CN102154110A - High-yield microalgae cultivating method - Google Patents
High-yield microalgae cultivating method Download PDFInfo
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- CN102154110A CN102154110A CN201110029154XA CN201110029154A CN102154110A CN 102154110 A CN102154110 A CN 102154110A CN 201110029154X A CN201110029154X A CN 201110029154XA CN 201110029154 A CN201110029154 A CN 201110029154A CN 102154110 A CN102154110 A CN 102154110A
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Abstract
The invention relates to a high-yield microalgae cultivating method. The method comprises a step of carrying out heterotrophic cultivation on microalgae algae species and a step of carrying out photoautotrophic cultivation by using algae cells obtained by heterotrophic cultivation as seeds. For the microalgae capable of growing heterotrophically, due to the adoption of the method, the advantage that the algae cells grow rapidly in the heterotrophic phase of the microalgae can be sufficiently played, not only a great amount of algae species can be provided in time for large scale photoautotrophic cultivation of the microalgae, but also the photoautotrophic growth of the microalgae and a forming rate of target products (such as oil, proteins and the like) can be quickened. An important technical means is provided for solving the problems of long algae specie spreading cultivation period, low cell growing speed and low yield of the target products in the large scale photoautotrophic cultivation process of the microalgae.
Description
Technical field
The invention belongs to bioenergy field and/or little algae biological technical field, relate to a kind of microalgae culture method of high yield.
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, feed, medicine, 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.
The mixotrophism of little algae 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.Compare with the cultivation of light autotrophy, the frustule quality (as albumen and pigment content etc.) that heterotrophism is cultivated is low, often is difficult to use.Therefore, in above-mentioned three kinds of little algae training modes, the frustule quality height that the light autotrophy is cultivated enjoys people's attention.At present, the investigator hankers after adopting the grease of little algae light autotrophy culturing process accumulation to produce thing diesel oil (Attilio Converti next life, Alessandro A.Casazza, Erika Y.Ortiz, Patrizia Perego, Marco Del Borghi., Effect of temperature and nitrogen concentration on the growthand lipid content of Nannochloropsis oculata and Chlorella vulgaris for biodeselproduction[J] .Chemical Engineering and Processing, 2009,48:1146～1151).
Though the light autotrophy culturing process of little algae can accumulate a large amount of useful matter (as grease, albumen etc.), still have following 3 aspect problems: 1) inoculum density hangs down and causes being vulnerable to assorted algae and protozoic pollution; 2) at present seed spreads cultivation and all adopts the light autotrophy to cultivate, and its cycle very very long (generally needing 1～2 month) and being polluted easily, the algae kind needs a large amount of culture apparatuses and equipment and floor space big in constantly the spreading cultivation of some months; 3) little algae light autotrophy culturing process cell growth productive rate slow, the purpose product is low.
Therefore, this area still needs a kind of microalgae culture method of high yield.
Summary of the invention
At the problems referred to above, the invention provides a kind of effective solution, but, carry out the heterotrophism of algae kind and cultivate for the heterotrophism cultured microalgae, cultivate the frustule that is obtained with heterotrophism subsequently and carry out the cultivation of light autotrophy as seed.Adopt the inventive method can give full play to the advantage that little algae grew fast in the heterotrophism stage, can not only cultivate for the extensive light autotrophy of little algae a large amount of algae kinds in time are provided, but also can accelerate little algae light autophyting growth and purpose product (as grease, albumen etc.) formation speed, for solving the algae kind that exists in the extensive light autotrophy of the little algae culturing process long, the slow and low problem of purpose product productive rate of cell growth of cycle that spreads cultivation, a kind of important techniques means are provided.
Therefore, first aspect present invention provides a kind of microalgae culture method, and this method comprises the heterotrophism culturing step of little algae algae kind and the light autotrophy culturing step of implementing as seed with the frustule that the heterotrophism cultivation is obtained.
Second aspect present invention provides a kind of grease production method, and described method comprises the heterotrophism culturing step of little algae algae kind, cultivate light autotrophy culturing step that the frustule obtained implements as seed and the step that frustule is gathered and grease extracts with heterotrophism.
Third aspect present invention provides a kind of method for producing protein, and described method comprises the heterotrophism culturing step of little algae algae kind, cultivate the light autotrophy culturing step that the frustule obtained implements as seed with heterotrophism and frustule is gathered and the step of proteins extraction.
In an embodiment, but described little algae is selected from the heterotrophism cultured microalgae.
In an embodiment, described little algae is selected from:
Chlorella pyrenoidesa in the Chlorophyta Chlorella (Chlorella pyrenoidosa), Chlorella vulgaris (Chlorella vulgaris), chlorella ellipsoidea (Chlorella ellipsoidea), Chlorella emersonii, Chlorella sorokiniana, Chlorella saccharophila, Chlorella regularis, Chlorellaminutissima, Chlorella protothecoides, Chlorella zofingiensis, and the Brachiomonas submarina in the Chlorophyta, Chlamydobonas reinhardtii, Chlamydomonasacidophila, Haematococcus pluvialis, Haematococcus lacustris, Scenedesmusobliquus, Spongiococcum exetriccium, Tetraselmis suecica, Tetraselmis chuii, Tetraselmis tetrathele, Tetraselmis verrucosa, Micractinium pusillum;
The Cylindrotheca fusiformis of Bacillariophyta, Nitzschia laevis, Nitzschia alba, Nitzschiafonticola, Navicula incerta, Navicula pelliculosa;
The Anabaena variabilis of Cyanophyta;
The Poterioochromonas malhamensis of Chrysophyta;
The Amphidinium carterae of Pyrrophyta, Crypthecodinium cohnii;
The Euglena gricilis of Euglenophyta; With
The Galdieria sulphuraria of rhodophyta.
