CN102154110B - A kind of microalgae culture method of high yield - Google Patents
A kind of microalgae culture method of high yield Download PDFInfo
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Abstract
The present invention relates to a kind of microalgae culture method of high yield, the Heterotrophic culture step that the method comprises micro-algae algae kind and the frustule that obtains using Heterotrophic culture are as the light autotrophy culturing step of seed.For can micro-algae of heterotrophic growth, employing the inventive method can give full play to the advantage that micro-algae grows fast at heterotrophism stage frustule, can not only cultivate for the extensive light autotrophy of micro-algae and a large amount of algae kind is provided in time, but also micro-algae photoautotrophic cultivation and object product (as grease, albumen etc.) synthesis speed can be accelerated, to spread cultivation the problem that the cycle is long, Growth of Cells slow and object products collection efficiency is low for solving the algae kind existed in micro-algae extensive light autotrophy culturing process, providing a kind of important technique means.
Description
Technical field
The invention belongs to field of biological energy source and/or technical field of microalga biology, relate to a kind of microalgae culture method of high yield.
Background technology
The multiple high value active substance such as rich in proteins, polysaccharide, lipid acid and carotenoid in microalgae cell.Therefore, current micro-algae has a wide range of applications in all many-sides such as food, feed, medicine, environmental protection and bioenergies.
The main training method of micro-algae has light autotrophy, mixotrophism and Heterotrophic culture.At present, the micro-algae (as chlorella, spirulina, salt algae, Haematocoocus Pluvialls etc.) having realized commercial application is nearly all adopt light autotrophy mode to cultivate.
The Combined hardening model of micro-algae needs cultivating in the bioreactor of sterilizing, and need to ensure sterile culture and sufficient illumination condition, high to the requirement of culture device, equipment is difficult to amplify simultaneously simultaneously.Therefore, in micro-algae large scale culturing of reality, mixotrophism mode is not almost adopted to cultivate.Compared with cultivating with light autotrophy, the frustule quality (as albumen and pigment content etc.) of Heterotrophic culture is low, is often difficult to application.Therefore, in above-mentioned three kinds of microdisk electrode patterns, the frustule quality that light autotrophy is cultivated is high, enjoys the concern of people.At present, the grease that investigator hankers after adopting micro-algae light autotrophy culturing process to accumulate carrys out production biofuel (AttilioConverti, AlessandroA.Casazza, ErikaY.Ortiz, PatriziaPerego, MarcoDelBorghi., Effectoftemperatureandnitrogenconcentrationonthegrowthan dlipidcontentofNannochloropsisoculataandChlorellavulgari sforbiodeselproduction [J] .ChemicalEngineeringandProcessing, 2009, 48:1146 ~ 1151).
Although the light autotrophy culturing process of micro-algae can accumulate a large amount of useful matteies (as grease, albumen etc.), still there are following 3 aspect problems: 1) inoculum density low causing is vulnerable to assorted algae and protozoic pollution; 2) at present seed spreads cultivation and all adopts light autotrophy to cultivate, its cycle very very long (generally needing 1 ~ 2 month) and easily being polluted, and algae kind in constantly the spreading cultivation of some months, need a large amount of culture apparatuses and equipment and floor space is large; 3) micro-algae light autotrophy culturing process Growth of Cells productive rate that is slow, object product is low.
Therefore, this area still needs a kind of microalgae culture method of high yield.
Summary of the invention
For the problems referred to above, the invention provides a kind of effective solution, for can micro-algae of Heterotrophic culture, carry out the Heterotrophic culture of algae kind, the frustule obtained using Heterotrophic culture subsequently carries out the cultivation of light autotrophy as seed.Employing the inventive method can give full play to the advantage that micro-algae grew fast in the heterotrophism stage, can not only cultivate for the extensive light autotrophy of micro-algae and a large amount of algae kind is provided in time, but also micro-algae photoautotrophic cultivation and object product (as grease, albumen etc.) synthesis speed can be accelerated, to spread cultivation the problem that the cycle is long, Growth of Cells slow and object products collection efficiency is low for solving the algae kind existed in micro-algae extensive light autotrophy culturing process, providing a kind of important technique means.
Therefore, first aspect present invention provides a kind of microalgae culture method, the Heterotrophic culture step that the method comprises micro-algae algae kind and the light autotrophy culturing step implemented as seed using the frustule that Heterotrophic culture obtains.
Second aspect present invention provides a kind of grease production method, the light autotrophy culturing step that the Heterotrophic culture step that described method comprises micro-algae algae kind, the frustule that obtains using Heterotrophic culture are implemented as seed and the step that frustule is gathered and grease extracts.
Third aspect present invention provides a kind of method for producing protein, the light autotrophy culturing step that the Heterotrophic culture step that described method comprises micro-algae algae kind, the frustule that obtains using Heterotrophic culture are implemented as seed and frustule is gathered and the step of proteins extraction.
In an embodiment, described micro-algae is selected from can micro-algae of Heterotrophic culture.
In an embodiment, described micro-algae is selected from:
Chlorella pyrenoidesa (Chlorellapyrenoidosa) in Chlorophyta Chlorella, Chlorella vulgaris (Chlorellavulgaris), chlorella ellipsoidea (Chlorellaellipsoidea), Chlorellaemersonii, Chlorellasorokiniana, Chlorellasaccharophila, Chlorellaregularis, Chlorellaminutissima, Chlorellaprotothecoides, Chlorellazofingiensis, and the 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 Pyrrophyta, Crypthecodiniumcohnii;
The Euglenagricilis of Euglenophyta; With
The Galdieriasulphuraria of rhodophyta.
In an embodiment, the step of described micro-algae algae kind heterotrophism comprises: in bio-reactor, add the substratum that pH is 4.0 ~ 9.0, batch culture, fed batch cultivation or Semi-continuous cultivation is carried out by the micro-algae algae kind of 0.1 ~ 30% access of working volume, culture temperature is 10 ~ 40 DEG C, control pH is less than 9.0, controls dissolved oxygen more than 1%.
In an embodiment, the step of described micro-algae light autotrophy comprises: heterotrophism energy microalgae algae kind be inoculated in light autotrophy culture apparatus and carry out light autotrophy, culture temperature is 5 ~ 50 DEG C, continuous illumination or intermittent illumination, intensity of illumination is 0.1 ~ 150klx, light autotrophy culture cycle is 5 ~ 500 hours, and Initial seeding density is 0.01 ~ 10.00 grams per liter, and pH is 4.0 ~ 12.0.
In an embodiment, Heterotrophic culture base is made up of nitrogenous source, organic carbon source, inorganic salt, trace element and water; Light autotrophy substratum is made up of nitrogenous source, inorganic salt and water.
