CN1837351A - Method for culturing chlorella with high-density and high-quality - Google Patents
Method for culturing chlorella with high-density and high-quality Download PDFInfo
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- CN1837351A CN1837351A CNA2006100256189A CN200610025618A CN1837351A CN 1837351 A CN1837351 A CN 1837351A CN A2006100256189 A CNA2006100256189 A CN A2006100256189A CN 200610025618 A CN200610025618 A CN 200610025618A CN 1837351 A CN1837351 A CN 1837351A
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- 241000195649 Chlorella <Chlorellales> Species 0.000 title claims description 78
- 238000000034 method Methods 0.000 title claims description 53
- 238000012258 culturing Methods 0.000 title description 7
- 238000010790 dilution Methods 0.000 claims abstract description 31
- 239000012895 dilution Substances 0.000 claims abstract description 31
- 241000195493 Cryptophyta Species 0.000 claims abstract description 28
- 239000007788 liquid Substances 0.000 claims abstract description 26
- 239000000243 solution Substances 0.000 claims description 37
- 240000009108 Chlorella vulgaris Species 0.000 claims description 34
- 235000007089 Chlorella vulgaris Nutrition 0.000 claims description 34
- 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 description 28
- 239000008103 glucose Substances 0.000 claims description 28
- 239000011573 trace mineral Substances 0.000 claims description 20
- 235000013619 trace mineral Nutrition 0.000 claims description 20
- 239000006052 feed supplement Substances 0.000 claims description 16
- 229910052760 oxygen Inorganic materials 0.000 claims description 16
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 14
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 14
- 239000001301 oxygen Substances 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 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 claims description 12
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 12
- 229910052799 carbon Inorganic materials 0.000 claims description 12
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 10
- 239000004202 carbamide Substances 0.000 claims description 10
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims description 10
- 238000011081 inoculation Methods 0.000 claims description 7
- 229910052757 nitrogen Inorganic materials 0.000 claims description 7
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 6
- 238000005286 illumination Methods 0.000 claims description 6
- 235000015097 nutrients Nutrition 0.000 claims description 6
- 239000001509 sodium citrate Substances 0.000 claims description 6
- 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 claims description 6
- 229940038773 trisodium citrate Drugs 0.000 claims description 6
- 235000013305 food Nutrition 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 238000012366 Fed-batch cultivation Methods 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims description 4
- 238000013019 agitation Methods 0.000 claims description 4
- 239000000654 additive Substances 0.000 claims description 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 3
- 239000003085 diluting agent Substances 0.000 claims description 3
- 230000001681 protective effect Effects 0.000 claims description 3
- 230000035425 carbon utilization Effects 0.000 abstract description 2
- 238000012364 cultivation method Methods 0.000 abstract 1
- 235000019804 chlorophyll Nutrition 0.000 description 26
- 229930002875 chlorophyll Natural products 0.000 description 26
- 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 26
- 235000018102 proteins Nutrition 0.000 description 24
- 108090000623 proteins and genes Proteins 0.000 description 24
- 102000004169 proteins and genes Human genes 0.000 description 24
- 239000000203 mixture Substances 0.000 description 12
- 239000001963 growth medium Substances 0.000 description 7
- 239000008399 tap water Substances 0.000 description 6
- 235000020679 tap water Nutrition 0.000 description 6
- 239000002028 Biomass Substances 0.000 description 4
- 229910017053 inorganic salt Inorganic materials 0.000 description 4
- 239000012452 mother liquor Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 229910003208 (NH4)6Mo7O24·4H2O Inorganic materials 0.000 description 3
- 241000195652 Auxenochlorella pyrenoidosa Species 0.000 description 3
- 235000007091 Chlorella pyrenoidosa Nutrition 0.000 description 3
- 235000013402 health food Nutrition 0.000 description 3
- 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
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 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
- 238000011161 development Methods 0.000 description 2
- 238000003113 dilution method Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 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
- 239000003513 alkali Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 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
- 230000003670 easy-to-clean Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method 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
- 238000011835 investigation Methods 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
- 238000011169 microbiological contamination Methods 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
- 150000003016 phosphoric acids Chemical class 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 230000037039 plant physiology Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000012549 training Methods 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 discloses a small ball algae cultivation method with high density and high quality, which comprises the following phases: high-density heterotrophy cultivation, high-density algae liquid dilution and photo-autotroph cultivation. The invention makes the small ball algae reach high-density level within shorter cultivation period, which improves the small ball algae quality.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of chlorella high-density and high-quality cultured method that is suitable for.
