WO2012065545A1 - Microalgae culturing method for oil and lutein rapid accumulation - Google Patents

Microalgae culturing method for oil and lutein rapid accumulation Download PDF

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WO2012065545A1
WO2012065545A1 PCT/CN2011/082261 CN2011082261W WO2012065545A1 WO 2012065545 A1 WO2012065545 A1 WO 2012065545A1 CN 2011082261 W CN2011082261 W CN 2011082261W WO 2012065545 A1 WO2012065545 A1 WO 2012065545A1
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culture
liter
microalgae
light
chlorella
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PCT/CN2011/082261
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French (fr)
Chinese (zh)
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李元广
范建华
黄建科
李淑兰
王伟良
魏鸿刚
沈国敏
李际军
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华东理工大学
上海泽元海洋生物技术有限公司
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Priority claimed from CN2010105458713A external-priority patent/CN102021208A/en
Priority claimed from CN201010567920.3A external-priority patent/CN102094061B/en
Application filed by 华东理工大学, 上海泽元海洋生物技术有限公司 filed Critical 华东理工大学
Publication of WO2012065545A1 publication Critical patent/WO2012065545A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil

Definitions

  • the invention belongs to the field of bioenergy and/or microalgae biotechnology, and relates to a microalgae cultivation method, in particular to a method for rapidly accumulating intracellular bioactive substances of microalgae, especially oil and lutein. Background technique
  • microalgae energy has broad prospects and unique advantages have been recognized at home and abroad. So far, the research and development work of the countries in this field has remained at the initial stage of experimental research and pilot demonstration (Li Yuanguang, Tan Tianwei, Huang Yingming, some scientific problems and analysis of microalgae biodiesel industrialization technology, China Basic Science, 2009, 5, 64-70), but they all encounter the bottleneck caused by the immature technology and high cost. Therefore, the microalgae energy has not been scaled up globally.
  • Microalgae cultivation includes three modes of autotrophic, heterotrophic and mixed nutrition (Yanna Liang, Nicolas Sarkany, Yi Cui. Biomass and lipid productivities of Chlorella vulgaris under autotrophic, heterotrophic and mixotrophic growth conditions. Biotechnology Letters, 2009, 31 : 1043- 1049). Heterotrophic culture mode (Han Xu, Xiaoling Miao, Qingyu Wu.
  • Closed photobioreactors include pipeline, flat, column, etc. (Ogbonna JC, Tanaka H. Industrial- size photobioreactors. Chemical technology, 1997, 27(7): 43 ⁇ 49), although with high cell density, growth
  • the advantages are quick, but due to high cost and immature amplification technology, it has not been applied to large-scale cultivation of microalgae.
  • (1) Mixed nutrition model refers to the culture method in which algae cells use light and organic matter as energy sources, and reuse organic matter and inorganic matter as carbon sources (Lee YK, Ding SY, Hoe CH, Low CS. Mixotrophic growth of Chlorella sorokiniana in outdoor Enclosed photobioreactor. Journal of applied phycology, 1996, 8: 163-169).
  • the photosynthesis and chemical heterotrophy of microalgae can be carried out simultaneously.
  • This model can reduce production costs by combining with wastewater treatment of organic matter rich in carbon and nitrogen, but the biggest problem is that it is easy to breed a large number of bacteria during large-scale cultivation, and it is difficult to achieve high-density cultivation of microalgae.
  • lutein is mainly extracted from plants such as marigold and calendula, but the use of these plants to produce lutein has certain disadvantages.
  • lutein is mainly present in esterified forms in these plants. Therefore, saponification is required for extracting lutein from plants such as marigold and calendula. This step not only reduces efficiency and yield, but also residual saponifier. It is easy to contaminate lutein products, which makes the purification process more difficult.
  • planting marigolds, calendulas and other plants requires a large amount of land. These plants usually have a growth cycle of 3 to 4 months (relative to the rapid growth of microalgae, the cycle is too long).
  • microalgae In comparison, the use of microalgae to produce lutein has certain advantages. First, lutein is mainly present in free form in microalgae, so there is no need for a saponification step in the production process. Secondly, the microalgae has a short growth cycle, a small production area, and the use of microalgae to produce lutein. Not limited by seasonal, climatic and geographical conditions, product quality and yield are relatively stable; finally, microalgae (such as chlorella, etc.) itself is a high-value product containing a large amount of active ingredients such as protein, oil, polysaccharide, etc. These substances are separated and extracted to realize the comprehensive utilization of microalgae cells.
  • active ingredients such as protein, oil, polysaccharide, etc.
  • microalgae energy is also urgently needed to realize the resource utilization of algae.
  • the non-fat components of the microalgae oil are rich in bioactive substances such as pigments, proteins and polysaccharides, and can be developed into high value-added products such as pharmaceuticals, foods and feed additives.
  • high value of algae residue such as extracting lutein
  • the comprehensive utilization of microalgae cells can also improve the comprehensive economic and environmental benefits of the microalgae energy production process and reduce the production cost of microalgae energy.
  • the cultivation modes of lutein produced by microalgae mainly include photoautotrophic and heterotrophic.
  • the disadvantage of microalgae autotrophic culture is that the microalgae cells grow slowly, the cell density and the lutein yield are low.
  • the highest volumetric yield of lutein in photoautotrophic culture of microalgae is 4.8 mg/L/d obtained by Sanchez JF and the cultivation of Scenedesmus almeriensis in a 2L cylindrical photobioreactor (Sanchez JF, Fernandez-Sevilla JM, Acien F G., et al. Biomass and lutein productivity of Scenedesmus almeriensis: influence of irradiance, dilution rate and temperature.
  • microalgae heterotrophic culture is that microalgae can be cultured in a high-density medium in a bioreactor, and the cell growth rate is fast, but there are disadvantages such as intracellular lutein, chlorophyll and the like, and low protein content.
  • Heterotrophic culture can achieve high cell density and high cell growth rate, so that the yield of lutein is greatly improved compared to photoautotrophic culture.
  • microalgae In addition to the heterotrophic culture and photoautotrophic culture mode, microalgae also has an uncommon culture mode, namely, mixed nutrient culture. So far, the highest volume yield (145mg/L/d) of lutein produced by microalgae culture and the highest content of intracellular lutein
  • the present invention designs a "heterotrophic-dilution-light-induced tandem culture” mode, which satisfies "available solar energy, high density, fixed C0 2 , low cost, Efficient" requirements.
  • chlorella Taking chlorella as an example, the process is as follows: First, the chlorella is cultured in a bioreactor to obtain high-density cells. After the organic carbon source in the culture solution is consumed, the algae is diluted with a medium containing no organic carbon source. The liquid, induced by light in a short time (8 ⁇ 24h), causes the oil and/or lutein in the algae cells to accumulate rapidly.
  • the heterotrophic phase in this mode is carried out in a heterotrophic culture bioreactor such as shake flask, mechanical agitation, airlift, bubbling, etc., in order to obtain higher density cells in a short time;
  • the induction phase can be used in any self-cultivation of microalgae In the system, the purpose is to increase the content of intracellular lipids and/or lutein by light induction; the heterotrophic phase and the light-inducing phase are separated and independent, and the algal fluid released in the heterotrophic phase is diluted with light-inducing medium. Then transfer to the light-induced culture stage.
  • this mode has high production efficiency, and there is no problem of contamination in the heterotrophic culture process caused by the combination of different systems (the bacteria in the photoautotrophic culture process does not affect the growth), and the culture system used
  • the advantages of flexible combination mode and low production cost can fully utilize the advantages of rapid accumulation of oil and/or lutein in the light-induced phase of chlorella.
  • the present invention is to solve the bottleneck and source of insufficient raw materials in the process of large-scale preparation of biodiesel. It provides an important technical means for the industrialization of lutein in microalgae and the high value of microalgae energy and algae residue to reduce the production cost. Summary of the invention
  • the present invention provides a microalgae cultivation method comprising the steps of heterotrophic culture of microalgae, and the step of diluting the microalgae culture liquid obtained by heterotrophic culture to perform light-induced culture.
  • the present invention also provides a method for rapidly accumulating intracellular fats and oils of microalgae, the method comprising the steps of heterotrophic culture of microalgae, the step of diluting the microalgae culture liquid obtained by heterotrophic culture, and performing light-induced culture, and optionally The steps of algae cell harvesting, oil separation and extraction.
  • the invention also provides a method for producing microalgae oil and fat, which comprises the steps of heterotrophic microalgae, the step of diluting the heterotrophic culture solution of the microalgae for light-induced culture, and the extraction of algae cells and the separation of oil and fat. step.
  • the method can realize the rapid accumulation of intracellular lipids, has a small footprint, high area yield, and low harvesting cost due to high cell density relative to microalgae self-cultivation (cell density at light induction) High, generally 2 ⁇ 5 g/L), which greatly improves production efficiency and reduces production costs.
  • the present invention also provides a method for rapidly accumulating intracellular lutein of microalgae, the method comprising the step of heterotrophic culture of microalgae, the step of diluting the obtained microalgae heterotrophic culture solution, and performing light-induced culture, and The selected algae cell harvesting, lutein separation and extraction steps.
  • the present invention also provides a method for producing lutein derived from microalgae, which comprises the steps of heterotrophic microalgae, the step of diluting the heterotrophic culture solution of the microalgae for light-induced culture, and the harvesting of algae cells, The step of separating and extracting lutein.
  • the method of the invention can achieve rapid accumulation of intracellular lutein, greatly improve production efficiency, reduce production cost, and provide high quality lutein.
  • the microalgae is selected from the group consisting of: Chlorella pyrenoidosa in the genus Chlorella, Chlorella vulgaris, Chlorella vulgaris Ellipsoidea), Chlorella emersonii, Chlorella sorokiniana, Chlorella saccharophila, Chlorella regular is, Chlorella minutissima, Chlorella protothecoides, Chlorella zofingiensis, and Brachiomonas submarina, Chlamydobonas reinhardtii, Chlamydomonas Acidophila, Haematococcus pluvialis, Haematococcus lacustris, Scenedesmus obliquus, Spongiococcum exetriccium Tetraselmis suecica, Tetraselmis chuii Tetraselmis tetrathele, Tetraselmis verrucosa, Micractinium pusillum; Cylindrot
  • the microalgae is selected from the group consisting of algae of the genus Chlorella.
  • the microalgae is selected from the group consisting of Ch re py idoscd, Chlorella vulgaris, of the genus Chlorella.
  • Chlorella ellipsoidea, Chlorella enter onii, Chlorella sorokiniana, Chlorella saccharophila, Chlorella regular is, Chlorella minutissima, Chlorella protothecoides and Chlorella zofingiemis
  • the microalgae is selected from the group consisting of Chrena pyrenoidoscO, Chlorella vulgaris, and Chlorella ellipsoidea.
  • the microalgae heterotrophic step comprises: adding a medium having a pH of 4.0 10.0 to the bioreactor, and accessing the microalgae algae according to a working volume of 0.1 to 30% for batch culture, Feeding batch culture, continuous culture or semi-continuous culture, the culture temperature is 10 ⁇ 50 °C, the control pH is less than 10.0, and the dissolved oxygen is controlled above 1%.
  • the step of heterogeneous culture of the microalgae may include: adding a culture having a pH of 4.0 10.0 (for example, 4.0 to 9.0) in the bioreactor.
  • a culture having a pH of 4.0 10.0 for example, 4.0 to 9.0
  • the culture temperature is 10 ⁇ 50 °C (for example, 10 ⁇ 40 °C)
  • control pH is less than 10.0 (for example, less than 9.0)
  • control dissolved oxygen above 1% for example, a culture having a pH of 4.0 10.0 (for example, 4.0 to 9.0) in the bioreactor.
  • the algal fluid dilution comprises diluting the heterotrophically obtained algal fluid to a cell density of 0.150 g/l with a medium having no organic carbon source and having a pH of 4.0 10.0.
  • the diluting the algae liquid may comprise diluting the heterotrophic obtained algae liquid to a cell density of 0.120 g/L with the medium,
  • the medium does not contain an organic carbon source and has a pH of 4.0 to 9.0.
  • the light-induced culture comprises transferring the diluted algal liquid into a light-inducing device for light-induced, continuous illumination or intermittent illumination, the culture temperature is 5 to 50 ° C, and the illumination intensity is 0.1 to 150 kk.
  • the light-induced culture period was 1 150 hours.
  • the heterotrophic medium consists of a nitrogen source, an organic carbon source, and a small amount of inorganic salts, trace elements, and water;
  • the photoinduction medium consists of a nitrogen source, an inorganic salt, and water.
  • the heterotrophic step is carried out in a shake flask, mechanically agitated, airlifted or bubbling heterotrophic culture bioreactor
  • the light inducing culture step is in a shake flask or selected from Open runway pool or round pool, Closed flat photobioreactor or ducted photobioreactor or column photobioreactor or film pouch and sling photobioreactor, any device that can be used for microalgae photoautotrophic culture, illumination
  • the condition is natural light or artificial light.
  • the medium used for heterotrophy consists essentially of the following components: KN0 3 5-15 g/l , glucose 10 ⁇ 60g/L, KH 2 P0 4 0.3-0.9g/L, Na 2 HP0 4 '12H 2 0 1.0-10.0 g/L, MgS0 4 '7H 2 0 0.2-1.0 g/L, CaCl 2 0.05 ⁇ 0.3g/L, FeS(V7H 2 0 0.01 0.05g/L; trace element 0.5 ⁇ 4ml, wherein the composition of trace elements is H 3 B0 3 5-15 g / liter, ZnS0 4 '7H 2 0 5.0-10.0 ⁇ / liter, MnCl 2 'H 2 0 1.0-2.0 g / liter, ( ⁇ 4 ) 6 ⁇ 7 0 24 ⁇ 4 ⁇ 2 0 0.5-1.5 g / liter, Cu
  • the medium used for heterotrophy is basically composed of the following components Composition: glucose 10 ⁇ 60 g/l, urea 2 ⁇ 8 g/l, KH 2 P0 4 1-2 g/l, Na 2 HP0 4 -12H 2 0 1.0-10.0 g/l, MgS0 4 '7H 2 0 1-2 g / liter, CaCl 2 0.05-0.1 g / liter, trisodium citrate 0.1 ⁇ 2.0 g / liter, Fe-EDTA solution 0.5 ⁇ 1 mL, A5 solution l ⁇ 5mL; Fe-EDTA solution formula is FeS0 4 -7H 2 0 20-30 g / liter and EDTA 20-40 g / liter; A5 solution formula is H 3 B0 3 2.5-4.0 ⁇ / liter, MnCl 2 -4H 2 0 1.0-2.0 g / liter, ZnS0 4 '7H 2 0
  • the medium used for heterotrophy consists essentially of the following components: KN0 3 5-15 g/ L, glucose 10 ⁇ 60 g/L, KH 2 P0 4 0.3-0.9 g/L, Na 2 HP0 4 '12H 2 0 1.0-10.0 g/L, MgS0 4 '7H 2 0 0.2-1.0 g/L, CaCl 2 0.05-0.3 g / liter, FeS (V7H 2 0 0.01-0.05 g / liter, trace elements 0.5 ⁇ 4ml and water, wherein the composition of trace elements is H 3 B0 3 5 ⁇ 15 g / liter, ZnS0 4 '7H 2 0 5.0 ⁇ 10.0 g/L, MnCl 2 'H 2 0 1.0 ⁇ 2.0 g/L, ( ⁇ 4) 6 ⁇ 7 0 24 ⁇ 4 ⁇ 2 0 0.5-1.5
  • the medium used for heterotrophy consists essentially of the following components: glucose 10 to 60 g / ⁇ , Urea 2 ⁇ 8g/L, KH 2 P0 4 1 ⁇ 2g/L, Na 2 HP0 4 '12H 2 0 1.0 ⁇ 10.0g/L, MgS0 4 '7H 2 0 1 ⁇ 2g/L, CaCl 2 0.05-0.1 1 liter, trisodium citrate 0.1 ⁇ 2.0 g/L, Fe-EDTA solution 0.5 ⁇ 1 mL, A5 solution 1-5 mL and water; wherein Fe-EDTA solution is FeS (V7H 2 0 20-30 ⁇ / liter and EDTA 20-40 g / liter; A5 solution formula is H 3 B0 3 2.5-4.0 g / liter, MnCl 2 '4H 2 0 1.0-2.0 g / liter, ZnS0 4 '
  • lutein is extracted by supercritical co 2 extraction, organic solvent extraction or ultrasonic assisted solvent extraction.
  • the method of the present invention further comprises: mixing the algal body after extracting lutein with other pigments, spray-drying to prepare algal flour, or separating and extracting the biologically active substance in the algal body.
  • the other pigment comprises chlorophyll.
  • the Biologically active substances include proteins, chlorophyll, polysaccharides, fatty acids, chlorella growth factors, and the like.
  • the oil separation and extraction method employs an organic solvent extraction method.
  • microalgae culture method further comprises the step of extracting microalgae oil.
  • the present application also provides a method for producing a fat or oil, the method comprising the steps of heterotrophic cultivation of microalgae, the step of diluting the heterotrophic cultured microalgae solution for light-induced culture, and the step of collecting algae cells and separating and extracting oils and fats. .
  • microalgae, the culture medium, and the culture conditions and the like used in the method for producing a fat or oil of the present application are as described in the above and the following detailed description of the specific embodiments.
  • the bacterium is cultured by the heterotrophic-dilution-light-inducing tandem technique disclosed in the present invention.
  • high-density heterotrophic culture of chlorella is carried out in a closed bioreactor. After the organic carbon source in the culture solution is consumed, the organic medium is not used. The carbon source medium dilutes the algae solution, and after a short period of time (8 ⁇ 24h), the oil content in the algae cells can be accumulated rapidly and the oil content is short-term (for example, 8 ⁇ 24 hours) than the heterotrophic
  • the algae cells in the stage are about doubled (the intracellular fat content in the heterotrophic stage is generally not more than 10%, and can reach 20 ⁇ 30% after light induction).
  • Figure 1 shows the results of heterotrophic-dilution-light-induced tandem culture of Chlorella pyrenoidosa for rapid accumulation of oil in a 50L bioreactor/3L flat photobioreactor tandem system (simultaneous determination of other major biochemical components in the cell) .
  • Figure 2 shows the variation of the natural light intensity and natural temperature of the 5T fermenter in series with the outdoor 30L plate and the 60L large basin for continuous cultivation of Chlorella vulgaris in the light-induced phase.
  • Figure 3 shows the process curve of the rapid accumulation of oil in the 30T fermenter in series to the outdoor 30L plate culture of Chlorella vulgaris (the other major biochemical components in the cell were also measured).