In an embodiment, described heterotrophism step can be carried out in the bio-reactor of Heterotrophic culture at shaking flask, mechanical agitation type, air lift type or bubbling style, described smooth autotrophy culturing step is in shaking flask or be selected from the raceway pond of open type or circle pond, enclosed flat plate photobioreactor or duct type bioreactor or pillar bioreactor, film stand any can be used for such as bag or Pig and carry out in the device that micro-algae light autotrophy cultivates, and illumination condition is that natural light or artificial light irradiate.
In an embodiment, when chlorella is Chlorella vulgaris, the substratum that heterotrophism uses consists of the following composition substantially: KNO
35 ~ 15 grams per liters, glucose 10 ~ 60 grams per liter, KH
2pO
40.3 ~ 0.9 grams per liter, Na
2hPO
412H
2o1.0 ~ 10.0 grams per liter, MgSO
47H
2o0.2 ~ 1.0 grams per liter, CaCl
20.05 ~ 0.3 grams per liter, FeSO
47H
2o0.01 ~ 0.05 grams per liter, trace element 0.5 ~ 4ml and water, its medium trace element consist of H
3bO
35 ~ 15 grams per liters, ZnSO
47H
2o5.0 ~ 10.0 grams per liter, MnCl
2h
2o1.0 ~ 2.0 grams per liter, (NH
4)
6mo
7o
244H
2o0.5 ~ 1.5 grams per liter, CuSO
45H
2o1.0 ~ 2.0 grams per liter, Co (NO
3)
26H
2o0.1 ~ 0.9 grams per liter.
In an embodiment, when chlorella is Chlorella pyrenoidesa, the substratum that heterotrophism uses consists of the following composition substantially: glucose 10 ~ 60 grams per liter, urea 2.0 ~ 8.0 grams per liter, KH
2pO
41.0 ~ 2.0 grams per liters, MgSO
47H
2o1.0 ~ 2.0 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 Fe-EDTA solution formula is FeSO
47H
2o20 ~ 30 grams per liter and EDTA20 ~ 40 grams per liter; A5 solution formula is H
3bO
32.5 ~ 4.0 grams per liters, MnCl
24H
2o1.0 ~ 2.0 grams per liter, ZnSO
47H
2o0.1 ~ 0.6 grams per liter, CuSO
45H
2o0.05 ~ 0.1 grams per liter, Na
2moO
40.01 ~ 0.05 grams per liter.
In a specific embodiment, after the glucose consumption in Heterotrophic culture liquid is complete, terminate Heterotrophic culture, and Heterotrophic culture gained frustule is implemented light autotrophy culturing step as seed.
In other embodiment of the application, the substratum described in the application can be used, culture condition implements above-mentioned microalgae culture method.
Cultivate production biofuel with the Heterotrophic culture of chlorella algae kind with for the frustule that Heterotrophic culture obtains as the light autotrophy of seed, describe advantage of the present invention in detail:
(1) algal species cultivation speed can greatly be improved.The Initial seeding density that general outdoor great Chi cultivates micro-algae is that the volume of 0.05 ~ 0.1g/l, great Chi is generally 5000L, if need meet this requirement, then needs micro-algae of 250 ~ 500g as seed.To spread cultivation algae kind according to traditional light autotrophy, then need 1 ~ February (algae cell density cultivated 10 days of algae kind light autotrophy is generally 0.2 ~ 0.5g/l, finally just need can enter great Chi through spread cultivation step by step (thinning ratios of about about 10 times)); And adopt 50L fermentor tank Heterotrophic culture algae kind, then only need 2 ~ 3 days.
(2), compared with cultivating with traditional algae kind light autotrophy, can reduce the device in algal species cultivation process and number of devices, and floor space is little, unit culture area (or volume) productive rate is high.Cultivate spreading cultivation of required algae kind for the great Chi light autotrophy of 5000L, the light autotrophy algae kind that spreads cultivation needs the culture apparatus of 5L, 50L and 500L, and algae kind Heterotrophic culture only needs the culture apparatus of 50L.
(3), compared with cultivating with algae kind light autotrophy, algae kind Heterotrophic culture is not subject to the impact of outdoor weather and environment.When algae kind light autotrophy is cultivated, if when running into rainy weather and cannot continue to cultivate, then need to move into indoor and add artificial light and cultivate algae kind to continue light autotrophy, thus add artificial lighting expense.In addition, algae kind light autotrophy is vulnerable to the pollution of the harmful organisms such as protozoon, assorted algae, thus causes algal species cultivation failure, has a strong impact on production.
(4), when outdoor great Chi cultivates, inoculum size is larger, is more not easy to be subject to other pollutions such as assorted algae or protozoon; The Heterotrophic culture of algae kind can provide a large amount of seed in time, thus meets the demand improving inoculum size when light autotrophy is cultivated.
(5) vigor of heterotrophism algae kind is better than the algae kind that light autotrophy is cultivated.Access identical frustule amount carry out light autotrophy cultivate time, utilize the cell of Heterotrophic culture as the algae cell density of the light autotrophy culturing process of algae kind, grease productive rate and protein yield etc., all higher than the analog value utilizing light autotrophy cultured cells as the light autotrophy culturing process of algae kind.In addition, higher owing to carrying out the algae cell density that the cultivation of light autotrophy obtains by heterotrophism algae kind, therefore, the cost of gathering of energy microalgae can be reduced.
In sum, the Heterotrophic culture of algae kind of the present invention and the frustule that obtains using Heterotrophic culture as the light autotrophy training mode of seed can solve when light autotrophy is cultivated due to algal species cultivation efficiency is low and the problems that vulnerable to pollution causes and micro-algae in light autotrophy culturing process growth velocity the slow and problem that object product (as grease, albumen etc.) productive rate is low.Therefore, the present invention solves the problem that in micro-algae light autotrophy culturing process, Growth of Cells is slow and object products collection efficiency is low to provide a kind of important technique means, especially utilizes the industrialization of micro-algae production biofuel to establish important basis.
Accompanying drawing explanation
Fig. 1 show Chlorella pyrenoidesa seed respectively in 500ml shaking flask Heterotrophic culture and in 2L triangular flask light autotrophy cultivate process of growth.
Fig. 2 show Chlorella vulgaris seed respectively in 500ml shaking flask Heterotrophic culture and in 2L triangular flask light autotrophy cultivate process of growth.
Fig. 3 show chlorella ellipsoidea seed respectively in 500ml shaking flask Heterotrophic culture and in 2L triangular flask light autotrophy cultivate process of growth.
