In addition, if the algae kind of outdoor light autotrophy is subject to the pollutions such as protozoon, planktonic organism, this batch of algae kind can be scrapped so, needs again to carry out the work that spreads cultivation of algae kind again, also can cause next step light autotrophy to be cultivated does not have the problem of algae kind for cultivating, and has a strong impact on production.
(4), when outdoor great Chi cultivates, inoculum size is larger, is more not easy to be subject to the pollutions such as other assorted algaes or protozoon.Therefore, the heterotrophism of algae kind or cultivation required seed can provide in time a large amount of light autotrophys to cultivate time is provided.
(5) heterotrophism or raise together with cell and be better than the vigor of light autotrophy algae kind as the vigor of algae kind, access identical frustule amount and carry out respectively light autotrophy while cultivating, while gathering, the algae cell density of heterotrophism algae kind and grease and total hydrocarbon productive rate are all higher than algae cell density and grease and the total hydrocarbon productive rate of light autotrophy algae kind.Therefore, in the situation that obtaining identical grease and total hydrocarbon productive rate, utilize heterotrophism or raise together with algae kind to make light autotrophy occupation area of equipment few, area productive rate is high.In addition, due to heterotrophism or raise together with cell to carry out as algae kind the algae cell density that the cultivation of light autotrophy obtains larger, therefore, greatly reduce the cost that grape algae is gathered.
In sum, the present invention using heterotrophism or raise together with frustule that cultivation obtained can solve light autotrophy completely and cultivate as the light autotrophy training mode of seed time due to algal species cultivation efficiency is low and vulnerable to pollution was caused problems and grape algae in light autotrophy growth period speed slow and grease and the problem such as total hydrocarbon productive rate is low.Therefore, the present invention, for cell and grease and the low problem of total hydrocarbon productive rate in solution grape algae light autotrophy culturing process provide important technique means, has established solid industrialization basis in particular for utilizing grape algae to produce liquid fuel.
Accompanying drawing explanation
Fig. 1 show grape frustule respectively in 500ml shaking flask heterotrophism, raise together with and light autotrophy incubation growth process.
Fig. 2 shows grape algae culturing process in 5L fermentor tank.In figure, the embodiment that ▲/■ represents to control pH and feeding strategy and added plant growth hormones; △/ represents only to control pH, not optimal feed substratum and feeding strategy and do not add the embodiment of plant growth hormones;
represent the embodiment that controls pH and feeding strategy, do not add plant growth hormones.
Fig. 3 shows grape algae heterotrophism seed, raise together with seed and light autotrophy seed carries out the frustule process of growth of light autotrophy cultivation in indoor 2L bioreactor.
Fig. 4 shows grape algae heterotrophism seed, raises together with seed and light autotrophy seed light autotrophy is cultivated in indoor 2L bioreactor grease and total hydrocarbon content and output.
Fig. 5 shows grape algae heterotrophism seed, raise together with the frustule process of growth that seed and light autotrophy seed carry out the cultivation of light autotrophy in 60L plastic tub out of doors.
Fig. 6 shows grape algae heterotrophism seed, raise together with seed and light autotrophy seed light autotrophy is cultivated in 60L plastic tub out of doors grease and total hydrocarbon content and output.
Embodiment
Term " substantially by ... form " represent in above-mentioned substratum except containing main ingredient KNO
3, outside glucose and inorganic salt, trace element and water, also can comprise some for the fundamental characteristics of composition or new characteristic (can maintain grape algae and reach higher level at shorter culture cycle inner cell density, hydrocarbons content is cultivated to compare with conventional heterotrophism and the had a more substantial increase) component of impact in fact not simultaneously.Term " by ... form " represent that above-mentioned substratum is comprised of pointed concrete component, there is no other components, but can be with content the impurity in scope conventionally.
Can adopt the known various grape algaes of prior art to implement technical scheme of the present invention, include but not limited to Botryococcus braunii (Botryococcus braunii), Botryococcus sudeticus, Botryococcus sp., Botryococcus spp..Botryococcus braunii comprises A, B and tri-strains of L.
In concrete embodiment, adopt Botryococcus braunii (Botryococcus braunii) A strain, the optimum growth temperature of A strain is 25 ℃, has surpassed 30 ℃ of speeds of growth just slack-off, A strain is produced C
23-C
33the unramified straight chain diolefine of odd number carbon and alkatrienes, also can be at thin intracellular accumulation grease.