Background technology
People are longer to the research history of chlorella, and wherein Chlorella vulgaris (Chlorella vulgaris) and Chlorella pyrenoidesa (Chlorella pyrenoidosa) are the more algae kinds of research, also are to commercially produce the 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 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.1 997,9,359～366).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 of producing Chlorella biomass for health food andanimal feed.J.Appl.Phycol.1997,9,359～366.).But from large-scale industrialization production angle, there are the insoluble problem in following three aspects in the culture systems of foundation such as Ogbonna and culture process: exist when 1) U type tubular type bioreactor is larger frangible, be not easy to clean, oxygen resolves problems such as slow, be difficult to amplify; 2) heterotrophism culture systems in the heterotrophism-light autotrophy series connection culturing process (can steam sterilizing on the throne) directly is connected with light autotrophy culture systems, and microbiological contamination easily can't realize aseptic technique when large scale culturing; 3) continuous culture process pollution microbes does not very easily use in suitability for industrialized production basically, and adopting more is the batch feeding culture process.Do not see the report of the high-density and high-quality cultivation of other kind in the bead genus in the document.
The present inventor at first adopts the substratum and the cultural method of uses such as Ogbonna that Chlorella vulgaris is carried out exploitative experiment, the result shows that the Chlorella vulgaris algae cell density only is 4.68g/L when cultivating end, frond protein and chlorophyll only are 23.3% and 1.2% respectively, can not realize the high-density high-quality ordinary chlorella cultivation.The substratum of uses such as this explanation employing Ogbonna and the high-density and high-quality of the chlorella that cultural method can not be common to other kind are cultivated.Therefore be necessary chlorella high-density and high-quality cultured method is launched further investigation.
Summary of the invention
Realization chlorella high-density and high-quality is cultivated and can be set about from three aspects: the one, select suitable medium, and the 2nd, select suitable culture systems, the 3rd, set up suitable culture process.The present invention is a research object with the Chlorella vulgaris mainly, has set up a kind of chlorella high-density and high-quality cultured method, and this method is applied to the cultivation of Chlorella pyrenoidesa high-density and high-quality.Another application for a patent for invention of applicant has provided and has been suitable for the culture media composition (Li Yuanguang that high-density high-quality ordinary chlorella is cultivated, Li Xingwu, Shen Guomin, Deng. a kind of culture media composition that is suitable for the high-density high-quality ordinary chlorella cultivation. Chinese invention patent, application number: 200610024004.9, this article is included this paper in as a reference), cultivate the substratum that Chlorella vulgaris adopts among the present invention and be this culture media composition, cultivate substratum that Chlorella pyrenoidesa adopts among the present invention and be the Endo substratum that Ogbonna etc. uses.
The purpose of this invention is to provide a kind of cultural method that is applicable to that the chlorella high-density and high-quality is cultivated.This cultural method can make chlorella 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 cultural method that is used for culturing chlorella with high-density and high-quality, described cultural method comprises the dilution and the light autotrophy cultivation three phases of bio-reactor high Density Heterotrophic, high-density algae liquid.
Particularly, the invention provides a kind of method of cultivating chlorella, it is characterized in that this cultural method may further comprise the steps:
A) carrying out heterotrophism in bio-reactor cultivates: adding pH is 6.0～7.0 substratum in bio-reactor, insert chlorella algae kind by 5～10% of working volume and carry out fed batch cultivation, culture temperature is 28～32 ℃, pH is less than 8.5 in control, the control dissolved oxygen finishes when the highest to cultivate to chlorella cells density more than 15%;
B) algae liquid dilution: it is the 5-25 grams per liter that the algae liquid that step a) is obtained with substratum is diluted to cell density, and described substratum does not contain organic carbon source, and its pH is 6.0～7.0;
C) the light autotrophy is cultivated: the diluent that step b) is obtained changes the cultivation of light autotrophy over to, and culture temperature is 17～42 ℃, and intensity of illumination is 5～60klx, and light autotrophy culture cycle is 30～40 hours.
In a preferable embodiment, described chlorella is selected from Chlorella vulgaris and Chlorella pyrenoidesa.
In a preferable embodiment, the substratum in the described step b) is the substratum that does not contain the step a) of organic carbon source, and wherein nitrogen concentration is 2～10 grams per liters.
In a preferable embodiment, step a) is carried out in the good and totally enclosed mechanical agitation type bio-reactor in the dissolved oxygen supply, and step c) is carried out in being selected from the light autotrophy culture apparatus of closed photo bioreactor, open Da Chi.
In a preferable embodiment, step a) finishes after the glucose completely consumed.
In a preferable embodiment, in step a), when being higher than 8.5, regulates the pH of substratum with acid.
