CN1807572A - Culture media composition suitable for cultivating high-density high-quality ordinary chlorella - Google Patents
Culture media composition suitable for cultivating high-density high-quality ordinary chlorella Download PDFInfo
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- CN1807572A CN1807572A CNA2006100240049A CN200610024004A CN1807572A CN 1807572 A CN1807572 A CN 1807572A CN A2006100240049 A CNA2006100240049 A CN A2006100240049A CN 200610024004 A CN200610024004 A CN 200610024004A CN 1807572 A CN1807572 A CN 1807572A
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- 239000000203 mixture Substances 0.000 title claims abstract description 29
- 239000001963 growth medium Substances 0.000 title claims abstract description 26
- 241000195649 Chlorella <Chlorellales> Species 0.000 title abstract description 26
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 20
- 239000008103 glucose Substances 0.000 claims abstract description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 14
- 229910017053 inorganic salt Inorganic materials 0.000 claims abstract description 7
- 240000009108 Chlorella vulgaris Species 0.000 claims description 53
- 235000007089 Chlorella vulgaris Nutrition 0.000 claims description 53
- 238000000034 method Methods 0.000 claims description 28
- 235000013619 trace mineral Nutrition 0.000 claims description 28
- 239000011573 trace mineral Substances 0.000 claims description 28
- 235000013305 food Nutrition 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 5
- 239000000654 additive Substances 0.000 claims description 4
- 230000001681 protective effect Effects 0.000 claims description 4
- 241000195493 Cryptophyta Species 0.000 abstract description 6
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 abstract description 4
- 235000010333 potassium nitrate Nutrition 0.000 abstract description 2
- 235000019804 chlorophyll Nutrition 0.000 description 17
- 229930002875 chlorophyll Natural products 0.000 description 17
- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 description 17
- 235000018102 proteins Nutrition 0.000 description 16
- 108090000623 proteins and genes Proteins 0.000 description 16
- 102000004169 proteins and genes Human genes 0.000 description 16
- 229910052799 carbon Inorganic materials 0.000 description 6
- 229910003208 (NH4)6Mo7O24·4H2O Inorganic materials 0.000 description 4
- 239000002028 Biomass Substances 0.000 description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 239000002054 inoculum Substances 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- CKUAXEQHGKSLHN-UHFFFAOYSA-N [C].[N] Chemical compound [C].[N] CKUAXEQHGKSLHN-UHFFFAOYSA-N 0.000 description 3
- 238000005286 illumination Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000012452 mother liquor Substances 0.000 description 3
- LMSDCGXQALIMLM-UHFFFAOYSA-N 2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid;iron Chemical compound [Fe].OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O LMSDCGXQALIMLM-UHFFFAOYSA-N 0.000 description 2
- 241000195652 Auxenochlorella pyrenoidosa Species 0.000 description 2
- 235000007091 Chlorella pyrenoidosa Nutrition 0.000 description 2
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 2
- 241000195648 Pseudochlorella pringsheimii Species 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 230000001651 autotrophic effect Effects 0.000 description 2
- 235000013402 health food Nutrition 0.000 description 2
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 238000005588 Kraus reaction Methods 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 101100010166 Mus musculus Dok3 gene Proteins 0.000 description 1
- XUYPXLNMDZIRQH-LURJTMIESA-N N-acetyl-L-methionine Chemical compound CSCC[C@@H](C(O)=O)NC(C)=O XUYPXLNMDZIRQH-LURJTMIESA-N 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 230000035425 carbon utilization Effects 0.000 description 1
- 235000021466 carotenoid Nutrition 0.000 description 1
- 150000001747 carotenoids Chemical class 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 239000002075 main ingredient Substances 0.000 description 1
- 238000003808 methanol extraction Methods 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 150000003016 phosphoric acids Chemical class 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 230000037039 plant physiology Effects 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 229940038773 trisodium citrate Drugs 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
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Abstract
The invention relates to a culture media composition suitable for cultivating high-density high-quality ordinary chlorella, wherein the culture medium mainly comprises KNO3 7-11g/l, glucose 25-35g/l and small amount of inorganic salt, minor elements and water. The culture medium can substantially improve the quality of globule algae.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of culture media composition of high-density high-quality ordinary chlorella cultivation and method that high-density and high-quality is cultivated Chlorella vulgaris of being suitable for.