Heterotrophism or raise together with
Can adopt various substratum well known in the art carry out the heterotrophism of grape algae seed or raise together with cultivation.Do not have illumination different from heterotrophism, raising together with is to have illumination.In heterotrophism and the substratum of raising together with, all need to add organic carbon source.Conventionally, heterotrophism, with to raise together with substratum identical, all contains nitrogenous source, organic carbon source, inorganic salt, trace element and water.Being applicable to nitrogenous source that grape algae cultivates, organic carbon source, inorganic salt, trace element etc. is that this area is known.For example, as nitrogenous source, spendable have urea or various nitrate, as KNO
3deng; As organic carbon source, can make any carbon source that can be used as microorganism culturing, include but not limited to glucose, maltose, sucrose, lactose, glycerine, starch.But there are no at heterotrophism or add the report of plant growth hormones in raising together with substratum, the present invention has creatively added significantly Promote cell's growth of plant growth hormones in grape algae culture medium.
For the remarkable Promote cell's growth of the plant growth hormones of substratum of the present invention (comprise heterotrophism, raise together with substratum and light autotrophy substratum), this parahormone includes but not limited to 2,4 dichlorophenoxyacetic acid, benzyladenine, dormin, Plant hormones regulators,gibberellins, 3-indolebutyric acid, naphthylacetic acid and rapin etc.In substratum, can contain one or more plant growth hormoness.In substratum, the total content of plant growth hormones can be 0.001-35 mg/litre substratum, conventionally in 0.001-20 mg/litre, is not more typically 0.001-15 mg/litre, 0.005-10 mg/litre, 0.01-10 mg/litre, 0.1-5 mg/litre not etc.
In specific embodiment, if each plant growth hormones exists, its concentration can be, for example 2,4 dichlorophenoxyacetic acid 0.001-5 mg/litre, benzyladenine 0.001-5 mg/litre, dormin 0.001-5 mg/litre, Plant hormones regulators,gibberellins 0.001-5 mg/litre, 3-indolebutyric acid 0.001-5 mg/litre, naphthylacetic acid 0.001-5 mg/litre, rapin 0.001-5 mg/litre.The concentration of each plant growth hormones is preferably separately, such as 0.01-4 mg/litre, 0.1-4 mg/litre, 0.3-4 mg/litre, 0.3-3 mg/litre, 0.5-2.5 mg/litre not etc.
Can obtain described plant growth hormones from commercially available approach, then directly add in heterotrophism disclosed in this invention/raise together with and substratum that light autotrophy is used, the example of this class substratum as mentioned below.
The present invention's heterotrophism used and raise together with substratum substantially by KNO
3, glucose, plant growth hormones, 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
2one or more in O or whole.
In this substratum, each component of substratum can change within the specific limits and can not have very large materially affect to microalgae cell density and quality.Therefore, the consumption of these components should not be subject to the strict restriction of embodiment.As known to those skilled in the art, in substratum, also can add inorganic salt, such as magnesium sulfate, calcium chloride, ferrous sulfate and phosphoric acid salt etc., and trace element is as Mn, Zn, B, I, M, Cu, Co etc.
In the present invention, preferably 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
2one or more in O.Inorganic salt and micro-consumption can be determined according to conventional knowledge.
Heterotrophism of the present invention and raise together with substratum and substantially consist of the following composition: KNO
30.1～15 grams per liter, glucose 0.1～50 grams per liter, K
2hPO
40.1～10.0 grams per liter, MgSO
47H
2o0.1～10 grams per liter, CaCl
22H
2o0.01～5 grams per liter, ironic citrate 0.01～5 grams per liter, citric acid 0.1～15 grams per liter, 2,4-dichlorphenoxyacetic acid 0.001-5 mg/litre, benzyladenine 0.001-5 mg/litre, dormin 0.001-5 mg/litre, Plant hormones regulators,gibberellins 0.001-5 mg/litre, 3-indolebutyric acid 0.001-5 mg/litre, naphthylacetic acid 0.001-5 mg/litre, rapin 0.001-5 mg/litre, trace element 0.5～20ml and water, wherein trace element consists of H
3bO
30.1～10 grams per liter, ZnSO
47H
2o0.1～10.0 grams per liter, MnCl
24H
2o0.5～10.0 grams per liter, (NH
4)
6mo
7o
244H
2o0.01～5 grams per liter, CuSO
45H
2o0.01～5.0 grams per liter, Co (NO
3)
26H
2o0.01～5 grams per liter.
In one embodiment, heterotrophism of the present invention and raise together with substratum and substantially consist of the following composition: KNO
30.2～5 grams per liter, glucose 1～15 grams per liter, K
2hPO
40.1～2.0 grams per liter, MgSO
47H
2o0.1～2.0 grams per liter, CaCl
22H
2o0.05～1.0 grams per liter, ironic citrate 0.05～0.5 grams per liter, citric acid 0.1～1.5 grams per liter, dormin 0.001-5 mg/litre, Plant hormones regulators,gibberellins 0.001-5 mg/litre, 3-indolebutyric acid 0.001-5 mg/litre, rapin 0.001-5 mg/litre, trace element 0.5～10ml and water, wherein trace element consists of H
3bO
31～5 grams per liter, ZnSO
47H
2o0.1～1.5 grams per liter, MnCl
24H
2o0.5～5.0 grams per liter, (NH
4)
6mo
7o
244H
2o0.01～0.2 grams per liter, CuSO
45H
2o0.01～0.5 grams per liter and Co (NO
3)
26H
2o0.05～0.5 grams per liter.