In an embodiment, the heterotrophic step of described little algae algae kind comprises: adding pH is 4.0～9.0 substratum in bio-reactor, insert little algae algae kind by 0.1～30% of working volume and carry out batch culture, fed batch cultivation or semicontinuous cultivation, culture temperature is 10～40 ℃, pH is less than 9.0 in control, and the control dissolved oxygen is more than 1%.
In an embodiment, the step of described little algae light autotrophy comprises: the little algae algae of heterotrophism energy kind is inoculated in the light autotrophy culture apparatus carries out the light autotrophy, culture temperature is 5～50 ℃, continuous illumination or intermittent illumination, intensity of illumination is 0.1～150klx, light autotrophy culture cycle is 5～500 hours, and initial inoculation density is 0.01～10.00 grams per liter, and pH is 4.0～12.0.
In an embodiment, the heterotrophism substratum 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, but described heterotrophism step is carried out in the bio-reactor that shakes bottle, mechanical agitation type, air lift type or the cultivation of bubbling style heterotrophism, described smooth autotrophy culturing step is shaking bottle or is being selected from the runway pond of open type or any can be used for such as circle pond, enclosed flat bioreactor or duct type bioreactor or pillar bioreactor, the upright bag of film or Pig etc. carries out in the device that little algae light autotrophy cultivates, and illumination condition is natural light or artificial light irradiation.
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 and water, wherein trace element consists of H
3BO
35～15 grams per liters, ZnSO
47H
2O 5.0～10.0 grams per liters, MnCl
2H
2O1.0～2.0 grams per liters, (NH
4)
6Mo
7O
244H
2O 0.5～1.5 grams per liter, CuSO
45H
2O 1.0～2.0 grams per liters, Co (NO
3)
26H
2O 0.1～0.9 grams per liter.
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 2.0～8.0 grams per liters, KH
2PO
41.0～2.0 grams per liters, MgSO
47H
2O 1.0～2.0 grams per liters, CaCl
20.05～0.1 grams per liter, trisodium citrate 0.1～2.0 grams per liter, Fe-EDTA solution 0.5～1mL, A5 solution 1～5mL 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 0.05～0.1 grams per liter, Na
2MoO
40.01～0.05 grams per liter.
In a specific embodiment, after the glucose consumption in the heterotrophism nutrient solution is intact, finishes heterotrophism and cultivate, and heterotrophism is cultivated the gained frustule implement light autotrophy culturing step as seed.
In other embodiment of the application, can use the described substratum of the application, culture condition to implement above-mentioned microalgae culture method.
Cultivating and cultivate the production biofuel with the frustule that the heterotrophism cultivation is obtained as the light autotrophy of seed with the heterotrophism of chlorella algae kind is example, describes advantage of the present invention in detail:
(1) can improve algal species cultivation speed greatly.The initial inoculation density that general outdoor Da Chi cultivates little algae is 0.05～0.1g/l, and the volume of Da Chi is generally 5000L, if need satisfy this requirement, the little algae that then needs 250～500g is as seed.The algae kind if the traditional light autotrophy of employing spreads cultivation then needs for 1～February (10 days algae cell density of algae kind light autotrophy cultivation is generally 0.2～0.5g/l, needs just can enter Da Chi at last through spread cultivation step by step (about about 10 times thinning ratio)); And adopt 50L fermentor tank heterotrophism to cultivate the algae kind, then only need 2～3 days.
(2) compare with traditional algae kind light autotrophy cultivation, can reduce device and number of devices in the algal species cultivation process, and floor space is little, unit culture area (or volume) productive rate height.Cultivating spreading cultivation of required algae kind with the Da Chi light autotrophy of 5000L is example, and the light autotrophy algae kind that spreads cultivation needs the culture apparatus of 5L, 50L and 500L, and algae kind heterotrophism is cultivated and only needed the culture apparatus of 50L to get final product.
(3) compare with the cultivation of algae kind light autotrophy, algae kind heterotrophism is cultivated the influence that is not subjected to outdoor weather and environment.When algae kind light autotrophy is cultivated, in the time of can't continuing to cultivate if run into rainy weather, then need to move into indoor and add artificial light and continue the light autotrophy and cultivate the algae kind, thereby increased the artificial lighting expense.In addition, algae kind light autotrophy is vulnerable to the pollution of harmful organisms such as protozoon, assorted algae, thereby causes the algal species cultivation failure, has a strong impact on production.
When (4) outdoor Da Chi cultivated, inoculum size was big more, is not easy to be subjected to pollutions such as other assorted algaes or protozoon more; The heterotrophism of algae kind is cultivated can in time provide a large amount of seeds, thereby satisfies the demand that improves inoculum size when the light autotrophy is cultivated.
(5) vigor of heterotrophism algae kind is better than the algae kind that the light autotrophy is cultivated.Insert identical frustule amount and carry out the light autotrophy when cultivating, utilize algae cell density, grease productive rate and the albumen productive rate etc. of heterotrophism cultured cells, all be higher than and utilize the analog value of light autotrophy cultured cells as the light autotrophy culturing process of algae kind as the light autotrophy culturing process of algae kind.In addition, because it is higher to carry out the algae cell density that light autotrophy cultivation obtained with heterotrophism algae kind, therefore, can reduce the cost of gathering of the little algae of the energy.