Background technology
People are longer to the research history of chlorella, and wherein Chlorella vulgaris (Chlorella vulgaris) is a more algae kind of research, also are to commercially produce one of algae kind that generally adopts at present.
At present, the training method that chlorella is commonly used has two kinds: open outdoor Da Chi light autotrophy is cultivated and the fermentor tank heterotrophism is cultivated.It is present commercialization scale operation method commonly used that open outdoor Da Chi cultivates, though its cost is low, because shortcoming such as low, the easy pollution of cell density, floor space be big is very limited its development.Heterotrophism is cultivated and is in the laboratory development at present basically, though it can improve algae cell density greatly, but the frond that obtains is cultivated the content of comparing its protein and pigment with the light autotrophy and is obviously descended, and promptly quality reduces, and does not have the using value of the Chlorella vulgaris of light autotrophy cultivation.It is low when the content of discovery born of the same parents inner chlorophyll, protein, carotenoid, nicotinic acid was all than the light autophyting growth when Atev.A (1981) heterotrophism was cultivated Chlorella vulgaris A2 (Chlorella vulgaris A2), the interior amino acid of born of the same parents removes Methionin and tyrosine content is higher than the light autophyting growth, other aminoacids content all is lower than the light autotrophy and cultivates (Atev A., Manova A.Heterotrophic cultivation of Chlorella vulgaris A2.Dokl.Bolg.Akad.Nauk, (English) 1981,34 (5): 687～690).Magnifying the soldier waits (1996) to find that Chlorella pyrenoidesa (Chlorella prototuecoides) cell inner chlorophyll disappears when the autotrophy mode-conversion is the heterotrophism pattern, total protein content reduces, only (magnify the soldier for 1/5 of light autotrophy cultivation, Wu Qingyu. the heterotrophism of chlorella cells transforms. the plant physiology circular, 1996,32 (2): 140～144).James.C.Ogbonna etc. (1997) heterotrophism is cultivated Chlorella pyrenoidesa C-212 (Chlorella pyrenoidosa C-212) and has also been confirmed similar experimental result (Ogbonna J.C., Masui.H., Tanaka.H.Sequential heterotrophic/autotrophic cultivation-an efficient method ofproducing Chlorella biomass for health food and animal feed.J.Appl.Phycol.1997,9,359～366.).Pan Xin etc. (2002) heterotrophism is cultivated chlorella ellipsoidea, also find protein content reduce (Dai Chuanchao is etc. the research that chlorella heterotrophy is cultivated for Pan Xin, Li Jianhong. Food science, 2002,23 (4): 28～33).Therefore, when cultivating, heterotrophism all has quality decline problem for three kinds (being Chlorella vulgaris, Chlorella pyrenoidesa, chlorella ellipsoidea) commonly used in the Chlorella.Therefore, how keeping it high-quality when realizing high-density culture, is major issue that needs to be resolved hurrily in the chlorella culture technique.
James.C.Ogbonna etc. (1997) adopt Endo substratum heterotrophism-light autotrophy series connection to cultivate Chlorella pyrenoidesa C-212 (Chlorella pyrenoidosa C-212) can to make frond protein and chlorophyll content obtain by a relatively large margin raising (Ogbonna J.C., Masui.H., Tanaka.H.Sequential heterotrophic:autotrophiccultivation-an efficient method ofproducing Chlorella biomass for health food and animalfeed.J.Appl.Phycol.1997,9,359～366.).The heterotrophism of relevant Chlorella vulgaris-light autotrophy series connection is cultivated in the document and is not appeared in the newspapers, and the research that therefore will carry out this respect at first must solve the substratum problem.
The present invention at first adopts the Endo substratum heterotrophism that the Chlorella pyrenoidesa heterotrophism-series connection of light autotrophy is cultivated-light autotrophy series connection of reporting in the document to cultivate Chlorella vulgaris, finds that this substratum is not suitable for Chlorella vulgaris growth and frond protein and chlorophyllous accumulation; Adopt Chlorella vulgaris Sorokin-Krauss substratum (hereinafter to be referred as the SK substratum) (1958) heterotrophism-light autotrophy series connection commonly used of having added glucose to cultivate Chlorella vulgaris then, find that frond protein and chlorophyll content are not significantly improved.The SK substratum that this explanation utilizes the Endo substratum and added glucose can not realize that all the high-density and high-quality of this algae cultivates, and therefore, is necessary to solve the substratum problem that high-density high-quality ordinary chlorella is cultivated.