At one preferably in embodiment, heterotrophism of the present invention and raise together with culture media composition and should consist of the following composition: KNO
31.2 grams per liters, glucose 5 grams per liters, K
2hPO
40.6 grams per liter, MgSO
47H
2o0.8 grams per liter, CaCl
20.2 grams per liter, ironic citrate 0.1 grams per liter, citric acid 0.6 grams per liter, dormin 1 mg/litre, Plant hormones regulators,gibberellins 0.8 mg/litre, rapin 2 mg/litre, micro-6ml and 1000ml water, wherein trace element consists of H
3bO
32～3 grams per liters, ZnSO
47H
2o0.1～0.3 grams per liter, MnCl
24H
2o1～2.5 grams per liter, (NH
4)
6mo
7o
244H
2o0.01～0.04 grams per liter, CuSO
45H
2o0.05～0.1 grams per liter, Co (NO
3)
26H
2o0.5～0.1 grams per liter.
According to after above-mentioned formulated substratum, available conventional means as acid or alkali by as described in the pH of substratum be adjusted to 5.0～11.0, preferably 7.0～9.0, and at 115～120 ℃ autoclaving 15～20 minutes.Can adopt batch culture, fed batch cultivation, repeated fed-batch culture, semicontinuous cultivation or cultured continuously isotype implement described heterotrophism or raise together with cultivation.
When carrying out seed heterotrophism or raise together with cultivation, the corresponding substratum preparing is joined in bio-reactor, benefit adds water to working volume, conventionally coefficient is 0.6～0.8, then steam sterilizing is (121 ℃, maintain approximately 20 minutes), when temperature is down to 20～30 ℃, by 1～15% access grape algae of working volume, starts heterotrophism or raise together with cultivation.
When grape algae heterotrophism is cultivated, do not need illumination.When grape algae is raised together with cultivation, need illumination, illumination can utilize natural light (sunlight), can be also artificial light, as luminescent lamp, LED lamp etc.Source of artificial light can or also can be deep in the outside of nutrient solution (external light source) nutrient solution inside (internal light source).Intensity of illumination scope 1～2500 μ molm
-2s
-1, in embodiment preferably, light intensity is 1～900 μ molm
-2s
-1.
No matter adopt any training method in batch culture, fed batch cultivation, repeated fed-batch culture, semicontinuous cultivation or cultured continuously, in culturing process, must control applicable culture condition and make micro-algae seed normal growth.Conventionally, controlling temperature is 20～35 ℃, and for example 22～30 ℃, dissolved oxygen is not less than 5% air saturation concentration, and pH is controlled at 6.0～9.0.In a preferred embodiment, temperature is 24～28 ℃, and dissolved oxygen is not less than 10% air saturation concentration, and pH is controlled at 7.5～8.0.
Heterotrophism is cultivated and can in the bio-reactor of heterotrophism cultivation, be carried out at shaking flask, mechanical agitation type, air lift type, bubbling style etc.Algae kind raise together with can shaking flask, can steam sterilizing closed photo bioreactor (transparent), and carry out in the bio-reactor (containing external light source and/or internal light source) containing illumination system such as mechanical agitation type.
When seed heterotrophism and raise together with glucose consumption in nutrient solution complete after, seed heterotrophism or raise together with to cultivate and finish.Heterotrophism or time of raising together with are conventionally at about 3-20 days.
In the method for the invention, heterotrophism or raise together with and cultivate in grape algae process, by control of additive raw material pH, the element such as constant and carbon, nitrogen and/or phosphorus is stabilized within the scope of finite concentration, and heterotrophism or raise together with is cultivated, and while finishing, to control the concentration of the nutritive ingredients such as carbon, nitrogen and/or phosphorus lower be even zero.
Heterotrophism or raise together with in process, is controlled at the pH of algae liquid by feed supplement steady state value, for example a pH8.0 in the scope of 6.0-10.0.Conventionally the pH of algae liquid is controlled to a steady state value within the scope of 7.5-9.0, is more preferably controlled in the scope of 7.8-8.5.
The a little change that should be understood that pH value allows.For example, can allow the change of have ± Y of pH, wherein Y≤1.0, for example Y≤0.2, Y≤0.1.In certain embodiments, Y=0.Therefore, in a specific embodiment, by feed supplement, the pH of algae liquid is controlled as X ± Y, wherein, these 5.0≤X ± Y≤9.0.For example, in an embodiment of the invention, within the pH of algae liquid being controlled at by feed supplement to 8.0 ± 0.3 scope.