When in sum, the heterotrophism of algae kind of the present invention is cultivated and is cultivated with heterotrophism the frustule that obtained and can solve the light autotrophy and cultivate as the light autotrophy training mode of seed since the problems that algal species cultivation efficient is low and vulnerable to pollution was caused and little algae in light autotrophy culturing process growth velocity the slow and low problem of purpose product (as grease, albumen etc.) productive rate.Therefore, the present invention provides a kind of important techniques means for solving in little algae light autotrophy culturing process the slow and low problem of purpose product productive rate of cell growth, has established important basis in particular for the industrialization that utilizes little algae production biofuel.
Description of drawings
Fig. 1 shows that the Chlorella pyrenoidesa seed shakes the process of growth that heterotrophism is cultivated and the light autotrophy is cultivated in the 2L triangular flask in the bottle at 500ml respectively.
Fig. 2 shows that the Chlorella vulgaris seed shakes the process of growth that heterotrophism is cultivated and the light autotrophy is cultivated in the 2L triangular flask in the bottle at 500ml respectively.
Fig. 3 shows that the chlorella ellipsoidea seed shakes the process of growth that heterotrophism is cultivated and the light autotrophy is cultivated in the 2L triangular flask in the bottle at 500ml respectively.
Fig. 4 shows Chlorella pyrenoidesa heterotrophism seed and the light autotrophy seed frustule growth curve that the light autotrophy is cultivated in indoor 2L bioreactor.
Fig. 5 shows Chlorella pyrenoidesa heterotrophism seed and the light autotrophy seed grease productive rate that the light autotrophy is cultivated in indoor 2L bioreactor.
Fig. 6 shows Chlorella vulgaris heterotrophism seed and the light autotrophy seed frustule growth curve that the light autotrophy is cultivated in indoor 2L bioreactor.
Fig. 7 shows Chlorella vulgaris heterotrophism seed and the light autotrophy seed grease productive rate that the light autotrophy is cultivated in indoor 2L bioreactor.
Fig. 8 shows chlorella ellipsoidea heterotrophism seed and the light autotrophy seed frustule growth curve that the light autotrophy is cultivated in indoor 2L bioreactor.
Fig. 9 shows chlorella ellipsoidea heterotrophism seed and the light autotrophy seed grease productive rate that the light autotrophy is cultivated in indoor 2L bioreactor.
Figure 10 shows Chlorella pyrenoidesa heterotrophism seed the light autotrophy is cultivated in the 2L bioreactor out of doors frustule growth curve and maximum oil fat productive rate.
Figure 11 shows Chlorella pyrenoidesa heterotrophism seed the light autotrophy is cultivated in the 60L plastic tub out of doors frustule growth curve and maximum oil fat productive rate.
Embodiment
The little algae that is applicable to the application comprises that all can carry out the heterotrophism cultured microalgae, include but not limited to the Chlorella pyrenoidesa (Chlorella pyrenoidosa) in the Chlorophyta Chlorella, Chlorella vulgaris (Chlorellavulgaris), chlorella ellipsoidea (Chlorella ellipsoidea), Chlorella emersonii, Chlorellasorokiniana, Chlorella saccharophila, Chlorella regularis, Chlorella minutissima, Chlorella protothecoides, Chlorella zofingiensis, and the Brachiomonassubmarina in the Chlorophyta, Chlamydobonas reinhardtii, Chlamydomonas acidophila, Haematococcus pluvialis, Haematococcus lacustris, Scenedesmus obliquus, Spongiococcum exetriccium, Tetraselmis suecica, Tetraselmis chuii, Tetraselmistetrathele, 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 Galdieriasulphuraria of rhodophyta.
In preferred embodiment, the present invention adopts chlorella enforcement.In preferred embodiment, the present invention adopts Chlorella pyrenoidesa, Chlorella vulgaris or chlorella ellipsoidea to implement.In other preferred embodiment, little algae that the present invention adopts Chlorella pyrenoidesa, Chlorella vulgaris or chlorella ellipsoidea to implement high grease productive rate and high protein productive rate is cultivated.
The heterotrophism that can adopt various substratum well known in the art to carry out little algae seed is cultivated.Usually, the heterotrophism substratum contains nitrogenous source, organic carbon source, inorganic salt, trace element and water.Be applicable to that nitrogenous source that little algae cultivates, organic carbon source, inorganic salt, trace element etc. are that this area is known.For example, as nitrogenous source, spendable have urea or various nitrate, as KNO
3Deng; As organic carbon source, can use glucose, sucrose, glycerine etc.
This class substratum comprises HA-SK substratum (Chinese patent ZL 200610024004.9), Endo substratum (Ogbonna J.C., Masui.H., Tanaka.H.Sequential heterotrophic:autotrophiccultivation-an efficient method of producing Chlorella biomass for health foodand animal feed.J.Appl.Phycol.1997,9,359～366) etc.
The used HA-SK substratum of the present invention is by KNO basically
3, glucose and inorganic salt, trace element and water forms.In described technical scheme, described trace element should be selected from H
3BO
3, ZnSO
47H
2O, MnCl
2H
2O, (NH
4)
6Mo
7O
244H
2O, CuSO
45H
2O, Co (NO
3)
26H
2Among the O one or more or whole.