  • Figure 4 shows the process curve of the 5T fermenter connected in series to the outdoor 60L large pot cultured Chlorella vulgaris to accumulate oil quickly (the other major biochemical components in the cell were also measured).
  • Figure 5 shows the variation of the outdoor natural light intensity and natural temperature during the light induction phase of the conventional chlorella in the 5T fermentor connected to the outdoor 20m 2 runway pool and the 3.14m 2 round pool.
  • Figure 6 shows the process curve of the rapid accumulation of oil in the culturing of the chlorella by the 5T fermentor in series to the outdoor 20m 2 runway pool (the other major biochemical components in the cell were also determined).
  • Figure 7 shows the process curve of the rapid accumulation of oil in the 5T fermenter in series to the outdoor 3.14m 2 round cell culture of common chlorella (simultaneous determination of other major biochemical components in the cell).
  • Figure 8 shows the variation of the main biochemical components in the cells cultured in a 500 mL shake flask / 3 L cylindrical photoreactor.
  • Figures 9 and 10 show the process of producing lutein by heterotrophic-dilution-light-induced tandem culture of Chlorella pyrenoidosa in a 5L bioreactor/3L flat photobioreactor tandem system.
  • Figure 9 shows the heterotrophic culture process of Chlorella pyrenoids in a 5L bioreactor;
  • Figure 10 shows light-induced culture of Chlorella pyrenoids in a 3L flat photobioreactor Raise the process.
  • Figure 11 and Figure 12 show the process of producing lutein by heterotrophic-dilution-light-induced tandem culture of Chlorella pyrenoids in a 50L bioreactor/10L cylindrical photobioreactor tandem system.
  • Figure 11 shows the heterotrophic culture process of Chlorella pyrenoids in a 50L bioreactor
  • Figure 12 shows the photoinduced culture process of Chlorella pyrenoidosa in a 10L cylindrical photobioreactor.
  • Figures 13 and 15 show the process of producing lutein by heterotrophic-dilution-light-induced tandem culture of Chlorella vulgaris in a 50L bioreactor/30L flat photobioreactor cascade system.
  • Fig. 13 shows the heterotrophic culture process of the common chlorella in the 50L bioreactor
  • Fig. 15 shows the photoinduced culture of the ordinary chlorella in the outdoor 30L flat photobioreactor.
  • Figure 14 and Figure 16 show the process of producing lutein by heterotrophic-dilution-light-induced tandem culture of N. glabrata in a 5L bioreactor/3L flat biophotobioreactor cascade system.
  • Figure 14 shows the heterotrophic culture process of Chlorella ellipses in a 5L bioreactor;
  • Figure 16 shows the photoinduced process of Chlorella ellipses in a 3L flat photobioreactor.
  • Microalgae suitable for use in the present application include, but are not limited to, Chlorella pyrenoidosa in the genus Chlorella, Chlorella vulgaris, # Chlorella ellipsoidea , Chlorella emersonii, Chlorella sorokiniana, Chlorella saccharophila, Chlorella regularis, Chlorella minutissima, Chlorella protothecoides, Chlorella zofingiensis, and Brachiomonas submarina, Chlamydobonas reinhardtii, Chlamydomonas acidophila, Haematococcus pluvialis, Haematococcus lacustris, Scenedesmus obliquus, Spongiococcum exetriccium, Tetraselmis Suecica, Tetraselmis chuii, Tetraselmis tetrathele, Tetraselmis verrucosa, Micractinium pusillum-
  • the present invention utilizes algae of the genus Chlorella Chlorella to produce oils and fats.
  • the present invention employs Chlorella pyrenoidosa, Chlorella vulgaris or Chlorella ellipses to produce oils and fats.
  • the invention employs Chlorella pyrenoidosa, Chlorella vulgaris, and Chlorella ellipsoid to produce lutein.
  • the purpose of this step is to rapidly obtain a large number of algae cells for rapid synthesis and accumulation of various active ingredients in the light-inducing stage, including oils, pigments, proteins, polysaccharides and the like.
  • the microalgae heterotrophic culture can be carried out using various media well known in the art.
  • the heterotrophic medium contains a nitrogen source, an organic carbon source, a small amount of inorganic salts, trace elements, and water.
  • Nitrogen sources, organic carbon sources, inorganic salts, trace elements, and the like suitable for microalgae culture are well known in the art.
  • the nitrogen source urea or various nitrates such as KN0 3 may be used, and as the organic carbon source, for example, glucose or the like may be used.
  • Such media include HA-SK medium (Chinese patent ZL 200610024004.9), Endo medium (Ogbonna JC, Masui. H., Tanaka. H. Sequential heterotrophic: autotrophic cultivation an effective method of producing Chlorella biomass for health food and Animal feed. J. Appl. Phycol. 1997, 9, 359-366) and so on.
  • the HA-SK medium used in the present invention consists essentially of KNO 3 , glucose, and a small amount of inorganic salts, trace elements, and water.
  • the trace element is preferably selected from the group consisting of H 3 B0 3 , ZnS0 4 -7H 2 0, MnCl 2 -H 2 0, ( ⁇ 4 ) 6 ⁇ 7 0 24 ⁇ 4 ⁇ 2 0, CuS0 4 - One or more or all of 5H 2 0, Co(N0 3 ) 2 ⁇ 6 ⁇ 2 0 .
  • composition of the present invention may contain some essential properties or new to the composition in addition to the main component KNO 3 glucose and a small amount of inorganic salts, trace elements and water.
  • the characteristics ie, maintaining the microalgae at a higher cell density in a shorter culture period, while the active substance content is significantly increased compared to conventional heterotrophic culture) have no substantially affected components.
  • Consisting of as used herein means that the composition of the present invention consists of the specific components indicated, without other components, but may carry impurities in a usual range.
  • the components of the medium can be varied within a certain range without greatly affecting the density and quality of the microalgae cells. Therefore, the amounts of these components should not be strictly limited by the examples.
  • a small amount of inorganic salts such as magnesium sulfate, calcium chloride, ferrous sulfate, and phosphate may be added to the medium, and a small amount of trace elements such as ⁇ , ⁇ , ⁇ , I, M, Cu, Co, etc.
  • a preferred trace element component is preferably selected from the group consisting of H 3 B0 3 , ZnS0 4 -7H 2 0, MnCl 2 -H 2 0, ( ⁇ 4 ) 6 ⁇ 7 0 24 ⁇ 4 ⁇ 2 0, CuS0 4 One or more of -5H 2 0, Co(N0 3 ) 2 -6H 2 0 .
  • the amount of inorganic salts and trace elements can be determined based on conventional knowledge.
  • the HA-SK medium used in the present invention consists essentially of the following components: KN0 3 5 ⁇ 15 g/L, glucose 10-60 g/L, KH 2 P0 4 0.3-0.9 g/L, Na 2 HP0 4 ' 12H 2 0 1.0-10.0 g / liter, MgS0 4 '7H 2 0 0.2-1.0 g / liter, CaCl 2 0.05 0.3 g / liter, FeS0 4 '7H 2 0 0.01 ⁇ 0.05 g / liter; trace elements 0.5 ⁇ 4ml, The composition of trace elements is H 3 B0 3 5 ⁇ 15 g / liter, ZnS0 4 -7H 2 0 5.0 ⁇ 10.0 g / liter, MnCl 2 -H 2 0 1.0 ⁇ 2.0 g / liter, ( ⁇ 4 ) 6 ⁇ 7 0 24 ⁇ 4 ⁇ 2 0 0.5-1.5 g/l, CuS0 4 '5H 2
  • the HA-SK medium composition of the present invention is preferably composed of the following components: KN0 3 7 g/L, glucose 40 g/L, KH 2 P0 4 0.6 g/L, Na 2 HP (V 12H 2 0 2.0 g / liter, MgS0 4 '7H 2 0 0.8 g / liter, CaCl 2 0.2 g / liter, FeS (V7H 2 O 0.03 g / liter; trace element 1.5mL, wherein the trace element composition is H 3 B0 3 11-12 g/l, ZnS0 4 -7H 2 0 8.5-9.5 g/l, MnCl 2 0 1.4-1.5 g/l, ( ⁇ 4 ) 6 ⁇ 7 0 24 ⁇ 4 ⁇ 2 0 0.8-0.9 g/l, CuS0 4 '5H 2 0 1.5-1.6 g/l, Co(N0 3 ) 2 '6H 2 0 0.45-0.55
  • the Endo medium used in the present invention consists essentially of the following components: glucose 10 60 g/l, urea 2-8 g/l, KH 2 P0 4 1-2 g/l, Na 2 HP0 4 ' 12H 2 0 1.0- 10.0 g / liter, MgS0 4 '7H 2 0 1-2 g / liter, CaCl 2 0.05 ⁇ 0.1 g / liter, trisodium citrate 0.1 ⁇ 2.0 g / liter, Fe-EDTA solution 0.5 ⁇ 1 mL, A5 solution ⁇ 5mL; wherein the Fe-EDTA solution is FeS (V7H 2 0 20-30 g / liter and EDTA 20-40 g / liter; A5 solution formula is H 3 B0 3 2.5 ⁇ 4.0 g / liter, MnCl 2 '4H 2 0 1.0 ⁇ 2.0 g / liter, ZnS0 4 '7H 2 0 0. ⁇ 0.6
  • the Endo medium consists of the following components: glucose 40 g/l, urea 6.0 g/l, KH 2 P0 4 1.5 g/l, Na 2 HP0 4 ' 12H 2 0 5.0 g /L, MgS0 4 '7H 2 0 1.8 g / liter, CaCl 2 0.05 g / liter, trisodium citrate 0.4 g / liter, Fe-EDTA solution 0.8 mL, A5 solution 2.0 mL, of which Fe-EDTA solution is FeS (V7H 2 0 25 g / liter and EDTA 33.5 g / liter, A5 solution formulation is H 3 B0 3 2.86 g / liter, MnCl 2 -4H 2 0 1.81 g / liter, ZnS0 4 '7H 2 0 0.222 g / liter, CuS0 4 '5H 2 0 0.07 g/l, Na 2 Mo0 4
  • the pH of the medium can be adjusted to 4.0-9.0 by a conventional means such as an acid or a base, and autoclaved at 115 to 120 ° C for 15 to 20 minutes.
  • Heterotrophic culture can be carried out in four ways, such as batch culture, fed-batch culture, semi-continuous culture (with release) or continuous culture.
  • the corresponding prepared medium is added to the bioreactor, and water is added to the working volume, usually with a charging coefficient of 0.6 0.8, and then steam sterilized (121 ° C, maintained for about 20 minutes).
  • the temperature drops to 30 ⁇ 35 °C, the microalgae are connected to the heterotrophic culture according to 1 ⁇ 15% of the working volume.
  • the glucose in the medium is consumed (usually 27 ⁇ 45 hours), it is necessary to supplement the feed, supplementing the carbon source (such as glucose) and nitrogen source (for example, culturing ordinary chlorella
  • the nitrogen source is KN0 3
  • the nitrogen source of the culture protein chlorella is urea
  • the nutrient salt such as inorganic salt
  • the supplemental nutrient salt is the corresponding medium which is concentrated to promote the growth of the microalgae. It can be fed every 5 ⁇ 8 hours.
  • the additional concentration of glucose can be 15 25 g / liter
  • the additional concentration of nitrogen source solution can be 2 ⁇ 5 g / liter.
  • suitable culture conditions must be controlled to allow the microalgae to grow normally.
  • the control temperature is 20 ⁇ 35 °C, for example, 28 ⁇ 30 °C
  • the dissolved oxygen is not less than 5% of the air saturation concentration
  • the pH is not higher than 9.0.
  • the dissolved oxygen is not less than 10% of the air saturation concentration and the pH is not higher than 8.5.
  • the dissolved oxygen is not less than 15% of the air saturation concentration, and the pH is not higher than 8.
  • the pH should not be too high or too low. Generally, as the culture progresses, the pH will rise slowly (this phenomenon is particularly obvious for ordinary chlorella). If the pH is too high, the growth of algae cells will be adversely affected.
  • the acid for example, 10% sulfuric acid
  • the acid is adjusted so that the pH is not higher than 9.0, and the preferred pH is 6.5 to 7.5.
  • the bioreactor is organic at the end of the heterotrophic culture stage.
  • the carbon source needs to be completely consumed.
  • the stripping operation is performed when the organic carbon source is completely consumed in the bioreactor.
  • the organic carbon source such as glucose in the algae solution should be zero or close to zero.
  • Heterotrophic can be carried out in a heterotrophic culture bioreactor such as shake flask, mechanical agitation, airlift, or bubbling.
  • a heterotrophic culture bioreactor such as shake flask, mechanical agitation, airlift, or bubbling.
  • the purpose of this step is to enable light-inducing cultured chlorella to efficiently absorb light energy, improve light energy utilization efficiency, and reduce algal cell mortality. Because the light intensity is "time, empty, nonlinear" decay in the algae liquid, at high cell density, most of the algae cells in the reactor are in the dark area, and almost no light is received, so that the algae cells are easy to die and will Affects the efficiency of light induction.
  • the high-density algae liquid obtained by heterotrophic culture should be diluted.
  • the high-density algae solution is diluted with water and a medium containing no organic carbon source to maintain the cell density at 0.1 10 g / liter and adjust the pH to 5.0 8.0.
  • the algal fluid is diluted to maintain a cell density of 1-8 g/l.
  • the cell density is maintained at 1.0 5.0 g/l.
  • the cell density is maintained at 2 to 5 grams per liter or 2 to 4 grams per liter.
  • the pH of the diluted algae solution can be adjusted to 5.5 ⁇ 7.5, such as 6.0 7.5.
  • the light-inducing medium can be used for dilution.
  • the light-inducing medium usually contains a nitrogen source, an inorganic salt, and water, and does not contain an organic carbon source relative to the heterotrophic medium.
  • the medium has a nitrogen source concentration of 0.110 g/l, such as 2-10 g/l, 2-8 g/l, preferably 0.36 g/l, more preferably 0.5. 5 g / liter.
  • the nitrogen source may be the same as or different from the nitrogen source used in the heterotrophic cultivation step.
  • the high-density algal cells obtained by heterotrophic culture are preferably used in an initial medium having a nitrogen source concentration of 0.1 10 g/l (for example, 2 to 10 g/L) without an organic carbon source (for example,
  • the normal chlorella was cultured using HA-SK medium without glucose, and the nucleus chlorella was cultured with Endo medium without glucose for proper dilution.
  • the medium used for dilution does not need to be autoclaved. After preparation, adjust the pH to 5.0 8.0 and use.
  • the dilution medium (light-inducing medium) consists of the following components:
  • the dilution medium (light-induced medium) consists of the following components: urea 0.1 5.0 g / liter, MgS0 4 -7H 2 0, 0.5 ⁇ 5.0 g / liter, CaCl 2 0.01 ⁇ 0.06 g / liter , FeS0 4 -7H 2 0 0.01 ⁇ 0.06, EDTA
  • the dilution medium (light-inducing medium) consists of the following components:
  • the purpose of this step is to allow the microalgae to receive light, and to accelerate the mass synthesis of the algae cells to accumulate various active ingredients by light induction.
  • the resulting dilution is transferred to a light-inducing device for light-induced culture.
  • the added light-inducing medium is as described above, the temperature is controlled at 5 to 50 ° C, the light intensity is 0.11 to 150 kk, continuous illumination or intermittent illumination, the light-induced culture period is 1 to 150 hours, and the ventilation is 0.1 2.0 wm.
  • the photobioreactor described therein includes all closed photobioreactors (bottle shakers, tubing, flat plates, columns, film stand pouches and sling bags, etc.) and all open photobioreactors (runway cells) , round pools and bubbling basins, etc.).
  • the culture temperature can be controlled within the range of 15 to 35 ° C, for example, 18 to 35 ° C, 20 to 35 ° C, 20 to 30 ° C, and the like.
  • the light intensity is l ⁇ 70kk, for example, 1 ⁇ 60, 1 ⁇ 50, 1 ⁇ 40, 1 ⁇ 30, 1 ⁇ 20, l ⁇ 10kk, etc., depending on the specific production situation.
  • the ventilation can be controlled to be 0.15 to 2.0 wm, for example, 0.2-1.8 0.5-1.5 0.8-1.5 1.0 1.5 wm or the like.
  • the culture temperature is controlled at 10 to 50 ° C
  • the light intensity is 1 to 10 kk
  • the gas flow rate is 0.05 to 2.0 wm.
  • the light-induced culture period is 8 100 hours.
  • the light-induced culture period may be 8 to 90 hours, 8 to 80 hours, 8 to 60 hours, 8 to 48 hours, 8 ⁇ 24 hours vary; or, the light-induced culture period can be 12 72 hours, 12 60 hours, 12 48 hours, 12 36 hours, 12 24 hours, or 24 60 hours, 24 to 48 hours.
  • the "light-induced culture period” includes the entire light-induced culture process, for example, the outdoor culture-time light-induced culture period includes the time when there is no light at night.
  • lighting time refers to the time during which light-induced culture of microalgae is carried out using the light intensity described herein, i.e., the time does not include the time when no light is illuminated at night.
  • the photoinduction culture step has an illumination time of 8-48 hours, such as 8 to 36 hours, 8-24 hours, 8-18 hours, 8-12 hours, 12-36 hours, 12-24 hours. Etc., and any length of time within the above range.
  • the light-induced culture step of the present application also includes a light-induced culture step in the range of 8 to 48 hours of illumination.
  • Light-induced culture can be carried out by artificial light, or light-induced culture can be carried out outdoors by natural light.
  • the chlorella is generally about 20 ⁇ 30%)
  • the light-induced culture can be ended, and the algal cells are harvested for subsequent separation and extraction of the oil.
  • the concentration of lutein in the culture solution reaches the highest, the light-induced culture is terminated, and the algal cells are harvested for separation and extraction of lutein or directly harvested algae cells for algal powder preparation. 4. Algae cell harvesting, oil extraction and comprehensive utilization of algae
  • algae cell wall breaking methods include, but are not limited to, algae autolysis, high pressure homogenization, enzymatic hydrolysis, aqueous phase pyrolysis, etc. Broken wall method.
  • the method for extracting intracellular fats includes, but is not limited to, organic solvent extraction, that is, drying the algae at 80-105 ° C to a constant weight, grinding the algal powder, and extracting the oil from the dry algae powder using a chloroform methanol standard extraction solvent.
  • the extraction solvent is repeatedly extracted until the color of the algal powder turns white, and the solvent is removed by rotary evaporation.
  • the extraction of lutein from chlorella can be carried out by a conventional organic solvent extraction method.
