CN101979498B - A kind of method that micro-algae high-yield heterotrophic is cultivated - Google Patents
A kind of method that micro-algae high-yield heterotrophic is cultivated Download PDFInfo
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Abstract
The present invention relates to the new method that the semicontinuous heterotrophism of a kind of micro-algae is cultivated. Adopt this method heterotrophism to cultivate micro-algae, can realize micro-algae high-yield heterotrophic and cultivate. The present invention can be applicable to heterotrophic microalgae or and the extensive high efficiency production of active material.
Description
Technical field
The invention belongs to Microalgae biotechnology field, relate to a kind of method that micro-algae high-yield heterotrophic is cultivated.
Background technology
In microalgae cell, be rich in the multiple high value active materials such as protein, polysaccharide, aliphatic acid and carotenoid. Therefore,Current micro-algae has a wide range of applications in all many-sides such as food, medicine, feed, environmental protection and bioenergies.
The main training method of micro-algae has light autotrophy, mixotrophism and heterotrophism to cultivate. At present, realized commercial applicationMicro-algae (as chlorella, spirulina, salt algae, haematococcus pluvialis etc.) is nearly all to adopt light autotrophy mode to cultivate, but also has minorityThe large-scale culture of micro-algae (as the hidden dinoflagellate of Kou Shi etc.) adopts heterotrophism method.
The light autotrophy of micro-algae is cultivated and is mainly existed that cell density is low, production efficiency is low, the cost of gathering is high, be vulnerable to miscellaneous bacteriaWith shortcomings such as protozoic pollution and the impacts of natural environment and climate condition. Mixotrophism is cultivated need to be anti-at photo-biological that can sterilizingAnswer in device and cultivate, need to ensure sterile culture and sufficient illumination condition simultaneously, to culture device require high, simultaneouslyEquipment is difficult to amplify. Therefore, in micro-algae large-scale culture of reality, almost do not adopt mixotrophism mode to cultivate.
Except above-mentioned two kinds of training methods, the micro-algae of part can carry out heterotrophism cultivation, has by interpolation glucose etc.Machine carbon source makes micro-algae heterotrophic growth. This training method has the following advantages: (1) is not subject to the restriction of the conditions such as natural environment and climate;(2) can keep pure culture, thereby ensure the homogeneity of product quality; (3) cultivate with respect to light autotrophy, can obtain high thinBorn of the same parents' concentration and production efficiency; (4) can use for reference and utilize industrial fermentation technology and the production equipment etc. of comparative maturity.
Therefore,, in above-mentioned three kinds of micro-algae training modes, heterotrophism is cultivated and is the most easily realized industrialization, enjoys people's passNote. The mode that many researchers cultivate by heterotrophism is produced high value added product, as lutein, astaxanthin, vitamin, anti-badHematic acid etc.; In addition, also there is researcher to adopt micro-algae heterotrophism training method to carry out production biodiesel.
Existing micro-algae heterotrophism training method mainly contains in batches and fed-batch culture, has no and utilizes semicontinuous method to carry outThe report of the heterotrophism cultivation aspect of micro-algae. Batch culture is a kind of mode more generally adopting during heterotrophism is cultivated. At present, by dividingChlorella is cultivated in batch mode, and algae cell density is about 20g/L left and right (ShiXM, ZhangXW, ChenF.HeterotrophicproductionofbiomassandluteinbyChlorellaprotothecoidesonvariousNitrogensources.Enzymeandmicrobialtechnology.2000,27:312-318). Fed-batch trainingSupport and can obtain higher frustule concentration and productive rate with respect to batch culture. In fed-batch culture, mainly contain flow feedingWith batch (-type) feed supplement mode. Wu Zhengyun etc. cultivate chlorella pyrenoidosa, chlorella the highest at 19L fermentation tank flow feeding heterotrophismCell concentration and growth rate reach 116.2g/L and 1.02g/ (Lh) (WuZY, ShiXM.Optimizationforhigh-densitycultivationofheterotrophicChlorellabasedonahybridneuralNetworkmodel.LettersinAppliedMicrobiology.2007,44 (1): 13-18). And promulgated by the State Council in adoptingFed-batch process in bright patent (ZL200610025618.9) is cultivated chlorella pyrenoidosa in 50L tank, cultivates a 90h left sideThe right side, algae cell density can reach 150g/L.