Term " be basically by ... form " in the above-mentioned substratum of expression except containing main ingredient KNO
3, outside glucose and inorganic salt, trace element and the water, also can comprise some for the fundamental characteristics of composition or new characteristic (can keep little algae and reach higher level at short culture cycle inner cell density, activity substance content is cultivated to compare with conventional heterotrophism and the had a more substantial increase simultaneously) component of influence in fact not.Term " by ... form " the above-mentioned substratum of expression is made up of pointed concrete component, do not have other components, but can have the impurity of content in common scope.
In this substratum, each component of substratum can change within the specific limits and can very big materially affect do not arranged to microalgae cell density and quality.Therefore, the consumption of these components is not limited by the strictness of embodiment should.As known to those skilled in the art, also can add inorganic salt in the substratum, for example sal epsom, 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, preferable micro-component should be selected from H
3BO
3, ZnSO
47H
2O, MnCl
2H
2O, (NH
4)
6Mo
7O
244H
2O, CuSO
45H
2O, Co (NO
3)
26H
2Among the O one or more.The consumption of inorganic salt and trace element can be determined according to conventional knowledge.
HA-SK substratum of the present invention is grouped into by following one-tenth basically: KNO
35～15 grams per liters, glucose 10～60 grams per liters, KH
2PO
40.3～0.9 grams per liter, Na2HPO
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 and water, wherein trace element consists of H
3BO
35～15 grams per liters, ZnSO
47H
2O 5.0～10.0 grams per liters, MnCl
2H
2O 1.0～2.0 grams per liters, (NH
4)
6Mo
7O
244H
2O0.5～1.5 grams per liters, CuSO
45H
2O 1.0～2.0 grams per liters, Co (NO
3)
26H
2O 0.1～0.9 grams per liter.
In a preferable embodiment, HA-SK culture media composition of the present invention should be grouped into by following one-tenth: KNO
37 grams per liters, glucose 40 grams per liters, KH
2PO
40.6 grams per liter, Na
2HPO
412H
2O 2.0 grams per liters, MgSO
47H
2O 0.8 grams per liter, CaCl
20.2 grams per liter, FeSO
47H
2O 0.03 grams per liter, micro-1.5mL and water 1000mL, wherein trace element consists of H
3BO
311～12 grams per liters, ZnSO
47H
2O 8.5～9.5 grams per liters, MnCl
2H
2O 1.4～1.5 grams per liters, (NH
4)
6Mo
7O
244H
2O0.8～0.9 grams per liter, CuSO
45H
2O 1.5～1.6 grams per liters, Co (NO
3)
26H
2O 0.45～0.55 grams per liter.
The used Endo substratum of the present invention is grouped into by following one-tenth basically: glucose 10～60 grams per liters, urea 2～8 grams per liters, KH
2PO
41～2 grams per liter, Na
2HPO
412H
2O 1.0～10.0 grams per liters, MgSO
47H
2O1～2 grams per liters, CaCl
20.05～0.1 grams per liter, trisodium citrate 0.1～2.0 grams per liter, Fe-EDTA solution 0.5～1mL, A5 solution 1～5mL 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
2O1.0～2.0 grams per liters, ZnSO
47H
2O 0.1～0.6 grams per liter, CuSO
45H
2O 5～10 grams per liters, Na
2MoO
40.01～0.05 grams per liter.
In a preferable embodiment, described Endo substratum is grouped into by following one-tenth: glucose 40 grams per liters, urea 6.0 grams per liters, KH
2PO
41.5 grams per liter, Na2HPO
412H
2O 5.0 grams per liters, MgSO
47H
2The O1.8 grams per liter, CaCl
20.05 grams per liter, trisodium citrate 0.4 grams per liter, Fe-EDTA solution 0.8mL, A5 solution 2.0mL and water, wherein the Fe-EDTA solution formula is FeSO
47H
2O 25 grams per liters and EDTA33.5 grams per liter, A5 solution formula are H
3BO
32.86 grams per liter, MnCl
24H
2O 1.81 grams per liters, ZnSO
47H
2O 0.222 grams per liter, CuSO
45H
2O 0.07 grams per liter, Na
2MoO
40.021 grams per liter.
After according to above-mentioned formulated substratum, available conventional means as acid or alkali with as described in the pH of substratum be adjusted to 4.0～9.0, and 115～120 ℃ of following autoclavings 15～20 minutes.Can adopt four kinds of modes such as batch culture, fed batch cultivation, semicontinuous cultivation (band is put) or cultured continuously to implement the seed heterotrophism cultivates.
When carrying out the cultivation of seed heterotrophism, corresponding prepared culture medium is joined in the bio-reactor, benefit adds water to working volume, usually coefficient is 0.6～0.8, steam sterilizing is (121 ℃ then, kept about 20 minutes), when temperature is reduced to 30～35 ℃, insert little algae seed by 1～15% of working volume and begin the heterotrophism cultivation.
No matter adopt which kind of training method, in culturing process, must make little algae seed normal growth by the suitable culture condition of control.Usually, controlled temperature is 20～35 ℃, and for example 28～30 ℃, dissolved oxygen is not less than 5% air saturation concentration, and pH is not higher than 9.0.In a preferred embodiment, dissolved oxygen is not less than 10% air saturation concentration, and pH is not higher than 8.5.In other preferred embodiment, dissolved oxygen is not less than 15% air saturation concentration, and pH is not higher than 8.
In culturing process, pH is unsuitable too high or too low, generally along with the carrying out of cultivating, pH can slowly rise (obvious especially) for this phenomenon of Chlorella vulgaris, growth has a negative impact the too high meeting of pH to frustule, regulate so use acid (for example 10% sulfuric acid), make pH not be higher than 9.0, preferable pH should be 6.5～7.5.