Fig. 4 shows Chlorella pyrenoidesa heterotrophism seed and the light autotrophy seed frustule growth curve that light autotrophy is cultivated in indoor 2L bioreactor.
Fig. 5 shows Chlorella pyrenoidesa heterotrophism seed and the light autotrophy seed grease productive rate that light autotrophy is cultivated in indoor 2L bioreactor.
Fig. 6 shows Chlorella vulgaris heterotrophism seed and the light autotrophy seed frustule growth curve that light autotrophy is cultivated in indoor 2L bioreactor.
Fig. 7 shows Chlorella vulgaris heterotrophism seed and the light autotrophy seed grease productive rate that light autotrophy is cultivated in indoor 2L bioreactor.
Fig. 8 shows chlorella ellipsoidea heterotrophism seed and the light autotrophy seed frustule growth curve that light autotrophy is cultivated in indoor 2L bioreactor.
Fig. 9 shows chlorella ellipsoidea heterotrophism seed and the light autotrophy seed grease productive rate that light autotrophy is cultivated in indoor 2L bioreactor.
Figure 10 shows the Chlorella pyrenoidesa heterotrophism seed frustule growth curve that light autotrophy is cultivated in 2L bioreactor out of doors and the highest grease productive rate.
Figure 11 shows the Chlorella pyrenoidesa heterotrophism seed frustule growth curve that light autotrophy is cultivated in 60L plastic tub out of doors and the highest grease productive rate.
Embodiment
The micro-algae being applicable to the application comprises all micro-algaes carrying out Heterotrophic culture, include but not limited to the Chlorella pyrenoidesa (Chlorellapyrenoidosa) in Chlorophyta Chlorella, Chlorella vulgaris (Chlorellavulgaris), chlorella ellipsoidea (Chlorellaellipsoidea), Chlorellaemersonii, Chlorellasorokiniana, Chlorellasaccharophila, Chlorellaregularis, Chlorellaminutissima, Chlorellaprotothecoides, Chlorellazofingiensis, and the 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 Pyrrophyta, Crypthecodiniumcohnii, the Euglenagricilis of Euglenophyta, the Galdieriasulphuraria of rhodophyta.
In a preferred embodiment, the present invention adopts chlorella to implement.In preferred embodiment, the present invention adopts Chlorella pyrenoidesa, Chlorella vulgaris or chlorella ellipsoidea to implement.In other preferred embodiment, the present invention adopts Chlorella pyrenoidesa, Chlorella vulgaris or chlorella ellipsoidea to implement the microdisk electrode of high grease productive rate and high protein productive rate.
Various substratum well known in the art can be adopted to carry out the Heterotrophic culture of micro-algae seed.Usually, Heterotrophic culture base contains nitrogenous source, organic carbon source, inorganic salt, trace element and water.Being applicable to the nitrogenous source of microdisk electrode, organic carbon source, inorganic salt, trace element etc. is that this area is known.Such as, as nitrogenous source, spendable have urea or various nitrate, as KNO
3deng; As organic carbon source, glucose, sucrose, glycerine etc. can be used.
This kind of substratum comprises HA-SK substratum (Chinese patent ZL200610024004.9), Endo substratum (OgbonnaJ.C., Masui.H., Tanaka.H.Sequentialheterotrophic:autotrophiccultivation-anefficientmethodofproducingChlorellabiomassforhealthfoo dandanimalfeed.J.Appl.Phycol.1997,9,359 ~ 366) etc.
The present invention's HA-SK substratum used is by KNO substantially
3, glucose and inorganic salt, trace element and water composition.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
2in O one or more or all.
Term " being by ... composition substantially " represents in above-mentioned substratum except containing main ingredient KNO
3, outside glucose and inorganic salt, trace element and water, also can comprise the component that some fundamental characteristics for composition or new characteristic (can maintain micro-algae and reach higher level at shorter culture cycle inner cell density, activity substance content has a more substantial increase compared with conventional Heterotrophic culture simultaneously) do not affect in fact.Term " by ... composition " represent that above-mentioned substratum is made up of pointed concrete component, there is no other components, but can with the impurity of content in usual scope.
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 by the strict restriction of embodiment.As known to those skilled in the art, in substratum, also inorganic salt can be added, 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.The consumption of inorganic salt and trace element can be determined according to Conventional wisdom.
HA-SK substratum of the present invention consists of the following composition substantially: KNO
35 ~ 15 grams per liters, glucose 10 ~ 60 grams per liter, KH
2pO
40.3 ~ 0.9 grams per liter, Na2HPO
412H
2o1.0 ~ 10.0 grams per liter, MgSO
47H
2o0.2 ~ 1.0 grams per liter, CaCl
20.05 ~ 0.3 grams per liter, FeSO
47H
2o0.01 ~ 0.05 grams per liter, trace element 0.5 ~ 4ml and water, its medium trace element consist of H
3bO
35 ~ 15 grams per liters, ZnSO
47H
2o5.0 ~ 10.0 grams per liter, MnCl
2h
2o1.0 ~ 2.0 grams per liter, (NH
4)
6mo
7o
244H
2o0.5 ~ 1.5 grams per liter, CuSO
45H
2o1.0 ~ 2.0 grams per liter, Co (NO
3)
26H
2o0.1 ~ 0.9 grams per liter.
In one preferably embodiment, HA-SK culture media composition of the present invention should consist of the following composition: KNO
37 grams per liters, glucose 40 grams per liter, KH
2pO
40.6 grams per liter, Na
2hPO
412H
2o2.0 grams per liter, MgSO
47H
2o0.8 grams per liter, CaCl
20.2 grams per liter, FeSO
47H
2o0.03 grams per liter, micro-1.5mL and water 1000mL, its medium trace element consist of H
3bO
311 ~ 12 grams per liters, ZnSO
47H
2o8.5 ~ 9.5 grams per liter, MnCl
2h
2o1.4 ~ 1.5 grams per liter, (NH
4)
6mo
7o
244H
2o0.8 ~ 0.9 grams per liter, CuSO
45H
2o1.5 ~ 1.6 grams per liter, Co (NO
3)
26H
2o0.45 ~ 0.55 grams per liter.
The present invention's Endo substratum used consists of the following composition substantially: glucose 10 ~ 60 grams per liter, urea 2 ~ 8 grams per liter, KH
2pO
41 ~ 2 grams per liter, Na
2hPO
412H
2o1.0 ~ 10.0 grams per liter, MgSO
47H
2o1 ~ 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 Fe-EDTA solution formula is FeSO
47H
2o20 ~ 30 grams per liter and EDTA20 ~ 40 grams per liter; A5 solution formula is H
3bO
32.5 ~ 4.0 grams per liters, MnCl
24H
2o1.0 ~ 2.0 grams per liter, ZnSO
47H
2o0.1 ~ 0.6 grams per liter, CuSO
45H
2o5 ~ 10 grams per liter, Na
2moO
40.01 ~ 0.05 grams per liter.