Micro-algae heterotrophism is cultivated has lot of advantages, but exist, heterotrophism frustule quality is low, the interior activity substance content of born of the same parentsLow and be only suitable in can heterotrophism cultivating the shortcomings such as algae kind. Micro-algae of Chinese invention patent (ZL200610025618.9) invention is highly denseThe method that degree high-quality is cultivated can solve micro-algae heterotrophism and cultivate the low problem of quality. But, Chinese invention patent (ZL200610025618.9) adopt batch feeding culture technique in realizing the high Density Heterotrophic of micro-algae, still existFollowing problem: the postvaccinal lag phase of (1) batch culture is long, productive rate is low; (2) late stage of culture is because cell density is high, oxygen consumptionAmount is large, and the oxygen delivery capacity wretched insufficiency of bioreactor, causes late growing stage speed obviously to reduce; (3) heterotrophism batch culture knotShu Shi, as lower in overcast and rainy outdoor photoinduction culture efficiency, therefore cultivate with micro-algae heterotrophism the photoinduction matching and cultivateThe number of devices of system and illuminating area must increase considerably, and cause disposable apparatus investment large; (4) for batch feedingHeterotrophism is cultivated, and the photoinduction time is shorter, and the utilization rate of the photoinduction culture systems therefore matching with heterotrophism culture device is lower;(5) cultivate for batch feeding heterotrophism, cannot carry out photoinduction out of doors when running into exceedingly odious weather time, biological respinseMicro-algae heterotrophism nutrient solution in device cannot be emitted and must be continued to cultivate, but can cause even this batch of cultivation of frustule mortalityLiquid is all scrapped.
For the problems referred to above, the invention provides a kind of effectively solution, in heterotrophism incubation, adopt and partly connectThe continuous mode of operation of cultivating,, in heterotrophism incubation, emits part algae liquid, then the culture medium of supplementary respective volume or asepticWater, makes frustule in heterotrophism culture apparatus, continue to cultivate. The method has improved the productive rate of frustule greatly, has reduced biologyThe oxygen supply requirement of reactor, can be at any time photoinduction cultivate provide microalgae cell, also can be can heterotrophic growth the micro-algae of the energyScale light autotrophy is cultivated provides a large amount of seeds.
Semicontinuous heterotrophism taking chlorella is cultivated as example, describes advantage of the present invention in detail as follows:
(1) can greatly improve frustule productive rate. Calculate the frustule speed of on average growing in one-period by each band cycle of puttingRate can reach 3.0g/ (Lh); And the fed-batch culture technology of employing Chinese invention patent (ZL200610025618.9),Frustule average growth rate is 0.867g/ (Lh) left and right; The average growth rate of batch culture is only 0.487g/ (Lh)Left and right.
(2), compared with fed-batch culture, semicontinuous cultivation has reduced the oxygen supply requirement to bioreactor. As 50L tankWhen middle fed-batch culture, speed of agitator need to be up to 800rpm; And adopting semicontinuous cultivation of the present invention, speed of agitator is the highestBe no more than 700rpm.
(3) can be according to outdoor weather condition, select tape is put the time flexibly: as select to emit morning part algae liquid, thisSample can make full use of daylong sunlight and carry out photoinduction; Meet when overcast and rainy, can adjust flexibly band and put the time, effectivelyWhile having overcome actual outdoor production, adopt batch feeding cultivation to run into the overcast and rainy algae liquid of cannot emitting and cause heterotrophism nutrient solution to be scrappedAnd cause direct economic loss and environmental issue;
(4) can be at any time cultivate a large amount of algae kinds is provided for energy micro-algae large-scale light autotrophy that can heterotrophic growth: the energy is micro-When algae large-scale culture, algae kind spreads cultivation the time long (cultivate for outdoor great Chi, generally reach about 1-2 month), has a strong impact onThe production efficiency of whole cultivation stage, semicontinuous Heterotrophic Culture provided by the invention, can provide highdensity at any timeAlgae liquid, the problem that spreads cultivation fast of the algae kind when effectively having solved energy micro-algae large-scale and cultivating.
In sum, the semicontinuous heterotrophism training mode of micro-algae of the present invention can solve completely in heterotrophism incubation due toThe high problems that cause of density and the problem of mutually mating on time and equipment with photoinduction. Therefore, this invention is micro-algaeHeterotrophism cultivate and the production of active material, in particular for micro-algae heterotrophism-dilution-photoinduction series connection cultivate production algae powder andActive material has been established solid industrialization basis.
