CN101979498A - High-yield heterotrophic culture method for microalgae - Google Patents
High-yield heterotrophic culture method for microalgae Download PDFInfo
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Abstract
The invention relates to a new semi-continuous heterotrophic culture method for microalgae. The heterotrophic culture method for the microalgae can realize high-yield heterotrophic culture of the microalgae. The method can be applied to large-scale efficient production of heterotrophic microalgae or/and active substances thereof.
Description
Technical field
The invention belongs to little algae biological technical field, relate to a kind of little algae high yield heterotrophism cultured method.
Background technology
Multiple high value active substances such as rich in proteins, polysaccharide, lipid acid and carotenoid in the microalgae cell.Therefore, present little algae has application widely in all many-sides such as food, medicine, feed, environmental protection and bioenergies.
The main training method of little algae has light autotrophy, mixotrophism and heterotrophism to cultivate.At present, little algae (as chlorella, spirulina, salt algae, Haematocoocus Pluvialls etc.) of having realized commercial application nearly all is to adopt light autotrophy mode to cultivate, but also has the large scale culturing of the little algae of minority (as the Kou Shi Crypthecodinium cohnii etc.) to adopt the heterotrophism method.
The light autotrophy of little algae is cultivated and is existed mainly that cell density is low, production efficiency is low, the cost height of gathering, be vulnerable to assorted bacterium and shortcomings such as protozoic pollution and natural environment and climate condition influence.Mixotrophism is cultivated and need be cultivated in the bioreactor that can sterilize, need ensure sterile culture and competent illumination condition simultaneously, to culture device require highly, equipment is difficult to amplify simultaneously.Therefore, in little algae large scale culturing of reality, almost do not adopt the mixotrophism mode to cultivate.
Except above-mentioned two kinds of training methods, the little algae of part can carry out heterotrophism to be cultivated, and promptly makes little algae heterotrophic growth by adding organic carbon sources such as glucose.This training method has the following advantages: (1) is not subjected to the restriction of conditions such as natural environment and climate; (2) can keep pure culture, thereby guarantee product quality homogeneity; (3) cultivate with respect to the light autotrophy, can obtain high cell concn and production efficiency; (4) can use for reference and utilize the industrial fermentation technology of comparative maturity and production unit etc.
Therefore, in above-mentioned three kinds of little algae training modes, heterotrophism is cultivated easy realization of industrialization, enjoys people's attention.Many investigators produce high value added product by the heterotrophism best cultivation, as xenthophylls, astaxanthin, VITAMIN, xitix etc.; In addition, also there is the investigator to adopt little algae heterotrophism training method to produce thing diesel oil next life.
Existing little algae heterotrophism training method mainly contains in batches and fed batch cultivation, does not see and utilizes semicontinuous method to carry out the report of the heterotrophism cultivation aspect of little algae.Batch culture is a kind of mode that more generally adopts during heterotrophism is cultivated.At present, cultivate chlorella by batch mode, algae cell density is (ShiXM about about 20g/L, Zhang XW, Chen F.Heterotrophic production of biomass and lutein by Chlorella protothecoides on various nitrogen sources.Enzyme and microbial technology.2000,27:312-318).Fed batch cultivation can obtain higher frustule concentration and productive rate with respect to batch culture.In the fed batch cultivation, mainly contain flow feeding and intermittent type feed supplement mode.Wu Zhengyun etc. cultivate Chlorella pyrenoidesa at 19L fermentor tank flow feeding heterotrophism, high cell concentration and the growth velocity of chlorella reach 116.2g/L and 1.02g/ (Lh) (Wu ZY, Shi XM.Optimization for high-density cultivation of heterotrophic Chlorella based on a hybrid neural network model.Letters in Applied Microbiology.2007,44 (1): 13-18).And the fed-batch process in the employing Chinese invention patent (ZL 200610025618.9) is cultivated Chlorella pyrenoidesa in the 50L jar, cultivates about 90h, and algae cell density can reach 150g/L.