In one preferably embodiment, described Endo substratum consists of the following composition: glucose 40 grams per liter, urea 6.0 grams per liter, KH
2pO
41.5 grams per liters, Na2HPO
412H
2o5.0 grams per liter, MgSO
47H
2o1.8 grams per liter, CaCl
20.05 grams per liter, trisodium citrate 0.4 grams per liter, Fe-EDTA solution 0.8mL, A5 solution 2.0mL and water, wherein Fe-EDTA solution formula is FeSO
47H
2o25 grams per liter and EDTA33.5 grams per liter, A5 solution formula is H
3bO
32.86 grams per liters, MnCl
24H
2o1.81 grams per liter, ZnSO
47H
2o0.222 grams per liter, CuSO
45H
2o0.07 grams per liter, Na
2moO
40.021 grams per liter.
After according to above-mentioned formulated substratum, available conventional means as acid or alkali by as described in the pH of substratum be adjusted to 4.0 ~ 9.0, and at 115 ~ 120 DEG C autoclaving 15 ~ 20 minutes.Four kinds of modes such as batch culture, fed batch cultivation, Semi-continuous cultivation (band is put) or cultured continuously can be adopted to implement seed Heterotrophic culture.
When carrying out seed Heterotrophic culture, the corresponding substratum prepared is joined in bio-reactor, benefit adds water to working volume, usual coefficient is 0.6 ~ 0.8, then steam sterilizing (121 DEG C, maintain about 20 minutes), when temperature is down to 30 ~ 35 DEG C, start Heterotrophic culture by the micro-algae seed of 1 ~ 15% access of working volume.
No matter adopt which kind of training method, in culturing process, the culture condition that must control to be applicable to makes micro-algae seed normal growth.Usually, control temperature is 20 ~ 35 DEG C, such as 28 ~ 30 DEG C, and dissolved oxygen is not less than the air saturation concentration of 5%, and pH is not higher than 9.0.In a preferred embodiment, dissolved oxygen is not less than the air saturation concentration of 10%, and pH is not higher than 8.5.In other preferred embodiment, dissolved oxygen is not less than the air saturation concentration of 15%, and pH is not higher than 8.
In culturing process, pH is unsuitable too high or too low, generally along with the carrying out cultivated, pH can slowly rise (obvious especially for this phenomenon of Chlorella vulgaris), the too high meeting of pH has a negative impact to frustule growth, so application acid (sulfuric acid of such as 10%) regulates, make pH not higher than 9.0, preferably pH should be 6.5 ~ 7.5.
Heterotrophism can carry out in the bio-reactor of Heterotrophic culture at shaking flask, mechanical agitation type, air lift type, bubbling style etc.
After the glucose consumption in seed Heterotrophic culture liquid is complete, then seed Heterotrophic culture terminates.Subsequently, be inoculated in light autotrophy culture apparatus and carry out the cultivation of light autotrophy.The Initial seeding density that light autotrophy is cultivated is generally 0.01 ~ 1 grams per liter, and temperature is 10 ~ 40 DEG C, and intensity of illumination is 0.1 ~ 100klx, and light dark period is 24:0 ~ 6:18, pH is 4.0 ~ 9.0, and air flow is 0.05 ~ 5vvm, passes into CO
2concentration is 0.03 ~ 5%.
When frustule growth is in stationary phase (when algae cell density does not increase), then terminates light autotrophy and cultivate, frustule is gathered.
Various smooth autotrophy substratum well known in the art can be adopted to carry out the cultivation of light autotrophy.Usually, light autotrophy substratum contains nitrogenous source, phosphorus source, inorganic carbon source, inorganic salt, trace element and water.Being applicable to the nitrogenous source of microdisk electrode, phosphorus source, inorganic carbon source, inorganic salt, trace element etc. is that this area is known.Such as, as nitrogenous source, spendable have urea or various nitrate, as KNO
3deng; As phosphorus source, spendable have such as NaH
2pO
4; As inorganic carbon source, spendable have such as CO
2deng.
This kind of substratum is substratum based on F-Si substratum.
The present invention's improvement F-Si substratum used is by NaNO substantially
3, NaH
2pO
4and trace element, a small amount of VITAMIN and water composition.In described technical scheme, described trace element should be selected from FeC
6h
5o
75H
2o, ZnSO
47H
2o, MnCl
2h
2o, Na
2moO
42H
2o, CuSO
45H
2o, Na
2eDTA, CoCl
2in one or more or all.
Term " being by ... composition substantially " represents in above-mentioned substratum except containing main ingredient NaNO
3, NaH
2pO
4and outside trace element, a small amount of VITAMIN and water, also can comprise some fundamental characteristics for composition or new characteristic (micro-algae can be maintained and reach higher level at shorter culture cycle inner cell density, simultaneously activity substance content cultivate with conventional light autotrophy compared with have a more substantial increase) component that do not affect in fact.
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 by the strict restriction of embodiment.As known to those skilled in the art, in substratum, also inorganic salt can be added, such as magnesium sulfate, calcium chloride and ferrous sulfate 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 FeC
6h
5o
75H
2o, ZnSO
47H
2o, MnCl
24H
2o, Na
2moO
42H
2o, CuSO
45H
2o, Na
2eDTA, CoCl
2in one or more or all.The consumption of inorganic salt and trace element can be determined according to Conventional wisdom.
Improvement F/2-Si substratum of the present invention consists of the following composition substantially: NaNO
30.1 ~ 1.0 grams per liter, NaH
2pO
42H
2o0.01 ~ 0.1 grams per liter; Trace element 0.5 ~ 4ml, its medium trace element consist of ZnSO
47H
2o0.02 ~ 0.2 grams per liter, MnCl
24H
2o0.2 ~ 2.0 grams per liter, CuSO
45H
2o0.01 ~ 0.1 grams per liter, FeC
6h
5o
75H
2o1.0 ~ 10 grams per liter, Na
2moO
42H
2o0.05 ~ 0.5 grams per liter, Na
2eDTA2.0 ~ 20 grams per liter, CoCl
20.01 ~ 0.1 grams per liter, VITAMIN 0.5 ~ 4ml and water, its medium trace element consist of vitamin B12 0.1 ~ 1.0 mg/litre, vitamin B15 0 ~ 1000 mg/litre, vitamin H 0.1 ~ 1.0 mg/litre.