Summary of the invention
The invention provides a kind of method that micro-algae high-yield heterotrophic is cultivated, can solve micro-algae high Density Heterotrophic mistakeThe problem existing in journey, realizes the high yield serialization of micro-algae and active material thereof and produces.
In a detailed description of the invention, adopt semicontinuous training method to carry out heterotrophism cultivation,, in incubation, putGo out part algae liquid, heterotrophism culture medium and the sterilized water of adding respective volume are cultivated rear continuation simultaneously.
In a detailed description of the invention, described micro-algae is selected from: the chlorella pyrenoidosa in Chlorophyta Chlorella(Chlorellapyrenoidosa), chlorella vulgaris (Chlorellavulgaris), chlorella ellipsoidea (Chlorellaellipsoidea)，Chlorellaemersonii，Chlorellasorokiniana，Chlorellasaccharophila，Chlorellaregularis，Chlorellaminutissima，ChlorellaProtothecoides, Chlorellazofingiensis, and Brachiomonassubmarina in Chlorophyta,Chlamydobonasreinhardtii，Chlamydomonasacidophila，Haematococcuspluvialis，Haematococcuslacustris，Scenedesmusobliquus，Spongiococcumexetriccium，Tetraselmissuecica，Tetraselmischuii，Tetraselmistetrathele，Tetraselmisverrucosa，Micractiniumpusillum；
The Cylindrothecafusiformis of Bacillariophyta, Nitzschialaevis, Nitzschiaalba,Nitzschiafonticola，Naviculaincerta，Naviculapelliculosa；
The Anabaenavariabilis of Cyanophyta;
The Poterioochromonasmalhamensis of Chrysophyta;
The Amphidiniumcarterae of Pyrrhophyta, Crypthecodiniumcohnii;
The Euglenagricilis of Euglenophyta;
The Galdieriasulphuraria of Rhodophyta.
In a detailed description of the invention, when described micro-algae heterotrophism is cultivated: in bioreactor, add pH be 4.0～9.0 culture medium, carries out semicontinuous cultivation cultivation by the micro-algae algae kind of 0.1～30% access of working volume, and cultivation temperature is 10～40 DEG C, controlling pH is 4.0～9.0, controls dissolved oxygen more than 1%.
In a detailed description of the invention, heterotrophism culture medium is by nitrogenous source, organic carbon source and inorganic salts, trace element and waterComposition.
In a detailed description of the invention, described heterotrophism is cultivated can at shaking flask, mechanical agitation type, gas-lifting type or bubble typeIn the bioreactor that heterotrophism is cultivated, carry out.
In a detailed description of the invention, described method also comprises, the microalgae cell that band in heterotrophism process is released and/Or the microalgae cell directly processed algae powder of heterotrophism after finishing, or for extracting active material in born of the same parents, or proceed to photoinduction and cultivate, orProceeding to light autotrophy cultivates.
In a detailed description of the invention, in the time that chlorella is chlorella vulgaris, the culture medium that heterotrophism uses substantiallyConsist of the following composition: KNO35～15 grams per liters, glucose 10～60 grams per liters, KH2PO40.3～0.9 grams per liter, Na2HPO4·12H2O1.0～10.0 grams per liter, MgSO4·7H2O0.2～1.0 grams per liter, CaCl20.05～0.3 grams per liter, FeSO4·7H2O0.01～0.05 grams per liter, trace element 0.5～4ml, He Shui, wherein trace element consist of H3BO35～15 grams per liters,ZnSO4·7H2O5.0～10.0 grams per liter, MnCl2·H2O1.0～2.0 grams per liter, (NH4)6Mo7O24·4H2O0.5～1.5Grams per liter, CuSO4·5H2O1.0～2.0 grams per liter and Co (NO3)2·6H2O0.1～0.9 grams per liter.