In a preferable embodiment, in step a), after inoculation, carried out feed supplement in 27～33 hours, carried out feed supplement afterwards every 5～8 hours, feed supplement is to add glucose and nitrogenous source solution (nitrogenous source is KNO when cultivating Chlorella vulgaris as previously mentioned
3, be urea when cultivating Chlorella pyrenoidesa), glucose concn reaches the 15-25 grams per liter to the concentration of adding of glucose in the nutrient solution in order to make, and nitrogen concentration reaches the 3-4 grams per liter.
In a preferable embodiment, when chlorella was Chlorella vulgaris, the substratum that the described heterotrophism in the step a) is cultivated was grouped into by following one-tenth basically: 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
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
2O0.45～0.55 grams per liter; Water.
In a preferable embodiment, when chlorella was Chlorella pyrenoidesa, the substratum that the described heterotrophism in the step a) is cultivated was grouped into by following one-tenth basically: glucose 27-30 grams per liter, urea 4.5-5 grams per liter, KH
2PO
41-1.5 grams per liter, MgSO
47H
2O 1-1.5 grams per liter, CaCl
20.07-0.1 grams per liter, trisodium citrate 0.1-0.3 grams per liter, Fe-EDTA solution 0.5-1mL, A5 solution 3-3.5mL, wherein the Fe-EDTA solution formula is FeSO
47H
2O24-26 grams per liter and EDTA 32-35 grams per liter, the A5 solution formula is H
3BO
32.8-2.9 grams per liter, MnCl
24H
2The O1.7-1.9 grams per liter, ZnSO
47H
2O 0.2-0.25 grams per liter, CuSO
45H
2O 0.05-0.1 grams per liter, Na
2MoO
40.02-0.25 grams per liter; Water.
A further aspect of the invention relates to the purposes of chlorella culture in preparation protective foods or fodder additives that obtains with aforesaid method.
Cultivate chlorella with method disclosed by the invention, can make chlorella cell density in short culture cycle reach the level (cell density can reach 45～55 grams per liters when heterotrophism was cultivated end) of high-density culture on the one hand, another prior aspect is that its quality that can make chlorella 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 chlorella high-density and high-quality.
The accompanying drawing summary
Fig. 1 has shown that heterotrophism is cultivated the result that cultivate with substratum dilution and undiluted Chlorella vulgaris with dilution the end back in the open dull and stereotyped bioreactor series system of 5L bio-reactor/1L.
Fig. 2 has shown the result that heterotrophism-dilution under the different extension rates-series connection of light autotrophy is cultivated in the dull and stereotyped bioreactor of 5L bio-reactor/1L, wherein, H represents the heterotrophism cultivation stage, A-10 represents light autotrophy cultivation stage dry cell weight 11.45g/L, A-20 represents light autotrophy cultivation stage dry cell weight 21.89g/L, and A-30 represents light autotrophy cultivation stage dry cell weight 32.56g/L.
Fig. 3 has shown the result of heterotrophism-dilution in the flat bioreactor series system of 50L bio-reactor/30L-light autotrophy series connection batch feeding cultivation Chlorella vulgaris.
Fig. 4 has shown that heterotrophism-dilution in the flat bioreactor series system of 50L bio-reactor/30L-the flat bioreactor of 30L placed outdoor cultivation results when Chlorella vulgaris was cultivated in the series connection of light autotrophy.
Fig. 5 has shown the cultivation results that Chlorella vulgaris light autotrophy cultivation stage carries out out of doors among the flat bioreactor of 30L, 5L plastic tub, the open Da Chi.
Fig. 6 has shown the result of heterotrophism-dilution in the flat bioreactor series system of 50L bio-reactor/30L-light autotrophy series connection batch feeding cultivation Chlorella pyrenoidesa.
Specific embodiments
The invention provides a kind of method of culturing chlorella with high-density and high-quality, this cultural method comprises the dilution and the light autotrophy cultivation three phases of bio-reactor high Density Heterotrophic, high-density algae liquid:
A) carrying out heterotrophism in bio-reactor cultivates: adding pH is 6.0～7.0 substratum in bio-reactor, insert chlorella algae kind by 5～10% of working volume and carry out fed batch cultivation, culture temperature is 28～32 ℃, pH is less than 8.5 in control, the control dissolved oxygen finishes when the highest to cultivate to chlorella cells density more than 15%;
B) algae liquid dilution: it is the 5-25 grams per liter that the algae liquid that step a) is obtained with substratum is diluted to cell density, and described substratum does not contain organic carbon source, and its pH is 6.0～7.0;
C) the light autotrophy is cultivated: the diluent that step b) is obtained changes the cultivation of light autotrophy over to, and culture temperature is 17～42 ℃, and intensity of illumination is 5～60klx, and light autotrophy culture cycle is 30～40 hours.