Summary of the invention
The purpose of this invention is to provide a kind of culture media composition that is applicable to that high-density high-quality ordinary chlorella is cultivated.This substratum can make Chlorella vulgaris reach under the prerequisite of high-density culture, and the frond quality is cultivated to compare with heterotrophism and had a more substantial increase, and cultivates the level when reaching the cultivation of light autotrophy when finishing.
For achieving the above object, the invention provides a kind of culture media composition that is used to cultivate Chlorella vulgaris, described substratum is basically by KNO
3, glucose and small amounts of inorganic salt, trace element and water forms.
In a preferable embodiment, described trace element is 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.
In a good especially embodiment, described substratum is grouped into by following one-tenth basically or is grouped into by following one-tenth: KNO
37～11 grams per liters, glucose 25～35 grams per liters, KH
2PO
40.6～0.8 grams per liter, Na
2HPO
412H
2O1.7～2.0 grams per liters, MgSO
47H
2O 0.6～0.8 grams per liter, CaCl
20.1～0.2 grams per liter, FeSO
47H
2O 0.01～0.03 grams per liter; Trace element 0.5～2ml, 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
2O 0.8～0.9 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; Water 1000ml.
The present invention relates to above-mentioned culture media composition on the other hand in the purposes that is used for cultivating Chlorella vulgaris.
The present invention relates to a kind of method of cultivating Chlorella vulgaris on the other hand, and this method adopts heterotrophism-light autotrophy series connection to cultivate Chlorella vulgaris, it is characterized in that described method has adopted culture media composition of the present invention.
The present invention relates to a kind of Chlorella vulgaris culture that obtains with aforesaid method on the other hand.
The present invention relates to a kind of composition that contains the Chlorella vulgaris culture of aforesaid method acquisition on the other hand, as protective foods or fodder additives etc.
A further aspect of the invention relates to the purposes of Chlorella vulgaris culture in preparation protective foods or fodder additives that obtains with aforesaid method.
Come heterotrophism-light autotrophy series connection to cultivate Chlorella vulgaris with culture media composition disclosed by the invention, can make Chlorella vulgaris cell density in short culture cycle reach the level of high-density culture on the one hand, another prior aspect is that its quality that can make Chlorella vulgaris and heterotrophism are cultivated to compare and has a more substantial increase, reach the level when the light autotrophy is cultivated, thereby realized the cultivation of high-density high-quality ordinary chlorella.
The accompanying drawing summary
Fig. 1 has shown with different substratum by heterotrophism-resulting result of light autotrophy series connection cultivation Chlorella vulgaris, wherein solid mark is represented to cultivate the result that the back obtains with substratum of the present invention, and hollow mark represents that the SK substratum through adjusting slightly that is used as contrast cultivates the result of back acquisition.
Fig. 2 has shown that the Endo substratum that is used as contrast carries out heterotrophism-light autotrophy series connection and cultivates the resulting result of Chlorella vulgaris.
Fig. 3 has shown the dry cell weight when cultivating Chlorella vulgaris with the series connection of different carbon-nitrogen ratio heterotrophism-light autotrophy.
Fig. 4 has shown chlorophyll content and the protein content when heterotrophism under different carbon-nitrogen ratios-light autotrophy is connected the cultivation Chlorella vulgaris.
Fig. 5 is presented at the result who cultivates in the open dull and stereotyped bioreactor series system of 5L bio-reactor/1L.
Specific embodiments
Adopt Endo substratum and the adjusted slightly SK substratum that the Chlorella pyrenoidesa heterotrophism-series connection of light autotrophy is cultivated reported in the document (to consider that mainly primary SK substratum is a light autotrophy substratum, be not easy to the growth of Chlorella vulgaris heterotrophism cultivation stage) heterotrophism-light autotrophy series connection cultivation Chlorella vulgaris, found that algae cell density is lower in the Endo substratum, protein and chlorophyll content are also lower, though and the frustule growth is very fast in the SK substratum, the frond protein when cultivating end is cultivated to compare with heterotrophism with chlorophyll content and be there is no obvious raising.It is the Chlorella vulgaris heterotrophism-light autotrophy series connection cultivation of purpose that this explanation Endo substratum and primary SK substratum all are not suitable for the high-density and high-quality.