But heterotrophism can carry out in the bio-reactor that shakes heterotrophism cultivations such as bottle, mechanical agitation type, air lift type, bubbling style.
After the glucose consumption in the seed heterotrophism nutrient solution was intact, then the seed heterotrophism was cultivated and is finished.Subsequently, be inoculated into and carry out the light autotrophy in the light autotrophy culture apparatus and cultivate.The initial inoculation density that the light autotrophy is cultivated is generally 0.01～1 grams per liter, and temperature is 10～40 ℃, and intensity of illumination is 0.1～100klx, and light dark period is 24:0～6:18, and pH is 4.0～9.0, and air flow is 0.05～5vvm, feeds CO
2Concentration is 0.03～5%.
When the frustule growth is in stationary phase (when algae cell density does not increase), then finish the light autotrophy and cultivate, frustule is gathered.
Can adopt various smooth autotrophy substratum well known in the art to carry out the light autotrophy cultivates.Usually, light autotrophy substratum contains nitrogenous source, phosphorus source, inorganic carbon source, inorganic salt, trace element and water.Be applicable to that nitrogenous source that little algae cultivates, phosphorus source, inorganic carbon source, inorganic salt, trace element etc. are that this area is known.For example, as nitrogenous source, spendable have urea or various nitrate, as KNO
3Deng; As the phosphorus source, spendable have a for example NaH
2PO
4As inorganic carbon source, spendable have a for example CO
2Deng.
This class substratum is a basic medium with the F-Si substratum.
The used improvement F-Si substratum of the present invention is by NaNO basically
3, NaH
2PO
4And trace element, a small amount of VITAMIN and water are formed.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 " be basically by ... form " in the above-mentioned substratum of expression except containing main ingredient NaNO
3, NaH
2PO
4And outside trace element, a small amount of VITAMIN and the water, also can comprise some for the fundamental characteristics of composition or new characteristic (can keep little algae and reach higher level at short culture cycle inner cell density, activity substance content is cultivated to compare with conventional light autotrophy and the had a more substantial increase simultaneously) component of influence in fact not.
In this substratum, each component of substratum can change within the specific limits and can very big materially affect do not arranged to microalgae cell density and quality.Therefore, the consumption of these components is not limited by the strictness of embodiment should.As known to those skilled in the art, also can add inorganic salt in the substratum, for example sal epsom, calcium chloride and ferrous sulfate etc., and trace element is as Mn, Zn, B, I, M, Cu, Co etc.
In the present invention, preferable 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 knowledge.
Improvement F/2-Si substratum of the present invention is to be grouped into by following one-tenth basically: NaNO
30.1～1.0 grams per liters, NaH
2PO
42H
2O 0.01～0.1 grams per liter; Trace element 0.5～4ml, wherein trace element consists of ZnSO
47H
2O 0.02～0.2 grams per liter, MnCl
24H
2O 0.2～2.0 grams per liter, CuSO
45H
2O0.01～0.1 grams per liter, FeC
6H
5O
75H
2O 1.0～10 grams per liters, Na
2MoO
42H
2O 0.05～0.5 grams per liter, Na
2EDTA 2.0～20 grams per liters, CoCl
20.01～0.1 grams per liter, VITAMIN 0.5～4ml and water, wherein trace element consists of vitamin B12 0.1～1.0 mg/litre, vitamin B15 0～1000 mg/litre, vitamin H 0.1～1.0 mg/litre.
In a preferable embodiment, the improved F/2-Si substratum of the present invention should be grouped into by following one-tenth: NaNO
30.5 grams per liter, NaH
2PO
42H
2O 0.05 grams per liter; Trace element 1.5ml, wherein trace element consists of ZnSO
47H
2O 0.1～0.15 grams per liter, MnCl
24H
2O1.0～1.5 grams per liters, CuSO
45H
2O 0.03～0.07 grams per liter, FeC
6H
5O
75H
2O 3.0～4.0 grams per liters, Na
2MoO
42H
2O0.1～0.3 grams per liter, Na
2EDTA 10～12 grams per liters, CoCl
20.02～0.06 grams per liter, VITAMIN 2ml and water 1000mL, wherein trace element consists 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 with as described in the pH of substratum be adjusted to 4.0～9.0.Can adopt four kinds of modes such as batch culture, fed batch cultivation, semicontinuous cultivation (band is put) or cultured continuously to implement the light autotrophy cultivates.
Frustule is gathered, the comprehensive utilization of greasy extraction and frond
The light autotrophy is carried out centrifugal gathering to little algae after cultivating and finishing, and obtains wet frond.The collecting method of frustule includes but not limited to technology such as high speed centrifugation, flocculation, air supporting or filtration; The frustule wall-breaking method includes but not limited to Wet-process wall breaking methods such as frond self-dissolving, high-pressure homogenization, enzymic hydrolysis, water pyrolysis.
Greasy extracting method includes but not limited to organic solvent extractionprocess in the born of the same parents, that is: frond is dried to constant weight under 80～105 ℃, adopt chloroform methanol standard extraction solvent from dry algae powder, to extract grease after grinding the algae powder, extraction solvent extracts repeatedly until algae powder color and becomes white, and rotary evaporation is removed solvent.
Other compositions in the supernatant liquor progressively separation and Extraction obtain lipid acid, chlorophyll etc., or directly with all the components in the supernatant liquor and the dry chlorella powder that obtains of frond precipitation mixed atomizing.