In one preferably embodiment, the F/2-Si substratum that the present invention improves should consist of the following composition: NaNO
30.5 grams per liter, NaH
2pO
42H
2o0.05 grams per liter; Trace element 1.5ml, its medium trace element consist of ZnSO
47H
2o0.1 ~ 0.15 grams per liter, MnCl
24H
2o1.0 ~ 1.5 grams per liter, CuSO
45H
2o0.03 ~ 0.07 grams per liter, FeC
6h
5o
75H
2o3.0 ~ 4.0 grams per liter, Na
2moO
42H
2o0.1 ~ 0.3 grams per liter, Na
2eDTA10 ~ 12 grams per liter, CoCl
20.02 ~ 0.06 grams per liter, VITAMIN 2ml and water 1000mL, its medium trace element consist of vitamin B12 0.03 ~ 0.05 mg/litre, VB11 00 ~ 200 mg/litre, vitamin H 0.5 ~ 1.0 mg/litre.
After according to above-mentioned formulated substratum, available conventional means as acid or alkali by as described in the pH of substratum be adjusted to 4.0 ~ 9.0.Four kinds of modes such as batch culture, fed batch cultivation, Semi-continuous cultivation (band is put) or cultured continuously can be adopted to implement light autotrophy cultivate.
Frustule is gathered, the extraction of grease and frond comprehensive utilization
Light autotrophy carries out centrifugal gathering to micro-algae after cultivating and terminating, and obtains wet frond.The collecting method of frustule includes but not limited to the technology such as high speed centrifugation, flocculation, 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, aqueous phase pyrolysis.
In born of the same parents, the extracting method of grease includes but not limited to organic solvent extractionprocess, that is: frond is dried to constant weight at 80 ~ 105 DEG C, chloroform methanol standard extraction solvent is adopted to extract grease from dry algae powder after grinding algae powder, extraction solvent extracts repeatedly until algae powder color becomes white, and rotary evaporation removes solvent.
Other compositions in supernatant liquor progressively separation and Extraction can obtain lipid acid, chlorophyll etc., or directly all the components in supernatant liquor and frond are precipitated that mixed atomizing is dry obtains chlorella powder.
In the present invention, can the micro-algae cultivating gained be fully utilized, extract the various activeconstituents such as pigment (such as xenthophylls), protein, polysaccharide wherein.The sequence of extraction of activeconstituents there is no particular restriction, but usually will meet this prerequisite of component damages that the step first extracted can not cause rear extraction.The method extracting protein, polysaccharide etc. is also that this area is known.
The measuring method relating to biochemical component in frustule dry weight, fat content and born of the same parents is herein as follows:
Frustule dry weight measures: in micro-algae (as chlorella) culturing process, get nutrient solution V milliliter, centrifugal 10 minutes of 8000rpm, by the frond deionized water wash after centrifugal 3 times, be transferred in weighing bottle (W1 (gram)), dry to constant weight W2 (gram) in 105 DEG C of baking ovens.Frond dry weight Cx can calculate according to following formula: Cx (grams per liter)=(W2-W1)/V/1000.
Grease measures: get the frustule of a certain amount of each cultivation stage oven dry to constant weight, be ground to Powdered in mortar, take appropriate algae powder (0.2 ~ 0.5g) to be 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.Rotate evaporate to dryness after merging supernatant, weigh and calculate fat content.
Fat content (%) is calculated as follows:
Grease (%)=(W2-W0)/W1 × 100
In formula: W1--is algae grain weight amount, g; W0--dries the rotary evaporation bottle weight to constant weight, g; W2--is the weight of evaporative flask after oil extraction liquid evaporate to dryness, g.
Protein content determination: in micro-algae (as chlorella) cell total protein content mensuration adopt Kjeldahl determination (Ning Zhengxiang. food composition analysis handbook. Beijing: China Light Industry Press, 1998,76-78).
Below will be further described related content of the present invention by embodiment.Unless otherwise described, the present invention adopt substratum in each component concentration all represent with grams per liter (g/L).Should be understood that in the application " contain ", " comprising " also comprise " by ... composition ", " by ... form " implication.
Embodiment 1: the research of frustule growth in Chlorella pyrenoidesa seed heterotrophism and light autotrophy culturing process
The Chlorella pyrenoidesa of the present embodiment carries out carrying out the cultivation of light autotrophy in the triangular flask of Heterotrophic culture and 2L respectively in the shaking flask of 500ml.As the seed that next step light autotrophy is cultivated, determine the frustule growth curve of Chlorella pyrenoidesa heterotrophism and light autotrophy cultivation seed respectively.Inoculum density during Chlorella pyrenoidesa seed Heterotrophic culture is 0.20g/l, and temperature is 30 DEG C, and rotating speed is 150rmp, and when cultivating 72h, the glucose in nutrient solution has consumed, and algae cell density is 6.8g/l, for the seed that next step light autotrophy is cultivated; Inoculum density when Chlorella pyrenoidesa seed light autotrophy is cultivated is 0.20g/l, temperature is 25 DEG C, intensity of illumination is 2000lx, light dark period is 24:0, every day, timing shook 3 times, and when cultivating 120h, frustule is in exponential phase of growth, algae cell density is 0.56g/l, for the seed (Fig. 1) that next step light autotrophy is cultivated.As can be seen here, compared with the seed cultivated with light autotrophy, the seed cell density high (being 12.1 times of light autotrophy seed) of Heterotrophic culture and culture cycle short (than light autotrophy seed shorten 48 little time).
Embodiment 2: the research of frustule growth in Chlorella vulgaris seed heterotrophism and light autotrophy culturing process
The Chlorella vulgaris of the present embodiment carries out carrying out the cultivation of light autotrophy in the triangular flask of Heterotrophic culture and 2L respectively in the shaking flask of 500ml, as the seed that next step light autotrophy is cultivated, determine the frustule growth curve of Chlorella vulgaris heterotrophism and light autotrophy cultivation seed respectively.Inoculum density during Chlorella vulgaris seed Heterotrophic culture is 0.30g/l, and temperature is 30 DEG C, and rotating speed is 150rmp, and when cultivating 72h, the glucose in nutrient solution has consumed, and algae cell density is 8.1g/l, for the seed that next step light autotrophy is cultivated; Inoculum density when Chlorella vulgaris seed light autotrophy is cultivated is 0.30g/l, temperature is 25 DEG C, intensity of illumination is 2000lx, light dark period is 24:0, every day, timing shook 3 times, and when cultivating 120h, frustule is in exponential phase of growth, algae cell density is 0.65g/l, for the seed (Fig. 2) that next step light autotrophy is cultivated.As can be seen here, compared with the seed cultivated with light autotrophy, the seed cell density high (being 12.5 times of light autotrophy seed) of Heterotrophic culture and culture cycle short (than light autotrophy seed shorten 48 little time).