  • an organic solvent is added to the algal mud for extraction, and then the supernatant and the algal body precipitate are obtained by stirring and centrifuging, and the supernatant is concentrated under reduced pressure, stirred with water, and filtered to obtain lutein crystals.
  • the chlorella is isolated and extracted from the chlorella using a supercritical CO 2 extraction technique.
  • the obtained chlorella solution is concentrated and directly spray dried to obtain chlorella powder.
  • the chlorella powder thus obtained can be used for development into animal feed, aquaculture bait, food, food additives, medicines and nutraceuticals, and the like.
  • the microalgae obtained by the culture can be comprehensively utilized to extract various active ingredients such as polyunsaturated fatty acids, proteinaceous substances, chlorophyll, and polysaccharides.
  • active ingredients such as polyunsaturated fatty acids, proteinaceous substances, chlorophyll, and polysaccharides.
  • the order of extraction of the active ingredient is not particularly limited, but it is usually the premise that the step of first extraction cannot cause loss of the component to be extracted later.
  • the other components in the supernatant may be gradually separated and extracted to obtain fatty acids, chlorophyll, etc., or directly mixed with all the components in the supernatant and sprayed with the algal body to obtain chlorella powder.
  • the obtained algal flour can be used for development into animal feed, aquaculture bait, food, food additives, medicines and nutrients.
  • the microalgae obtained by the culture can be comprehensively utilized to extract various active ingredients such as a pigment, a protein, and a polysaccharide.
  • active ingredients such as a pigment, a protein, and a polysaccharide.
  • the order of extraction of the active ingredient is not particularly limited, but it is usually the premise that the step of first extraction does not result in loss of the component to be extracted.
  • Determination of dry weight of algae cells Take 50 ml of culture medium during the culture of microalgae (such as chlorella), centrifuge at 8000 rpm for 10 minutes, and wash the algae after centrifugation 3 times with deionized water, and transfer to a weighing bottle ( In Wi (g), dry in an oven at 105 °C to constant weight W 2 (g).
  • Grease (%) (W 2 -W.) / Wi X 100
  • - is the weight of algae powder, g; Wo - the weight of the rotary evaporation bottle for drying to constant weight, g; W 2 - the weight of the evaporation bottle after evaporation of the oil extract, g.
  • Carbohydrate Refer to the Pharmacopoeia 2005 edition, select the anthrone-sulfuric acid color reaction, use anhydrous glucose as a control, and measure by colorimetry at 625 nm.
  • chloroplast pigment content microalgae (such as chlorella) Determination of chloroplast pigment content by Lichtenthaler et al., Determination of total carotenoids and chlorophylls a and b of leaf extracts in different Sovents. Biochemical Society Transactions, 1983, 11 :591-592).
  • Microalgae (such as chlorella) Ash is measured by burning at 550 °C to constant weight, which is the national standard GB6438-86.
  • the method of the invention has the following advantages: high-density microalgae cells can be obtained in a short time, and the oil content in the microalgae cells can be obtained in a short time. Improve quickly.
  • the invention divides the microalgae culture synthetic oil into two stages of algae cell growth and oil fat synthesis accumulation, namely heterotrophic culture and light induction culture stage.
  • the purpose of heterotrophic cultivation of microalgae is to obtain a large number of algae cells quickly, and the purpose of light-induced culture is to induce rapid synthesis and accumulation of large amounts of oil and fat in microalgae cells, and to promote microalgae cultivation by heterotrophic-dilution-light-induced tandem culture.
  • the yield of oil and fat has solved the contradiction between energy microalgae cell growth and oil and fat synthesis to a certain extent, greatly reducing the cost, and providing the problem of over-costing caused by insufficient raw materials in the large-scale preparation process of biofuels. New technical means.
  • microalgae could be used to produce oil on a large scale only by a mode of photoautotrophicity.
  • photoautotrophic culture is completely different from the light-induced culture of the present invention.
  • the medium does not contain an organic carbon source.
  • the microalgae utilizes C0 2 , water, light, and a small amount of inorganic nutrients in the atmosphere for photoautotrophic growth to maintain their own growth.
  • the general seeding density is low (0.1g).
  • the medium for photoinduction of the present application is different from the photoautotrophic medium. Since the heterotrophic stage is rich in nutrients such as inorganic salts and trace elements, the desired light-inducing medium is diluted.
  • the photoautotrophic culture medium It is much simpler than the photoautotrophic culture medium, for example, compared with the microalgae photoautotrophic medium BG-11 commonly used in the literature, the light-induced culture for dilution. Phosphate, sodium carbonate, ammonium ferric citrate and trace elements are not required in the nutrient base, and the amount of nitrate added is also much less than that of the photoautotrophic medium.
  • Phosphate, sodium carbonate, ammonium ferric citrate and trace elements are not required in the nutrient base, and the amount of nitrate added is also much less than that of the photoautotrophic medium.
  • the heterotrophic stage Through the cultivation in the heterotrophic stage, a large number of algae cells can be accumulated in a short period of time. When scaled up, the light-induced algal cell density is entered by dilution (in the case of chlorella, the density ranges from 2 to 5 g/L).
  • the inoculation density of the photoautotrophic culture method is 20 times higher than that of the light autotrophic culture method, which solves the shortcomings such as low seeding density of the photoautotrophic culture method and long seed expansion period, and is also in a short period of light induction period (general 8 ⁇ 24 hours), the density of algae cells is almost unchanged (that is, it no longer grows), the intracellular fat content can accumulate rapidly, and the content increases exponentially (from 10% to 20 ⁇ 30%).
  • the area is greatly reduced, the area yield is greatly improved, the entire culture period is significantly shortened, the harvesting cost is reduced, and the production efficiency is greatly improved.
  • the highest volume yield (145 mg/L/d) of lutein produced by microalgae and the highest content of intracellular lutein (7.6 mg/g) have been under continuous light-mixed nutrient culture conditions.
  • the culture mode can only be carried out in a steam-sterilizable closed photobioreactor, and the culture process must ensure absolute sterility, and a reasonable configuration of the light source is required, which cannot be achieved in actual production. Therefore, the use of a mixed nutrient model to culture microalgae to produce lutein does not have industrial value.
  • the invention divides the production of lutein by microalgae culture into two stages of algae growth and product accumulation (lutein), namely heterotrophic culture and light-induced culture.
  • lutein a large number of lutein-producing microalgae cells can be obtained in a short time.
  • the algae solution is diluted and transferred to light-induced culture, and the lutein content in the algae is rapidly increased to twice or more than the initial amount.
  • the invention has the following advantages: (1) The light-induced algal cell density is very high (2 ⁇ 10g/L), which is about 10 times of the conventional photoautotrophic culture algae cell density (about 0.2 ⁇ lg / L); (2) The light induction time is very short (about ld ⁇ 2d), while the microalgae self-cultivation time is very long (about 7d ⁇ 14d), so the production efficiency of lutein is greatly improved; (3) relative to microalgae light self In culture, the higher density of algae cells in light induction makes the area required for light induction small, while the high cell density makes the harvesting cost greatly reduced. (4) Heterotrophic culture is almost independent of climate and weather. The light-induced culture can be carried out in a glass room under natural light or artificial light.
  • the continuous production of lutein can be achieved by the method of the invention; (5) the algae rich in oil and lutein are carried out.
  • Step-by-step extraction, coupling of microalgae energy and high value-added product development and resource utilization of algae, can obtain additional products and economic benefits, and reduce the production cost of microalgae energy.
  • heterotrophic medium and tap water were added to a 50 L bioreactor to 25 L and then sterilized when the temperature was lowered to 30 °.
  • Heterotrophic culture conditions The temperature is 30 ° C, the initial rotation speed is 150 r / min, the air flow rate is lvvm, the pH is less than 8.0, and the dissolved oxygen is controlled by 15% or more by adjusting the rotation speed during the cultivation.
  • the high-density algal solution depleted of glucose in the heterotrophic culture process was diluted to about 2.70 g/L, and the following light-inducing medium was added, and transferred to a 3 L flat-plate photoreactor for light-induced culture at a temperature of 30 ° C.
  • the light intensity is 8000k
  • the air flow is lvvm.
  • the cell density decreased from 2.70 g/L to 2.65 g/L
  • the oil content increased from 9.70% to 19.70% (see Figure 1).
  • Heterotrophic medium glucose 60.0 g, urea 8.0 g, MgS0 4 ⁇ 7H 2 0 2.0 g, KH 2 P0 4 1.1 g, Na 2 HP0 4 ⁇ 12H 2 0 9.0 g, CaCl 2 0.02 g, trisodium citrate 1.8 ⁇ , Fe-EDTA solution 1.0ml trace element solution 4.5ml, and water 1000ml; where Fe-EDTA solution is FeS0 4 ⁇ 7H 2 0 15 g / liter and EDTA 1.4 g / liter, trace element solution formula is H 3 B0 3 2.11 g/l, MnCl 2 ⁇ 43 ⁇ 40 0.81 g/l, ZnS0 4 ⁇ 7H 2 0 0.11 g/l, CuS0 4 ⁇ 5H 2 0 10.0 g/l, Na 2 Mo 0 4 0.05 g/l.
  • Light-induced medium 0.5 g of urea, 1.0 g of MgS0 4 '7H 2 0, 0.05 g of trisodium citrate, 0.01 g of CaCl 2 , 0.4 ml of Fe-EDTA solution, and 1000 ml of water; the formulation of Fe-EDTA solution is 15 g. / liter and EDTA 1.4 g / liter.
  • Example 2
  • the 500 L fermenter is used as a seed tank to culture the common chlorella, and the cells grow according to the culture process.
  • the dissolved oxygen level in the fermentation broth to adjust the tank pressure, aeration and agitation speed, maintain sufficient oxygen content in the culture solution to ensure the rapid growth of algae cells.
  • the high-density algae solution depleted of glucose in the pre-feeding batch heterotrophic culture process was diluted with light-inducing medium, and then transferred to a 30 L flat-plate photoreactor and a 60 L bubbling large-scale pot for outdoor light induction. to cultivate.
  • Light-induced culture conditions natural temperature, temperature between 13 and 30 ° C, natural light, light intensity between 0 and 36 kk (see Figure 2), air flow of 1 vvm.
  • the density of algae cells in the 30 L flat reactor decreased from 1.60 g/L to 1.46 g/L, and the oil content increased from 7.87% to 13.27%.
  • the density of algae cells from the night was 1.46 g.
  • the high-density common chlorella algae solution after the semi-continuous heterotrophic culture was diluted with the light-inducing medium, transferred to a 3 T runway pool and a 3.14 m 2 oblique-leaf stirred round cell for outdoor light-induced culture.
  • Light-induced culture conditions lower natural temperature, temperature between 5 and 18 ° C, natural light, light intensity between 0 and 34 kk (see Figure 5), air flow of l vvm. Because the semi-continuous culture of the batch 5 T fermenter ended in the evening, the algae cells entered the night after the light was induced, and the light induction stage was carried out for 90 h. The weather was fine in the first 2 days, but the temperature was lower.
  • the evening of the algae liquid the lowest gas
  • the temperature is only 5 °C, and the maximum temperature is 14 °C in the next two days.
  • the weather turns cloudy with light rain, and the temperature starts to rise slightly.
  • the third day starts to be hot and humid in the evening, with thunderstorms throughout the night, and the fourth day is cloudy. There is light rain, rain stops in the afternoon, and the cultivation and harvesting of algae cells is finished.
  • the heterotrophic medium is: glucose 60.0 g, potassium nitrate 10.0 g, MgS0 4 ⁇ 7 ⁇ 2 0 0.2 g, KH 2 P0 4 0.3 g, Na 2 HP0 4 ⁇ 12 ⁇ 2 0 8.8 g, CaC12 0.02 g, Fe- 1.0ml of EDTA solution, 3.5ml of trace element solution, 1000ml of water; the formulation of Fe-EDTA solution is FeS0 4 ⁇ 7H 2 0 15g / liter and EDTA 1.4g / liter, the formula of trace element solution is H 3 B0 3 2.86 g / Liter, MnCl 2 ⁇ 4H 2 0 0.11 g / liter, ZnS0 4 ⁇ 7H 2 0 9.22 g / liter, CuS0 4 ⁇ 5H 2 0 1.00 g / liter, ( ⁇ 4 ) 6 ⁇ 7 0 24 ⁇ 4 ⁇ 2 0 0.1 g /L
  • the light-inducing medium is: potassium nitrate 0.5 g, MgS0 4 ⁇ 7H 2 0 0.6 g, CaCl 2 0.03 g, Fe-EDTA solution 1.5 ml, water 1000 ml; wherein Fe-EDTA solution is 8 g/L and EDTA 10.4 G/L.
  • Example 3 Study on the Change of Main Biochemical Compositions of Chlorella ellipses during Heterotrophic-Dilution-Light Induced Tandem Culture
  • the changes of main biochemical components in the heterotrophic-dilution-light-induced tandem culture of Chlorella ellipsoid were determined at the level of a 500 mL shake flask / 3 L cylindrical photobioreactor.
  • the ellipsoidal culture was cultured in a heterotrophic-dilution-light-induced tandem culture technique, a 500 mL shake flask, a liquid volume of 200 mL, 28 ° C, 150 rpm, and the algal cell density reached 10 g/L. Above and when glucose is depleted, it is transferred to a 3L cylindrical photobioreactor for light-induced culture.
  • the algae cell density is about 2g/L, the temperature is 30°C, and the light intensity is 10Kk.
  • the culture medium used for heterotrophic and light induction is consistent with the corresponding medium of Chlorella pyrenoidosa.
  • Example 4 The following heterotrophic medium and water were added to a 5 L bioreactor to 2.8 L, followed by steam sterilization, and then when the temperature was lowered to 30 ° C, Chlorella pyrenoidosa was introduced to start heterotrophic culture. Heterotrophic culture conditions: The temperature is 30 ⁇ 1 °C, the air flow is lvvm, the pH is less than 8.0, and the dissolved oxygen is controlled by more than 15%.
  • the first feeding was carried out 53.9 hours after inoculation, and then the feeding was carried out every 5 ⁇ 8h, supplemented 4 times, and the dry weight of the cells reached 88.40h reached 132.2g/L (see Figure 9).
  • the culture is transferred to light-induced culture.
  • the high-density algal solution after the heterotrophic culture was diluted to 2.55 g/L, and the following light-inducing medium was added, and transferred to a 3 L flat photobioreactor for light-induced culture.
  • Light-induced culture conditions The temperature was maintained at 28-33 °C, the air flow was lvvm, and the illumination was bilateral, with a light intensity of 15 kk per side. After 27 h of light-induced culture, the dry weight of the cells decreased from 2.55 g/L to 1.82 g/L, lutein increased from 1.57 mg/g Dcw to 3.64 mg/g Dcw, and the yield of lutein was 5.88 mg at 27 h after light induction.
  • the heterotrophic and feeding medium was: glucose 60.0 g, urea 8.0 g, MgS (V7H 2 0 2.0 g, KH 2 P0 4 1.1 g, Na 2 HP0 4 - 12H 2 0 9.0 g, CaCl 2 0.02 g, citric acid 1.8 g of trisodium, 1.0 ml of Fe-EDTA solution, 4.5 ml of trace element solution, 1000 ml of water; the formulation of Fe-EDTA solution is FeS (V7H 2 0 15 g / liter and EDTA 1.4 g / liter, the formulation of trace element solution is H 3 B0 3 2.11 g / liter, MnCl 2 4H 2 0 0.81 g / liter, ZnS0 4 -7H 2 0 0.11 g / liter, CuS0 4 -5H 2 0 10.0 g / liter, Na 2 Mo0 4 0.05 g / liter.
  • the light-inducing medium is: urea 0.5 g, MgS0 4 -7H 2 0 1.0 g, trisodium citrate 0.05 g, CaCl 2 0.01 g, Fe-EDTA solution 0.4 ml, water 1000 ml; wherein the Fe-EDTA solution is FeS0 4 '7H 2 0 15 g / liter and EDTA 1.4 g / liter.
  • heterotrophic medium Add the following heterotrophic medium and tap water to 25L in a 50L bioreactor and sterilize at 121 °C for 20 min. Then, when the temperature drops to about 30 °C, access the chlorella chlorella at 10% of the working volume. Start heterotrophic culture.
  • Heterotrophic culture conditions temperature is 30 ° C, air flow is lvvm, pH is 6.0 8.0, and dissolved oxygen is controlled by more than 15%.
  • a supplementary carbon source is added, and when the urea is consumed, a nitrogen source is added.
  • the density of algae cells reached 130.5g/L at 98.89h after supplementing the nitrogen source three times (see Figure 11).
  • the high-density algae after the heterotrophic culture was diluted to 2.01 g/L, and the light-inducing medium was added, and transferred to a 10 L cylindrical photobioreactor for light induction.
  • Light-induced culture conditions natural temperature, temperature at 30 ° C, light intensity at 3 kk, air flow at lvvm.
  • the dry weight of the cells decreased from 2.01 g/L to 1.66 g/L
  • lutein increased from 2.55 mg/g Dcw to 3.47 mg/g Dcw
  • photoinduced for 30 h and the lutein yield was 4.61 mg/L. /d;
  • the yield of lutein reached 5.73 mg/L/d if only light was induced for 24 h (see Figure 12).
  • the medium was identical to the medium of Example 4.
  • Example 6 Add the following heterotrophic medium and tap water to the 25L bioreactor to 25L and sterilize at 121 °C for 20min, then connect to the common chlorella at 13% of the working volume when the temperature drops to about 30 °C. Heterotrophic culture.
  • Heterotrophic culture conditions temperature is 30 ° C, air flow is lvvm, pH is less than 9.0.
  • carbon source when the carbon source is consumed, glucose is added, and when the nitrogen source is consumed, potassium nitrate is added.
  • the cell density of 58.20h algae is up to 54.5g/L (see Figure 13).
  • the heterotrophic cultured high-density algae was diluted to about 3.2 g/L, and light-inducing medium was added, and transferred to a 30-liter flat photobioreactor for outdoor light-induced culture.
  • Light-induced culture conditions natural temperature, natural light, and air flow rate of 1.0 wm. After light induction for 28h, lutein increased from the initial 1.10mg/gDcw to 1.82mg/gDcw; when photoinduced for 23h, the lutein content was 1.67mg/gDcw, and the lutein yield reached 5.23mg/L/dC. 15).