Among the present invention, can little algae of cultivating gained be fully utilized, extract various activeconstituentss such as wherein pigment (for example xenthophylls), protein, polysaccharide.Extraction of active ingredients there is no particular restriction in proper order, can not cause the composition of back extraction to lose this prerequisite but will satisfy the step of extracting earlier usually.The method of extracting protein, polysaccharide etc. also is that this area is known.
The measuring method that relates to biochemical component in frustule dry weight, fat content and the born of the same parents herein is as follows:
The frustule dry weight is measured: get nutrient solution V milliliter in little algae (as chlorella) culturing process, centrifugal 10 minutes of 8000rpm, with deionized water wash 3 times of the frond after centrifugal, 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.
Grease is measured: get a certain amount of each cultivation stage and dry frustule to constant weight, in mortar, be ground to Powdered, (0.2～0.5g) carefully is transferred in the centrifuge tube to take by weighing an amount of algae powder, add an amount of extraction solvent (chloroform: sonic oscillation 30min in ultrasonator methyl alcohol=2: 1), the centrifugal 10min of 8000rpm, supernatant is transferred in the dry rotary evaporation bottle of known weight, it is colourless until supernatant to repeat above-mentioned steps.Rotate evaporate to dryness after merging supernatant, weigh and calculate fat content.
Fat content (%) is calculated as follows:
Grease (%)=(W2-W0)/W1 * 100
In the formula: W1--is an algae grain weight amount, g; W0--is the rotary evaporation bottle weight of drying to constant weight, g; W2--is the weight of evaporative flask behind the oil extraction liquid evaporate to dryness, g.
Protein content determination: the mensuration of total protein content employing Kjeldahl determination in little algae (as chlorella) cell (Ning Zhengxiang. the food composition analysis handbook. Beijing: China Light Industry Press, 1998,76-78).
Below will be further described related content of the present invention by embodiment.Unless have describedly in addition, each component concentration is all used grams per liter (g/L) expression in the substratum that the present invention adopts.Should be understood that among the application " contain ", " comprising " also comprise " by ... form ", " by ... constitute " implication.
Embodiment 1: frustule Study on Growth in Chlorella pyrenoidesa seed heterotrophism and the light autotrophy culturing process
The Chlorella pyrenoidesa of present embodiment respectively shaking of 500ml carry out in the bottle that heterotrophism is cultivated and the triangular flask of 2L in carry out the cultivation of light autotrophy.As the seed that next step light autotrophy is cultivated, measured the frustule growth curve that Chlorella pyrenoidesa heterotrophism and light autotrophy are cultivated seed respectively.Inoculum density when Chlorella pyrenoidesa seed heterotrophism is cultivated is 0.20g/l, and temperature is 30 ℃, and rotating speed is 150rmp, and when cultivating 72h, the glucose in the nutrient solution has consumed, and algae cell density is 6.8g/l, is used 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 ℃, intensity of illumination is 2000lx, light dark period is 24:0, regularly shake every day 3 times, when cultivating 120h, frustule is in exponential phase of growth, algae cell density is 0.56g/l, is used for the seed (Fig. 1) that next step light autotrophy is cultivated.This shows, compare, seed cell density height that heterotrophism is cultivated (be light autotrophy seed 12.1 times) and culture cycle short (having shortened 48 hours) than light autotrophy seed with the seed that the light autotrophy is cultivated.
Embodiment 2: frustule Study on Growth in Chlorella vulgaris seed heterotrophism and the light autotrophy culturing process
The Chlorella vulgaris of present embodiment respectively shaking of 500ml carry out in the bottle that heterotrophism is cultivated and the triangular flask of 2L in carry out the cultivation of light autotrophy, as the seed that next step light autotrophy is cultivated, measured the frustule growth curve that Chlorella vulgaris heterotrophism and light autotrophy are cultivated seed respectively.Inoculum density when Chlorella vulgaris seed heterotrophism is cultivated is 0.30g/l, and temperature is 30 ℃, and rotating speed is 150rmp, and when cultivating 72h, the glucose in the nutrient solution has consumed, and algae cell density is 8.1g/l, is used 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 ℃, intensity of illumination is 2000lx, light dark period is 24:0, regularly shake every day 3 times, when cultivating 120h, frustule is in exponential phase of growth, algae cell density is 0.65g/l, is used for the seed (Fig. 2) that next step light autotrophy is cultivated.This shows, compare, seed cell density height that heterotrophism is cultivated (be light autotrophy seed 12.5 times) and culture cycle short (having shortened 48 hours) than light autotrophy seed with the seed that the light autotrophy is cultivated.
Embodiment 3: frustule Study on Growth in chlorella ellipsoidea seed heterotrophism and the light autotrophy culturing process
The chlorella ellipsoidea of present embodiment respectively shaking of 500ml carry out in the bottle that heterotrophism is cultivated and the triangular flask of 2L in carry out the cultivation of light autotrophy, as the seed that next step light autotrophy is cultivated, measured the frustule growth curve that chlorella ellipsoidea heterotrophism and light autotrophy are cultivated seed respectively.Inoculum density when chlorella ellipsoidea seed heterotrophism is cultivated is 0.25g/l, and temperature is 30 ℃, and rotating speed is 150rmp, and when cultivating 72h, the glucose in the nutrient solution has consumed, and algae cell density is 8.8g/l, is used 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 ℃, intensity of illumination is 2000lx, light dark period is 24:0, regularly shake every day 3 times, when cultivating 120h, frustule is in exponential phase of growth, algae cell density is 0.71g/l, is used for the seed (Fig. 3) that next step light autotrophy is cultivated.This shows, compare, seed cell density height that heterotrophism is cultivated (be light autotrophy seed 12.4 times) and culture cycle short (having shortened 48 hours) than light autotrophy seed with the seed that the light autotrophy is cultivated.