Heterotrophism or raise together with and cultivate in micro-algae process, can be controlled at the content of carbon in algae liquid in the scope of 2-20mM by feed supplement, and the content of nitrogen is controlled in the scope of 0.5-10mM, and the content of phosphorus is controlled in the scope of 0.05-3.5mM.
In one embodiment, can the content of carbon in algae liquid be controlled in the scope of 2-12mM by feed supplement, the content of nitrogen is controlled in the scope of 1-6mM, and the content of phosphorus is controlled in the scope of 0.1-2.5mM.
Also comprise by feed supplement the content of magnesium in algae liquid is controlled in the scope of 0.05-3mM.In a specific embodiment, by feed supplement, the content of magnesium in algae liquid is controlled in the scope of 0.10-2.0mM.
Heterotrophism or raise together with while cultivate finishing, it is even zero for being substantially exhausted that the concentration of the nutritive ingredients such as carbon, nitrogen and/or phosphorus is controlled, for example, the concentration of carbon source is zero, nitrogenous source and/or phosphorus source/or the concentration in magnesium source be controlled at below 0.01mM.
Light autotrophy
Can be first to obtained grape algae heterotrophism or raise together with and add light autotrophy substratum in nutrient solution, and carry out the cultivation of light autotrophy after adding thin up.
Or, can be directly to heterotrophism or raise together with and cultivate the grape frustule obtaining and carry out the cultivation of light autotrophy, directly add light autotrophy substratum and carry out the cultivation of light autotrophy.
Subsequently, be inoculated in light autotrophy culture apparatus and carry out the cultivation of light autotrophy, be not added with organic compounds, the in the situation that of essential inorganic nutrients and luminous energy existence, to the CO as carbon source
2reduce assimilation, the training mode that in synthetic cell, all organic metabolites carry out.The Initial seeding density that light autotrophy is cultivated is generally 0.01～1 grams per liter, and temperature is 5～50 ℃, and intensity of illumination is 1-2500 μ molm
-2s
-1, continuous illumination or intermittent illumination, pH is 4.0～9.0, air flow is 0.05～5vvm, passes into CO
2concentration is 0.03～10%.
When frustule is grown in stationary phase (when algae cell density does not increase), finish light autotrophy and cultivate, frustule is gathered.The light autotrophy time is generally 1-50 days.
Can adopt various smooth autotrophy substratum well known in the art to carry out the cultivation of light autotrophy.Conventionally, light autotrophy substratum contains nitrogenous source, phosphorus source, inorganic carbon source, inorganic salt, trace element and water.Being applicable to nitrogenous source that micro-algae cultivates, phosphorus source, inorganic carbon source, inorganic salt, trace element etc. is that this area is known.For example, as nitrogenous source, spendable have urea or various nitrate, as KNO
3deng; As phosphorus source, spendable have a for example K
2hPO
4; As inorganic carbon source, spendable have a for example CO
2deng.In order to promote cell Fast Growth, the present invention has creatively added plant growth hormones in light autotrophy substratum.
Smooth autotrophy substratum of the present invention consists of the following composition substantially: KNO
30.1～15 grams per liter, K
2hPO
40.1～10.0 grams per liter, MgSO
47H
2o0.1～10 grams per liter, CaCl
22H
2o0.01～5 grams per liter, ironic citrate 0.01～5 grams per liter, citric acid 0.1～15 grams per liter, 2,4-dichlorphenoxyacetic acid 0.001-5 mg/litre, benzyladenine 0.001-5 mg/litre, dormin 0.001-5 mg/litre, Plant hormones regulators,gibberellins 0.001-5 mg/litre, 3-indolebutyric acid 0.001-5 mg/litre, naphthylacetic acid 0.001-5 mg/litre, rapin 0.001-5 mg/litre, trace element 0.5～20ml and water, wherein trace element consists of H
3bO
30.1～10 grams per liter, ZnSO
47H
2o0.1～10.0 grams per liter, MnCl
24H
2o0.5～10.0 grams per liter, (NH
4)
6mo
7o
244H
2o0.01～5 grams per liter, CuSO
45H
2o0.01～5.0 grams per liter, Co (NO
3)
26H
2o0.01～5 grams per liter.