A) bio-reactor high Density Heterotrophic
Stage/step a) is carried out in bio-reactor.Described bio-reactor can be done suitable selection according to conventional technical ability by those skilled in the art, for example can be the good and totally enclosed mechanical agitation type bio-reactor of dissolved oxygen supply.
Reading hereinafter, especially behind the embodiment, it will be appreciated by those skilled in the art that essence of the present invention and do not lie in substratum itself.In other words, those skilled in the art also can adopt other substratum of being used always in the chlorella cultivation field in the present invention.In preferred version of the present invention, two kinds of substratum have specifically been used, it is a kind of that (this culture media composition applicant has applied for a Chinese invention patent for the HA-SK substratum, application number: 200610024004.9), be used to cultivate Chlorella vulgaris, another kind is the Endo substratum that Ogbonna etc. uses, and is used to cultivate Chlorella pyrenoidesa.Therefore, in preferable embodiment, method of the present invention is applicable to cultivates Chlorella vulgaris and Chlorella pyrenoidesa.
The used HA-SK substratum of the present invention 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 very big materially affect do not arranged to 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, HA-SK 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.
The used Endo substratum of the present invention is grouped into by following one-tenth basically: glucose 27-30 grams per liter, urea 4.5-5 grams per liter, KH
2PO
41-1.5 grams per liter, MgSO
47H
2O 1-1.5 grams per liter, CaCl
20.07-0.1 grams per liter, trisodium citrate 0.1-0.3 grams per liter, Fe-EDTA solution 0.5-1mL, A5 solution 3-3.5mL, wherein the Fe-EDTA solution formula is FeSO
47H
2O 24-26 grams per liter and EDTA 32-35 grams per liter, the A5 solution formula is H
3BO
32.8-2.9 grams per liter, MnCl
24H
2O 1.7-1.9 grams per liter, ZnSO
47H
2O 0.2-0.25 grams per liter, CuSO
45H
2O 0.05-0.1 grams per liter, Na
2MoO
40.02-0.25 grams per liter; Water.
In a preferable embodiment, described Endo substratum is grouped into by following one-tenth: glucose 28 grams per liters, urea 4.8 grams per liters, KH
2PO
41.2 grams per liter, MgSO
47H
2O 1.2 grams per liters, CaCl
20.08 grams per liter, trisodium citrate 0.2 grams per liter, Fe-EDTA solution 0.64mL, A5 solution 3.2mL, wherein the Fe-EDTA solution formula is FeSO
47H
2O 25 grams per liters and EDTA 33.5 grams per liters, the A5 solution formula is H
3BO
32.86 grams per liter, MnCl
24H
2The O1.81 grams per liter, ZnSO
47H
2O 0.222 grams per liter, CuSO
45H
2O 0.07 grams per liter, Na
2MoO
40.021 grams per liter; Water.
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 6.0～7.0, and 115-120 ℃ of following autoclaving 15～20 minutes.In addition, the urea in the Endo substratum is mixed with behind the highly concentrated solution autoclaving separately.
Above-mentioned substratum being added back in the bio-reactor (coefficient is generally 0.6～0.8, preferably 0.7), add water (should be tap water) to working volume, then sterilization.Then, when temperature is reduced to 25～30 ℃, insert chlorella by 5～15% (being more preferred from 5-10%) of working volume and begin the heterotrophism cultivation, culture condition is: temperature: 25～30 ℃, the control dissolved oxygen is more than 15%, and pH is less than 8.5.
The heterotrophism cultivation stage adopts the mode of fed batch cultivation to carry out, can be in (normally 27-38 hour after) feed supplement after inoculation for some time, and afterwards every feed supplement in 5～8 hours, feed supplement is to add carbon source glucose and nitrogenous source (nitrogenous source of cultivating Chlorella vulgaris is KNO
3, the nitrogenous source of cultivating Chlorella pyrenoidesa is a urea) and solution, the concentration of adding of glucose is the 15-25 grams per liter, the concentration of adding of nitrogenous source solution is the 3-4 grams per liter, cultivates and finishes to cultivate when chlorella cells density is the highest.The concentration of feed supplement liquid is suitable higher, and purpose is to make the nutrient solution volume be unlikely to feed supplement and too big.The feed supplement mother liquor will separately be sterilized.
In the feed supplement process if the rapidly phenomenon of drop of dissolved oxygen occurs, can regulate rotating speed, dissolved oxygen is remained on the certain level, understand growth that suppresses frustule even the phenomenon that the frond self-dissolving occurs, thereby influence the further raising of chlorella cells density because dissolved oxygen is not enough.When cultivating, should be noted that pH is unsuitable too high because pH too high frustule be difficult for growth.When pH is higher than 8.5, should use for example 10% sulfuric acid adjusting pH of acid.