Embodiment 4: the research that the light autotrophy is cultivated in indoor 2L bioreactor of Chlorella pyrenoidesa heterotrophism seed and light autotrophy seed
Present embodiment light autotrophy in indoor 2L cylinder shape airlift photobioreactor is cultivated the Chlorella pyrenoidesa seed of heterotrophism and light autotrophy, has measured heterotrophism and light autotrophy seed frustule growth and the grease productive rate in its light autotrophy process respectively.The inoculum density of heterotrophism and light autotrophy seed is 0.3g/l, and light autotrophy culture temperature is 30 ℃, all feeds 2% CO
2, air flow is 0.5vvm, and intensity of illumination is 10000lx, and light dark period is 24:0.The light autotrophy was cultivated 96 hours, and the algae cell density of heterotrophism seed is 2.03g/l, and the grease productive rate is 149.55mg/l/d; The algae cell density of light autotrophy seed only is 0.93g/l, and the grease productive rate only is 87.68mg/l/d (Fig. 4 and Fig. 5).This shows that compare with light autotrophy seed, the vigor of heterotrophism seed is stronger, the algae cell density of identical incubation time higher (be light autotrophy seed 2.2 times), grease productive rate higher (be light autotrophy seed 1.7 times).
Embodiment 5: the research that the light autotrophy is cultivated in indoor 2L bioreactor of Chlorella vulgaris heterotrophism seed and light autotrophy seed
Present embodiment light autotrophy in indoor 2L cylinder shape airlift photobioreactor cultivate heterotrophism and light autotrophy the Chlorella vulgaris seed, measured heterotrophism and light autotrophy seed frustule growth and the grease productive rate in its light autotrophy process respectively.The inoculum density of heterotrophism and light autotrophy seed is 0.3g/l, and light autotrophy culture temperature is 30 ℃, all feeds 2% CO
2, air flow is 0.5vvm, and intensity of illumination is 10000lx, and light dark period is 24:0.The light autotrophy was cultivated 96 hours, and the algae cell density of heterotrophism seed is 2.03g/l, and the grease productive rate is 138.49mg/l/d; The algae cell density of light autotrophy seed only is 1.25g/l, and the grease productive rate only is 82.81mg/l/d (Fig. 6 and Fig. 7).This shows that compare with light autotrophy seed, the vigor of heterotrophism seed is stronger, the algae cell density of identical incubation time higher (be light autotrophy seed 1.6 times), grease productive rate higher (be light autotrophy seed 1.7 times).
Embodiment 6: the research that the light autotrophy is cultivated in indoor 2L bioreactor of chlorella ellipsoidea heterotrophism seed and light autotrophy seed
Present embodiment light autotrophy in indoor 2L cylinder shape airlift photobioreactor cultivate heterotrophism and light autotrophy the chlorella ellipsoidea seed, measured heterotrophism and light autotrophy seed frustule growth and the grease productive rate in its light autotrophy process respectively.The inoculum density of heterotrophism and light autotrophy seed is 0.3g/l, and light autotrophy culture temperature is 30 ℃, all feeds 2% CO
2, air flow is 0.5vvm, and intensity of illumination is 10000lx, and light dark period is 24:0.The light autotrophy was cultivated 96 hours, and the algae cell density of heterotrophism seed is 1.65g/l, and the grease productive rate is 121.48mg/l/d; The algae cell density of light autotrophy seed only is 0.91g/l, and the grease productive rate only is 72.29mg/l/d (Fig. 8 and Fig. 9).This shows that compare with light autotrophy seed, the vigor of heterotrophism seed is stronger, the algae cell density of identical incubation time higher (be light autotrophy seed 1.8 times), grease productive rate higher (be light autotrophy seed 1.7 times).
Embodiment 7: the research that the light autotrophy is cultivated in the 2L bioreactor out of doors of Chlorella pyrenoidesa heterotrophism seed
Present embodiment out of doors in the 2L cylinder shape airlift photobioreactor light autotrophy cultivate Chlorella pyrenoidesa heterotrophism seed, measured frustule process of growth and the maximum oil fat productive rate of heterotrophism seed in its outdoor light autotrophy process.Initial inoculation density is 0.06g/l, and temperature and light intensity are outdoor actual temperature and light intensity, and air flow is 0.3vvm.The light autotrophy was cultivated 83 hours, and algae cell density and grease productive rate all reach maximum, was respectively 1.44g/l and 123.43mg/l/d (Figure 10).This shows that heterotrophic seed is not only cultivated and reached high grease productive rate in indoor flash of light preceding an earthquake autotrophy fast, and still flash of light preceding an earthquake autotrophy cultivation and reach high grease productive rate fast out of doors.