  • the heterotrophic and feeding medium was: glucose 60.0 g, potassium nitrate 10.0 g, MgS (V7H 2 0 0.2 g, KH 2 P0 4 0.3 g, Na 2 HP (V 12H 2 0 8.8 g, CaC12 0.02 g, Fe-) 1.0ml of EDTA solution, 3.5ml of trace element solution, 1000ml of water;
  • the formula of Fe-EDTA solution is FeS (V7H 2 15 15g / liter and EDTA 1.4g / liter, the formula of trace element solution is H 3 B0 3 2.86 g / Liter, MnCl 2 -4H 2 0 0.11 g / liter, ZnS0 4 -7H 2 0 9.22 g / liter, CuS0 4 -5H 2 0 1.00 g / liter, ( ⁇ 4 ) 6 ⁇ 7 0 24 ⁇ 4 ⁇ 2 0 0.1 g /L and Co(N0 3 )
  • the light-inducing medium was: potassium nitrate 0.5 g, MgS (V7H 2 0.6 g, CaCl 2 0.03 g, Fe-EDTA solution 1.5 ml, water 1000 ml; wherein the Fe-EDTA solution formulation was FeS0 4 '7H 2 0 8 g/ l and EDTA 10.4 g / l.
  • Example 7 potassium nitrate 0.5 g, MgS (V7H 2 0.6 g, CaCl 2 0.03 g, Fe-EDTA solution 1.5 ml, water 1000 ml; wherein the Fe-EDTA solution formulation was FeS0 4 '7H 2 0 8 g/ l and EDTA 10.4 g / l.
  • Heterotrophic culture conditions temperature is 30 ⁇ 1 °C, air flow is lvvm, pH is less than 8.5, and dissolved oxygen is controlled at 5% or more.
  • the algal cell density was 53.0 g/L (see Figure 14), at which point the heterotrophic culture was transferred to light-induced culture.
  • the high-density algal solution after the heterotrophic culture was diluted to 4.0 g/L, and the following light-inducing medium was added, and transferred to a 3 L flat photobioreactor for light-induced culture.
  • Light-induced culture conditions the temperature was maintained at 28 ⁇ 33 °C, the air flow rate was l.Ovvm, and the bilateral light intensity was 15klx per side.
  • the dry weight of the cells decreased from 4.0 g/L to 2.8 g/L, lutein increased from 1.5 mg/g Dcw to 3.6 mg/g Dcw, and the lutein yield was 5.04 mg/L/d.

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Abstract

A microalgae culturing method, especially a method for rapidly accumulating intracellular oil and/or lutein and other intracellular material of microalgae is disclosed in present invention, which includes the steps of heterotrophic culture, dilution, photoindusive culture, collecting microalae, and extracting oil and/or lutein, etc. Using present method can have full advantages to rapidly accumulate oil and/or lutein and other intracellular material from microalgae during photoindusive phase, and provide important technical means for solving the problems of the scarcity of raw materials in the scale preparation process of biofuels, and reducing production cost the lutein industrialization of microalgae and the coupling of microalgae energy development and high valuation of microalgae residue

Description

一种快速积累油脂和叶黄素的微藻培养方法 技术领域  Microalgae cultivation method for rapidly accumulating oil and lutein
本发明属于生物能源领域和 /或微藻生物技术领域， 涉及一种微藻培养方法， 尤其是 快速积累微藻胞内生物活性物质， 特别是油脂和叶黄素的方法。 背景技术  The invention belongs to the field of bioenergy and/or microalgae biotechnology, and relates to a microalgae cultivation method, in particular to a method for rapidly accumulating intracellular bioactive substances of microalgae, especially oil and lutein. Background technique
微藻能源发展前景广阔、 优势独特已获国内外公认。 迄今， 世界各国在该领域的研 发工作还停留在实验研究和中试论证的起步阶段（李元广、 谭天伟、 黄英明， 微藻生物 柴油产业化技术中的若干科学问题及其分析， 中国基础科学， 2009， 5， 64-70 ) ， 但均 遇到技术不成熟而导致成本高这一瓶颈， 因而微藻能源在全球尚未实现规模化制备。  The development of microalgae energy has broad prospects and unique advantages have been recognized at home and abroad. So far, the research and development work of the countries in this field has remained at the initial stage of experimental research and pilot demonstration (Li Yuanguang, Tan Tianwei, Huang Yingming, some scientific problems and analysis of microalgae biodiesel industrialization technology, China Basic Science, 2009, 5, 64-70), but they all encounter the bottleneck caused by the immature technology and high cost. Therefore, the microalgae energy has not been scaled up globally.
掌握能源微藻的高效培养模式是实现微藻能源规模化制备的基础。 现有生长快的藻 种一般油脂含量较低， 而含油量高的藻种生长大多比较缓慢。 这一矛盾是能源微藻高效 培养模式开发的现实障碍。 微藻培养包括自养、 异养和混合营养三种模式 （Yanna Liang, Nicolas Sarkany, Yi Cui. Biomass and lipid productivities of Chlorella vulgaris under autotrophic, heterotrophic and mixotrophic growth conditions. Biotechnology Letters, 2009, 31 : 1043-1049 )。异养培养模式 (Han Xu, Xiaoling Miao, Qingyu Wu. High quality biodiesel production from a microalga Chlorella protothecoides by heterotrophic growth in fermenters. Journal of Biotechnology, 2006, 126:499-507 )存在 "不能直接利用太阳能、不能固定 C02、 能耗大"等问题， 因而难以用于能源微藻的大规模培养。 美国 1998年的 ASP计划工作 总结报告指出： 相对低成本的开放式光自养培养是最有前景的培养模式 （Sheehan J, Dunahay T, Benemann J, et al. A Look Back at the U. S. Department of Energy's Aquatic Species Program—- Biodiesel from Algae. National Renewable Energy Laboratory, 1998 ) 。 但 是， 开放式光自养存在的 "培养密度低、 易被污染、 水分蒸发、 受环境因素影响大"等 问题， 也使其难以满足能源生产的规模需求。 Mastering the efficient culture mode of energy microalgae is the basis for the large-scale preparation of microalgae energy. The existing fast growing algae species generally have lower oil content, while the algae species with higher oil content are mostly slower to grow. This contradiction is a practical obstacle to the development of an efficient culture model for energy microalgae. Microalgae cultivation includes three modes of autotrophic, heterotrophic and mixed nutrition (Yanna Liang, Nicolas Sarkany, Yi Cui. Biomass and lipid productivities of Chlorella vulgaris under autotrophic, heterotrophic and mixotrophic growth conditions. Biotechnology Letters, 2009, 31 : 1043- 1049). Heterotrophic culture mode (Han Xu, Xiaoling Miao, Qingyu Wu. High quality biodiesel production from a microalga Chlorella protothecoides by heterotrophic growth in fermenters. Journal of Biotechnology, 2006, 126:499-507) exists "cannot directly use solar energy, can not be fixed Problems such as C0 2 and high energy consumption are difficult to use for large-scale cultivation of energy microalgae. The United States 1998 ASP work summary report pointed out: Relatively low-cost open photoautotrophic culture is the most promising training model (Sheehan J, Dunahay T, Benemann J, et al. A Look Back at the US Department of Energy's Aquatic Species Program—Biodiesel from Algae. National Renewable Energy Laboratory, 1998). However, open photoautotrophic problems such as low cultivation density, easy contamination, evaporation of water, and large environmental factors have made it difficult to meet the scale requirements of energy production.
针对开放式光自养培养存在的问题， 人们试图采用封闭式光生物反应器进行微藻的 光自养培养。封闭式光生物反应器包括管道式、平板式、柱式等（Ogbonna J C, Tanaka H. Industrial- size photobioreactors. Chemical technology, 1997, 27(7):43~49 )，虽然具有细胞密 度高、 生长快等优点， 但由于成本高、 放大技术不成熟等原因， 目前尚未应用于微藻的 大规模培养。  In view of the problems existing in open photoautotrophic culture, people have tried to use the closed photobioreactor to carry out photoautotrophic cultivation of microalgae. Closed photobioreactors include pipeline, flat, column, etc. (Ogbonna JC, Tanaka H. Industrial- size photobioreactors. Chemical technology, 1997, 27(7): 43~49), although with high cell density, growth The advantages are quick, but due to high cost and immature amplification technology, it has not been applied to large-scale cultivation of microalgae.
此外， 近年来人们试图采用封闭式光生物反应器和开放池组合进行微藻的光自养培 养： 即先利用密闭式光生物反应器实现微藻的高密度培养， 再利用开放池降低生产成本。 该模式基本具备 "可利用太阳能、 高密度、 固定 co2、 低成本、 高效" 的特征， 但如何 实现全过程的有效控制和***工程优化， 还有待深入研究。 In addition, in recent years, people have tried to use the closed photobioreactor and open cell combination to carry out photoautotrophic cultivation of microalgae: firstly, the high-density culture of microalgae is realized by closed photobioreactor, and the production cost is reduced by using open pool. . This model basically has the characteristics of "available solar energy, high density, fixed co 2 , low cost, high efficiency", but how to achieve effective control and system engineering optimization of the whole process remains to be further studied.
学者公认能源微藻培养模式应具备 "可利用太阳能、 高密度、 固定 co2、 低成本、 高效" 的特征， 为实现这一目标， 研究者先后又探索推出了 "混合营养"、 "先自养， 后异养"等培养模式： Scholars acknowledge that the energy microalgae culture model should have the characteristics of "available solar energy, high density, fixed co 2 , low cost, high efficiency". To achieve this goal, the researchers have successively explored the introduction of "mixed nutrition" and "first" Cultivation, post-heterotrophic" and other training modes:
( 1 ) 混合营养模式： 是指藻细胞利用光和有机物作为能源、 再利用有机物和无机 物作为碳源的培养方式 （Lee YK, Ding SY, Hoe CH, Low CS. Mixotrophic growth of Chlorella sorokiniana in outdoor enclosed photobioreactor. Journal of applied phycology, 1996, 8: 163-169)。在微藻的混合营养培养过程中，微藻的光合自养和化能异养可以同时进行。 该模式可以通过与富含碳、 氮等有机物废水处理结合而降低生产成本， 但其最大问题是 大规模培养过程中容易滋生大量杂菌， 难以实现微藻的高密度培养。  (1) Mixed nutrition model: refers to the culture method in which algae cells use light and organic matter as energy sources, and reuse organic matter and inorganic matter as carbon sources (Lee YK, Ding SY, Hoe CH, Low CS. Mixotrophic growth of Chlorella sorokiniana in outdoor Enclosed photobioreactor. Journal of applied phycology, 1996, 8: 163-169). In the mixed nutrient culture process of microalgae, the photosynthesis and chemical heterotrophy of microalgae can be carried out simultaneously. This model can reduce production costs by combining with wastewater treatment of organic matter rich in carbon and nitrogen, but the biggest problem is that it is easy to breed a large number of bacteria during large-scale cultivation, and it is difficult to achieve high-density cultivation of microalgae.
(2) "先自养， 后异养"模式： 是先利用密闭式光生物反应器自养以固定 C02， 后进行异养培养 ( Xiong W, Gao C, Yan D, et al. Double C02 fixation in photosynthesis - fermentation model enhances algal lipid synthesis for biodiesel production. Bioresource Technology, 2010, 101 :2287-2293 ) 。 该模式存在的最大问题是光诱导培养过程放大后无 法 ί故至 lj无菌培养 ( Scott SA, Davey MP, Dennis JS, et al. Biodiesel from algae: challenges and prospects. Current Opinion in Biotechnology, 2010,21 : 1-10) ， 由于微藻的异养培养要求藻 种必须不带任何杂菌， 因此该模式无法放大， 不具有规模化应用价值。 (2) "Self-autotrophic, post-heterotrophic" mode: It is self-supporting with a closed photobioreactor to fix C0 2 and then heterotrophic culture ( Xiong W, Gao C, Yan D, et al. Double C0 2 fixation in photosynthesis - fermentation model enhances algal lipid synthesis for biodiesel production. Bioresource Technology, 2010, 101 : 2287-2293 ). The biggest problem with this model is that the light-induced culture process cannot be amplified until it is sterile (Scott SA, Davey MP, Dennis JS, et al. Biodiesel from algae: challenges and prospects. Current Opinion in Biotechnology, 2010, 21 : 1-10) , Because the heterotrophic culture of microalgae requires algae species to be free of any bacteria, the model cannot be amplified and does not have a large-scale application value.
叶黄素在医药和保健品、 食品和饲料、 化妆品及水产养殖行业等方面广泛的应用前 景， 使许多国内外研究机构和公司对叶黄素的生物合成、 分离提取、 生理生化功能等进 行研究。 目前， 叶黄素主要从万寿菊、 金盏花等植物中提取获得， 但是利用这些植物生 产叶黄素存在着某些缺点。 首先， 叶黄素在这些植物中主要是酯化形式存在， 因此从万 寿菊、 金盏花等植物中提取叶黄素时需要进行皂化处理， 该步骤不但降低了效率和收率， 同时残余的皂化剂容易污染叶黄素产品， 给纯化过程增加了难度。 其次， 种植万寿菊、 金盏花等植物需要大量的土地， 这些植物的生长周期一般为 3~4个月 （相对于可快速生 长的微藻而言， 周期太长） 。  The broad application prospects of lutein in medicine and health care products, food and feed, cosmetics and aquaculture industries have enabled many domestic and foreign research institutions and companies to study the biosynthesis, separation and extraction, physiological and biochemical functions of lutein. . Currently, lutein is mainly extracted from plants such as marigold and calendula, but the use of these plants to produce lutein has certain disadvantages. First, lutein is mainly present in esterified forms in these plants. Therefore, saponification is required for extracting lutein from plants such as marigold and calendula. This step not only reduces efficiency and yield, but also residual saponifier. It is easy to contaminate lutein products, which makes the purification process more difficult. Secondly, planting marigolds, calendulas and other plants requires a large amount of land. These plants usually have a growth cycle of 3 to 4 months (relative to the rapid growth of microalgae, the cycle is too long).
相比较而言， 利用微藻来生产叶黄素具有某些优势。 首先叶黄素在微藻中主要是以 游离的形式存在， 因此在生产过程中不需要皂化步骤； 其次， 微藻的生长周期短、 生产 设备占地面积小， 同时利用微藻生产叶黄素不受季节、 气候及地域条件限制， 产品质量 和产量相对稳定； 最后， 微藻 （如小球藻等） 本身就是一种高价值的产品， 含有大量的 蛋白质、 油脂、 多糖等活性成分， 可以分离提取这些物质， 实现微藻细胞的综合利用。 此外， 微藻能源的产业化开发也迫切需要实现藻体的资源化利用。 微藻提油后的非油脂 组分中含有丰富的色素、 蛋白质和多糖等生物活性物质， 可被开发成为医药、 食品及饲 料添加剂等高附加值产品。 通过对藻渣的高值化利用 （如提取叶黄素） ， 不仅可以实现 微藻细胞的综合利用， 还能够提高微藻能源生产过程的综合经济效益和环保效益， 降低 微藻能源的生产成本。 In comparison, the use of microalgae to produce lutein has certain advantages. First, lutein is mainly present in free form in microalgae, so there is no need for a saponification step in the production process. Secondly, the microalgae has a short growth cycle, a small production area, and the use of microalgae to produce lutein. Not limited by seasonal, climatic and geographical conditions, product quality and yield are relatively stable; finally, microalgae (such as chlorella, etc.) itself is a high-value product containing a large amount of active ingredients such as protein, oil, polysaccharide, etc. These substances are separated and extracted to realize the comprehensive utilization of microalgae cells. In addition, the industrialization of microalgae energy is also urgently needed to realize the resource utilization of algae. The non-fat components of the microalgae oil are rich in bioactive substances such as pigments, proteins and polysaccharides, and can be developed into high value-added products such as pharmaceuticals, foods and feed additives. By using the high value of algae residue (such as extracting lutein), not only can it be realized The comprehensive utilization of microalgae cells can also improve the comprehensive economic and environmental benefits of the microalgae energy production process and reduce the production cost of microalgae energy.
至今， 利用微藻产叶黄素的培养模式主要有光自养和异养两种。 微藻光自养培养的 缺点是微藻细胞生长速度慢、 细胞密度及叶黄素产率低。 目前光自养培养微藻的叶黄素 最高体积产率为 Sanchez J F等在 2L圆柱式光生物反应器中培养 Scenedesmus almeriensis 所获得的 4.8mg/L/d ( Sanchez J F, Fernandez-Sevilla J M, Acien F G.， et al. Biomass and lutein productivity of Scenedesmus almeriensis: influence of irradiance, dilution rate and temperature. Appl Microbiol Biotechnol , 2008, 79:719-729) ， 叶黄素的最高面积产率为 Fernandez等在 4000L管道式反应器中培养 Scenedesmus almeriensis所达到的 290mg/m2/d Up to now, the cultivation modes of lutein produced by microalgae mainly include photoautotrophic and heterotrophic. The disadvantage of microalgae autotrophic culture is that the microalgae cells grow slowly, the cell density and the lutein yield are low. At present, the highest volumetric yield of lutein in photoautotrophic culture of microalgae is 4.8 mg/L/d obtained by Sanchez JF and the cultivation of Scenedesmus almeriensis in a 2L cylindrical photobioreactor (Sanchez JF, Fernandez-Sevilla JM, Acien F G., et al. Biomass and lutein productivity of Scenedesmus almeriensis: influence of irradiance, dilution rate and temperature. Appl Microbiol Biotechnol, 2008, 79:719-729), the highest area yield of lutein is Fernandez et al. at 4000L 290mg/m2/d achieved by the cultivation of Scenedesmus almeriensis in a pipeline reactor
( Del Campo J A, Mercedes Garcia Gonzalez, Guerrero M G. Outdoor cultivation of microalgae for carotenoid production: current state and perspectives. Appl Microbiol Biotechnol, 2007, 74: 1163-1174) 。 ( Del Campo J A, Mercedes Garcia Gonzalez, Guerrero M G. Outdoor cultivation of microalgae for carotenoid production: current state and perspectives. Appl Microbiol Biotechnol, 2007, 74: 1163-1174).
微藻异养培养的优点是微藻能够在生物反应器中进行高密度培养， 细胞生长速率 快， 但存在细胞内叶黄素、 叶绿素等色素和蛋白质含量低等缺点。 异养培养能够获得高 细胞密度和高细胞生长速度， 从而使得叶黄素的产率相对于光自养培养有很大提高。  The advantage of microalgae heterotrophic culture is that microalgae can be cultured in a high-density medium in a bioreactor, and the cell growth rate is fast, but there are disadvantages such as intracellular lutein, chlorophyll and the like, and low protein content. Heterotrophic culture can achieve high cell density and high cell growth rate, so that the yield of lutein is greatly improved compared to photoautotrophic culture.