The present inventor is based on the SK substratum, by adding Na therein
2HPO
412H
2O, the concentration that changes carbon-nitrogen ratio (C/N) wherein, selects specific carbon source and nitrogenous source and adjust other nutritive ingredient in the substratum, obtain to be applicable to the substratum of Chlorella vulgaris heterotrophism-light autotrophy series connection cultivation, used this substratum can realize the high-density and high-quality cultivation of Chlorella vulgaris.
In this article, the quality parameter of described frond is unit frond protein and chlorophyll content.The biomass index of described frond is a frond dry weight in the unit nutrient solution.Those skilled in the art can adopt routine techniques to described unit frond protein, chlorophyll content with measure.Particularly, the mensuration of protein content adopt Kjeldahl determination (Ning Zhengxiang. food composition analysis handbook [M]. Beijing: China Light Industry Press, 1998,76～78.).Measuring chlorophyll content adopts methanol extraction colorimetry (Ogbonna J.C., Masui.H., Tanaka.H.Sequential heterotrophic:autotrophic cultivation-an efficient method ofproducing Chlorella biomass for health foodand animal feed.J.Appl.Phycol.1997,9,359～366.).Get the nutrient solution V (ml) in a certain moment in the culturing process when frond dry weight is measured, the centrifugal 10min of 7200r/min with deionized water wash 3 times of the frond after centrifugal, is transferred in the weighing bottle (W1 (g)) and dries to constant weight W2 (g) in 80 ℃ of baking ovens.Frond dry weight C
XCan calculate according to following formula: C
X(g/L)=(W2-W1)/V/1000.
The present invention at first provides a kind of culture media composition that is used to cultivate Chlorella vulgaris, and described substratum is basically by KNO
3, glucose and small amounts of 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 used herein " basically by ... form " in the expression composition of the present invention except containing main ingredient KNO
3, outside glucose and small amounts of inorganic salt, trace element and the water, also can comprise some for the fundamental characteristics of composition or new characteristic (can keep the level that Chlorella vulgaris cell density in short culture cycle reaches high-density culture, chlorophyll is cultivated to compare with conventional heterotrophism with protein level and the had a more substantial increase simultaneously) component of influence in fact not.Term used herein " by ... form " expression composition of the present invention 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 not have a significant impact Chlorella vulgaris 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 should add small amounts of inorganic salt in the substratum, for example sal epsom, calcium chloride, ferrous sulfate and phosphoric acid salt etc., and a small amount of 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.
KNO as nitrogenous source
3Scope be 7.0～11.0 grams per liters, can be between 25～35 grams per liters as the glucose of the carbon source and the energy.Particularly, KNO
3Concentration can be the 7-10 grams per liter, 7-9 grams per liter or about 8 grams per liters.The concentration of glucose can be 26-34 grams per liter, 27-33 grams per liter, 28-32 grams per liter, 29-31 grams per liter or about 30 grams per liters.KH
2PO
4Concentration can be 0.65-0.75 grams per liter or about 0.7 grams per liter.Na
2HPO
412H
2The concentration of O can be 1.75-1.95 grams per liter, 1.8-1.9 grams per liter, 1.83-1.85 grams per liter or about 1.84 grams per liters.MgSO
47H
2The concentration of O can be 0.65-0.75 grams per liter or about 0.7 grams per liter.CaCl
2Concentration can be the 0.13-0.18 grams per liter, 0.14-0.15 grams per liter, or about 0.142 grams per liter.FeSO
47H
2The concentration of O can be 0.015-0.025 grams per liter or about 0.02 grams per liter.
For trace element, a preferable embodiment is to add the micro-mother liquor that 0.5～2ml has following composition: H in culture media composition
3BO
311～12 grams per liters, ZnSO
47H
2O 8.5～9.5 grams per liters, MnCl
2H
2O1.4～1.5 grams per liters, (NH
4)
6Mo
7O
244H
2O 0.8～0.9 grams per liter, CuSO
45H
2O 1.5～1.6g, Co (NO
3)
26H
2O 0.45～0.55 grams per liter.This trace element mother liquor can preparation in advance before preparation culture media composition of the present invention.The add-on of trace element mother liquor can be 0.6,0.8,1,1.2,1.4,1.6,1.8 and 2 milliliter.