Heterotrophism be cultured to chlorella cells density when the highest (can reach 45 grams per liters-55 grams per liter or higher usually) finish to cultivate, better can reaching in chlorella cells density finishes when glucose is by completely consumed in the highest and the nutrient solution to cultivate.
B) dilution of high-density algae liquid
In stage/step b), adopting pH is that 6.0～7.0 the substratum that does not contain organic carbon source is cultivated the high-density algae liquid that obtains to heterotrophism and diluted.Described substratum can just wherein not contain organic carbon source, and its pH is 6.0-7.0 corresponding to the conventional substratum that is used to cultivate chlorella in the prior art.In a more preferred embodiment, the nitrogen concentration of described substratum is 2～10 grams per liters, is preferably 2～8 grams per liters, is more preferred from 3～6 grams per liters.Described nitrogenous source can be identical or different with used nitrogenous source in the step a).
In a preferred concrete scheme, the high-density frustule that heterotrophism cultivate to obtain should be that the initial medium (adopt the HA-SK substratum that does not contain glucose when promptly cultivating Chlorella vulgaris, adopt the Endo substratum that does not contain glucose when cultivating Chlorella pyrenoidesa) of 2～10 grams per liters suitably dilutes with not containing organic carbon source, nitrogen concentration.
The substratum that dilution is adopted need not autoclaving, and preparing adjusting pH to 6.0～7.0, back can use.Cell density after the dilution is preferably 2～25 grams per liters, is more preferred from 5～20 grams per liters.
In a better scheme, the nitrogen concentration in the substratum that dilution is adopted is 3～5 grams per liters, and the cell density after the dilution is 10～16 grams per liters.
C) the light autotrophy is cultivated
Subsequently, after light autotrophy culture apparatus cleaned up with tap water, carry out the light autotrophy after changing over to the enchylema after the dilution of step b) wherein and cultivate.The light autotrophy is cultivated with the heterotrophism cultivation and is carried out in different bio-reactors.Usually, the light autotrophy is cultivated and can be carried out in light autotrophy culture apparatuses such as closed photo bioreactor, flat bioreactor, open Da Chi.Culture temperature is controlled at 17～42 ℃, and intensity of illumination is 5～60klx, and light autotrophy culture cycle is 30～40 hours.
The crucial part of the present invention is, the heterotrophism cultivation stage carries out in different bio-reactors with light autotrophy cultivation stage, heterotrophism is cultivated in the dissolved oxygen supply and is carried out in the good and totally enclosed mechanical agitation type bio-reactor, and the light autotrophy is cultivated and carried out in light autotrophy culture apparatuses such as closed photo bioreactor, open Da Chi.And the high-density algae liquid that the heterotrophism cultivation obtains must adopt the substratum that does not contain organic carbon source suitably to change the cultivation of light autotrophy over to after the dilution, just can realize the high-density and high-quality cultivation.
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.
The mensuration employing Kjeldahl determination of protein content (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 efficientmethod of producing Chlorella biomass for health food and 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.
Not only can make chlorella 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 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 chlorella high-density and high-quality.Therefore, the chlorella 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 that the present invention adopts.Chlorella vulgaris of the present invention and Chlorella pyrenoidesa are available from Wuhan hydrobiont institute of the Chinese Academy of Sciences.
Embodiment 1
Add the substratum that following heterotrophism adopts in the 5L bio-reactor, add tap water and sterilize to 3.4L, insert Chlorella vulgaris by 6% of working volume then when temperature is reduced to 30 ℃, the beginning heterotrophism is cultivated.The heterotrophism culture condition: temperature is 30 ℃, and rotating speed is adjusted to the 400r/min that cultivates when finishing gradually from the 200r/min in when inoculation, and air flow quantity is 1vvm, and pH is less than 8.5, and the control dissolved oxygen is more than 15%.Begin to add feed supplement liquid (20g/L glucose and 3.33g/LKNO during the 30h of inoculation back
3Mixing solutions), added 1 time every 5～8 hours afterwards, add altogether 4 times.Be cultured to 56.34h dry cell weight () and reach 47.91g/L, frond protein (zero) and chlorophyll () content are respectively 33.42% and the 15.65mg/gDcw (see figure 1), and the heterotrophism cultivation stage finishes.