Little algae heterotrophism is cultivated has lot of advantages, and heterotrophism frustule quality is low, the interior activity substance content of born of the same parents is low and only be suitable for heterotrophism cultivates shortcomings such as algae kind but still exist.Little algae high-density and high-quality cultured method of Chinese invention patent (ZL200610025618.9) invention can solve little algae heterotrophism and cultivate the low problem of quality.Yet Chinese invention patent (ZL 200610025618.9) adopts the batch feeding culture technique in the high Density Heterotrophic that realizes little algae, still has following problem: the postvaccinal lag phase of (1) batch culture is long, productive rate is low; (2) late stage of culture is owing to cell density height, oxygen-consumption are big, and the oxygen delivery capacity wretched insufficiency of bio-reactor causes late growing stage speed obviously to reduce; When (3) the heterotrophism batch culture finished, lower as overcast and rainy then outdoor photoinduction culture efficiency, therefore number of devices and the illuminating area of cultivating the photoinduction culture systems that is complementary with little algae heterotrophism must increase considerably, and causes the disposable apparatus investment big; (4) cultivate for the heterotrophism of batch feeding, the photoinduction time is shorter, and therefore the utilization ratio of the photoinduction culture systems that is complementary with the heterotrophism culture device is lower; (5) cultivate for the batch feeding heterotrophism, in the time of can't carrying out photoinduction out of doors when running into exceedingly odious weather, then the little algae heterotrophism nutrient solution in the bio-reactor can't be emitted and must be continued to cultivate, but can cause frustule mass mortality even this batch nutrient solution all to be scrapped.
At the problems referred to above, the invention provides a kind of effective solution, in the heterotrophism culturing process, adopt semicontinuous cultivation operating method, promptly in the heterotrophism culturing process, emit part algae liquid, replenish the substratum or the sterilized water of respective volume then, make frustule in the heterotrophism culture apparatus, continue to cultivate.This method has improved the productive rate of frustule greatly, has reduced the oxygen supply requirement to bio-reactor, cultivates mass-producing light autotrophy microalgae cell is provided, also can be the little algae of the energy of heterotrophic growth and cultivates a large amount of seeds are provided but can be photoinduction at any time.
Cultivating with the semicontinuous heterotrophism of chlorella is example, and it is as follows to describe advantage of the present invention in detail:
(1) can improve the frustule productive rate greatly.Calculate by each band cycle of putting, the average growth velocity of frustule can reach 3.0g/ (Lh) in the one-period; And the fed batch cultivation technology of employing Chinese invention patent (ZL200610025618.9), the average growth velocity of frustule is about 0.867g/ (Lh); The average growth velocity of batch culture only is about 0.487g/ (Lh).
(2) compare with fed batch cultivation, semicontinuous cultivation has reduced the oxygen supply requirement to bio-reactor.As in the 50L jar during fed batch cultivation, mixing speed need be up to 800rpm; And adopt semicontinuous cultivation of the present invention, the highest 700rpm that is no more than of mixing speed.
(3) can be according to the outdoor weather condition, select tape is put the time flexibly: as select emit part algae liquid morning, can make full use of daylong sunlight like this and carry out photoinduction; Meet when overcast and rainy, can adjust band flexibly and put the time, when having overcome actual outdoor production effectively, adopt batch feeding to cultivate to run into the overcast and rainy algae liquid of can't emitting to cause the heterotrophism nutrient solution to be scrapped and cause direct economic loss and environmental issue;
(4) but can be at any time cultivate a large amount of algae kinds be provided for the energy micro-algae large-scale light autotrophy of heterotrophic growth: during the little algae large scale culturing of the energy; the algae kind time that spreads cultivation is grown and (cultivates for outdoor Da Chi; generally reach about 1-2 month); had a strong impact on the production efficiency of whole cultivation stage; semicontinuous heterotrophism culture technique provided by the invention; can provide highdensity algae liquid at any time, the problem that spreads cultivation fast of the algae kind when having solved energy micro-algae large-scale effectively and cultivating.
In sum, the semicontinuous heterotrophism training mode of little algae of the present invention can solve in the heterotrophism culturing process fully because the problems that the density height causes and and photoinduction mutual problem of mating on time and equipment.Therefore, this invention is the heterotrophism cultivation of little algae and the production of active substance thereof, in particular for little algae heterotrophism-dilution-the algae powder is produced in photoinduction series connection cultivation and active substance has been established solid industrialization basis.
Summary of the invention
The invention provides a kind of little algae high yield heterotrophism cultured method, can solve the problem that exists in little algae high Density Heterotrophic process, realize that the high yield serialization of little algae and active substance thereof is produced.