Embodiment 8: the research that the light autotrophy is cultivated in the 60L plastic tub out of doors of Chlorella pyrenoidesa heterotrophism seed
Present embodiment out of doors in the 60L plastic tub light autotrophy cultivate Chlorella pyrenoidesa heterotrophism seed, measured frustule process of growth and the maximum oil fat productive rate of heterotrophism seed in its outdoor light autotrophy process.Initial inoculation density is 0.10g/l, and temperature and light intensity are outdoor actual temperature and light intensity, and air flow is 0.3vvm.The light autotrophy was cultivated 108 hours, and algae cell density and grease productive rate all reach maximum, was respectively 0.75g/l and 55.34mg/l/d (Figure 11).Volume of culture in 60L plastic tub is generally 50L, if want initial inoculation density to reach 0.10g/l, then needs the algae kind of 5g; The seed (see figure 1) of cultivating with heterotrophism only needs the bottle (liquid amount is 200ml) that shakes of 4 500ml to cultivate the seed that can obtain 5.44g in 3 days, and the seed (see figure 1) of cultivating with the light autotrophy then needs the triangular flask (liquid amount is 1L) of 9 2L to cultivate the seed that just can obtain 5.04g in 5 days.This shows that cultivate with the seed light autotrophy and compare, the seed heterotrophism is cultivated the incubation time that can not only shorten seed, improves the efficient of whole culturing process, and the required culture apparatus consumption of seed culture is less and floor space is less.Therefore, if want outdoor extensive light autotrophy to cultivate little algae, have only so and adopt heterotrophism to cultivate the demand that seed could in time satisfy a large amount of algae kinds.
Although object lesson of the present invention described above, having a bit is significantly to those skilled in the art, promptly can do various variations and change to the present invention under the premise without departing from the spirit and scope of the present invention.Therefore, claims have covered all these changes within the scope of the present invention.
Claims (10)
1. a microalgae culture method is characterized in that, this method comprises the heterotrophism culturing step of little algae algae kind and the light autotrophy culturing step of implementing as seed with the frustule that the heterotrophism cultivation is obtained.
2. a grease production method is characterized in that, described method comprises the heterotrophism culturing step of little algae algae kind, cultivate light autotrophy culturing step that the frustule obtained implements as seed and the step that frustule is gathered and grease extracts with heterotrophism.
3. a method for producing protein is characterized in that, described method comprises the heterotrophism culturing step of little algae algae kind, cultivate the light autotrophy culturing step that the frustule obtained implements as seed with heterotrophism and frustule is gathered and the step of proteins extraction.
4. as each described method among the claim 1-3, it is characterized in that the heterotrophism that adopts batch culture, fed batch cultivation, semicontinuous cultivation and cultured continuously isotype to implement described little algae algae kind is cultivated.
5. as method as described in each among the claim 1-4, it is characterized in that described little algae is selected from:
Chlorella pyrenoidesa in the Chlorophyta Chlorella (Chlorella pyrenoidosa) Chlorella vulgaris (Chlorella vulgaris), chlorella ellipsoidea (Chlorella ellipsoidea), Chlorella emersonii, Chlorella sorokiniana, Chlorella saccharophila, Chlorella regularis, Chlorellaminutissima, Chlorella protothecoides, Chlorella zofingiensis, and the Brachiomonas submarina in the Chlorophyta, Chlamydobonas reinhardtii, Chlamydomonasacidophila, Haematococcus pluvialis, Haematococcus lacustris, Scenedesmusobliquus, Spongiococcum exetriccium, Tetraselmis suecica, Tetraselmis chuii, Tetraselmis tetrathele, Tetraselmis verrucosa, Micractinium pusillum;
The Cylindrotheca fusiformis of Bacillariophyta, Nitzschia laevis, Nitzschia alba, Nitzschiafonticola, Navicula incerta, Navicula pelliculosa;
The Anabaena variabilis of Cyanophyta;
The Poterioochromonas malhamensis of Chrysophyta;
The Amphidinium carterae of Pyrrophyta, Crypthecodinium cohnii;
The Euglena gricilis of Euglenophyta; With
The Galdieria sulphuraria of rhodophyta.
6. as each described method among the claim 1-5, it is characterized in that, the step that described little algae algae kind heterotrophism is cultivated comprises: adding pH is 4.0～9.0 substratum in bio-reactor, insert little algae algae kind by 0.1～30% of working volume and carry out batch culture, fed batch cultivation, semicontinuous cultivation or cultured continuously, culture temperature is 10～40 ℃, pH is less than 9.0 in control, and the control dissolved oxygen is more than 1%.
7. as each described method among the claim 1-6, it is characterized in that, cultivate the frustule that is obtained with heterotrophism and carry out the cultivation of light autotrophy as seed, comprise: the algae kind of heterotrophism cultivation is received carried out the cultivation of light autotrophy in the light autotrophy culture apparatus, culture temperature is 5～50 ℃, continuous illumination or intermittent illumination, intensity of illumination is 0.1～150klx, light autotrophy culture cycle is 5～500 hours, and initial inoculation density is 0.01～10.00 grams per liter, and pH is 4.0～12.0.
8. as each described method among the claim 1-7, it is characterized in that the heterotrophism substratum 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.
9. as each described method among the claim 1-8, it is characterized in that, described algae kind heterotrophism culturing step carries out in shaking bottle, mechanical agitation type, air lift type or bubbling style bio-reactor, described smooth autotrophy culturing step is shaking bottle or is being selected from the runway pond of open type or circle pond, enclosed flat bioreactor or duct type bioreactor or pillar bioreactor, the upright bag of film or Pig etc. are used for the device that little algae light autotrophy cultivates and carry out, and illumination condition is natural light or artificial light.
10. as each described method in the claim 1～9, it is characterized in that, after the organic carbon source in the heterotrophism nutrient solution runs out of, finish heterotrophism and cultivate, and heterotrophism is cultivated the gained frustule implement light autotrophy culturing step as seed.
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