微藻除异养培养和光自养培养模式外， 还有一种不常用的培养模式即混合营养培 养。 迄今， 微藻培养产叶黄素的最高体积产率 （145mg/L/d) 和胞内叶黄素的最高含量 In addition to the heterotrophic culture and photoautotrophic culture mode, microalgae also has an uncommon culture mode, namely, mixed nutrient culture. So far, the highest volume yield (145mg/L/d) of lutein produced by microalgae culture and the highest content of intracellular lutein
( 7.6mg/g) 均是在连续光照的混合营养培养条件下获得的 （Martin, Lucia. Process for obtaining lutein from algae. EP180843A1, 2007) 。 但该培养模式只能在可蒸汽灭菌的封闭 式光生物反应器中进行， 且培养过程必须保证绝对的无菌， 同时需要光源的合理配置， 这在实际生产中无法实现。 因此， 利用混合营养模式培养微藻生产叶黄素不具有产业化 价值。 (7.6 mg/g) were obtained under continuous light mixed nutrient culture conditions (Martin, Lucia. Process for obtaining lutein from algae. EP180843A1, 2007). However, this culture mode can only be carried out in a steam-sterilizable closed photobioreactor, and the culture process must ensure absolute sterility, and at the same time requires a reasonable configuration of the light source, which cannot be achieved in actual production. Therefore, the use of a mixed nutrient model to culture microalgae to produce lutein does not have industrial value.
由上述可见， 无论是采用光自养培养模式还是异养培养模式， 较低的胞内叶黄素含 量和叶黄素产率， 同时加上微藻大规模培养较高的成本， 制约了应用微藻培养来生产叶 黄素的产业化进程。 因此， 有必要探索新的微藻培养工艺或方法使叶黄素产率及含量大 幅度提高， 同时又使微藻大规模培养的成本大幅度下降， 这样才能够满足利用微藻培养 产叶黄素的产业化要求。  It can be seen from the above that whether using the photoautotrophic culture mode or the heterotrophic culture mode, the lower intracellular lutein content and lutein yield, coupled with the high cost of large-scale cultivation of microalgae, restricts the application. Microalgae cultivation to produce the industrialization process of lutein. Therefore, it is necessary to explore a new microalgae culture process or method to greatly increase the yield and content of lutein, and at the same time greatly reduce the cost of large-scale cultivation of microalgae, so as to satisfy the cultivation of yellow leaves by using microalgae. Industrialization requirements.
综合上述几种微藻培养模式的优缺点， 本发明设计了一种 "异养 -稀释 -光诱导串联 培养"模式， 该模式满足了 "可利用太阳能、 高密度、 固定 C02、 低成本、 高效" 的要 求。 以小球藻为例， 其流程如下： 首先利用生物反应器异养培养小球藻以获得高密度细 胞， 待培养液中有机碳源消耗完毕后， 用不含有机碳源的培养基稀释藻液， 经短时间内 ( 8~24h) 的光照诱导， 使藻细胞内的油脂和 /或叶黄素快速大量积累。 该模式中的异养 阶段是在摇瓶、 机械搅拌式、 气升式、 鼓泡式等可异养培养的生物反应器中进行， 目的 是为了在短时间内获得较高密度的细胞； 光诱导阶段可在任何可用于微藻光自养培养的 ***中进行， 目的是通过光诱导作用提高胞内油脂和 /或叶黄素的含量； 异养阶段与光诱 导阶段分开独立进行， 异养阶段放出的藻液先用光诱导培养基进行稀释后再转入光诱导 培养阶段。 过程中需确保进入光诱导阶段的藻液中不含有机碳源， 这样可避免光诱导阶 段滋生过多的杂菌； 而通过稀释作用可以确保光诱导阶段的藻细胞能获得充足的光照， 实现胞内油脂和 /或叶黄素含量的快速提高。 该模式和上述其他模式相比， 具有生产效率 高、 不存在因不同***的组合而造成的异养培养过程染菌问题 （光自养培养过程有杂菌 并不影响生长） 、 所用培养***的组合方式灵活和生产成本低等优点， 可充分发挥小球 藻在光诱导阶段油脂和 /或叶黄素快速积累的优势， 本发明为解决生物柴油规模化制备过 程中的原料不足的瓶颈、 源于微藻的叶黄素产业化以及微藻能源和藻渣高值化利用耦合 降低生产成本等问题提供了重要的技术手段。 发明内容 Combining the advantages and disadvantages of the above several microalgae culture modes, the present invention designs a "heterotrophic-dilution-light-induced tandem culture" mode, which satisfies "available solar energy, high density, fixed C0 2 , low cost, Efficient" requirements. Taking chlorella as an example, the process is as follows: First, the chlorella is cultured in a bioreactor to obtain high-density cells. After the organic carbon source in the culture solution is consumed, the algae is diluted with a medium containing no organic carbon source. The liquid, induced by light in a short time (8~24h), causes the oil and/or lutein in the algae cells to accumulate rapidly. The heterotrophic phase in this mode is carried out in a heterotrophic culture bioreactor such as shake flask, mechanical agitation, airlift, bubbling, etc., in order to obtain higher density cells in a short time; The induction phase can be used in any self-cultivation of microalgae In the system, the purpose is to increase the content of intracellular lipids and/or lutein by light induction; the heterotrophic phase and the light-inducing phase are separated and independent, and the algal fluid released in the heterotrophic phase is diluted with light-inducing medium. Then transfer to the light-induced culture stage. In the process, it is necessary to ensure that the algae liquid entering the light-inducing stage does not contain an organic carbon source, thereby avoiding the growth of excessive bacteria in the light-inducing stage; and the dilution can ensure that the algae cells in the light-inducing stage can obtain sufficient light to achieve Rapid increase in intracellular fat and/or lutein content. Compared with the other modes mentioned above, this mode has high production efficiency, and there is no problem of contamination in the heterotrophic culture process caused by the combination of different systems (the bacteria in the photoautotrophic culture process does not affect the growth), and the culture system used The advantages of flexible combination mode and low production cost can fully utilize the advantages of rapid accumulation of oil and/or lutein in the light-induced phase of chlorella. The present invention is to solve the bottleneck and source of insufficient raw materials in the process of large-scale preparation of biodiesel. It provides an important technical means for the industrialization of lutein in microalgae and the high value of microalgae energy and algae residue to reduce the production cost. Summary of the invention
本发明提供一种微藻培养方法， 该方法包括微藻异养培养的步骤， 和将异养培养所 获得的微藻培养液稀释后进行光诱导培养的步骤。  The present invention provides a microalgae cultivation method comprising the steps of heterotrophic culture of microalgae, and the step of diluting the microalgae culture liquid obtained by heterotrophic culture to perform light-induced culture.
本发明还提供一种快速积累微藻胞内油脂的方法， 该方法包括微藻异养培养的步 骤， 将异养培养所获得的微藻培养液稀释后进行光诱导培养的步骤， 以及任选的藻细胞 采收、 油脂分离提取的步骤。  The present invention also provides a method for rapidly accumulating intracellular fats and oils of microalgae, the method comprising the steps of heterotrophic culture of microalgae, the step of diluting the microalgae culture liquid obtained by heterotrophic culture, and performing light-induced culture, and optionally The steps of algae cell harvesting, oil separation and extraction.
本发明还提供一种微藻油脂的生产方法， 该方法包括微藻异养的步骤， 将微藻的异 养培养液稀释后进行光诱导培养的步骤， 以及藻细胞采收、 油脂分离提取的步骤。 该方 法能够实现胞内油脂的快速积累， 具有占地面积少、 面积产率高、 相对微藻光自养而言 因细胞密度高而带来的采收成本低 （光诱导时的细胞密度较高， 一般为 2~5 g/L) ， 因而 大大提高了生产效率， 降低了生产成本。  The invention also provides a method for producing microalgae oil and fat, which comprises the steps of heterotrophic microalgae, the step of diluting the heterotrophic culture solution of the microalgae for light-induced culture, and the extraction of algae cells and the separation of oil and fat. step. The method can realize the rapid accumulation of intracellular lipids, has a small footprint, high area yield, and low harvesting cost due to high cell density relative to microalgae self-cultivation (cell density at light induction) High, generally 2~5 g/L), which greatly improves production efficiency and reduces production costs.
本发明还提供一种快速积累微藻胞内叶黄素的方法， 该方法包括微藻的异养培养步 骤， 将所获得的微藻异养培养液稀释后进行光诱导培养的步骤， 以及任选的藻细胞采收、 叶黄素分离提取的步骤。  The present invention also provides a method for rapidly accumulating intracellular lutein of microalgae, the method comprising the step of heterotrophic culture of microalgae, the step of diluting the obtained microalgae heterotrophic culture solution, and performing light-induced culture, and The selected algae cell harvesting, lutein separation and extraction steps.
本发明还提供一种源于微藻的叶黄素生产方法， 该方法包括微藻异养的步骤， 将微 藻的异养培养液稀释后进行光诱导培养的步骤， 以及藻细胞采收、 叶黄素分离提取的步 骤。 本发明的方法能够实现胞内叶黄素的快速积累， 大大提高了生产效率， 降低了生产 成本， 且提供了高品质的叶黄素。  The present invention also provides a method for producing lutein derived from microalgae, which comprises the steps of heterotrophic microalgae, the step of diluting the heterotrophic culture solution of the microalgae for light-induced culture, and the harvesting of algae cells, The step of separating and extracting lutein. The method of the invention can achieve rapid accumulation of intracellular lutein, greatly improve production efficiency, reduce production cost, and provide high quality lutein.
在一个具体实施方式中， 所述的微藻选自： 绿藻门小球藻属中的蛋白核小球藻 ( Chlorella pyrenoidosa) ， 普通小球藻 ( Chlorella vulgaris ， #有圆小球藻 ( Chlorella ellipsoidea)， Chlorella emersonii, Chlorella sorokiniana, Chlorella saccharophila, Chlorella regular is, Chlorella minutissima, Chlorella protothecoides, Chlorella zofingiensis, 以及绿 藻门中的 Brachiomonas submarina， Chlamydobonas reinhardtii， Chlamydomonas acidophila, Haematococcus pluvialis , Haematococcus lacustris, Scenedesmus obliquus , Spongiococcum exetriccium Tetraselmis suecica, Tetraselmis chuii Tetraselmis tetrathele, Tetraselmis verrucosa, Micractinium pusillum; 藻门的 Cylindrotheca fusiformis, Nitzschia laevis , Nitzschia alba, Nitzschia fonticola, Navicula incerta, Navicula pelliculosa 蓝藻门 的 Anabaena variabilis; 金藻门的 Poterioochromonas malhame is; 甲藻门的 Amphidinium carterae， Crypthecodinium cohnii； 裸藻门的 Euglena gricilis； 红藻门的 Galdieria sulphur aria In a specific embodiment, the microalgae is selected from the group consisting of: Chlorella pyrenoidosa in the genus Chlorella, Chlorella vulgaris, Chlorella vulgaris Ellipsoidea), Chlorella emersonii, Chlorella sorokiniana, Chlorella saccharophila, Chlorella regular is, Chlorella minutissima, Chlorella protothecoides, Chlorella zofingiensis, and Brachiomonas submarina, Chlamydobonas reinhardtii, Chlamydomonas Acidophila, Haematococcus pluvialis, Haematococcus lacustris, Scenedesmus obliquus, Spongiococcum exetriccium Tetraselmis suecica, Tetraselmis chuii Tetraselmis tetrathele, Tetraselmis verrucosa, Micractinium pusillum; Cylindrotheca fusiformis, Nitzschia laevis, Nitzschia alba, Nitzschia fonticola, Navicula incerta, Navicula pelliculosa Anabaena variabilis; Poterioochromonas malhame is of the genus Algae; Amphidinium carterae, Crypthecodinium cohnii of the genus Algae; Euglena gricilis of the genus Eucalyptus; Galdieria sulphur aria of the red algae
在一个具体实施方式中， 所述微藻选自绿藻门小球藻属的藻类。 尤其是， 所述微藻 选自绿藻门小球藻属中的蛋白核小球藻 Ch re py idoscd， 普通小球藻 ( Chlorella vulgaris)，
Figure imgf000006_0001
( Chlorella ellipsoidea ， Chlorella enter onii, Chlorella sorokiniana, Chlorella saccharophila, Chlorella regular is, Chlorella minutissima, Chlorella protothecoides 禾口 Chlorella zofingiemis In a specific embodiment, the microalgae is selected from the group consisting of algae of the genus Chlorella. In particular, the microalgae is selected from the group consisting of Ch re py idoscd, Chlorella vulgaris, of the genus Chlorella.
Figure imgf000006_0001
(Chlorella ellipsoidea, Chlorella enter onii, Chlorella sorokiniana, Chlorella saccharophila, Chlorella regular is, Chlorella minutissima, Chlorella protothecoides and Chlorella zofingiemis
在一个具体实施方式中， 所述微藻选自蛋白核小球藻 Ch rena pyrenoidoscO ， 普 通小球藻 ( Chlorella vulgaris) 禾口椭圆小球藻 ( Chlorella ellipsoidea) 。  In a specific embodiment, the microalgae is selected from the group consisting of Chrena pyrenoidoscO, Chlorella vulgaris, and Chlorella ellipsoidea.
在一个具体实施方式中， 所述微藻异养的步骤包括： 在生物反应器中加入 pH 为 4.0 10.0的培养基， 按工作体积的 0.1~30%接入微藻藻种进行分批培养、 补料分批培养、 连续培养或半连续培养， 培养温度为 10~50°C， 控制 pH小于 10.0， 控制溶氧在 1 %以上。  In a specific embodiment, the microalgae heterotrophic step comprises: adding a medium having a pH of 4.0 10.0 to the bioreactor, and accessing the microalgae algae according to a working volume of 0.1 to 30% for batch culture, Feeding batch culture, continuous culture or semi-continuous culture, the culture temperature is 10~50 °C, the control pH is less than 10.0, and the dissolved oxygen is controlled above 1%.
在另一具体实施方式中， 例如， 在生产叶黄素的实施例中， 所述微藻异养培养的步 骤可包括： 在生物反应器中加入 pH为 4.0 10.0 (例如 4.0〜9.0 ) 的培养基， 按工作体积 的 0.1~30%接入微藻藻种进行分批培养、 补料分批培养、 半连续培养或连续培养， 培养 温度为 10~50°C (例如 10〜40°C ) ， 控制 pH小于 10.0 (例如， 小于 9.0 ) ， 控制溶氧在 1 %以上。  In another embodiment, for example, in the embodiment for producing lutein, the step of heterogeneous culture of the microalgae may include: adding a culture having a pH of 4.0 10.0 (for example, 4.0 to 9.0) in the bioreactor. Base, according to the working volume of 0.1~30% access to the microalgae species for batch culture, fed-batch culture, semi-continuous culture or continuous culture, the culture temperature is 10~50 °C (for example, 10~40 °C) , control pH is less than 10.0 (for example, less than 9.0), control dissolved oxygen above 1%.
在一个具体实施方式中， 所述藻液稀释包括用培养基将异养获得的藻液稀释至细胞 密度为 0.1 50克 /升， 所述培养基不含有机碳源， 其 pH为 4.0 10.0。  In a specific embodiment, the algal fluid dilution comprises diluting the heterotrophically obtained algal fluid to a cell density of 0.150 g/l with a medium having no organic carbon source and having a pH of 4.0 10.0.
在另一具体实施方式中， 例如， 在生产叶黄素的实施例中， 所述藻液稀释可包括用 培养基将异养获得的藻液稀释至细胞密度为 0.1 20克 /升， 所述培养基不含有机碳源， 其 pH为 4.0~9.0。  In another embodiment, for example, in the embodiment for producing lutein, the diluting the algae liquid may comprise diluting the heterotrophic obtained algae liquid to a cell density of 0.120 g/L with the medium, The medium does not contain an organic carbon source and has a pH of 4.0 to 9.0.
在一个具体实施方式中， 所述光诱导培养包括将稀释后的藻液转入光诱导装置中进 行光诱导， 连续光照或间歇光照， 培养温度为 5~50°C， 光照强度为 0.1~150kk， 光诱导 培养周期为 1 150小时。  In a specific embodiment, the light-induced culture comprises transferring the diluted algal liquid into a light-inducing device for light-induced, continuous illumination or intermittent illumination, the culture temperature is 5 to 50 ° C, and the illumination intensity is 0.1 to 150 kk. The light-induced culture period was 1 150 hours.
在一个具体实施方式中， 异养培养基由氮源、 有机碳源以及少量无机盐、 微量元素 和水组成； 光诱导培养基由氮源、 无机盐和水组成。  In a specific embodiment, the heterotrophic medium consists of a nitrogen source, an organic carbon source, and a small amount of inorganic salts, trace elements, and water; the photoinduction medium consists of a nitrogen source, an inorganic salt, and water.
在一个具体实施方式中， 所述异养步骤在摇瓶、 机械搅拌式、 气升式或鼓泡式可异 养培养的生物反应器中进行，所述光诱导培养步骤在摇瓶或选自敞开式的跑道池或圆池、 封闭式的平板式光生物反应器或管道式光生物反应器或柱式光生物反应器或薄膜立袋与 吊袋光生物反应器等任何可用于微藻光自养培养的装置中进行， 光照条件为自然光或人 工光照射。 In a specific embodiment, the heterotrophic step is carried out in a shake flask, mechanically agitated, airlifted or bubbling heterotrophic culture bioreactor, the light inducing culture step is in a shake flask or selected from Open runway pool or round pool, Closed flat photobioreactor or ducted photobioreactor or column photobioreactor or film pouch and sling photobioreactor, any device that can be used for microalgae photoautotrophic culture, illumination The condition is natural light or artificial light.