Embodiment 3: the research of frustule growth in chlorella ellipsoidea seed heterotrophism and light autotrophy culturing process
The chlorella ellipsoidea of the present embodiment carries out carrying out the cultivation of light autotrophy in the triangular flask of Heterotrophic culture and 2L respectively in the shaking flask of 500ml, as the seed that next step light autotrophy is cultivated, determine the frustule growth curve of chlorella ellipsoidea heterotrophism and light autotrophy cultivation seed respectively.Inoculum density during chlorella ellipsoidea seed Heterotrophic culture is 0.25g/l, and temperature is 30 DEG C, and rotating speed is 150rmp, and when cultivating 72h, the glucose in nutrient solution has consumed, and algae cell density is 8.8g/l, for the seed that next step light autotrophy is cultivated; Inoculum density when chlorella ellipsoidea seed light autotrophy is cultivated is 0.25g/l, temperature is 25 DEG C, intensity of illumination is 2000lx, light dark period is 24:0, every day, timing shook 3 times, and when cultivating 120h, frustule is in exponential phase of growth, algae cell density is 0.71g/l, for the seed (Fig. 3) that next step light autotrophy is cultivated.As can be seen here, compared with the seed cultivated with light autotrophy, the seed cell density high (being 12.4 times of light autotrophy seed) of Heterotrophic culture and culture cycle short (than light autotrophy seed shorten 48 little time).
Embodiment 4: Chlorella pyrenoidesa heterotrophism seed and the light autotrophy seed research that light autotrophy is cultivated in indoor 2L bioreactor
The Chlorella pyrenoidesa seed of the present embodiment light autotrophy cultivation heterotrophism and light autotrophy in indoor 2L cylinder shape airlift photobioreactor, determines heterotrophism respectively and the frustule of light autotrophy seed in its light autotrophy process grows and grease productive rate.The inoculum density of heterotrophism and light autotrophy seed is 0.3g/l, and light autotrophy culture temperature is 30 DEG C, all passes into the CO of 2%
2, air flow is 0.5vvm, and intensity of illumination is 10000lx, and light dark period is 24:0.Light autotrophy cultivates 96 hours, and the algae cell density of heterotrophism seed is 2.03g/l, and grease productive rate is 149.55mg/l/d; The algae cell density of light autotrophy seed is only 0.93g/l, and grease productive rate is only 87.68mg/l/d (Fig. 4 and Fig. 5).As can be seen here, compared with light autotrophy seed, the vigor of heterotrophism seed is stronger, and the algae cell density of identical incubation time is higher (being 2.2 times of light autotrophy seed), grease productive rate higher (being 1.7 times of light autotrophy seed).
Embodiment 5: Chlorella vulgaris heterotrophism seed and the light autotrophy seed research that light autotrophy is cultivated in indoor 2L bioreactor
The present embodiment light autotrophy in indoor 2L cylinder shape airlift photobioreactor cultivate heterotrophism and light autotrophy Chlorella vulgaris seed, determine the frustule growth in its light autotrophy process of heterotrophism and light autotrophy seed and grease productive rate respectively.The inoculum density of heterotrophism and light autotrophy seed is 0.3g/l, and light autotrophy culture temperature is 30 DEG C, all passes into the CO of 2%
2, air flow is 0.5vvm, and intensity of illumination is 10000lx, and light dark period is 24:0.Light autotrophy cultivates 96 hours, and the algae cell density of heterotrophism seed is 2.03g/l, and grease productive rate is 138.49mg/l/d; The algae cell density of light autotrophy seed is only 1.25g/l, and grease productive rate is only 82.81mg/l/d (Fig. 6 and Fig. 7).As can be seen here, compared with light autotrophy seed, the vigor of heterotrophism seed is stronger, and the algae cell density of identical incubation time is higher (being 1.6 times of light autotrophy seed), grease productive rate higher (being 1.7 times of light autotrophy seed).
Embodiment 6: chlorella ellipsoidea heterotrophism seed and the light autotrophy seed research that light autotrophy is cultivated in indoor 2L bioreactor
The present embodiment light autotrophy in indoor 2L cylinder shape airlift photobioreactor cultivate heterotrophism and light autotrophy chlorella ellipsoidea seed, determine the frustule growth in its light autotrophy process of heterotrophism and light autotrophy seed and grease productive rate respectively.The inoculum density of heterotrophism and light autotrophy seed is 0.3g/l, and light autotrophy culture temperature is 30 DEG C, all passes into the CO of 2%
2, air flow is 0.5vvm, and intensity of illumination is 10000lx, and light dark period is 24:0.Light autotrophy cultivates 96 hours, and the algae cell density of heterotrophism seed is 1.65g/l, and grease productive rate is 121.48mg/l/d; The algae cell density of light autotrophy seed is only 0.91g/l, and grease productive rate is only 72.29mg/l/d (Fig. 8 and Fig. 9).As can be seen here, compared with light autotrophy seed, the vigor of heterotrophism seed is stronger, and the algae cell density of identical incubation time is higher (being 1.8 times of light autotrophy seed), grease productive rate higher (being 1.7 times of light autotrophy seed).
Embodiment 7: the Chlorella pyrenoidesa heterotrophism seed research that light autotrophy is cultivated in 2L bioreactor out of doors
The present embodiment out of doors in 2L cylinder shape airlift photobioreactor light autotrophy cultivate Chlorella pyrenoidesa heterotrophism seed, determine heterotrophism seed at the frustule process of growth of its outdoor light autotrophy process and the highest grease productive rate.Initial seeding density is 0.06g/l, and temperature and light intensity are outdoor actual temperature and light intensity, and air flow is 0.3vvm.Light autotrophy cultivates 83 hours, and algae cell density and grease productive rate all reach maximum, is respectively 1.44g/l and 123.43mg/l/d (Figure 10).As can be seen here, the seed of heterotrophism not only can in indoor flash of light preceding an earthquake autotrophy be cultivated and reach high grease productive rate fast, and still can flash of light preceding an earthquake autotrophy cultivate and reach high grease productive rate fast out of doors.