In one embodiment, smooth autotrophy substratum of the present invention consists of the following composition substantially: KNO
30.2～5 grams per liter, K
2hPO
40.1～2.0 grams per liter, MgSO
47H
2o0.1～2.0 grams per liter, CaCl
22H
2o0.05～1.0 grams per liter, ironic citrate 0.05～0.5 grams per liter, citric acid 0.1～1.5 grams per liter, dormin 0.001-5 mg/litre, Plant hormones regulators,gibberellins 0.001-5 mg/litre, 3-indolebutyric acid 0.001-5 mg/litre, rapin 0.001-5 mg/litre, trace element 0.5～10ml and water, wherein trace element consists of H
3bO
31～5 grams per liter, ZnSO
47H
2o0.1～1.5 grams per liter, MnCl
24H
2o0.5～5.0 grams per liter, (NH
4)
6mo
7o
244H
2o0.01～0.2 grams per liter, CuSO
45H
2o0.01～0.5 grams per liter and Co (NO
3)
26H
2o0.05～0.5 grams per liter.
At one, preferably in embodiment, smooth autotrophy culture media composition of the present invention should consist of the following composition: KNO
31.2 grams per liter, K
2hPO
40.6 grams per liter, MgSO
47H
2o0.8 grams per liter, CaCl
20.2 grams per liter, ironic citrate 0.1 grams per liter, citric acid 0.6 grams per liter, dormin 1 mg/litre, Plant hormones regulators,gibberellins 0.8 mg/litre, rapin 2 mg/litre, micro-6ml and 1000ml water, wherein trace element consists of H
3bO
32～3 grams per liters, ZnSO
47H
2o0.1～0.3 grams per liter, MnCl
24H
2o1～2.5 grams per liter, (NH
4)
6mo
7o
244H
2o0.01～0.04 grams per liter, CuSO
45H
2o0.05～0.1 grams per liter, Co (NO
3)
26H
2o0.5～0.1 grams per liter.
According to after above-mentioned formulated substratum, available conventional means as acid or alkali by as described in the pH of substratum be adjusted to 6.0～9.0, and at 115～120 ℃ autoclaving 15～20 minutes or adopt clorox sterilization 2～20h, then with Sulfothiorine, neutralize.Can adopt four kinds of modes such as batch culture, fed batch cultivation, semicontinuous cultivation (band is put) or cultured continuously to implement light autotrophy cultivates.
Frustule gather and cell in the extraction of total hydrocarbon
Light autotrophy is carried out centrifugal gathering to grape algae after cultivating and finishing, and obtains wet frond.The collecting method of frustule includes but not limited to high speed centrifugation, flocculation, the technology such as air supporting or filtration; Frustule wall-breaking method includes but not limited to the Wet-process wall breaking methods such as frond self-dissolving, high-pressure homogenization, enzymic hydrolysis, water pyrolysis.
In born of the same parents, the extraction and determination method of grease includes but not limited to organic solvent extractionprocess, that is: frond is dried to constant weight at 80～105 ℃, after grinding algae powder, adopt chloroform methanol standard extraction solvent from dry algae powder, to extract grease, extraction solvent extracts repeatedly until algae powder color becomes white, and rotary evaporation is removed solvent.
In born of the same parents, the extraction and determination method of total hydrocarbon includes but not limited to organic solvent extractionprocess, that is: frond is dried to constant weight at 80～105 ℃, after grinding algae powder, adopt normal hexane from dry algae powder, to extract total hydrocarbon, extraction solvent extracts repeatedly until algae powder color becomes white, with nitrogen, dries up removal solvent.The extracting method of total hydrocarbon also comprises the hydrocarbon of directly secreting in isolated cell from nutrient solution.
The measuring method that relates to frustule dry weight and total hydrocarbon content is herein as follows:
Frustule dry weight is measured: in grape algae culturing process, get nutrient solution V milliliter, centrifugal 10 minutes of 8000rpm, by deionized water wash 3 times of the frond after centrifugal, is transferred to weighing bottle (W1(gram)) in, in 80 ℃ of baking ovens, dry to constant weight W2(gram).Frond dry weight Cx can calculate according to following formula: Cx(grams per liter)=(W2-W1)/V/1000.
Grease is measured: get a certain amount of each cultivation stage and dry the frustule to constant weight, in mortar, be ground to Powdered, taking appropriate algae powder (0.2～0.5g) is carefully transferred in centrifuge tube, add appropriate extraction solvent (chloroform: methyl alcohol=2:1) sonic oscillation 30min in ultrasonator, the centrifugal 10min of 8000rpm, supernatant is transferred in the dry rotary evaporation bottle of known weight, repeats above-mentioned steps until supernatant is colourless.After merging supernatant, rotate evaporate to dryness, weigh and calculate fat content.
Fat content (%) is calculated as follows:
Grease (%)=(W2-W0)/W1 * 100
In formula: W1---be algae grain weight amount, g; W0---for drying the rotary evaporation bottle weight to constant weight, g; W2---be the weight of evaporative flask after oil extraction liquid evaporate to dryness, g.