In an embodiment, adopt semicontinuous training method to carry out heterotrophism and cultivate, promptly in culturing process, emit part algae liquid, heterotrophism substratum and the sterilized water of adding respective volume simultaneously make the back continue to cultivate.
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, when described little algae heterotrophism was cultivated: adding pH was 4.0～9.0 substratum in bio-reactor, insert little algae algae kind by 0.1～30% of working volume and carry out semicontinuous cultivation cultivation, culture temperature is 10～40 ℃, control pH is 4.0～9.0, and the control dissolved oxygen is more than 1%.
In an embodiment, the heterotrophism substratum is made up of nitrogenous source, organic carbon source and inorganic salt, trace element and water.
In an embodiment, but described heterotrophism is cultivated and can be carried out in the bio-reactor that shakes bottle, mechanical agitation type, air lift type or the cultivation of bubbling style heterotrophism.
In an embodiment, described method also comprises, the microalgae cell after microalgae cell that band in the heterotrophism process is released and/or heterotrophism finish is directly made algae powder, or is used to extract active substance in the born of the same parents, or change the photoinduction cultivation over to, or change the cultivation of light autotrophy over to.
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, He Shui, 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 and Co (NO
3)
26H
2O 0.1～0.9 grams per liter.
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 and water; 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 and Na
2MoO
40.01～0.05 grams per liter.
In an embodiment, when the density of heterotrophic little algae reaches 5g/L when above, emit part algae liquid, replenish the substratum and the sterilized water of respective volume then, make frustule in the heterotrophism culture apparatus, continue cultivation.
In an embodiment, when emitting algae liquid, the algae liquid of emitting accounts for heterotrophism and cultivates 20～80% of algae liquid cumulative volume.
Description of drawings
Fig. 1 shows that Chlorella pyrenoidesa is in 50L bio-reactor batch feeding culturing process.
Fig. 2 shows that Chlorella pyrenoidesa is in the semicontinuous heterotrophism culturing process of 50L bio-reactor.
Fig. 3 shows that Chlorella vulgaris is in the semicontinuous heterotrophism culturing process of 50L bio-reactor.
Fig. 4 shows that chlorella ellipsoidea is in the semicontinuous heterotrophism culturing process of 5L bio-reactor.
Embodiment
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 algae door, Crypthecodinium cohnii; The Euglena gricilis of Euglenophyta; The Galdieria sulphuraria of rhodophyta.
In preferred embodiment, the present invention adopts Chlorella pyrenoidesa, Chlorella vulgaris and chlorella ellipsoidea.
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, inorganic salt, trace element and water.Those skilled in the art can determine nitrogenous source, organic carbon source, the inorganic salt in the heterotrophism substratum, the consumption of trace element according to the conventional knowledge in this area.
In a preferable embodiment, select HA-SK substratum (Chinese patent ZL200610024004.9) and Endo substratum (Ogbonna J.C. for use, Masui.H., Tanaka.H.Sequential heterotrophic:autotrophic cultivation-an efficient method of producing Chlorella biomass for health food and animal feed.J.Appl.Phycol.1997,9,359～366).
Corresponding prepared culture medium is joined in the bio-reactor, mend and add water to working volume, coefficient is 0.6～0.8 usually, 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.
Using before semicontinuous cultivation band puts, can adopt in batches or fed batch cultivation makes frustule reach certain density, 5～30g/L for example, 30～60g/L, 60～120g/L etc.