Embodiment 8: the Chlorella pyrenoidesa heterotrophism seed research that light autotrophy is cultivated in 60L plastic tub out of doors
The present embodiment out of doors in 60L plastic tub light autotrophy cultivate Chlorella pyrenoidesa heterotrophism seed, determine heterotrophism seed at the frustule process of growth of its outdoor light autotrophy process and the highest grease productive rate.Initial seeding density is 0.10g/l, and temperature and light intensity are outdoor actual temperature and light intensity, and air flow is 0.3vvm.Light autotrophy cultivates 108 hours, and algae cell density and grease productive rate all reach maximum, is respectively 0.75g/l and 55.34mg/l/d (Figure 11).Volume of culture in a 60L plastic tub is generally 50L, reaches 0.10g/l to Initial seeding density, then need the algae kind of 5g; Only need the shaking flask (liquid amount is 200ml) of 4 500ml to cultivate the seed that 3 days can obtain 5.44g with the seed (see Fig. 1) of Heterotrophic culture, then need the triangular flask of 9 2L (liquid amount is 1L) to cultivate the seed that 5 days just can obtain 5.04g with the seed (see Fig. 1) that light autotrophy is cultivated.As can be seen here, compared with cultivating with seed light autotrophy, seed Heterotrophic culture can not only shorten the incubation time of seed, improves the efficiency of whole culturing process, and culture apparatus consumption needed for seed culture is less and floor space is less.Therefore, cultivate micro-algae to open air extensive light autotrophy, so only have the demand adopting Heterotrophic culture seed could meet a large amount of algae kind in time.
Although be described above object lesson of the present invention, having a bit is obvious to those skilled in the art, namely can make various changes the present invention and change under the premise without departing from the spirit and scope of the present invention.Therefore, claims cover all these variations within the scope of the present invention.
Claims (24)
1. a microalgae culture method, it is characterized in that, the Heterotrophic culture step that the method comprises micro-algae algae kind and the light autotrophy culturing step implemented as seed using the frustule that Heterotrophic culture obtains, wherein, the Initial seeding density that light autotrophy is cultivated is 0.01 ~ 1 grams per liter.
2. the method for claim 1, is characterized in that, adopts batch culture, fed batch cultivation, Semi-continuous cultivation or cultured continuously pattern to implement the Heterotrophic culture of described micro-algae algae kind.
3. the method for claim 1, is characterized in that, described micro-algae is selected from:
Chlorella pyrenoidesa (Chlorellapyrenoidosa) in Chlorophyta Chlorella, Chlorella vulgaris (Chlorellavulgaris), chlorella ellipsoidea (Chlorellaellipsoidea), Chlorellaemersonii, Chlorellasorokiniana, Chlorellasaccharophila, Chlorellaregularis, Chlorellaminutissima, Chlorellaprotothecoides, Chlorellazofingiensis, and the 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 Pyrrophyta, Crypthecodiniumcohnii;
The Euglenagricilis of Euglenophyta; With
The Galdieriasulphuraria of rhodophyta.
4. the method for claim 1, it is characterized in that, the step of described micro-algae algae kind Heterotrophic culture comprises: in bio-reactor, add the substratum that pH is 4.0 ~ 9.0, batch culture, fed batch cultivation, Semi-continuous cultivation or cultured continuously is carried out by the micro-algae algae kind of 0.1 ~ 30% access of working volume, culture temperature is 10 ~ 40 DEG C, control pH is less than 9.0, controls dissolved oxygen more than 1%.
5. the method for claim 1, it is characterized in that, the cultivation of light autotrophy is carried out as seed using the frustule that Heterotrophic culture obtains, comprise: the algae kind of Heterotrophic culture is received in light autotrophy culture apparatus and carries out the cultivation of light autotrophy, culture temperature is 5 ~ 50 DEG C, continuous illumination or intermittent illumination, and intensity of illumination is 0.1 ~ 150klx, light autotrophy culture cycle is 5 ~ 500 hours, and pH is 4.0 ~ 12.0.
6. the method for claim 1, is characterized in that, the Heterotrophic culture base of algae kind is made up of nitrogenous source, organic carbon source, inorganic salt, trace element and water; Light autotrophy substratum is made up of nitrogenous source, inorganic salt and water.
7. the method for claim 1, it is characterized in that, described algae kind Heterotrophic culture step is carried out in shaking flask, mechanical agitation type, air lift type or Sparged bioreactors, described smooth autotrophy culturing step is in shaking flask or be selected from the raceway pond of open type or circle pond, enclosed flat plate photobioreactor or duct type bioreactor or pillar bioreactor, film stand in the device cultivated for micro-algae light autotrophy of bag or Pig and carry out, and illumination condition is natural light or artificial light.
8. the method for claim 1, is characterized in that, after the organic carbon source in Heterotrophic culture liquid runs out of, terminates Heterotrophic culture, and Heterotrophic culture gained frustule is implemented light autotrophy culturing step as seed.
9. a grease production method, it is characterized in that, the light autotrophy culturing step that the Heterotrophic culture step that described method comprises micro-algae algae kind, the frustule that obtains using Heterotrophic culture are implemented as seed and the step that frustule is gathered and grease extracts, wherein, the Initial seeding density that light autotrophy is cultivated is 0.01 ~ 1 grams per liter.
10. method as claimed in claim 9, is characterized in that, adopts batch culture, fed batch cultivation, Semi-continuous cultivation or cultured continuously pattern to implement the Heterotrophic culture of described micro-algae algae kind.
11. methods as claimed in claim 9, it is characterized in that, described micro-algae is selected from:
Chlorella pyrenoidesa (Chlorellapyrenoidosa) in Chlorophyta Chlorella, Chlorella vulgaris (Chlorellavulgaris), chlorella ellipsoidea (Chlorellaellipsoidea), Chlorellaemersonii, Chlorellasorokiniana, Chlorellasaccharophila, Chlorellaregularis, Chlorellaminutissima, Chlorellaprotothecoides, Chlorellazofingiensis, and the 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 Pyrrophyta, Crypthecodiniumcohnii;
The Euglenagricilis of Euglenophyta; With
The Galdieriasulphuraria of rhodophyta.
12. methods as claimed in claim 9, it is characterized in that, the step of described micro-algae algae kind Heterotrophic culture comprises: in bio-reactor, add the substratum that pH is 4.0 ~ 9.0, batch culture, fed batch cultivation, Semi-continuous cultivation or cultured continuously is carried out by the micro-algae algae kind of 0.1 ~ 30% access of working volume, culture temperature is 10 ~ 40 DEG C, control pH is less than 9.0, controls dissolved oxygen more than 1%.