在一个具体实施方式中， 例如， 在产油脂的实施例中， 当小球藻为普通小球藻时， 异养所使用的培养基基本上由以下成分组成： KN03 5-15克 /升、 葡萄糖 10~60克 /升、 KH2P04 0.3-0.9克 /升、 Na2HP04'12H20 1.0-10.0克 /升、 MgS04'7H20 0.2-1.0克 /升、 CaCl2 0.05~0.3克 /升、 FeS(V7H20 0.01 0.05克 /升； 微量元素 0.5~4ml， 其中微量元素的组成为 H3B03 5-15 克 /升， ZnS04'7H20 5.0-10.0 克 /升， MnCl2'H20 1.0-2.0 克 /升， ( Η4)6Μο7024·4Η20 0.5-1.5克 /升， CuS04'5H20 1.0-2.0克 /升， Co(N03)2'6H20 0.1-0.9克 / 升； 水。 在一个具体实施方式中， 当小球藻为蛋白核小球藻时， 异养所使用的培养基基 本上由以下成分组成： 葡萄糖 10~60 克 /升， 尿素 2~8 克 /升， KH2P04 1-2 克 /升， Na2HP04-12H20 1.0-10.0克 /升， MgS04'7H20 1-2克 /升， CaCl2 0.05-0.1克 /升， 柠檬酸三 钠 0.1~2.0克 /升， Fe-EDTA溶液 0.5~1 mL, A5溶液 l~5mL; 其中 Fe-EDTA溶液配方为 FeS04-7H20 20-30克 /升和 EDTA 20-40克 /升； A5溶液配方为 H3B03 2.5-4.0克 /升， MnCl2-4H20 1.0-2.0克 /升， ZnS04'7H20 0.1-0.6克 /升， CuS04'5H20 5-10克 /升， Na2Mo04 0.01-0.05克 /升； 水。 In a specific embodiment, for example, in the case of producing oil and fat, when the chlorella is common chlorella, the medium used for heterotrophy consists essentially of the following components: KN0 3 5-15 g/l , glucose 10~60g/L, KH 2 P0 4 0.3-0.9g/L, Na 2 HP0 4 '12H 2 0 1.0-10.0 g/L, MgS0 4 '7H 2 0 0.2-1.0 g/L, CaCl 2 0.05~0.3g/L, FeS(V7H 2 0 0.01 0.05g/L; trace element 0.5~4ml, wherein the composition of trace elements is H 3 B0 3 5-15 g / liter, ZnS0 4 '7H 2 0 5.0-10.0克 / liter, MnCl 2 'H 2 0 1.0-2.0 g / liter, ( Η 4 ) 6 Μο 7 0 24 ·4Η 2 0 0.5-1.5 g / liter, CuS0 4 '5H 2 0 1.0-2.0 g / liter, Co(N0 3 ) 2 '6H 2 0 0.1-0.9 g / liter; water. In a specific embodiment, when the chlorella is Chlorella chlorella, the medium used for heterotrophy is basically composed of the following components Composition: glucose 10~60 g/l, urea 2~8 g/l, KH 2 P0 4 1-2 g/l, Na 2 HP0 4 -12H 2 0 1.0-10.0 g/l, MgS0 4 '7H 2 0 1-2 g / liter, CaCl 2 0.05-0.1 g / liter, trisodium citrate 0.1 ~ 2.0 g / liter, Fe-EDTA solution 0.5~1 mL, A5 solution l~5mL; Fe-EDTA solution formula is FeS0 4 -7H 2 0 20-30 g / liter and EDTA 20-40 g / liter; A5 solution formula is H 3 B0 3 2.5-4.0克 / liter, MnCl 2 -4H 2 0 1.0-2.0 g / liter, ZnS0 4 '7H 2 0 0.1-0.6 g / liter, CuS0 4 '5H 2 0 5-10 g / liter, Na 2 Mo0 4 0.01-0.05 g/l; water.
在另一具体实施方式中， 例如在产叶黄素的实施例中， 当藻种为普通小球藻时， 异 养所使用的培养基基本上由以下成分组成: KN03 5~15克 /升、葡萄糖 10~60克 /升、 KH2P04 0.3-0.9克 /升、 Na2HP04'12H20 1.0-10.0克 /升、 MgS04'7H20 0.2-1.0克 /升、 CaCl2 0.05-0.3 克 /升、 FeS(V7H20 0.01-0.05克 /升、微量元素 0.5~4ml和水，其中微量元素的组成为 H3B03 5~15克 /升、 ZnS04'7H20 5.0~10.0克 /升、 MnCl2'H20 1.0~2.0克 /升、 （ Η4)6Μο7024·4Η20 0.5-1.5克 /升、 CuS04'5H20 1.0-2.0克 /升和 Co(N03)2'6H20 0.1-0.9克 /升。 In another embodiment, for example, in the embodiment of producing lutein, when the algae species is Chlorella vulgaris, the medium used for heterotrophy consists essentially of the following components: KN0 3 5-15 g/ L, glucose 10~60 g/L, KH 2 P0 4 0.3-0.9 g/L, Na 2 HP0 4 '12H 2 0 1.0-10.0 g/L, MgS0 4 '7H 2 0 0.2-1.0 g/L, CaCl 2 0.05-0.3 g / liter, FeS (V7H 2 0 0.01-0.05 g / liter, trace elements 0.5 ~ 4ml and water, wherein the composition of trace elements is H 3 B0 3 5 ~ 15 g / liter, ZnS0 4 '7H 2 0 5.0~10.0 g/L, MnCl 2 'H 2 0 1.0~2.0 g/L, ( Η4) 6 Μο 7 0 24 ·4Η 2 0 0.5-1.5 g/L, CuS0 4 '5H 2 0 1.0-2.0 g /L and Co(N0 3 ) 2 '6H 2 0 0.1-0.9 g/L.
在一个具体实施方式中， 例如在产叶黄素的实施例中， 当小球藻为蛋白核小球藻时， 异养所使用的培养基基本上由以下成分组成：葡萄糖 10~60克 /升、尿素 2~8克 /升、 KH2P04 1~2克 /升、 Na2HP04'12H20 1.0~10.0克 /升、 MgS04'7H20 1~2克 /升、 CaCl2 0.05-0.1 1 升、 柠檬酸三钠 0.1~2.0克 /升、 Fe-EDTA溶液 0.5~1 mL、 A5溶液 l~5mL和水； 其中 Fe-EDTA溶液配方为 FeS(V7H20 20-30克 /升和 EDTA 20-40克 /升； A5溶液配方为 H3B03 2.5-4.0克 /升、 MnCl2'4H20 1.0-2.0克 /升、 ZnS04'7H20 0.1-0.6克 /升、 CuS04'5H20 5-10克 /升和 Na2Mo04 0.01-0.05克 /升。 In a specific embodiment, for example, in the embodiment of producing lutein, when the chlorella is Chlorella chlorella, the medium used for heterotrophy consists essentially of the following components: glucose 10 to 60 g /升, Urea 2~8g/L, KH 2 P0 4 1~2g/L, Na 2 HP0 4 '12H 2 0 1.0~10.0g/L, MgS0 4 '7H 2 0 1~2g/L, CaCl 2 0.05-0.1 1 liter, trisodium citrate 0.1~2.0 g/L, Fe-EDTA solution 0.5~1 mL, A5 solution 1-5 mL and water; wherein Fe-EDTA solution is FeS (V7H 2 0 20-30克 / liter and EDTA 20-40 g / liter; A5 solution formula is H 3 B0 3 2.5-4.0 g / liter, MnCl 2 '4H 2 0 1.0-2.0 g / liter, ZnS0 4 '7H 2 0 0.1-0.6 g /L, CuS0 4 '5H 2 0 5-10 g / liter and Na 2 Mo0 4 0.01-0.05 g / liter.
在一个具体实施方式中， 采用超临界 co2萃取法、 有机溶剂提取法或超声辅助溶剂 提取法提取叶黄素。 In a specific embodiment, lutein is extracted by supercritical co 2 extraction, organic solvent extraction or ultrasonic assisted solvent extraction.
在一个具体实施方式中， 本发明的方法还包括： 将提取叶黄素后的藻体与其他色素 混合进行喷雾干燥制成藻粉， 或对藻体中的生物活性物质进行分离提取。  In a specific embodiment, the method of the present invention further comprises: mixing the algal body after extracting lutein with other pigments, spray-drying to prepare algal flour, or separating and extracting the biologically active substance in the algal body.
在一个具体实施方式中， 所述其他色素包括叶绿素。 在一个具体实施方式中， 所述 生物活性物质包括蛋白质、 叶绿素、 多糖、 脂肪酸、 小球藻生长因子等。 In a specific embodiment, the other pigment comprises chlorophyll. In a specific embodiment, the Biologically active substances include proteins, chlorophyll, polysaccharides, fatty acids, chlorella growth factors, and the like.
在一个具体的实施方式中， 所述油脂分离提取方法采用有机溶剂提取法。  In a specific embodiment, the oil separation and extraction method employs an organic solvent extraction method.
在另一具体实施例中， 所述微藻培养方法还包括提取微藻油脂的步骤。  In another specific embodiment, the microalgae culture method further comprises the step of extracting microalgae oil.
本申请还提供一种油脂生产方法， 该方法包括异养培养微藻的步骤， 将异养培养的 微藻藻液稀释后进行光诱导培养的步骤， 以及藻细胞采收、 油脂分离提取的步骤。  The present application also provides a method for producing a fat or oil, the method comprising the steps of heterotrophic cultivation of microalgae, the step of diluting the heterotrophic cultured microalgae solution for light-induced culture, and the step of collecting algae cells and separating and extracting oils and fats. .
本申请的油脂生产方法中使用到的微藻、 培养基以及培养条件等如本文上文以及下 文具体实施方式部分所详述。  The microalgae, the culture medium, and the culture conditions and the like used in the method for producing a fat or oil of the present application are as described in the above and the following detailed description of the specific embodiments.
用本发明公开的异养-稀释-光诱导串联技术培养小球藻， 首先在密闭式生物反应器 中高密度异养培养小球藻， 待培养液中有机碳源消耗完毕后， 用不含有机碳源的培养基 稀释藻液， 经短时间内 （8~24h) 的光照诱导， 可使藻细胞内的油脂含量快速大量积累， 油脂含量在短时间内 （例如 8~24小时） 比异养阶段的藻细胞提高约一倍（异养阶段胞内 油脂含量一般不超过 10%， 经光诱导后可达 20~30%) 。 附图简述  The bacterium is cultured by the heterotrophic-dilution-light-inducing tandem technique disclosed in the present invention. First, high-density heterotrophic culture of chlorella is carried out in a closed bioreactor. After the organic carbon source in the culture solution is consumed, the organic medium is not used. The carbon source medium dilutes the algae solution, and after a short period of time (8~24h), the oil content in the algae cells can be accumulated rapidly and the oil content is short-term (for example, 8~24 hours) than the heterotrophic The algae cells in the stage are about doubled (the intracellular fat content in the heterotrophic stage is generally not more than 10%, and can reach 20~30% after light induction). BRIEF DESCRIPTION OF THE DRAWINGS
图 1显示在 50L生物反应器 /3L平板式光生物反应器串联***中采用异养-稀释-光 诱导串联培养蛋白核小球藻快速积累油脂的结果 （同时测定了细胞内其他主要生化成 分） 。  Figure 1 shows the results of heterotrophic-dilution-light-induced tandem culture of Chlorella pyrenoidosa for rapid accumulation of oil in a 50L bioreactor/3L flat photobioreactor tandem system (simultaneous determination of other major biochemical components in the cell) .
图 2显示 5T发酵罐串联至户外 30L平板和 60L大盆连续培养普通小球藻光诱导阶 段户外自然光强和自然温度的变化曲线。  Figure 2 shows the variation of the natural light intensity and natural temperature of the 5T fermenter in series with the outdoor 30L plate and the 60L large basin for continuous cultivation of Chlorella vulgaris in the light-induced phase.
图 3显示 5T发酵罐串联至户外 30L平板培养普通小球藻快速积累油脂的过程曲线 (同时测定了细胞内其他主要生化成分） 。  Figure 3 shows the process curve of the rapid accumulation of oil in the 30T fermenter in series to the outdoor 30L plate culture of Chlorella vulgaris (the other major biochemical components in the cell were also measured).
图 4显示 5T发酵罐串联至户外 60L大盆培养普通小球藻快速积累油脂的过程曲线 (同时测定了细胞内其他主要生化成分） 。  Figure 4 shows the process curve of the 5T fermenter connected in series to the outdoor 60L large pot cultured Chlorella vulgaris to accumulate oil quickly (the other major biochemical components in the cell were also measured).
图 5显示 5T发酵罐串联至户外 20m2跑道池和 3.14m2圆池连续培养普通小球藻光 诱导阶段户外自然光强和自然温度的变化曲线。 Figure 5 shows the variation of the outdoor natural light intensity and natural temperature during the light induction phase of the conventional chlorella in the 5T fermentor connected to the outdoor 20m 2 runway pool and the 3.14m 2 round pool.
图 6显示 5T发酵罐串联至户外 20m2跑道池培养普通小球藻快速积累油脂的过程曲 线 （同时测定了细胞内其他主要生化成分） 。 Figure 6 shows the process curve of the rapid accumulation of oil in the culturing of the chlorella by the 5T fermentor in series to the outdoor 20m 2 runway pool (the other major biochemical components in the cell were also determined).
图 7显示 5T发酵罐串联至户外 3.14m2圆池培养普通小球藻快速积累油脂的过程曲 线 （同时测定了细胞内其他主要生化成分） 。 Figure 7 shows the process curve of the rapid accumulation of oil in the 5T fermenter in series to the outdoor 3.14m 2 round cell culture of common chlorella (simultaneous determination of other major biochemical components in the cell).
图 8显示 500mL摇瓶 /3L圆柱式光反应器串联培养椭圆小球藻胞内主要生化成分的 变化曲线。  Figure 8 shows the variation of the main biochemical components in the cells cultured in a 500 mL shake flask / 3 L cylindrical photoreactor.
图 9和图 10显示 5L生物反应器 /3L平板式光生物反应器串联***中采用异养-稀释- 光诱导串联培养蛋白核小球藻生产叶黄素的过程。其中， 图 9显示蛋白核小球藻在 5L生 物反应器异养培养过程； 图 10显示蛋白核小球藻在 3L平板式光生物反应器内光诱导培 养过程。 Figures 9 and 10 show the process of producing lutein by heterotrophic-dilution-light-induced tandem culture of Chlorella pyrenoidosa in a 5L bioreactor/3L flat photobioreactor tandem system. Figure 9 shows the heterotrophic culture process of Chlorella pyrenoids in a 5L bioreactor; Figure 10 shows light-induced culture of Chlorella pyrenoids in a 3L flat photobioreactor Raise the process.
图 11和图 12显示 50L生物反应器 /10L圆柱式光生物反应器串联***中采用异养- 稀释-光诱导串联培养蛋白核小球藻生产叶黄素的过程。 其中， 图 11 显示蛋白核小球藻 在 50L生物反应器异养培养过程；图 12显示蛋白核小球藻在 10L圆柱式光生物反应器内 光诱导培养过程。  Figure 11 and Figure 12 show the process of producing lutein by heterotrophic-dilution-light-induced tandem culture of Chlorella pyrenoids in a 50L bioreactor/10L cylindrical photobioreactor tandem system. Among them, Figure 11 shows the heterotrophic culture process of Chlorella pyrenoids in a 50L bioreactor; Figure 12 shows the photoinduced culture process of Chlorella pyrenoidosa in a 10L cylindrical photobioreactor.
图 13和图 15显示 50L生物反应器 /30L平板式光生物反应器串联***中采用异养- 稀释-光诱导串联培养普通小球藻生产叶黄素的过程。其中， 图 13显示普通小球藻在 50L 生物反应器中异养培养过程；图 15显示普通小球藻在户外 30L平板式光生物反应器内光 诱导培养。  Figures 13 and 15 show the process of producing lutein by heterotrophic-dilution-light-induced tandem culture of Chlorella vulgaris in a 50L bioreactor/30L flat photobioreactor cascade system. Among them, Fig. 13 shows the heterotrophic culture process of the common chlorella in the 50L bioreactor; Fig. 15 shows the photoinduced culture of the ordinary chlorella in the outdoor 30L flat photobioreactor.
图 14和图 16显示 5L生物反应器 /3L平板式生物光生物反应器串联***中采用异养 -稀释-光诱导串联培养椭圆小球藻生产叶黄素的过程。其中， 图 14显示椭圆小球藻在 5L 生物反应器异养培养过程； 图 16显示椭圆小球藻在 3L平板式光生物反应器中光诱导过 程。 具体实施方案  Figure 14 and Figure 16 show the process of producing lutein by heterotrophic-dilution-light-induced tandem culture of N. glabrata in a 5L bioreactor/3L flat biophotobioreactor cascade system. Figure 14 shows the heterotrophic culture process of Chlorella ellipses in a 5L bioreactor; Figure 16 shows the photoinduced process of Chlorella ellipses in a 3L flat photobioreactor. Specific implementation
适用于本申请的微藻包括但不限于绿藻门小球藻属中的蛋白核小球藻 （ Chlorella pyrenoidosa) ， 普通小球藻 ( Chlorella vulgaris ) ， #有圆小球藻 (. Chlorella ellipsoidea) ， Chlorella emersonii, Chlorella sorokiniana, Chlorella saccharophila, Chlorella regularis, Chlorella minutissima, Chlorella protothecoides, Chlorella zofingiensis , 以及绿藻门中的 Brachiomonas submarina， Chlamydobonas reinhardtii， Chlamydomonas acidophila， Haematococcus pluvialis, Haematococcus lacustris, Scenedesmus obliquus, Spongiococcum exetriccium, Tetraselmis suecica, Tetraselmis chuii, Tetraselmis tetrathele , Tetraselmis verrucosa, Micractinium pusillum-,
Figure imgf000009_0001
Cylindrotheca fusiformis, Nitzschia laevis, Nitzschia alba , Nitzschia fonticola , Navicula incerta , Navicula pelliculosa； 蓝藻门的 Anabaena variabilis-, 金藻门的 Poterioochromonas malhamensis; 甲藻门的 Amphidinium carterae， Crypthecodinium cohnii； 裸藻门的 Euglena gricilis； 红藻门的 Galdieria sulphur aria 0 Microalgae suitable for use in the present application include, but are not limited to, Chlorella pyrenoidosa in the genus Chlorella, Chlorella vulgaris, # Chlorella ellipsoidea , Chlorella emersonii, Chlorella sorokiniana, Chlorella saccharophila, Chlorella regularis, Chlorella minutissima, Chlorella protothecoides, Chlorella zofingiensis, and Brachiomonas submarina, Chlamydobonas reinhardtii, Chlamydomonas acidophila, Haematococcus pluvialis, Haematococcus lacustris, Scenedesmus obliquus, Spongiococcum exetriccium, Tetraselmis Suecica, Tetraselmis chuii, Tetraselmis tetrathele, Tetraselmis verrucosa, Micractinium pusillum-,
Figure imgf000009_0001
Cylindrotheca fusiformis, Nitzschia laevis, Nitzschia alba, Nitzschia fonticola, Navicula incerta, Navicula pelliculosa; Anabaena variabilis- of Cyanophyta, Poterioochromonas malhamensis of the genus Cymbidium; Amphidinium carterae, Crypthecodinium cohnii of the genus Algae; Euglena gricilis of the genus Eucalyptus; Algae's Galdieria sulphur aria 0
在优选的实施方式中， 本发明采用绿藻门小球藻属的藻类来生产油脂。 在更优选的 实施方式中， 本发明采用蛋白核小球藻、 普通小球藻或椭圆小球藻来生产油脂。 在优选 的实施方式中， 本发明采用蛋白核小球藻、 普通小球藻和椭圆小球藻来生产叶黄素。  In a preferred embodiment, the present invention utilizes algae of the genus Chlorella Chlorella to produce oils and fats. In a more preferred embodiment, the present invention employs Chlorella pyrenoidosa, Chlorella vulgaris or Chlorella ellipses to produce oils and fats. In a preferred embodiment, the invention employs Chlorella pyrenoidosa, Chlorella vulgaris, and Chlorella ellipsoid to produce lutein.