In a detailed description of the invention, in the time that chlorella is chlorella pyrenoidosa, the culture medium that heterotrophism uses is basicOn consist of the following composition: glucose 10～60 grams per liters, urea 2～8 grams per liters, KH2PO41～2 grams per liter, Na2HPO4·12H2O1.0～10.0 grams per liter, MgSO4·7H2O1～2 grams per liter, CaCl20.05～0.1 grams per liter, trisodium citrate 0.1～2.0 grams per liters, Fe-EDTA solution 0.5～1mL, A5 solution 1～5mL and water; Wherein Fe-EDTA solution formula is FeSO4·7H2O20～30 grams per liters and EDTA20～40 grams per liter; A5 solution formula is H3BO32.5～4.0 grams per liters, MnCl2·4H2O1.0～2.0 grams per liters, ZnSO4·7H2O0.1～0.6 grams per liter, CuSO4·5H2O5～10 grams per liter and Na2MoO40.01～0.05Grams per liter.
In a detailed description of the invention, when the density of micro-algae of heterotrophism reaches 5g/L when above, emit part algae liquid, thenSupplement culture medium and the sterilized water of respective volume, make frustule in heterotrophism culture apparatus, continue to cultivate.
In a detailed description of the invention, while emitting algae liquid, the algae liquid of emitting account for heterotrophism cultivate 20 of algae liquid cumulative volume～80％。
Brief description of the drawings
Fig. 1 shows that chlorella pyrenoidosa is in 50L bioreactor batch feeding incubation.
Fig. 2 shows that chlorella pyrenoidosa is in the semicontinuous heterotrophism incubation of 50L bioreactor.
Fig. 3 shows that chlorella vulgaris is in the semicontinuous heterotrophism incubation of 50L bioreactor.
Fig. 4 shows that chlorella ellipsoidea is in the semicontinuous heterotrophism incubation of 5L bioreactor.
Detailed description of the invention
The micro-algae that is applicable to the application includes but not limited to the chlorella pyrenoidosa in Chlorophyta Chlorella(Chlorellapyrenoidosa), chlorella vulgaris (Chlorellavulgaris), chlorella ellipsoidea (Chlorellaellipsoidea)，Chlorellaemersonii，Chlorellasorokiniana，Chlorellasaccharophila，Chlorellaregularis，Chlorellaminutissima，ChlorellaProtothecoides, Chlorellazofingiensis, and Brachiomonassubmarina in Chlorophyta,Chlamydobonasreinhardtii，Chlamydomonasacidophila，Haematococcuspluvialis，Haematococcuslacustris，Scenedesmusobliquus，Spongiococcumexetriccium，Tetraselmissuecica，Tetraselmischuii，Tetraselmistetrathele，TetraselmisVerrucosa, Micractiniumpusillum; The Cylindrothecafusiformis of Bacillariophyta, Nitzschialaevis，Nitzschiaalba，Nitzschiafonticola，Naviculaincerta，NaviculaPelliculosa; The Anabaenavariabilis of Cyanophyta; The Poterioochromonas of ChrysophytaMalhamensis; The Amphidiniumcarterae of algae door, Crypthecodiniumcohnii; The Euglena of EuglenophytaGricilis; The Galdieriasulphuraria of Rhodophyta.
In a preferred embodiment, the present invention adopts chlorella pyrenoidosa, chlorella vulgaris and chlorella ellipsoidea.
Can adopt various culture medium well known in the art to carry out micro-algae heterotrophism cultivates. Conventionally, heterotrophism culture medium contains nitrogenSource, organic carbon source, inorganic salts, trace element and water. Those skilled in the art can determine heterotrophism according to the conventional knowledge in this areaThe consumption of nitrogenous source in culture medium, organic carbon source, inorganic salts, trace element.
At one preferably in embodiment, select HA-SK culture medium (Chinese patent ZL200610024004.9) andEndo culture medium (OgbonnaJ.C., Masui.H., Tanaka.H.Sequentialheterotrophic:autotrophiccultivation-anefficientmethodofproducingChlorellabiomassforhealthfoodandanimalfeed.J.Appl.Phycol.1997，9，359～366)。
The corresponding culture medium preparing is joined in bioreactor, mend and add water to working volume, conventionally coefficientBe 0.6～0.8, then steam sterilizing (121 DEG C, maintain approximately 20 minutes), in the time that temperature is down to 30～35 DEG C, by working volumeThe micro-algae of 1～15% access starts heterotrophism and cultivates.
In application before semicontinuous cultivation band puts, can adopt in batches or fed-batch culture makes frustule reach certainDensity, for example 5～30g/L, 30～60g/L, 60～120g/L etc.