Total hydrocarbon is measured: get a certain amount of each cultivation stage and dry the frustule to constant weight, in mortar, be ground to Powdered, taking appropriate algae powder (0.2～0.5g) is carefully transferred in centrifuge tube, add appropriate extraction solvent (normal hexane) sonic oscillation 30min in ultrasonator, the centrifugal 10min of 5000rpm, supernatant is transferred in the screw-cap test tube of known weight, repeats above-mentioned steps until supernatant is colourless.After merging supernatant, at room temperature with nitrogen, normal hexane is dried up, weigh and calculate total hydrocarbon content.
Total hydrocarbon content (%) is calculated as follows:
Total hydrocarbon (%)=(W2-W0)/W1 * 100
In formula: W1---be algae grain weight amount, g; W0---for drying the screw-cap test tube weight to constant weight, g; W2---for extraction liquid dries up the weight of rear screw-cap test tube, g.
Below will to related content of the present invention, be further described by embodiment.Unless otherwise described, in the substratum that the present invention adopts, each component concentration all uses grams per liter (g/L) to represent.Should be understood that in the application " contain ", " comprising " also comprise " by ... form ", " by ... form " implication.
Embodiment 1: Botryococcus braunii (Botryococcus braunii) cell heterotrophism, raise together with and light autotrophy culturing process in the research of frustule growth
The grape algae of the present embodiment carries out respectively heterotrophism, raises together with and the cultivation of light autotrophy in the shaking flask of 500ml.The inoculum density that grape algae heterotrophism is cultivated is 0.05g/l, and temperature is 25 ℃, and rotating speed is 150rmp.The condition that grape algae is raised together with cultivation is consistent with heterotrophism cultivation, and except there is illumination outside, light intensity is 80 μ molm
-2s
-1.Grape algae heterotrophism is cultivated 12d with raising together with, and the glucose in nutrient solution has consumed, and algae cell density is 0.70g/l and 0.78g/L, can be used for the seed that next step light autotrophy is cultivated; Inoculum density when grape algae seed light autotrophy is cultivated is 0.05g/l, and temperature is 25 ℃, and intensity of illumination is 500 μ molm
-2s
-1, continuous illumination, while cultivating 12d, algae cell density is only 0.20g/l, the seed (Fig. 1) of cultivating for next step light autotrophy.As can be seen here, cultivate and to compare with light autotrophy, the frustule growth velocity that cultivation was cultivated and raised together with to heterotrophism is than light autotrophy cultured cells growth velocity fast (be respectively light autotrophy incubation growth speed 3.5 and 3.9 times)
Embodiment 2: control Botryococcus braunii (Botryococcus braunii) under different raise craft condition heterotrophism and raise together with culturing process comparison in 5L bio-reactor
In 5L bio-reactor, add described heterotrophism or raise together with substratum and water carries out steam sterilizing to 2.5L, then when temperature drops to 25 ℃, access grape algae, start heterotrophism or raise together with cultivation.Raise together with while cultivating, it is 700 μ molm that exterior light is shone
-2s
-1.At heterotrophism or raise together with while cultivating, start feed supplement, by controlling the Continuous Flow of supplemented medium, add pH constant in 8.0 ± 0.3.Supplemented medium comprises organic carbon source (glucose), nitrogenous source (KNO
3), the nutritive salt such as plant growth hormones and inorganic salt, the nutritive salt of adding is the above-mentioned corresponding substratum after concentrated, impel micro-algae continued growth, monitor in time the content of carbon, nitrogen, phosphorus, magnesium in fermented liquid simultaneously, suitably adjust this 4 class material at supplemented medium content, to guarantee that in fermented liquid, this 4 class material concentration is stable.In the situation that not adding hormone, other operations are all identical with experiment condition, only in substratum, do not contain plant growth hormones.And the in the situation that of optimal control not, only monitor the pH in fermented liquid, by supplemented medium, keep pH constant in 8.0 ± 0.3, other materials are not controlled as the content of carbon, nitrogen, phosphorus, magnesium, and hormonal substance does not also add, other experiment conditions are identical with operation.
The results are shown in Figure 2.When heterotrophism is cultivated end, to control pH and feeding strategy, and add the heterotrophism of plant hormone to cultivate, its dry cell weight reaches 15.6g/l; And only control pH and feeding strategy but do not add the heterotrophism of plant hormone to cultivate, its dry cell weight reaches 6.5g/l; And optimal feed strategy and do not add the heterotrophism of plant hormone to cultivate not, its dry cell weight is only 4.1g/l.Therefore, control feeding strategy optimization and add the heterotrophism of plant hormone to cultivate, with simple control pH but optimal feed strategy and the heterotrophism that do not add plant growth hormones are not cultivated and are compared, and cell density has improved 2.8 times; And compare with only controlling pH with feeding strategy but do not add the heterotrophism of plant hormone to cultivate, cell density has improved 1.4 times.When raising together with cultivation end, to control pH and feeding strategy, and add the heterotrophism of plant hormone to cultivate, its dry cell weight reaches 16.5g/l; And only control pH and feeding strategy but do not add the heterotrophism of plant hormone to cultivate, its dry cell weight reaches 7.6g/l; And optimal feed strategy and do not add the heterotrophism of plant hormone to cultivate not, its dry cell weight is only 4.8g/l.