1. 微藻在生物反应器中的高密度异养培养 1. High-density heterotrophic culture of microalgae in bioreactor
此步骤的目的是为了快速获得大量藻细胞， 以供光诱导阶段快速合成积累各种活性 成分， 尤其包括油脂、 色素、 蛋白质、 多糖等。 可采用本领域熟知的各种培养基来进行微藻异养培养。通常，异养培养基含有氮源、 有机碳源、 少量无机盐、 微量元素和水。 适用于微藻培养的氮源、 有机碳源、 无机盐、 微量元素等是本领域周知的。例如， 作为氮源， 可使用的有尿素或各种硝酸盐， 如 KN03 等； 作为有机碳源， 可使用的有例如葡萄糖等。 The purpose of this step is to rapidly obtain a large number of algae cells for rapid synthesis and accumulation of various active ingredients in the light-inducing stage, including oils, pigments, proteins, polysaccharides and the like. The microalgae heterotrophic culture can be carried out using various media well known in the art. Typically, the heterotrophic medium contains a nitrogen source, an organic carbon source, a small amount of inorganic salts, trace elements, and water. Nitrogen sources, organic carbon sources, inorganic salts, trace elements, and the like suitable for microalgae culture are well known in the art. For example, as the nitrogen source, urea or various nitrates such as KN0 3 may be used, and as the organic carbon source, for example, glucose or the like may be used.
这类培养基包括 HA-SK 培养基 （中国专利 ZL 200610024004.9 ) 、 Endo 培养基 ( Ogbonna J.C., Masui. H.， Tanaka.H. Sequential heterotrophic: autotrophic cultivation一 an efficient method of producing Chlorella biomass for health food and animal feed. J. Appl. Phycol.1997, 9, 359-366) 等。  Such media include HA-SK medium (Chinese patent ZL 200610024004.9), Endo medium (Ogbonna JC, Masui. H., Tanaka. H. Sequential heterotrophic: autotrophic cultivation an effective method of producing Chlorella biomass for health food and Animal feed. J. Appl. Phycol. 1997, 9, 359-366) and so on.
本发明所用的 HA-SK培养基是基本上由 KN03、葡萄糖以及少量无机盐、微量元素 和水组成。 在所述技术方案中， 所述微量元素宜选自 H3B03 , ZnS04-7H20, MnCl2-H20, ( Η4)6Μο7024·4Η20, CuS04-5H20, Co(N03)2 ·6Η20中的一种或多种或全部。 The HA-SK medium used in the present invention consists essentially of KNO 3 , glucose, and a small amount of inorganic salts, trace elements, and water. In the above technical solution, the trace element is preferably selected from the group consisting of H 3 B0 3 , ZnS0 4 -7H 2 0, MnCl 2 -H 2 0, ( Η 4 ) 6 Μο 7 0 24 ·4Η 2 0, CuS0 4 - One or more or all of 5H 2 0, Co(N0 3 ) 2 ·6Η 2 0 .
本文所用的术语 "基本上由……组成"表示本发明的组合物中除了含有主要组分 KN03 葡萄糖以及少量无机盐、 微量元素和水外， 还可包含一些对于组合物的基本特性 或新的特性 （即可维持微藻在较短的培养周期内细胞密度达到较高的水平， 同时活性物 质含量与常规异养培养相比有较大幅度提高） 没有实质上影响的组分。 本文所用的术语 "由……组成"表示本发明的组合物由所指出的具体组分组成， 没有其他组分， 但是可 以带有含量在通常范围内的杂质。 The term "consisting essentially of" as used herein means that the composition of the present invention may contain some essential properties or new to the composition in addition to the main component KNO 3 glucose and a small amount of inorganic salts, trace elements and water. The characteristics (ie, maintaining the microalgae at a higher cell density in a shorter culture period, while the active substance content is significantly increased compared to conventional heterotrophic culture) have no substantially affected components. The term "consisting of" as used herein means that the composition of the present invention consists of the specific components indicated, without other components, but may carry impurities in a usual range.
在该培养基中， 培养基的各组分可在一定范围内变化而不会对微藻细胞密度和品质 有很大的实质影响。 因此， 这些组分的用量不应受实施例的严格限制。 如本领域技术人 员所熟知的， 培养基中还可加入少量无机盐， 例如硫酸镁、 氯化钙、 硫酸亚铁和磷酸盐 等， 以及少量微量元素如 Μη、 Ζη、 Β、 I、 M、 Cu、 Co等。  In this medium, the components of the medium can be varied within a certain range without greatly affecting the density and quality of the microalgae cells. Therefore, the amounts of these components should not be strictly limited by the examples. As is well known to those skilled in the art, a small amount of inorganic salts such as magnesium sulfate, calcium chloride, ferrous sulfate, and phosphate may be added to the medium, and a small amount of trace elements such as Μη, Ζη, Β, I, M, Cu, Co, etc.
在本发明中， 较佳的微量元素组分宜选自 H3B03, ZnS04-7H20, MnCl2-H20, ( Η4)6Μο7024·4Η20, CuS04-5H20, Co(N03)2-6H20中的一种或多种。无机盐和微量元素的 用量可根据常规知识确定。 In the present invention, a preferred trace element component is preferably selected from the group consisting of H 3 B0 3 , ZnS0 4 -7H 2 0, MnCl 2 -H 2 0, ( Η 4 ) 6 Μο 7 0 24 ·4Η 2 0, CuS0 4 One or more of -5H 2 0, Co(N0 3 ) 2 -6H 2 0 . The amount of inorganic salts and trace elements can be determined based on conventional knowledge.
本发明所采用的 HA-SK培养基基本上由以下成分组成： KN03 5~15克 /升、 葡萄糖 10-60克 /升、 KH2P04 0.3-0.9克 /升、 Na2HP04' 12H20 1.0-10.0克 /升、 MgS04'7H20 0.2-1.0 克 /升、 CaCl2 0.05 0.3克 /升、 FeS04'7H20 0.01~0.05克 /升; 微量元素 0.5~4ml， 其中微量 元素的组成为 H3B03 5~15克 /升， ZnS04-7H20 5.0~10.0克 /升， MnCl2-H20 1.0~2.0克 /升, ( Η4)6Μο7024·4Η20 0.5-1.5克 /升， CuS04'5H20 1.0-2.0克 /升， Co(N03)2'6H20 0.1-0.9克 / 升； 水。 The HA-SK medium used in the present invention consists essentially of the following components: KN0 3 5~15 g/L, glucose 10-60 g/L, KH 2 P0 4 0.3-0.9 g/L, Na 2 HP0 4 ' 12H 2 0 1.0-10.0 g / liter, MgS0 4 '7H 2 0 0.2-1.0 g / liter, CaCl 2 0.05 0.3 g / liter, FeS0 4 '7H 2 0 0.01 ~ 0.05 g / liter; trace elements 0.5 ~ 4ml, The composition of trace elements is H 3 B0 3 5~15 g / liter, ZnS0 4 -7H 2 0 5.0~10.0 g / liter, MnCl 2 -H 2 0 1.0~2.0 g / liter, ( Η 4 ) 6 Μο 7 0 24 ·4Η 2 0 0.5-1.5 g/l, CuS0 4 '5H 2 0 1.0-2.0 g/l, Co(N0 3 ) 2 '6H 2 0 0.1-0.9 g/l; water.
在一个较佳的实施方案中，本发明的 HA-SK培养基组合物宜由以下成分组成: KN03 7克 /升、葡萄糖 40克 /升、 KH2P04 0.6克 /升、 Na2HP(V 12H20 2.0克 /升、 MgS04'7H20 0.8 克 /升、 CaCl2 0.2克 /升、 FeS(V7H2O 0.03克 /升； 微量元素 1.5mL， 其中微量元素的组成 为 H3B03 11-12 克 /升， ZnS04-7H20 8.5-9.5 克 /升， MnCl2 0 1.4-1.5 克 /升， ( Η4)6Μο7024·4Η20 0.8-0.9克 /升， CuS04'5H20 1.5-1.6克 /升， Co(N03)2'6H20 0.45-0.55克 /升； 水 1000mL。 In a preferred embodiment, the HA-SK medium composition of the present invention is preferably composed of the following components: KN0 3 7 g/L, glucose 40 g/L, KH 2 P0 4 0.6 g/L, Na 2 HP (V 12H 2 0 2.0 g / liter, MgS0 4 '7H 2 0 0.8 g / liter, CaCl 2 0.2 g / liter, FeS (V7H 2 O 0.03 g / liter; trace element 1.5mL, wherein the trace element composition is H 3 B0 3 11-12 g/l, ZnS0 4 -7H 2 0 8.5-9.5 g/l, MnCl 2 0 1.4-1.5 g/l, ( Η 4 ) 6 Μο 7 0 24 ·4Η 2 0 0.8-0.9 g/l, CuS0 4 '5H 2 0 1.5-1.6 g/l, Co(N0 3 ) 2 '6H 2 0 0.45-0.55 g/l; 1000 mL of water.
本发明所用的 Endo培养基基本上由以下成分组成： 葡萄糖 10 60克 /升， 尿素 2~8 克 /升， KH2P04 1-2克 /升， Na2HP04' 12H20 1.0-10.0克 /升， MgS04'7H20 1-2克 /升， CaCl2 0.05~0.1克 /升， 柠檬酸三钠 0.1~2.0克 /升， Fe-EDTA溶液 0.5~1 mL， A5溶液 l~5mL; 其中 Fe-EDTA溶液配方为 FeS(V7H20 20-30克 /升和 EDTA 20-40克 /升； A5溶液配方 为 H3B03 2.5~4.0克 /升， MnCl2'4H20 1.0~2.0克 /升， ZnS04'7H20 0. 〜 0.6克 /升， CuS04'5H20 5~10克 /升， Na2MoO4 0.01~0.05克 /升； 水。 The Endo medium used in the present invention consists essentially of the following components: glucose 10 60 g/l, urea 2-8 g/l, KH 2 P0 4 1-2 g/l, Na 2 HP0 4 ' 12H 2 0 1.0- 10.0 g / liter, MgS0 4 '7H 2 0 1-2 g / liter, CaCl 2 0.05 ~ 0.1 g / liter, trisodium citrate 0.1 ~ 2.0 g / liter, Fe-EDTA solution 0.5 ~ 1 mL, A5 solution ~5mL; wherein the Fe-EDTA solution is FeS (V7H 2 0 20-30 g / liter and EDTA 20-40 g / liter; A5 solution formula is H 3 B0 3 2.5 ~ 4.0 g / liter, MnCl 2 '4H 2 0 1.0~2.0 g / liter, ZnS0 4 '7H 2 0 0. ~ 0.6 g / liter, CuS0 4 '5H 2 0 5 ~ 10 g / liter, Na 2 MoO 4 0.01 ~ 0.05 g / liter;
在一个较佳的实施方案中， 所述 Endo培养基由以下成分组成： 葡萄糖 40克 /升， 尿 素 6.0克 /升， KH2P04 1.5克 /升， Na2HP04' 12H20 5.0克 /升， MgS04'7H20 1.8克 /升， CaCl2 0.05克 /升，柠檬酸三钠 0.4克 /升， Fe-EDTA溶液 0.8mL， A5溶液 2.0mL，其中 Fe-EDTA 溶液配方为 FeS(V7H20 25克 /升和 EDTA 33.5克 /升， A5溶液配方为 H3B03 2.86克 /升， MnCl2-4H20 1.81克 /升， ZnS04'7H20 0.222克 /升， CuS04'5H20 0.07克 /升， Na2Mo04 0.021 克 /升； 水。 In a preferred embodiment, the Endo medium consists of the following components: glucose 40 g/l, urea 6.0 g/l, KH 2 P0 4 1.5 g/l, Na 2 HP0 4 ' 12H 2 0 5.0 g /L, MgS0 4 '7H 2 0 1.8 g / liter, CaCl 2 0.05 g / liter, trisodium citrate 0.4 g / liter, Fe-EDTA solution 0.8 mL, A5 solution 2.0 mL, of which Fe-EDTA solution is FeS (V7H 2 0 25 g / liter and EDTA 33.5 g / liter, A5 solution formulation is H 3 B0 3 2.86 g / liter, MnCl 2 -4H 2 0 1.81 g / liter, ZnS0 4 '7H 2 0 0.222 g / liter, CuS0 4 '5H 2 0 0.07 g/l, Na 2 Mo0 4 0.021 g/l; water.
在根据上述配方配制培养基后， 可用常规手段如酸或碱将所述培养基的 pH调为 4.0-9.0, 并在 115~120°C下高压灭菌 15~20分钟。 可采用分批培养、 补料分批培养、 半 连续培养 （带放） 或连续培养等四种方式实施异养培养。  After the medium is formulated according to the above formulation, the pH of the medium can be adjusted to 4.0-9.0 by a conventional means such as an acid or a base, and autoclaved at 115 to 120 ° C for 15 to 20 minutes. Heterotrophic culture can be carried out in four ways, such as batch culture, fed-batch culture, semi-continuous culture (with release) or continuous culture.
当进行异养培养时， 将相应配制好的培养基加入到生物反应器中， 补加水至工作体 积，通常装料系数为 0.6 0.8，然后蒸汽灭菌（121 °C，维持约 20分钟），当温度降至 30~35 °C时， 按工作体积的 1~15%接入微藻开始异养培养。  When heterotrophic culture is carried out, the corresponding prepared medium is added to the bioreactor, and water is added to the working volume, usually with a charging coefficient of 0.6 0.8, and then steam sterilized (121 ° C, maintained for about 20 minutes). When the temperature drops to 30~35 °C, the microalgae are connected to the heterotrophic culture according to 1~15% of the working volume.
在异养培养一段时间后， 当培养基中葡萄糖被消耗完 （通常为 27~45小时） 时需要 进行补料， 补加碳源 （如葡萄糖） 、 氮源 （如， 培养普通小球藻的氮源为 KN03、 培养蛋 白核小球藻的氮源为尿素） 和无机盐等营养盐， 补加的营养盐是经浓缩后的上述相应培 养基， 促使微藻继续生长。 可每隔 5~8小时补料， 葡萄糖的补加浓度可为 15 25克 /升， 氮源溶液的补加浓度可为 2~5克 /升。 当补加一定次数 （一般为 4~7次） 的营养盐后， 微 藻细胞密度达到最高时， 异养培养阶段结束。 After a period of heterotrophic culture, when the glucose in the medium is consumed (usually 27~45 hours), it is necessary to supplement the feed, supplementing the carbon source (such as glucose) and nitrogen source (for example, culturing ordinary chlorella The nitrogen source is KN0 3 , the nitrogen source of the culture protein chlorella is urea, and the nutrient salt such as inorganic salt, and the supplemental nutrient salt is the corresponding medium which is concentrated to promote the growth of the microalgae. It can be fed every 5~8 hours. The additional concentration of glucose can be 15 25 g / liter, and the additional concentration of nitrogen source solution can be 2 ~ 5 g / liter. When the nutrient salt of a certain number of times (generally 4 to 7 times) is added, the cell density of the microalgae reaches the highest, and the heterotrophic culture stage ends.
无论采用何种培养方式， 在培养过程中， 须控制适合的培养条件使微藻正常生长。 通常， 控制温度为 20~35 °C， 例如 28~30°C， 溶氧不低于 5%的空气饱和浓度， pH不高于 9.0。 在优选的实施例中， 溶氧不低于 10%的空气饱和浓度， pH不高于 8.5。 在其它优选 的实施例中， 溶氧不低于 15%的空气饱和浓度， pH不高于 8。  Regardless of the culture method used, during the cultivation process, suitable culture conditions must be controlled to allow the microalgae to grow normally. Usually, the control temperature is 20~35 °C, for example, 28~30 °C, the dissolved oxygen is not less than 5% of the air saturation concentration, and the pH is not higher than 9.0. In a preferred embodiment, the dissolved oxygen is not less than 10% of the air saturation concentration and the pH is not higher than 8.5. In other preferred embodiments, the dissolved oxygen is not less than 15% of the air saturation concentration, and the pH is not higher than 8.
在培养过程中， pH不宜过高或过低， 一般随着培养的进行， pH会慢慢上升 （对于 普通小球藻此现象特别明显） ， pH过高会对藻细胞生长产生不利影响， 所以应用酸（例 如 10 %的硫酸） 进行调节， 使 pH不高于 9.0， 较佳的 pH应为 6.5~7.5。  During the cultivation process, the pH should not be too high or too low. Generally, as the culture progresses, the pH will rise slowly (this phenomenon is particularly obvious for ordinary chlorella). If the pH is too high, the growth of algae cells will be adversely affected. The acid (for example, 10% sulfuric acid) is adjusted so that the pH is not higher than 9.0, and the preferred pH is 6.5 to 7.5.
通常， 当采用分批培养、 补料分批培养时， 异养培养阶段结束时生物反应器中有机 碳源需完全消耗完。 当采用半连续培养方式时， 带放操作是在生物反应器中有机碳源完 全消耗完时进行。 无论采用何种异养培养方式， 转入光诱导的藻液， 在更佳的方案中应 使藻液中有机碳源例如葡萄糖含量为零或接近于零。 Usually, when batch culture or fed-batch culture is used, the bioreactor is organic at the end of the heterotrophic culture stage. The carbon source needs to be completely consumed. When the semi-continuous culture method is employed, the stripping operation is performed when the organic carbon source is completely consumed in the bioreactor. Regardless of the heterotrophic culture mode, the light-induced algae solution is transferred. In a better solution, the organic carbon source such as glucose in the algae solution should be zero or close to zero.
异养可以在摇瓶、机械搅拌式、气升式、鼓泡式等可异养培养的生物反应器中进行。  Heterotrophic can be carried out in a heterotrophic culture bioreactor such as shake flask, mechanical agitation, airlift, or bubbling.