Preferably in embodiment, adopt batch feeding to cultivate at one before band is put, heterotrophism was cultivated after a period of time,In the time that glucose in culture medium has been consumed (being generally 27～45 hours), need to carry out feed supplement, add carbon source (for example grapeSugar), (for example, cultivate the nitrogenous source of chlorella vulgaris is KNO to nitrogenous source3, the nitrogenous source of cultivating chlorella pyrenoidosa is urea) and inorganicThe nutritive salt such as salt, the nutritive salt of adding is the above-mentioned corresponding culture medium after concentrated, impels micro-algae continued growth. Can be every 5～Feed supplement in 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.For example, in the time that micro-algae (chlorella) cell density reaches a certain value (for example,, when the density of micro-algae of heterotrophism reaches 5g/L (preferably 20g/L, 30g/L or 40g/L) when above; In a preferred embodiment, for chlorella vulgaris, 45～65 grams per liters, for eggWhite nucleus chlorella, 90～110 grams per liters), from bioreactor, bleed off certain proportion (proportionality coefficient is 0.2～0.8)Algae liquid (this algae liquid can proceed to photoinduction cultivate or other process), then in bioreactor, add heterotrophism culture medium and nothingBacterium water, reaches its working volume can to carry out the requirement (in a preferred embodiment, return to band and put front volume) of heterotrophism cultivation,Now the algae cell density in bioreactor is reduced to band and is put preoperatively 0.2～0.8, and micro-algae continued growth is afterwards to a certain value(for example,, when the density of micro-algae of heterotrophism reaches 5g/L when above; In a preferred embodiment, for chlorella vulgaris, 45～65 grams per liters, for chlorella pyrenoidosa, 90～110 grams per liters), and then be with and put operation, circulation successively, when band is putAfter 10～20 times, finishing this heterotrophism cultivates.
In incubation, must control applicable condition of culture and make micro-algae normal growth. Conventionally, controlling temperature is 20～35DEG C, for example 28～32 DEG C, dissolved oxygen is not less than 5% saturation of the air concentration, and pH is not higher than 9.0. In a preferred embodiment, dissolved oxygenBe not less than 10% saturation of the air concentration, pH is not higher than 8.5. In other preferred embodiment, dissolved oxygen is not less than 15% skyGas saturated concentration, pH is not higher than 8.
Heterotrophism can enter in the bioreactor of heterotrophism cultivation at shaking flask, mechanical agitation type, gas-lifting type, bubble type etc.OK. In a preferred embodiment, adopt mechanical agitation type bioreactor.
Relate to frustule dry weight assay method herein as follows:
Frustule dry weight is measured: in micro-algae (as chlorella) incubation, get nutrient solution V milliliter, 8000rpm centrifugal 10Minute, by deionized water washing for the frond after centrifugal 3 times, be transferred to measuring cup (W1 (gram)) in, in 105 DEG C of baking ovens, dryTo 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 pyrenoidosa is in 50L tank fed-batch culture)
In 50L bioreactor, add following heterotrophism culture medium and running water to sterilizing after 25L, when temperature be down to 30 ±1 DEG C time, access chlorella pyrenoidosa, start heterotrophism and cultivate.
After cultivation starts, through feed supplement 5 times, in the time of 84.06h, cell density is 149.43g/L, then while continuing feed supplement 2 times, algaeThe obvious growth rate of cell is slow, is cultured to 97.60h heterotrophism and finishes, and cell density is 158.56g/L, and cell growth rate firstAfter increase, decline gradually, cell average yield is 1.63g/L/h (seeing Fig. 1). The batch culture later stage, speed of agitator was up to 800rpm.
Heterotrophism condition of culture: temperature is 30 ± 2 DEG C, and pH is less than 8.5, controls dissolved oxygen more than 5%.
Heterotrophism and supplemented medium:
8.0 grams of MgSO of 60.0 grams of urea of glucose4·7H2O2.0 gram
KH2PO41.1 grams of Na2HPO4·12H2O9.0 gram of CaCl20.02 gram
1.8 grams of trisodium citrates
Fe-EDTA solution 1.0ml trace element solution 4.5ml water 1000ml
Wherein Fe-EDTA solution formula is FeSO4·7H2O15 grams per liter and EDTA1.4 grams per liter, trace element solution is joinedSide is H3BO32.11 grams per liters, MnCl2·4H2O0.81 grams per liter, ZnSO4·7H2O0.11 grams per liter, CuSO4·5H2O10.0Grams per liter, Na2MoO40.05 grams per liter.