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
Heterotrophism is cultivated the frustule protein and the chlorophyll content that obtain and is in lower level, and the substratum that wherein a part of algae liquid adopts following dilution to adopt is diluted to about 20g/L with frustule, changes the flat bioreactor of 1L over to and carries out the cultivation of light autotrophy.Light autotrophy culture condition: temperature is 30 ℃, the long-pending 0.9L of dress liquid, and air flow quantity is 0.45L/min, and dull and stereotyped reactor adopts two-sided artificial lighting, and the light intensity of each face is 36klx.Continue to be cultured to 83.00h, dry cell weight (■) is constant basically, is 20.05g/L, and frond protein (●) and chlorophyll (▲) content are respectively 52.80% and the 36.45mg/gDcw (see figure 1).
KNO
3：4.95 FeSO
4·7H
2O：0.02
KH
2PO
4：0.7 Na
2HPO
4·12H
2O：1.84
MgSO
4·7H
2O：0.7 CaCl
2：0.142
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
Heterotrophism is cultivated another part of the algae liquid that obtains without dilution process, directly changes the flat bioreactor of 1L over to and carries out the cultivation of light autotrophy.Light autotrophy culture condition: temperature is 30 ℃, the long-pending 0.9L of dress liquid, and air flow quantity is 0.45L/min, and dull and stereotyped reactor adopts two-sided artificial lighting, and the light intensity of each face is 36klx.Continue to be cultured to 80.34h, dry cell weight descends significantly, is 38.45g/L, and frond protein and chlorophyll content are respectively 41.34% and the 29.32mg/gDcw (see figure 1), and it is starkly lower than above-mentioned result through dilution process.
The above results shows that heterotrophism is cultivated the high-density algae liquid that obtains must change the cultivation of light autotrophy over to by suitable dilution, has both kept cell density and has been in higher level, and the frond quality of Huo Deing also is in higher level simultaneously.
In the 5L bio-reactor, add the substratum that heterotrophism adopts among the embodiment 1, adopt the heterotrophism culture condition and the culture process that use among the embodiment 1 to cultivate Chlorella vulgaris.Be cultured to 60.08h dry cell weight () and reach 51.15g/L, frond protein (zero) and chlorophyll () content are respectively 33.65% and the 12.80mg/gDcw (see figure 2), and the heterotrophism cultivation stage finishes.
The substratum that adopts following dilution to adopt, wherein KNO
3Content is respectively 2.60g/L, 4.95g/L and 7.36g/L, and the high-density algae liquid that heterotrophism cultivate is obtained is diluted to respectively to change over to behind 11.45g/L (A-10), 21.89g/L (A-20) and the 32.56g/L (A-30) and carries out the cultivation of light autotrophy in the flat bioreactor of 1L.Identical among light autotrophy culture condition and the embodiment 1.Continue to be cultured to 87.08h, when changing light autotrophy initial cell dry weight over to and being 11.45g/L, dry cell weight slightly increases, and is 12.07g/L, and frond protein and chlorophyll content are respectively 51.49% and 33.51mg/gDcw; When changing light autotrophy initial cell dry weight over to and being 21.89g/L, dry cell weight is constant substantially, is 19.79g/L, and frond protein and chlorophyll content are respectively 52.54% and 36.15mg/gDcw; And when changing light autotrophy initial cell dry weight over to and being 32.56g/L, dry cell weight descends, and is 28.07g/L, and frond protein and chlorophyll content are respectively 51.16% and the 32.42mg/gDcw (see figure 2).
KNO
3：2.60、4.95、7.36 FeSO
4·7H
2O：0.02
KH
2PO
4：0.7 Na
2HPO
4·12H
2O：1.84
MgSO
4·7H
2O：0.7 CaCl
2：0.142
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
The above results shows, heterotrophism is cultivated the high-density algae liquid that obtains and cultivated the frond quality and all can reach higher level through changing the light autotrophy after the suitable dilution over to, but dilution back cell density when higher frustule dead by a relatively large margin.
Add the substratum that heterotrophism adopts among the embodiment 1 in the 50L bio-reactor, add tap water and sterilize to 34L, insert Chlorella vulgaris by 6% of working volume then when temperature is reduced to 30 ℃, the beginning heterotrophism is cultivated.Adopt the heterotrophism culture condition and the culture process that use among the embodiment 1 to cultivate Chlorella vulgaris, wherein the feed supplement number of times increases to 5 times.Be cultured to the 58.20h dry cell weight and reach 54.52g/L, frond protein and chlorophyll content are respectively 32.01% and the 12.70mg/gDcw (see figure 3), and the heterotrophism cultivation stage finishes.