In an especially good embodiment, culture media composition of the present invention should be grouped into by following one-tenth: KNO
37～11 grams per liters, glucose 25～35 grams per liters, KH
2PO
40.6～0.8 grams per liter, Na
2HPO
412H
2O 1.7～2.0 grams per liters, MgSO
47H
2O 0.6～0.8 grams per liter, CaCl
20.1～0.2 grams per liter, FeSO
47H
2O 0.01～0.03 grams per liter; Trace element 0.5～2ml, wherein trace element consists of H
3BO
311～12 grams per liters, ZnSO
47H
2O8.5～9.5 grams per liters, MnCl
2H
2O 1.4～1.5 grams per liters, (NH
4)
6Mo
7O
244H
2O 0.8～0.9 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; Water 1000ml.
After being mixed with culture media composition, regulate the pH of described substratum as required, for example be adjusted to 6.0～7.0 (being preferably 6.2-6.5), and sterilized 15～20 minutes down at 115-120 ℃.
The present invention relates to a kind of method of cultivating Chlorella vulgaris on the other hand, and this method adopts heterotrophism-light autotrophy series connection to cultivate Chlorella vulgaris, and described method adopts above-mentioned culture media composition.
The heterotrophism of Chlorella vulgaris-light autotrophy series connection is cultivated in shaking bottle or bio-reactor and is carried out.In shaking bottle or bio-reactor, carry out heterotrophism and cultivate, treat in the nutrient solution that the organic carbon source glucose consumption is intact after, carry out the light autotrophy in bottle or the flat bioreactor and cultivate but change it over to illumination shake.When adopting different substratum or culture vessel to cultivate in the culturing process, because Chlorella vulgaris growth velocity property of there are differences in different substratum or culture vessel causes intact different in size the causing of time of organic carbon source glucose consumption in the nutrient solution, the concrete time that therefore changes the cultivation of light autotrophy over to is also inconsistent.In a preferable embodiment, the heterotrophism culture condition is inoculum size 8-12% (being preferably 10%), and temperature is 28-32 ℃ (be preferably 29-31 ℃, be more preferred from 30 ℃); Light autotrophy culture condition is: temperature is 28-32 ℃ (be preferably 29-31 ℃, be more preferred from 30 ℃), outside light intensity 7-10klx (being preferably 8klx).
Therefore, the present invention also relates to the purposes that culture media composition of the present invention is used for cultivating Chlorella vulgaris on the other hand.
Not only can make Chlorella vulgaris cell density in short culture cycle reach the level of high-density culture with method of the present invention, another prior aspect is that its quality that can make Chlorella vulgaris and heterotrophism are cultivated to compare and has a more substantial increase, reach the level when the light autotrophy is cultivated, thereby realized the cultivation of high-density high-quality ordinary chlorella.Therefore, the Chlorella vulgaris culture of the present invention's acquisition can be used in many application such as preparation protective foods, fodder additives.
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.Chlorella vulgaris of the present invention is available from Wuhan hydrobiont institute of the Chinese Academy of Sciences.
Embodiment 1
Shake adding 100ml substratum as follows in the bottle at 250ml, inoculum density is 0.62g/L, heterotrophism is cultivated (liquid amount 100mL, 30 ℃ of temperature, shaking speed 150r/min) 48 hours, the result recorded cell density () and is 13.17g/L to the maximum, carried out the light autotrophy subsequently and cultivated (outside light intensity 8klx), (96h) frond protein () and chlorophyll () content reach the highest when cultivate finishing, and are respectively 49.75% and the 30.17mg/gDcw (see figure 1).
KNO
3: 9.25 glucose: 30
KH
2PO
4：0.7 Na
2HPO
4·12H
2O：1.84
MgSO
4·7H
2O：0.7 CaCl
2：0.142
FeSO
4·7H
2O：0.02
Trace element: 1ml; Trace element formula (g/L): H
3BO
311.42 ZnSO
47H
2O 8.82
MnCl
2·H
2O?1.42 (NH
4)
6Mo
7O
24·4H
2O?0.8707
CuSO
4·5H
2O?1.57?Co(NO
3)
2·6H
2O?0.49
Water: 1000ml
Adopt the method identical with embodiment 1, adopt Endo culture medium culturing Chlorella vulgaris as follows, maximum cell density () only is 4.68g/L, compare no considerable change through frond protein (zero) after the illumination cultivation and chlorophyll () content with the heterotrophism cultivation, only be respectively 23.34% and 12.35mg/gDcw (dry cell weight) (see figure 2).