Embodiment 2 (the semicontinuous cultivation of chlorella pyrenoidosa 50L tank)
In 50L bioreactor, add following heterotrophism culture medium and running water to sterilizing after 25L, when temperature be down to 30 ±The 12% access chlorella pyrenoidosa of pressing working volume 1 DEG C time, starts heterotrophism and cultivates.
Feed supplement for the first time in 47.4h hour after inoculation, 59.2h is feed supplement for the second time, is with and puts operation, from life at 67.91hIn thing reactor, bleed off a part of algae liquid and proceed to photoinduction and cultivate, then in 50L bioreactor, supplemented medium and water is extremelyWorking volume before band is put, now algae cell density is down to 52.5g/L from 82.2g/L, continues heterotrophism and cultivates. Afterwards every 6～9h feed supplement once, is with and is put operation after twice of feed supplement, and the volume bleeding off is about the half of working volume, cell density from110g/L is down to left and right 55g/L left and right, finishes this heterotrophism and cultivate after cultivation 172h. In whole semicontinuous incubation, cellGrowth 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 cultivatesIn journey, the highest speed of agitator is no more than 700rpm (seeing Fig. 2).
Heterotrophism condition of culture: temperature is 30 ± 2 DEG C, and pH is less than 8.5, controls dissolved oxygen more than 5%. Culture medium and embodiment 1Culture medium consistent.
Embodiment 3 (chlorella vulgaris is in the semicontinuous cultivation of 50L tank)
In 50L fermentation tank, add heterotrophism culture medium and running water to sterilizing after 25L, in the time that temperature is down to 31 ± 1 DEG C, connectEnter chlorella vulgaris seed, start heterotrophism and cultivate.
After feed supplement 5 times, reach 54.52g/L at 58.2h algae cell density, be then with and put operation, in fermentation tank, emit60% left and right algae liquid of working volume, supplementing culture medium and sterilized water, return to band and put front volume, and algae cell density is down to21.6g/L, then continues to cultivate. By after 3 feed supplements, algae cell density reaches 54.5g/L at 85.1h, now carries out afterwardsBand is put for the second time, and from emitting 60% left and right algae liquid of working volume in fermentation tank, supplementing culture medium and sterilized water return to band and putFront volume, algae cell density is down to 20.1g/L, is then continuing heterotrophism cultivation. Finish whole heterotrophism at 115.5h and cultivate, cultivateIn process, average frustule productive rate is 1.62g/L/d (seeing Fig. 3).
In semicontinuous incubation, temperature is controlled at 31 ± 1 DEG C, and pH maintains 6～8, by adjusting rotating speed, air streamAmount and tank pressure, make DO maintain 10～60%.
Heterotrophism and supplemented medium:
10.0 grams of MgSO of 60.0 grams of potassium nitrate of glucose4·7H2O0.2 gram
KH2PO40.3 gram of Na2HPO4·12H2O8.8 gram of CaCl20.02 gram
Fe-EDTA solution 1.0ml trace element solution 3.5ml water 1000ml
Wherein Fe-EDTA solution formula is FeSO4·7H2O15 grams per liter and EDTA1.4 grams per liter, trace element solution is joinedSide is H3BO32.86 grams per liters, MnCl2·4H2O0.11 grams per liter, ZnSO4·7H2O9.22 grams per liter, CuSO4·5H2O1.00Grams per liter, (NH4)6Mo7O24·4H2O0.1 grams per liter, Co (NO3)2·6H2O0.9 grams per liter.
Embodiment 4 (chlorella ellipsoidea is in the semicontinuous cultivation of 5L tank)
In 5L fermentation tank, add sterilizing after heterotrophism culture medium, 121 DEG C of sterilizing 20min left and right, when temperature is down to 30 ± 1DEG C time, access chlorella ellipsoidea start to carry out heterotrophism cultivation.
After 2 feed supplements, reach 52.3g/L (glucose has consumed simultaneously) at 66.0h cell density, now band is emitted work50% algae liquid of volume is then added glucose, KNO in tank3Deng nutriment, with sterilized water, volume is mended to original simultaneouslyLevel, cell density is down to 26.5g/L, continues feed supplement and cultivates. Then when algae cell density reaches 50 ± 5g/L, be with againPut. Heterotrophism is cultivated and is lasted 118.0h, and in process, total synteny is put 2 times, and frustule average yield is 1.23g/L/d (seeing Fig. 4).