The high-density frustule that 50L bio-reactor heterotrophism cultivate to obtain adopts the substratum (KNO wherein of dilution usefulness among the embodiment 1
3Content is 2.60g/L) be diluted to 14.88g/L, change the flat bioreactor of 30L then over to and carry out the cultivation of light autotrophy.Light autotrophy culture condition: temperature is 30 ℃, the long-pending 24L of dress liquid, and air flow quantity is 12L/min, and dull and stereotyped reactor adopts two-sided artificial lighting, and the light intensity of each face is 45klx.Continue to be cultured to 100h, dry cell weight is constant substantially, is 13.78g/L, and frond protein and chlorophyll content are respectively 55.79% and the 30.33mg/gDcw (see figure 3), has realized that basically the laboratory that high-density high-quality ordinary chlorella is cultivated amplifies.
Add the substratum that heterotrophism adopts among the embodiment 1 in the 50L bio-reactor, add tap water and sterilize to 34L, insert Chlorella vulgaris by 6% of working volume then when temperature is reduced to 30 ℃, the beginning heterotrophism is cultivated.Adopt the heterotrophism culture condition and the culture process that use among the embodiment 1 to cultivate Chlorella vulgaris.Be cultured to the 58.20h dry cell weight and reach 46.40g/L, frond protein and chlorophyll content are respectively 35.02% and the 15.46mg/gDcw (see figure 4), and the heterotrophism cultivation stage finishes.
The high-density frustule that heterotrophism cultivate to obtain adopts the substratum (KNO wherein of dilution usefulness among the embodiment 1
3Content is 2.60g/L) be diluted to 12.66g/L and change in the flat bioreactor of 30L, then the flat bioreactor of 30L is placed the open air to carry out the light autotrophy and cultivate.Light autotrophy culture condition: the long-pending 24L of dress liquid, air flow quantity is 12L/min, natural temperature, temperature is at 17.8～42.4 ℃, natural lighting, light intensity is at 0～65klx.Continue to be cultured to 113h out of doors, dry cell weight is 10.11g/L, and frond protein and chlorophyll content are respectively 57.40% and the 36.92mg/gDcw (see figure 4).
The The above results explanation, the influence that heterotrophism-dilution that the present invention sets up-light autotrophy series connection cultural method is not changed by natural light illumination mode that day alternates with night and natural temperature.
In the 1500L fermentor tank, add the substratum that heterotrophism adopts among the embodiment 1, and adopt culture condition and culture process among the embodiment 1 to cultivate Chlorella vulgaris, cultivate about 60 hour cell density and reach 50.80g/L, frond chlorophyll and protein content are respectively 10.98mg/gDcw and 30.37%.
The substratum of dilution usefulness among the high-density algae liquid embodiment 1 that obtains is diluted to 3.70g/L, respectively the flat bioreactor of 30L, 5L plastic tub (50 * 40 * 20cm), carry out outdoor light autotrophy among the open Da Chi and cultivate.Light autotrophy culture condition: natural temperature, temperature be at 17.8～42.4 ℃, natural lighting, and light intensity is at 0～65klx.The light autotrophy is cultivated 26.75h out of doors, and dry cell weight is 4.08g/L in the flat bioreactor of 30L, and frond protein and chlorophyll content are respectively 51.95% and 20.98mg/gDcw; Dry cell weight is 4.20g/L in the 5L plastic tub, and frond protein and chlorophyll content are respectively 55.02% and 21.85mg/gDcw; Dry cell weight is 3.90g/L among the open Da Chi, and frond protein and chlorophyll content are respectively 52.52% and the 22.83mg/gDcw (see figure 5).
The The above results explanation, heterotrophism-dilution that the present invention sets up-light autotrophy series connection cultural method is not subjected to the restriction of the culture apparatus of light autotrophy cultivation stage employing.
Add the substratum that following heterotrophism adopts in the 50L bio-reactor, add tap water and sterilize to 34L, insert Chlorella pyrenoidesa by 8% of working volume then when temperature is reduced to 30 ℃, the beginning heterotrophism is cultivated.The heterotrophism culture condition: temperature is 30 ℃, and rotating speed is adjusted to the 400r/min that cultivates when finishing gradually from the 150r/min in when inoculation, and air flow quantity is 1vvm, and pH is less than 8.5, and the control dissolved oxygen is more than 15%.Begin to add feed supplement liquid (18g/L glucose solution and 3.2g/L urea soln) during the 37h of inoculation back, added 1 time every 6～10 hours afterwards, add altogether 5 times.Be cultured to 71.48h dry cell weight () and reach 54.88g/L, frond protein (zero) and chlorophyll () content are respectively 46.18% and the 7.17mg/gDcw (see figure 6), and the heterotrophism cultivation stage finishes.
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 EDTA 33.5g/L; A5 solution formula: H
3BO
32.86g/L, MnCl
24H
2O 1.81g/L, ZnSO
47H
2O 0.222g/L, CuSO
45H
2O 0.07g/L, Na
2MoO
40.021g/L.