Embodiment 3: grape algae heterotrophism seed, raise together with seed and light autotrophy seed carries out the research of light autotrophy cultivation in indoor 2L bioreactor
The present embodiment light autotrophy in indoor 2L cylinder shape airlift photobioreactor cultivates heterotrophism, raise together with and the grape algae seed of light autotrophy, measured respectively heterotrophism, raised together with and algae cell density, final grease and total hydrocarbon content and the productive rate of light autotrophy seed in its light autotrophy process.Heterotrophism, raise together with and the inoculum density of light autotrophy seed is 0.3g/l, light autotrophy culture temperature is 25 ℃, all passes into 2% CO
2, air flow is 0.25vvm.Raising together with intensity of illumination while cultivating with light autotrophy, be 111 μ molm
-2s
-1, continuous illumination.Light autotrophy is cultivated 12d, and the algae cell density of heterotrophism seed is 1.98g/l, and total hydrocarbon productive rate is 50.58mg/l/d, grease productive rate 33.61mg/l/d; The algae cell density of raising together with seed is 2.1g/L, and total hydrocarbon productive rate is 58.13mg/l/d, and grease productive rate is 37.35mg/l/d; The algae cell density of light autotrophy seed is only 0.98g/l, and total hydrocarbon productive rate is only 21.08mg/l/d, grease productive rate 13.62mg/l/d(Fig. 3 and Fig. 4).As can be seen here, compare with light autotrophy seed, heterotrophism is stronger with the vigor of raising together with seed, the algae cell density of identical incubation time higher (be respectively light autotrophy seed 2.02 and 2.14 times), total hydrocarbon productive rate higher (be respectively light autotrophy seed 2.40 and 2.76 times), grease productive rate higher (be respectively light autotrophy seed 2.47 and 2.74 times).
Embodiment 4: grape algae heterotrophism seed, raise together with seed and light autotrophy seed 60L(liquid amount 40L out of doors) carry out the research of light autotrophy cultivation in plastic tub
The present embodiment out of doors in 60L plastic tub light autotrophy cultivate heterotrophism, raise together with and the grape algae seed of light autotrophy, measured respectively heterotrophism, raised together with and frustule growth and the total hydrocarbon productive rate of light autotrophy seed in its light autotrophy process.Initial seeding density is 0.15g/l, and temperature and light intensity are outdoor natural temperature and light intensity, and air flow is 0.3vvm.Light autotrophy is cultivated 12d.The algae cell density of heterotrophism seed is 0.92g/l, and total hydrocarbon productive rate is 21.85mg/l/d, grease productive rate 16.28mg/l/d; The algae cell density of raising together with seed is 0.95g/L, and total hydrocarbon productive rate is 23.32mg/l/d, grease productive rate 17.37mg/l/d; The algae cell density of light autotrophy seed is only 0.45g/l, and total hydrocarbon productive rate is only 9.49mg/l/d, and grease productive rate 7.5mg/l/d(is shown in Fig. 5 and Fig. 6).As can be seen here, compare with light autotrophy seed, heterotrophism is stronger with the vigor of raising together with seed, the algae cell density of identical incubation time higher (be respectively light autotrophy seed 1.92 and 1.98 times), total hydrocarbon productive rate higher (be respectively light autotrophy seed 2.30 and 2.46 times), grease productive rate higher (be respectively light autotrophy seed 2.17 and 2.31 times).
Although object lesson of the present invention described above, having is a bit significantly to those skilled in the art, can the present invention be made various changes and be changed 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. a grape algae cultural method, is characterized in that, the method comprises the heterotrophism of grape algae or raises together with culturing step and using heterotrophism or raise together with the light autotrophy culturing step that frustule that cultivation obtained is implemented as seed.
2. the production method of a grease and/or hydrocarbon, it is characterized in that, described method comprise the heterotrophism of grape algae algae kind or raise together with culturing step, light autotrophy culturing step that frustule that the heterotrophism or raise together with of usining is obtained is implemented as seed and frustule is gathered and the extraction step of grease and hydrocarbon.