In a preferable embodiment, before putting, adopt by band batch feeding to cultivate, after heterotrophism is cultivated for some time, when being consumed (being generally 27～45 hours), glucose in the substratum need carry out feed supplement, (for example, the nitrogenous source of cultivating Chlorella vulgaris is KNO to add carbon source (for example glucose), nitrogenous source
3, the nitrogenous source of cultivating Chlorella pyrenoidesa is a urea) and nutritive salt such as inorganic salt, the nutritive salt of adding is the above-mentioned corresponding substratum after concentrated, impels little algae continued growth.Can be every feed supplement in 5～12 hours, the concentration of adding of glucose can be 15～25 grams per liters, and the concentration of adding of nitrogenous source solution can be 2～10 grams per liters.When little algae (for example chlorella) when cell density reaches a certain value (for example, reaches 5g/L (preferred 20g/L, 30g/L or 40g/L) when above when the density of heterotrophic little algae; In a preferred embodiment, for Chlorella vulgaris, 45～65 grams per liters, for Chlorella pyrenoidesa, 90～110 grams per liters), promptly from bio-reactor, bleed off the algae liquid (this algae liquid can change photoinduction over to and cultivate or other processing) of certain proportion (scale-up factor is 0.2～0.8), in bio-reactor, add heterotrophism substratum and sterilized water then, its working volume is reached to carry out the heterotrophism culture requirement (in a preferred embodiment, return to band and put front volume), algae cell density in the bio-reactor is reduced to be with and is put preoperative 0.2～0.8 at this moment, little afterwards algae continued growth (for example, reaches 5g/L when above when the density of heterotrophic little algae to a certain value; In a preferred embodiment, for Chlorella vulgaris, 45～65 grams per liters, for Chlorella pyrenoidesa, 90～110 grams per liters), and then be with and put operation, circulation successively finishes this heterotrophism and cultivates after band is put 10～20 times.
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 28～32 ℃, 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.
But heterotrophism can carry out in the bio-reactor that shakes heterotrophism cultivations such as bottle, mechanical agitation type, air lift type, bubbling style.In a preferred embodiment, adopt the mechanical agitation type bio-reactor.
It is as follows to relate to frustule dry weight measuring method herein:
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, be transferred in the weighing bottle (W1 (gram)), in 105 ℃ 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.
Embodiment 1 (Chlorella pyrenoidesa is in 50L jar fed batch cultivation)
Following heterotrophism substratum of adding and tap water are sterilized to 25L in the 50L bio-reactor, insert Chlorella pyrenoidesa when temperature is reduced to 30 ± 1 ℃, and the beginning heterotrophism is cultivated.
Cultivate beginning after feed supplement 5 times, cell density is 149.43g/L when 84.06h, when continuing feed supplement 2 times again, the obvious growth velocity of frustule is slow, being cultured to the 97.60h heterotrophism finishes, cell density is 158.56g/L, and cell growth rate descends after increasing earlier gradually, and the cell average yield is the 1.63g/L/h (see figure 1).The batch culture later stage, mixing speed was up to 800rpm.
The heterotrophism culture condition: temperature is 30 ± 2 ℃, and pH is less than 8.5, and the control dissolved oxygen is more than 5%.
Heterotrophism and supplemented medium:
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.
Embodiment 2 (the semicontinuous cultivation of Chlorella pyrenoidesa 50L jar)
Following heterotrophism substratum of adding and tap water are sterilized to 25L in the 50L bio-reactor, press 12% of working volume and insert Chlorella pyrenoidesa when temperature is reduced to 30 ± 1 ℃, and the beginning heterotrophism is cultivated.
The feed supplement for the first time in 47.4h hour of inoculation back, 59.2h feed supplement for the second time, be with at 67.91h and put operation, promptly from bio-reactor, bleed off a part of algae liquid and change the photoinduction cultivation over to, working volume before supplemented medium and water to band is put in the 50L bio-reactor then, this moment, algae cell density was reduced to 52.5g/L from 82.2g/L, continued heterotrophism and cultivated.Afterwards every 6～9h feed supplement once, be with after twice of the feed supplement and put operation, the volume that bleeds off is about half of working volume, and cell density is reduced to about 55g/L from about 110g/L, cultivates to finish this heterotrophism behind the 172h and cultivate.In the whole semicontinuous culturing process, cell growth rate remains unchanged substantially, puts in the cycle at each band, and the average yield of frustule is up to 3.02g/L/d, and the highest mixing speed is no more than the 700rpm (see figure 2) in the culturing process.
The heterotrophism culture condition: temperature is 30 ± 2 ℃, and pH is less than 8.5, and the control dissolved oxygen is more than 5%.Substratum is consistent with the substratum of embodiment 1.
Embodiment 3 (Chlorella vulgaris is in the semicontinuous cultivation of 50L jar)
Adding heterotrophism substratum and tap water are sterilized to 25L in the 50L fermentor tank, when temperature is reduced to 31 ± 1 ℃, insert the Chlorella vulgaris seed, and the beginning heterotrophism is cultivated.