The high-density frustule that 50L bio-reactor heterotrophism cultivate to obtain adopts to change the dull and stereotyped bioreactor of 30L behind the substratum 15.20g/L of following dilution usefulness over to and carry out the light autotrophy and cultivate.Light autotrophy culture condition: temperature is 30 ℃, the long-pending 24L of dress liquid, and air flow quantity is 12L/min, and dull and stereotyped reactor adopts two-sided artificial lighting, and the light intensity of each face is 45klx.Be respectively 72.31% and the 20.97mg/gDcw (see figure 6) through the frustule protein of follow-up illumination cultivation and chlorophyll content.
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
2O 25g/L and EDTA 33.5g/L; A5 solution formula: H
3BO
32.86g/L, MnCl
24H
2O 1.81g/L, ZnSO
47H
2O 0.222g/L, CuSO
45H
2O 0.07g/L, Na
2MoO
40.021g/L.
The The above results explanation, heterotrophism-dilution that the present invention sets up-light autotrophy series connection cultural method is that a kind of chlorella high-density and high-quality is cultivated blanket method, not only can realize the high-density and high-quality cultivation of Chlorella vulgaris, and can realize the high-density and high-quality cultivation of Chlorella pyrenoidesa.
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 method of cultivating chlorella is characterized in that, this cultural method may further comprise the steps:
A) carrying out heterotrophism in bio-reactor cultivates: adding pH is 6.0～7.0 substratum in bio-reactor, insert chlorella algae kind by 5～10% of working volume and carry out fed batch cultivation, culture temperature is 28～32 ℃, pH is less than 8.5 in control, the control dissolved oxygen finishes when the highest to cultivate to chlorella cells density more than 15%;
B) algae liquid dilution: it is the 5-25 grams per liter that the algae liquid that step a) is obtained with substratum is diluted to cell density, and described substratum does not contain organic carbon source, and its pH is 6.0～7.0;
C) the light autotrophy is cultivated: the diluent that step b) is obtained changes the cultivation of light autotrophy over to, and culture temperature is 17～42 ℃, and intensity of illumination is 5～60klx, and light autotrophy culture cycle is 30～40 hours.
2. method according to claim 1 is characterized in that described chlorella is selected from Chlorella vulgaris and Chlorella pyrenoidesa.
3. method according to claim 1 is characterized in that, the substratum in the described step b) is the substratum that does not contain the step a) of organic carbon source, and wherein nitrogen concentration is 2～10 grams per liters.
4. method according to claim 1, it is characterized in that, step a) is carried out in the good and totally enclosed mechanical agitation type bio-reactor in the dissolved oxygen supply, and step c) is carried out in being selected from the light autotrophy culture apparatus of closed photo bioreactor, open Da Chi.
5. method according to claim 1 is characterized in that step a) finishes after the glucose completely consumed.
6. method according to claim 1 is characterized in that, in step a), regulates with acid when the pH of substratum is higher than 8.5.
7. method according to claim 1, it is characterized in that, in step a), after inoculation, carried out feed supplement in 27～33 hours, carried out feed supplement afterwards every 5～8 hours, feed supplement is to add carbon source glucose and nitrogenous source solution, and glucose concn reaches the 15-25 grams per liter to the concentration of adding of glucose in the nutrient solution in order to make, and nitrogen concentration reaches the 3-4 grams per liter.
8. method according to claim 2 is characterized in that, when chlorella was Chlorella vulgaris, the substratum that the described heterotrophism in the step a) is cultivated was grouped into by following one-tenth basically: 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
2O0.6～0.8 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.
9. method according to claim 2 is characterized in that, when chlorella was Chlorella pyrenoidesa, what the described heterotrophism in the step a) was cultivated was grouped into by following one-tenth basically: glucose 27-30 grams per liter, urea 4.5-5 grams per liter, KH
2PO
41-1.5 grams per liter, MgSO
47H
2O 1-1.5 grams per liter, CaCl
20.07-0.1 grams per liter, trisodium citrate 0.1-0.3 grams per liter, Fe-EDTA solution 0.5-1mL, A5 solution 3-3.5mL; Wherein the Fe-EDTA solution formula is FeSO
47H
2O 24-26 grams per liter and EDTA 32-35 grams per liter; The A5 solution formula is H
3BO
32.8-2.9 grams per liter, MnCl
24H
2O 1.7-1.9 grams per liter, ZnSO
47H
2O 0.2-0.25 grams per liter, CuSO
45H
2O 0.05-0.1 grams per liter, Na
2MoO
40.02-0.25 grams per liter; Water.
10. the purposes of chlorella culture in preparation protective foods or fodder additives of using the described method of claim 1 to obtain.
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