Glucose: 28g/L urea: 4.8g/L
KH
2PO
4：1.2g/L MgSO
4.7H
2O：1.2g/L
CaCl
20.08g/L trisodium citrate: 0.2g/L
Fe-EDTA solution: 0.64mL A5 solution: 3.2mL
Wherein the Fe-EDTA solution formula is FeSO
47H
2O25g/L and EDTA33.5g/L; A5 solution formula: H
3BO
32.86g/L, MnCl
2.4H
2O 1.81g/L, ZnSO
47H
2O 0.222g/L, CuSO
45H
2O 0.07g/L, Na
2MoO
40.021g/L.
Adopt the method identical with embodiment 1, adopt adjusted slightly SK culture medium culturing Chlorella vulgaris as follows, cultivating 60 hour cell density () and reach only up to 5.61g/L, only is respectively 29.30% and 17.88mg/gDcw through frond protein (zero) after the 36h illumination cultivation and chlorophyll () content.(see figure 1).
KNO
3: 2 glucose: 15
KH
2PO
4：1.25
MgSO
4·7H
2O：1.0 CaCl
2：0.083
FeSO
4·7H
2O：0.049
Trace element: 1ml trace element formula (g/L): H
3BO
311.42 ZnSO
47H
2O 8.82
MnCl
2·H
2O?1.42 (NH
4)
6Mo
7O
24·4H
2O?0.8707
CuSO
4·5H
2O?1.57?Co(NO
3)
2·6H
2O?0.49
Water: 1000ml
Can learn according to The above results, cultivate used substratum, can make cell density, protein and the chlorophyll content of Chlorella vulgaris all reach higher level by only changing heterotrophism-light autotrophy series connection.
Shake adding 100ml substratum as follows in the bottle, wherein KNO at 250ml
3Starting point concentration be respectively 3,5,7,9,11g/L, inoculum density are 0.60g/L, (wherein heterotrophism was cultivated 45 hours, liquid amount 100mL wherein, 30 ℃ of temperature, shaking speed 150r/min to cultivate 82 hours; The light autotrophy was cultivated 37 hours, outside light intensity 8klx).The result records KNO
3Cell density is 12.0g/L left and right sides (see figure 3) to the maximum (to be calculated as 10.20～13.12 with the C/N ratio) during 7～9g/L, and it is the highest that frond protein and chlorophyll content also reach, respectively 50.0% and 32.0mg/gDcw left and right sides (see figure 4).
KNO
3: 3,5,7,9,11 glucose: 30
KH
2PO
4：0.7 Na
2HPO
4·12H
2O：1.84
MgSO
4·7H
2O：0.7 CaCl
2：0.142
FeSO
4·7H
2O：0.02
Trace element: 1ml
Trace element formula (g/L): H
3BO
311.42 ZnSO
47H
2O 8.82
MnCl
2·H
2O?1.42 (NH
4)
6Mo
7O
24·4H
2O?0.8707
CuSO
4·5H
2O?1.57 Co(NO
3)
2·6H
2O?0.49
Water: 1000ml
In the open dull and stereotyped bioreactor series system of 5L bio-reactor/1L, add substratum as follows, liquid amount 75%, inoculum density is 0.60g/L, cultivates 65 hours (wherein cultivated 34 hours in the 5L bio-reactor, cultivated 31 hours in the open dull and stereotyped bioreactor of 1L).The result records when cultivating 34 hours cell density () and reaches and be 15.36g/L to the maximum, and frond protein (zero) and chlorophyll () content reach the highest respectively when cultivating 65 hours, is respectively 54.78% and the 31.23mg/gDcw (see figure 5).
KNO3:9.25 glucose: 30
KH
2PO
4：0.7 Na
2HPO
4·12H
2O：1.84
MgSO
4·7H
2O：0.7CaCl
2：0.142
FeSO
4·7H
2O：0.02
Trace element: 1ml
Trace element formula (g/L): H
3BO
311.42 ZnSO
47H
2O 8.82
MnCl
2·H
2O?1.42 (NH
4)
6Mo
7O
24·4H
2O?0.8707
CuSO
4·5H
2O?1.57?Co(NO
3)
2·6H
2O?0.49
Water: 1000ml
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 culture media composition that is used to cultivate Chlorella vulgaris is characterized in that, described substratum is basically by KNO
3, glucose and small amounts of inorganic salt, trace element and water forms.