In semicontinuous incubation, temperature is controlled at 31 ± 1 DEG C, and pH maintains 6～8. Used medium and embodiment 3Culture medium consistent.
Claims (4)
1. the method that micro-algae heterotrophism is cultivated, is characterized in that, adopts semicontinuous training method to carry out heterotrophism cultivation, wherein,Described micro-algae is selected from chlorella pyrenoidosa (Chlorellapyrenoidosa), chlorella vulgaris (ChlorellaOr chlorella ellipsoidea (Chlorellaellipsoidea) vulgaris);
Wherein, when the cell density of heterotrophic microalgae reaches 20g/L when above, emit part algae liquid, then supplement the training of respective volumeSupport base and sterilized water, make frustule in heterotrophism culture apparatus, continue to cultivate; While emitting algae liquid, the algae liquid of emitting accounts for heterotrophism and cultivatesAlgae liquid cumulative volume 20～80%;
And wherein,, in the time that algae kind is chlorella vulgaris or chlorella ellipsoidea, the culture medium that heterotrophism uses consists of the following composition:KNO35～15 grams per liters, glucose 10～60 grams per liters, KH2PO40.3～0.9 grams per liter, Na2HPO4·12H2O1.0～10.0Grams per liter, MgSO4·7H2O0.2～1.0 grams per liter, CaCl20.05～0.3 grams per liter, FeSO4·7H2O0.01～0.05 gram/Rise, trace element 0.5～4ml and water 1000ml, wherein trace element consist of H3BO35～15 grams per liters, ZnSO4·7H2O5.0～10.0 grams per liter, MnCl2·H2O1.0～2.0 grams per liter, (NH4)6Mo7O24·4H2O0.5～1.5 grams per liter,CuSO4·5H2O1.0～2.0 grams per liter and Co (NO3)2·6H2O0.1～0.9 grams per liter;
In the time that algae kind is chlorella pyrenoidosa, the culture medium that heterotrophism uses consists of the following composition: 10～60 grams of glucose/Liter, urea 2～8 grams per liters, KH2PO41～2 grams per liter, Na2HPO4·12H2O1.0～10.0 grams per liter, MgSO4·7H2O1～2 grams per liters, CaCl20.05～0.1 grams per liter, trisodium citrate 0.1～2.0 grams per liter, Fe-EDTA solution 0.5～1mL, A5 are moltenLiquid 1～5mL and water 1000ml; Wherein Fe-EDTA solution formula is FeSO4·7H2O20～30 grams per liter and EDTA20～40Grams per liter; A5 solution formula is H3BO32.5～4.0 grams per liters, MnCl2·4H2O1.0～2.0 grams per liter, ZnSO4·7H2O0.1～0.6 grams per liter, CuSO4·5H2O5～10 grams per liter and Na2MoO40.01～0.05 grams per liter.
2. the method for claim 1, is characterized in that, described heterotrophism cultivate in shaking flask, mechanical agitation type, gas-lifting type orBubble type can carry out in the bioreactor of heterotrophism cultivation.
3. method as claimed in claim 1, is characterized in that, described method also comprises, will in heterotrophism process, band be released micro-Microalgae cell after frustule and/or heterotrophism finish is algae powder processed directly, or for extracting active material in born of the same parents, or proceed to photo-inductionLead cultivation, or proceed to the cultivation of light autotrophy.
4. the method for claim 1, is characterized in that, described micro-algae is chlorella pyrenoidosa or chlorella vulgaris.
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| ES2825062T3 (en) | 2013-08-01 | 2021-05-14 | Fermentalg | Methods for the production of diatom biomass |
| CN104662162A (en) * | 2013-09-26 | 2015-05-27 | 华东理工大学 | Method using micro-algae for high-efficiency production of astaxanthin |
| CN103805514B (en) * | 2014-02-25 | 2016-04-20 | 中国科学院水生生物研究所 | A kind ofly photosynthetic the holding concurrently of inorganic nitrogen-sourced micro-algae is utilized to support high density fermentation cultural method and application |
| US9441197B2 (en) * | 2014-05-02 | 2016-09-13 | Entech, Llc | Methods of microalgae cultivation for increased resource production |
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| CN109153965B (en) * | 2016-08-25 | 2023-02-03 | 特里同阿盖亚创新公司 | Method for improving algae growth |
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