13. methods as claimed in claim 9, it is characterized in that, the cultivation of light autotrophy is carried out as seed using the frustule that Heterotrophic culture obtains, comprise: the algae kind of Heterotrophic culture is received in light autotrophy culture apparatus and carries out the cultivation of light autotrophy, culture temperature is 5 ~ 50 DEG C, continuous illumination or intermittent illumination, and intensity of illumination is 0.1 ~ 150klx, light autotrophy culture cycle is 5 ~ 500 hours, and pH is 4.0 ~ 12.0.
14. methods as claimed in claim 9, it is characterized in that, the Heterotrophic culture base of algae kind is made up of nitrogenous source, organic carbon source, inorganic salt, trace element and water; Light autotrophy substratum is made up of nitrogenous source, inorganic salt and water.
15. methods as claimed in claim 9, it is characterized in that, described algae kind Heterotrophic culture step is carried out in shaking flask, mechanical agitation type, air lift type or Sparged bioreactors, described smooth autotrophy culturing step is in shaking flask or be selected from the raceway pond of open type or circle pond, enclosed flat plate photobioreactor or duct type bioreactor or pillar bioreactor, film stand in the device cultivated for micro-algae light autotrophy of bag or Pig and carry out, and illumination condition is natural light or artificial light.
16. methods as claimed in claim 9, is characterized in that, after the organic carbon source in Heterotrophic culture liquid runs out of, terminate Heterotrophic culture, and Heterotrophic culture gained frustule is implemented light autotrophy culturing step as seed.
17. 1 kinds of method for producing protein, it is characterized in that, the light autotrophy culturing step that the Heterotrophic culture step that described method comprises micro-algae algae kind, the frustule that obtains using Heterotrophic culture are implemented as seed and frustule is gathered and the step of proteins extraction, wherein, the Initial seeding density that light autotrophy is cultivated is 0.01 ~ 1 grams per liter.
18. methods as claimed in claim 17, is characterized in that, adopt batch culture, fed batch cultivation, Semi-continuous cultivation or cultured continuously pattern to implement the Heterotrophic culture of described micro-algae algae kind.
19. methods as claimed in claim 17, it is characterized in that, described micro-algae is selected from:
Chlorella pyrenoidesa (Chlorellapyrenoidosa) in Chlorophyta Chlorella, Chlorella vulgaris (Chlorellavulgaris), chlorella ellipsoidea (Chlorellaellipsoidea), Chlorellaemersonii, Chlorellasorokiniana, Chlorellasaccharophila, Chlorellaregularis, Chlorellaminutissima, Chlorellaprotothecoides, Chlorellazofingiensis, and the 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 Pyrrophyta, Crypthecodiniumcohnii;
The Euglenagricilis of Euglenophyta; With
The Galdieriasulphuraria of rhodophyta.
20. methods as claimed in claim 17, it is characterized in that, the step of described micro-algae algae kind Heterotrophic culture comprises: in bio-reactor, add the substratum that pH is 4.0 ~ 9.0, batch culture, fed batch cultivation, Semi-continuous cultivation or cultured continuously is carried out by the micro-algae algae kind of 0.1 ~ 30% access of working volume, culture temperature is 10 ~ 40 DEG C, control pH is less than 9.0, controls dissolved oxygen more than 1%.
21. methods as claimed in claim 17, it is characterized in that, the cultivation of light autotrophy is carried out as seed using the frustule that Heterotrophic culture obtains, comprise: the algae kind of Heterotrophic culture is received in light autotrophy culture apparatus and carries out the cultivation of light autotrophy, culture temperature is 5 ~ 50 DEG C, continuous illumination or intermittent illumination, and intensity of illumination is 0.1 ~ 150klx, light autotrophy culture cycle is 5 ~ 500 hours, and pH is 4.0 ~ 12.0.
22. methods as claimed in claim 17, it is characterized in that, the Heterotrophic culture base of algae kind is made up of nitrogenous source, organic carbon source, inorganic salt, trace element and water; Light autotrophy substratum is made up of nitrogenous source, inorganic salt and water.
23. methods as claimed in claim 17, it is characterized in that, described algae kind Heterotrophic culture step is carried out in shaking flask, mechanical agitation type, air lift type or Sparged bioreactors, described smooth autotrophy culturing step is in shaking flask or be selected from the raceway pond of open type or circle pond, enclosed flat plate photobioreactor or duct type bioreactor or pillar bioreactor, film stand in the device cultivated for micro-algae light autotrophy of bag or Pig and carry out, and illumination condition is natural light or artificial light.
24. methods as claimed in claim 17, is characterized in that, after the organic carbon source in Heterotrophic culture liquid runs out of, terminate Heterotrophic culture, and Heterotrophic culture gained frustule is implemented light autotrophy culturing step as seed.
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| CN102154110B (en) * | 2011-01-27 | 2016-03-02 | 华东理工大学 | A kind of microalgae culture method of high yield |
| CN103103128A (en) * | 2011-11-10 | 2013-05-15 | 中国石油化工股份有限公司 | Method for high efficiency enrichment culture of microalgae |
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| CN104046567A (en) * | 2013-03-17 | 2014-09-17 | 中国石油化工股份有限公司 | Microalgae cultivation method and grease production method |
| CN103266062B (en) * | 2013-05-20 | 2014-11-19 | 中南大学 | Fresh water Chlorella pyrenoidosa XJ01 strain and application thereof in fixing CO2 and producing microalga grease |
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| WO2015168458A1 (en) * | 2014-05-02 | 2015-11-05 | John Whitman | Methods of microalgae cultivation for increased resource production |
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| MX2018009521A (en) * | 2016-02-08 | 2018-12-11 | Corbion Biotech Inc | Method for the protein enrichment of microalgal biomass. |
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| CN113136339B (en) * | 2020-01-19 | 2023-07-14 | 中国石油化工股份有限公司 | Method for continuously culturing photosynthetic microorganisms by mixotrophic-autotrophic culture, culture system and application thereof |
| CN113136345B (en) * | 2020-01-19 | 2023-05-05 | 中国石油化工股份有限公司 | Method for heterotrophic-autotrophic continuous cultivation of photosynthetic microorganisms, cultivation system and application thereof |
| CN113136321A (en) * | 2020-01-19 | 2021-07-20 | 中国石油化工股份有限公司 | Method and system for heterotrophic-autotrophic co-culture of photosynthetic microorganisms and method for production of biomass and bioenergy |
| CN113136341A (en) * | 2020-01-19 | 2021-07-20 | 中国石油化工股份有限公司 | Heterotrophic-autotrophic photosynthetic microorganism culture method and system and biomass and biological energy production method |
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| CN114032176A (en) * | 2021-11-26 | 2022-02-11 | 海南绿藻世界生物科技有限公司 | Culture system and culture method for promoting growth of microalgae |
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