2. culture media composition according to claim 1 is characterized in that described trace element is 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.
3. culture media composition according to claim 1 is characterized in that, described substratum is grouped into by following one-tenth:
KNO
37～11 grams per liters, glucose 25～35 grams per liters, KH
2PO
40.6～0.8 grams per liter, Na
2HPO
412H
2O1.7～2.0 grams per liters, MgSO
47H
2O 0.6～0.8 grams per liter, CaCl
20.1～0.2 grams per liter, FeSO
47H
2O 0.01～0.03 grams per liter; Trace element 0.5～2ml, 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
2O 0.8～0.9 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; Water 1000ml.
4. culture media composition according to claim 1 is characterized in that, the pH of described substratum is 6.0～7.0.
5. the described culture media composition of claim 1 is in the purposes that is used for cultivating Chlorella vulgaris.
6. method of cultivating Chlorella vulgaris, this method adopt heterotrophism-light autotrophy series connection to cultivate Chlorella vulgaris, it is characterized in that described method adopts the described culture media composition of claim 1.
7. method as claimed in claim 6 is characterized in that, described substratum is grouped into by following one-tenth: KNO
37～11 grams per liters, glucose 25～35 grams per liters, KH
2PO
40.6～0.8 grams per liter, Na
2HPO
412H
2O1.7～2.0 grams per liters, MgSO
47H
2O 0.6～0.8 grams per liter, CaCl
20.1～0.2 grams per liter, FeSO
47H
2O 0.01～0.03 grams per liter; Trace element 0.5～2ml, 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
2O 0.8～0.9 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; Water 1000ml.
8. Chlorella vulgaris culture that obtains with the described method of claim 6.
9. a composition is characterized in that, it comprises the described Chlorella vulgaris culture of claim 8.
10. the purposes of Chlorella vulgaris culture in preparation protective foods or fodder additives of using the described method of claim 6 to obtain.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101979498A (en) * | 2010-12-01 | 2011-02-23 | 华东理工大学 | High-yield heterotrophic culture method for microalgae |
| CN105433170A (en) * | 2015-11-25 | 2016-03-30 | 广西爱华方舟投资有限责任公司 | Multi-strain microorganism and chlorella vulgaris compound beverage preparation and preparation method thereof |
| CN105524836A (en) * | 2016-02-29 | 2016-04-27 | 通威股份有限公司 | Method for economically and efficiently cultivating chlorella |
| CN106282025A (en) * | 2016-08-31 | 2017-01-04 | 天津海友佳音生物科技股份有限公司 | A kind of method activating concentration algae solution |
| CN107198038A (en) * | 2017-05-24 | 2017-09-26 | 甘肃凯源生物技术开发中心 | The feed of cultivation health pig prepared by chlorella powder and spirulina powder composition and said composition |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1169941C (en) * | 2002-11-05 | 2004-10-06 | 清华大学 | Method for semiaseptic culturing heterotrophic chlorella |
| CN1213141C (en) * | 2003-08-11 | 2005-08-03 | 华东理工大学 | Heterotrophic medium composition for transgenenic chlorella |
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2006
- 2006-02-21 CN CNB2006100240049A patent/CN100386423C/en active Active
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101979498A (en) * | 2010-12-01 | 2011-02-23 | 华东理工大学 | High-yield heterotrophic culture method for microalgae |
| CN101979498B (en) * | 2010-12-01 | 2016-05-04 | 华东理工大学 | A kind of method that micro-algae high-yield heterotrophic is cultivated |
| CN105433170A (en) * | 2015-11-25 | 2016-03-30 | 广西爱华方舟投资有限责任公司 | Multi-strain microorganism and chlorella vulgaris compound beverage preparation and preparation method thereof |
| CN105524836A (en) * | 2016-02-29 | 2016-04-27 | 通威股份有限公司 | Method for economically and efficiently cultivating chlorella |
| CN106282025A (en) * | 2016-08-31 | 2017-01-04 | 天津海友佳音生物科技股份有限公司 | A kind of method activating concentration algae solution |
| CN107198038A (en) * | 2017-05-24 | 2017-09-26 | 甘肃凯源生物技术开发中心 | The feed of cultivation health pig prepared by chlorella powder and spirulina powder composition and said composition |
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| Publication number | Publication date |
|---|---|
| CN100386423C (en) | 2008-05-07 |
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