Reach 54.52g/L at the 58.2h algae cell density after the feed supplement 5 times, be with then and put operation, emit 60% left and right sides algae liquid of working volume in the fermentor tank, supplemental medium and sterilized water, return to band and put front volume, algae cell density is reduced to 21.6g/L, continues then to cultivate.Afterwards by after 3 feed supplements, algae cell density reaches 54.5g/L at 85.1h, carry out this moment being with the second time and put, emit 60% left and right sides algae liquid of working volume in the fermentor tank, supplemental medium and sterilized water return to band and put front volume, algae cell density is reduced to 20.1g/L, is continuing the heterotrophism cultivation then.Finish whole heterotrophism at 115.5h and cultivate, average frustule productive rate is the 1.62g/L/d (see figure 3) in the culturing process.
In semicontinuous culturing process, temperature is controlled at 31 ± 1 ℃, and pH maintains 6～8, by adjusting rotating speed, air flow quantity and tank pressure, makes DO maintain 10～60%.
Heterotrophism and supplemented medium:
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 CaCl20.02 grams
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 EDTA1.4 grams per liter, trace element solution prescription are H
3BO
32.86 grams per liter, MnCl
24H
2O 0.11 grams per liter, ZnSO
47H
2O 9.22 grams per liters, CuSO
45H
2O 1.00 grams per liters, (NH
4)
6Mo
7O
244H
2O 0.1 grams per liter, Co (NO
3)
26H
2The O0.9 grams per liter.
Embodiment 4 (chlorella ellipsoidea is in the semicontinuous cultivation of 5L jar)
Sterilize after in the 5L fermentor tank, adding the heterotrophism substratum, about 121 ℃ of sterilization 20min, when temperature is reduced to 30 ± 1 ℃, insert chlorella ellipsoidea and begin to carry out the heterotrophism cultivation.
After 2 feed supplements, reach 52.3g/L (glucose has consumed simultaneously) at the 66.0h cell density, this moment, band was emitted 50% algae liquid of working volume, added glucose, KNO then in jar
3Deng nutritive substance, with sterilized water volume to be mended to original level simultaneously, cell density is reduced to 26.5g/L, continues feed supplement and cultivates.Work as algae cell density then and reach 50 ± 5g/L, band is put again.Heterotrophism is cultivated and is lasted 118.0h, and total synteny is put 2 times in the process, and the frustule average yield is the 1.23g/L/d (see figure 4).
In semicontinuous culturing process, temperature is controlled at 31 ± 1 ℃, and pH maintains 6～8.Used substratum is consistent with the substratum of embodiment 3.
Claims (10)
1. a little algae heterotrophism cultured method is characterized in that, adopts semicontinuous training method to carry out heterotrophism and cultivates.
2. method according to claim 1 is characterized in that, in the heterotrophism culturing process, emits part algae liquid, replenishes the substratum and the sterilized water of respective volume then, makes frustule continue cultivation in the heterotrophism culture apparatus.
3. as method as described in each among the claim 1-2, it is characterized in that, 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.
4. as each described method among the claim 1-3, it is characterized in that described heterotrophism substratum is made up of nitrogenous source, organic carbon source and inorganic salt, trace element and water.
5. as each described method among the claim 1-4, it is characterized in that, but the cultivation of described heterotrophism is carried out in the bio-reactor that shakes bottle, mechanical agitation type, air lift type or the cultivation of bubbling style heterotrophism.
6. as method as described in each among the claim 1-5, it is characterized in that, described method also comprises, microalgae cell after band microalgae cell of releasing and/or heterotrophism finish in the heterotrophism process is directly made algae powder, or be used to extract active substance in the born of the same parents, or change the photoinduction cultivation over to, or change the cultivation of light autotrophy over to.
7. method as claimed in claim 2 is characterized in that, when the algae kind 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, He Shui, 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 and Co (NO
3)
26H
2O 0.1～0.9 grams per liter.
8. method as claimed in claim 2 is characterized in that, when the algae kind 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 and water; 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 and Na
2MoO
40.01～0.05 grams per liter.
9. the described method of each claim as described above is characterized in that, when the cell density of the little algae of heterotrophism reaches 5g/L when above, emits part algae liquid, replenishes the substratum and the sterilized water of respective volume then, makes frustule continue cultivation in the heterotrophism culture apparatus.
10. the described method of each claim as described above is characterized in that, when emitting algae liquid, the algae liquid of emitting accounts for 20～80% of algae liquid cumulative volume that heterotrophism cultivates.
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