3. efficient grape algae cultural method or a method of producing fast grease and/or hydrocarbon, is characterized in that, the method comprises:
(1) heterotrophism or raise together with and cultivate grape algae, wherein, culturing process, by control of additive raw material pH, the element such as constant and carbon, nitrogen and/or phosphorus is stabilized within the scope of finite concentration, and heterotrophism or raise together with cultivates that while finishing, to control the concentration of the nutritive ingredients such as carbon, nitrogen and/or phosphorus lower be even zero;
(2) using heterotrophism or raise together with gained frustule and implement light autotrophy as seed and cultivate, and
(3) gather frustule and separation and Extraction grease and/or hydrocarbon.
4. the method as described in any one in claim 1-3, is characterized in that, grape algae algae kind used includes but not limited to Botryococcus braunii, Botryococcus sudeticus, Botryococcus sp., Botryococcus spp..
5. the method as described in any one in claim 1-3, is characterized in that, adopts batch culture, fed batch cultivation, repeated fed-batch culture, semicontinuous cultivation or cultured continuously isotype implement described heterotrophism or raise together with cultivation.
6. the method as described in any one in claim 1-3, it is characterized in that, described heterotrophism or raise together with culturing step and comprise: adding pH in bio-reactor is 5.0～11.0 substratum, the micro-algae algae kind of 0.1～30% access by working volume is carried out batch culture, fed batch cultivation, repeated fed-batch culture, semicontinuous cultivation or cultured continuously, culture temperature is 10～40 ℃, control pH and be less than 10.0, control dissolved oxygen more than 1%.
7. the method as described in any one in claim 1-4, is characterized in that, when described grape algae algae kind heterotrophism is cultivated, does not need illumination; Described grape algae algae kind is raised together with while cultivating, and need to carry out illumination, range of light intensity 1～2500 μ molm
-2s
-1.
8. the method as described in any one in claim 1-3, it is characterized in that, using heterotrophism or raise together with frustule that cultivation obtained and carry out light autotrophy as seed and cultivate and comprise: heterotrophism or the algae kind of raising together with cultivation are received and in light autotrophy culture apparatus, carried out the cultivation of light autotrophy, culture temperature is 5～50 ℃, continuous illumination or intermittent illumination, intensity of illumination is 1～2500 μ molm
-2s
-1, light autotrophy culture cycle is 1～180 day, and Initial seeding density is 0.01～5.0 grams per liter, and described substratum is not containing organic carbon source, and its pH is 4.0～9.0.
9. the method as described in any one in claim 1-3, it is characterized in that, the heterotrophism of algae kind or raise together with substratum and contain nitrogenous source, organic carbon source, plant growth hormones, inorganic salt, trace element and water, or formed by described nitrogenous source, organic carbon source, plant growth hormones, inorganic salt, trace element and water; Described smooth autotrophy substratum contains plant growth hormones, nitrogenous source, inorganic salt and water, or is comprised of plant growth hormones, nitrogenous source, inorganic salt and water.
10. the method as described in any one in claim 1-3, is characterized in that, described heterotrophism culturing step carries out in shaking flask, mechanical agitation type, air lift type or Sparged bioreactors; Described raise together with step shaking flask, can steam sterilizing closed photo bioreactor (transparent), and mechanical agitation type etc. carries out containing in the bio-reactor of illumination system; Described smooth autotrophy culturing step is in shaking flask or be selected from the raceway pond of open type or the light autotrophy culture systems of circle pond, enclosed flat bioreactor or duct type bioreactor or pillar bioreactor or the vertical bag of film or open type and closed hybridization or adherent culture system etc. are carried out for any device of micro-algae light autotrophy cultivation, and illumination condition is natural light or artificial light.
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| CN104357501A (en) * | 2014-11-14 | 2015-02-18 | 暨南大学 | Method for promoting microalgae to accumulate fat quickly |
| CN104357501B (en) * | 2014-11-14 | 2018-03-20 | 暨南大学 | A kind of method for promoting microalgae frond accumulation grease |
| CN106929550A (en) * | 2015-12-30 | 2017-07-07 | 中国石油天然气股份有限公司 | A kind of method that biodiesel raw material is produced with Botryococcus braunii |
| CN105671095A (en) * | 2016-02-27 | 2016-06-15 | 昆明理工大学 | Cultivation method for promoting rapid accumulation of oil producing microalgae cells and grease |
| CN106190853A (en) * | 2016-04-18 | 2016-12-07 | 嘉兴泽元生物制品有限责任公司 | A kind of red algae cultural method of high yield phycocyanin |
| CN106190853B (en) * | 2016-04-18 | 2019-11-26 | 嘉兴泽元生物制品有限责任公司 | A kind of red algae cultural method of high yield phycocyanin |
| CN112391293A (en) * | 2020-11-25 | 2021-02-23 | 宁波大学 | Method for preparing squalene by autotrophic culture of botryococcus through heterotrophic biomembrane adherence |
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| WO2015085631A1 (en) | 2015-06-18 |
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