2. 高浓度藻液的稀释 2. Dilution of high concentration algae
此步骤的目的是为了使转入光诱导培养的小球藻高效地吸收光能， 提高光能利用效 率， 同时降低藻细胞的死亡率。 因为光强在藻液中呈 "时、 空、 非线性"衰减， 所以在 高细胞密度下， 反应器中藻细胞大部分处于暗区， 几乎接受不到光照， 这样藻细胞很容 易死亡而且会影响光诱导的效率。  The purpose of this step is to enable light-inducing cultured chlorella to efficiently absorb light energy, improve light energy utilization efficiency, and reduce algal cell mortality. Because the light intensity is "time, empty, nonlinear" decay in the algae liquid, at high cell density, most of the algae cells in the reactor are in the dark area, and almost no light is received, so that the algae cells are easy to die and will Affects the efficiency of light induction.
异养培养获得的高密度藻液应进行稀释操作， 用水和不含有机碳源的培养基对高密 度的藻液进行稀释， 使细胞密度维持在 0.1 10克 /升， 调节 pH至 5.0 8.0。 在其它实施例 中，稀释藻液，使细胞密度维持在 1~8克 /升。在优选的实施例中，使细胞密度维持在 1.0 5.0 克 /升。 在其它实施例中， 使细胞密度维持在 2~5克 /升或者 2~4克 /升。 稀释的藻液的 pH 可以调节为 5.5~7.5， 例如 6.0 7.5等。  The high-density algae liquid obtained by heterotrophic culture should be diluted. The high-density algae solution is diluted with water and a medium containing no organic carbon source to maintain the cell density at 0.1 10 g / liter and adjust the pH to 5.0 8.0. In other embodiments, the algal fluid is diluted to maintain a cell density of 1-8 g/l. In a preferred embodiment, the cell density is maintained at 1.0 5.0 g/l. In other embodiments, the cell density is maintained at 2 to 5 grams per liter or 2 to 4 grams per liter. The pH of the diluted algae solution can be adjusted to 5.5~7.5, such as 6.0 7.5.
可采用各种已知的稀释培养基来稀释藻液。 通常， 可使用光诱导培养基进行稀释。 光诱导培养基通常含有氮源、 无机盐和水， 相对于异养培养基不含有有机碳源。 在优选 的实施方案中， 所述培养基的氮源浓度为 0.1 10克 /升， 例如 2~10克 /升、 2~8克 /升， 较 佳为 0.3 6克 /升， 更佳为 0.5 5克 /升。 所述氮源可以与异养培养步骤中所用的氮源相同 或不同。  Various known dilution media can be used to dilute the algal fluid. Typically, the light-inducing medium can be used for dilution. The light-inducing medium usually contains a nitrogen source, an inorganic salt, and water, and does not contain an organic carbon source relative to the heterotrophic medium. In a preferred embodiment, the medium has a nitrogen source concentration of 0.110 g/l, such as 2-10 g/l, 2-8 g/l, preferably 0.36 g/l, more preferably 0.5. 5 g / liter. The nitrogen source may be the same as or different from the nitrogen source used in the heterotrophic cultivation step.
在一个优选的具体方案中， 异养培养获得的高密度藻细胞宜用氮源浓度为 0.1 10 克 /升 （例如 2~10克 /升） 而不含有机碳源的初始培养基 （例如， 培养普通小球藻时采用 不含葡萄糖的 HA-SK培养基， 培养蛋白核小球藻时采用不含葡萄糖的 Endo培养基） 进 行适当稀释。  In a preferred embodiment, the high-density algal cells obtained by heterotrophic culture are preferably used in an initial medium having a nitrogen source concentration of 0.1 10 g/l (for example, 2 to 10 g/L) without an organic carbon source (for example, The normal chlorella was cultured using HA-SK medium without glucose, and the nucleus chlorella was cultured with Endo medium without glucose for proper dilution.
稀释采用的培养基无需高压灭菌， 配制好后调节 pH至 5.0 8.0即可使用。  The medium used for dilution does not need to be autoclaved. After preparation, adjust the pH to 5.0 8.0 and use.
在具体实施方式中， 对普通小球藻， 稀释培养基（光诱导培养基） 由以下成分组成： In a specific embodiment, for ordinary Chlorella, the dilution medium (light-inducing medium) consists of the following components:
KN03 0.1-5 克 /升、 MgS04-7H20、 0.5-5.0 克 /升、 CaCl2 0.01-0.06 克 /升、 FeS04-7H20KN0 3 0.1-5 g / liter, MgS0 4 -7H 2 0, 0.5-5.0 g / liter, CaCl 2 0.01-0.06 g / liter, FeS0 4 -7H 2 0
0.01-0.06克 /升、 EDTA 0.020 0.052克 /升。 0.01-0.06 g / liter, EDTA 0.020 0.052 g / liter.
对蛋白核小球藻， 稀释培养基 （光诱导培养基） 由以下成分组成： 尿素 0.1 5.0克 / 升、 MgS04-7H20、 0.5~5.0克 /升、 CaCl2 0.01~0.06克 /升、 FeS04-7H20 0.01~0.06、 EDTAFor Chlorella pyrenoidosa, the dilution medium (light-induced medium) consists of the following components: urea 0.1 5.0 g / liter, MgS0 4 -7H 2 0, 0.5 ~ 5.0 g / liter, CaCl 2 0.01 ~ 0.06 g / liter , FeS0 4 -7H 2 0 0.01~0.06, EDTA
0.020-0.052克 /升、 柠檬酸钠 0.02 0.5克 /升。 0.020-0.052 g / liter, sodium citrate 0.02 0.5 g / liter.
在其它实施方式中， 对普通小球藻， 稀释培养基（光诱导培养基） 由以下成分组成： In other embodiments, for Oryza sativa, the dilution medium (light-inducing medium) consists of the following components:
KN03 1-8克 /升、 MgSO4'7H2O、0.5~1.0克 /升、 CaCl2 0.01-0.06克 /升、 FeS04'7H20 0.01-0.06 克 /升、 EDTA 0.020~0.052克 /升； 对蛋白核小球藻， 稀释培养基（光诱导培养基） 由以下 成分组成： 尿素 0.1-2.0 克 /升、 MgS04-7H20、 0.5-1.0 克 /升、 CaCl2 0.01-0.06 克 /升、 FeS04-7H20 0.01~0.06 EDTA 0.020~0.052克 /升、 柠檬酸钠 0.08~0.5克 /升。 KN0 3 1-8 g / liter, MgSO 4 '7H 2 O, 0.5 ~ 1.0 g / liter, CaCl 2 0.01 - 0.06 g / liter, FeS0 4 '7H 2 0 0.01 - 0.06 g / liter, EDTA 0.020 ~ 0.052 g /L; for chlorella chlorella, dilution medium (light-induced medium) by Composition: Urea 0.1-2.0 g/L, MgS0 4 -7H 2 0, 0.5-1.0 g/L, CaCl 2 0.01-0.06 g/L, FeS0 4 -7H 2 0 0.01~0.06 EDTA 0.020~0.052 g/L , sodium citrate 0.08 ~ 0.5 g / liter.
3. 光诱导培养 3. Light-induced culture
该步骤的目的是让微藻接受光照， 通过光诱导使藻细胞快速大量合成积累各种活性 成分。  The purpose of this step is to allow the microalgae to receive light, and to accelerate the mass synthesis of the algae cells to accumulate various active ingredients by light induction.
如上所述高密度微藻培养液经稀释后， 将所得稀释液转入光诱导装置中进行光诱导 培养。 添加的光诱导培养基如上文所述， 温度控制在 5~50°C， 光照强度为 0.11~150kk， 连续光照或间歇光照， 光诱导培养周期为 1~150小时， 通气量为 0.1 2.0 wm。 其中所述 的光生物反应器包括所有的封闭式光生物反应器 （摇瓶、 管道式、 平板式、 柱式、 薄膜 立袋与吊袋等） 和所有的开放式光生物反应器 （跑道池、 圆池和鼓泡式大盆等） 。  After the high-density microalgae culture solution is diluted as described above, the resulting dilution is transferred to a light-inducing device for light-induced culture. The added light-inducing medium is as described above, the temperature is controlled at 5 to 50 ° C, the light intensity is 0.11 to 150 kk, continuous illumination or intermittent illumination, the light-induced culture period is 1 to 150 hours, and the ventilation is 0.1 2.0 wm. The photobioreactor described therein includes all closed photobioreactors (bottle shakers, tubing, flat plates, columns, film stand pouches and sling bags, etc.) and all open photobioreactors (runway cells) , round pools and bubbling basins, etc.).
通常， 培养温度可控制在 15~35°C的范围内， 例如 18~35°C、 20-35 °C 20~30°C等。 通常， 光照强度为 l~70kk， 例如， 1~60、 1~50、 1~40、 1~30、 1~20、 l~10kk等， 可视 具体生产情况而定。通常，通气量可控制为 0.15~2.0 wm,例如， 0.2-1.8 0.5-1.5 0.8-1.5 1.0 1.5wm等。 在其它实施方式中， 培养温度控制在 10~50°C， 光照强度为 l~10kk， 通 气量为 0.05~2.0wm。  Usually, the culture temperature can be controlled within the range of 15 to 35 ° C, for example, 18 to 35 ° C, 20 to 35 ° C, 20 to 30 ° C, and the like. Usually, the light intensity is l~70kk, for example, 1~60, 1~50, 1~40, 1~30, 1~20, l~10kk, etc., depending on the specific production situation. Generally, the ventilation can be controlled to be 0.15 to 2.0 wm, for example, 0.2-1.8 0.5-1.5 0.8-1.5 1.0 1.5 wm or the like. In other embodiments, the culture temperature is controlled at 10 to 50 ° C, the light intensity is 1 to 10 kk, and the gas flow rate is 0.05 to 2.0 wm.
在其它实施例中， 光诱导培养周期为 8 100小时， 例如， 根据实际的天气情况， 光 诱导培养周期可以为 8~90小时、 8~80小时、 8~60小时、 8~48小时、 8~24小时不等； 或 者， 光诱导培养周期可以为 12 72小时、 12 60小时、 12 48小时、 12 36小时、 12 24 小时不等或 24 60小时、 24~48小时不等。  In other embodiments, the light-induced culture period is 8 100 hours. For example, according to actual weather conditions, the light-induced culture period may be 8 to 90 hours, 8 to 80 hours, 8 to 60 hours, 8 to 48 hours, 8 ~24 hours vary; or, the light-induced culture period can be 12 72 hours, 12 60 hours, 12 48 hours, 12 36 hours, 12 24 hours, or 24 60 hours, 24 to 48 hours.
在本申请中， "光诱导培养周期"包括了整个光诱导培养过程， 例如， 户外培养 时光诱导培养周期包括夜晚没有光照的时间。  In the present application, the "light-induced culture period" includes the entire light-induced culture process, for example, the outdoor culture-time light-induced culture period includes the time when there is no light at night.
在本申请中， "光照时间"指使用本申请所述光照强度对微藻实施光诱导培养的时 间， 即该时间不包括夜晚没有光照的时间。 在一些实施例中， 光诱导培养步骤的光照时 间为 8-48小时， 例如 8~36小时、 8-24小时、 8-18小时、 8-12小时、 12-36小时、 12-24 小时不等， 以及上述范围内的任意时长。  In the present application, "lighting time" refers to the time during which light-induced culture of microalgae is carried out using the light intensity described herein, i.e., the time does not include the time when no light is illuminated at night. In some embodiments, the photoinduction culture step has an illumination time of 8-48 hours, such as 8 to 36 hours, 8-24 hours, 8-18 hours, 8-12 hours, 12-36 hours, 12-24 hours. Etc., and any length of time within the above range.
因此， 本申请的光诱导培养步骤也包括光照时间为 8~48 小时范围内的光诱导培养 步骤。 可采用人工光照的方式进行光诱导培养， 也可在户外利用自然光照的方式进行光 诱导培养。  Therefore, the light-induced culture step of the present application also includes a light-induced culture step in the range of 8 to 48 hours of illumination. Light-induced culture can be carried out by artificial light, or light-induced culture can be carried out outdoors by natural light.
在光诱导过程中，当油脂含量达到最高值（不同微藻油脂含量达到的最大值不一样， 可通过测定光诱导过程中胞内油脂含量曲线判断， 小球藻一般为 20~30%左右）时可结束 光诱导培养， 收获藻细胞进行后续的油脂的分离提取。 在生产叶黄素时， 当培养液中叶 黄素浓度达到最高时， 结束光诱导培养， 收获藻细胞进行叶黄素的分离提取或直接采收 藻细胞进行藻粉制备。 4. 藻细胞采收、 油脂的提取及藻体综合利用 In the light-induced process, when the oil content reaches the highest value (the maximum value of different microalgae oil content is different, it can be judged by measuring the intracellular oil content curve in the light-induced process, the chlorella is generally about 20~30%) The light-induced culture can be ended, and the algal cells are harvested for subsequent separation and extraction of the oil. In the production of lutein, when the concentration of lutein in the culture solution reaches the highest, the light-induced culture is terminated, and the algal cells are harvested for separation and extraction of lutein or directly harvested algae cells for algal powder preparation. 4. Algae cell harvesting, oil extraction and comprehensive utilization of algae
光诱导培养结束后， 对小球藻进行离心采收， 获得湿藻体。 藻细胞的采收方法包括 但不限于高速离心、 絮凝， 气浮或过滤等技术； 藻细胞破壁方法包括但不限于藻体自溶、 高压匀浆、 酶水解、 水相热解等湿法破壁方法。  After the light-induced culture was completed, the chlorella was centrifuged to obtain a wet algae body. Methods for harvesting algae cells include, but are not limited to, high-speed centrifugation, flocculation, air flotation or filtration; algae cell wall breaking methods include, but are not limited to, algae autolysis, high pressure homogenization, enzymatic hydrolysis, aqueous phase pyrolysis, etc. Broken wall method.
胞内油脂的提取测定方法包括但不限于有机溶剂萃取法， 即： 将藻体于 80~105 °C下 干燥至恒重， 研磨藻粉后采用氯仿甲醇标准萃取溶剂从干藻粉中提取油脂， 萃取溶剂反 复提取直至藻粉颜色变为白色， 旋转蒸发去除溶剂。  The method for extracting intracellular fats includes, but is not limited to, organic solvent extraction, that is, drying the algae at 80-105 ° C to a constant weight, grinding the algal powder, and extracting the oil from the dry algae powder using a chloroform methanol standard extraction solvent. The extraction solvent is repeatedly extracted until the color of the algal powder turns white, and the solvent is removed by rotary evaporation.
小球藻内叶黄素的提取可采用传统的有机溶剂提取法进行。 首先将有机溶剂加入到 藻泥中进行萃取， 然后进行搅拌离心获得上清液和藻体沉淀， 对上清液进行减压浓缩、 搅拌加水、 过滤获得叶黄素晶体。 在一个较佳的方案中， 采用超临界 C02萃取技术对小 球藻进行叶黄素的分离提取。 在一个更佳的方案中， 将获得的小球藻液浓缩后直接喷雾 干燥获得小球藻粉。 由此获得的小球藻粉可用于开发成动物饲料、 水产养殖用饵料、 食 品、 食品添加剂、 药品和营养品等。 The extraction of lutein from chlorella can be carried out by a conventional organic solvent extraction method. First, an organic solvent is added to the algal mud for extraction, and then the supernatant and the algal body precipitate are obtained by stirring and centrifuging, and the supernatant is concentrated under reduced pressure, stirred with water, and filtered to obtain lutein crystals. In a preferred embodiment, the chlorella is isolated and extracted from the chlorella using a supercritical CO 2 extraction technique. In a more preferred embodiment, the obtained chlorella solution is concentrated and directly spray dried to obtain chlorella powder. The chlorella powder thus obtained can be used for development into animal feed, aquaculture bait, food, food additives, medicines and nutraceuticals, and the like.
本发明中， 可对培养所得的微藻进行综合利用， 提取其中的多不饱和脂肪酸、 蛋白 质、 叶绿素、 多糖等各种活性成分。 活性成分的提取顺序并无特殊限制， 但通常要满足 先提取的步骤不能导致后提取的成分损失这一前提。  In the present invention, the microalgae obtained by the culture can be comprehensively utilized to extract various active ingredients such as polyunsaturated fatty acids, proteinaceous substances, chlorophyll, and polysaccharides. The order of extraction of the active ingredient is not particularly limited, but it is usually the premise that the step of first extraction cannot cause loss of the component to be extracted later.
上清液中的其他成分可逐步分离提取获得脂肪酸、 叶绿素等， 或直接将上清液中的 所有成分与藻体沉淀混合喷雾干燥获得小球藻粉。 所得藻粉可用于开发成动物饲料、 水 产养殖用饵料、 食品、 食品添加剂、 药品和营养品等。  The other components in the supernatant may be gradually separated and extracted to obtain fatty acids, chlorophyll, etc., or directly mixed with all the components in the supernatant and sprayed with the algal body to obtain chlorella powder. The obtained algal flour can be used for development into animal feed, aquaculture bait, food, food additives, medicines and nutrients.
本发明中， 可对培养所得的微藻进行综合利用， 提取其中的色素、 蛋白质、 多糖等 各种活性成分。 活性成分的提取顺序并无特殊限制， 但通常要满足先提取的步骤不能导 致后提取的成分损失这一前提。  In the present invention, the microalgae obtained by the culture can be comprehensively utilized to extract various active ingredients such as a pigment, a protein, and a polysaccharide. The order of extraction of the active ingredient is not particularly limited, but it is usually the premise that the step of first extraction does not result in loss of the component to be extracted.
本文中涉及到藻细胞干重、 油脂含量及胞内生化成分的测定方法如下：  The methods for determining the dry weight, oil content and intracellular biochemical composition of algae cells are as follows:
藻细胞干重测定： 在微藻（如小球藻）培养过程中取培养液 V毫升， 8000 rpm离心 10分钟， 将离心后的藻体用去离子水洗涤 3次， 转移至称量瓶 （Wi (克） ） 中， 在 105 °C烘箱中烘干至恒重 W2 (克） 。 藻体干重 Cx可根据下式计算： Cx (克 /升） = (W2-Wi ) /V/lOOOo Determination of dry weight of algae cells: Take 50 ml of culture medium during the culture of microalgae (such as chlorella), centrifuge at 8000 rpm for 10 minutes, and wash the algae after centrifugation 3 times with deionized water, and transfer to a weighing bottle ( In Wi (g), dry in an oven at 105 °C to constant weight W 2 (g). The dry weight Cx of algae can be calculated according to the following formula: Cx (g/l) = (W 2 -Wi ) /V/lOOOo
油脂测定： 取一定量各培养阶段烘干至恒重的藻细胞， 在研钵中研磨至粉末状， 称 取适量藻粉 （0.2 0.5 g) 小心转移至离心管中， 加入适量萃取溶剂 （氯仿：甲醇 =2: 1 ) 于 超声振荡器中超声振荡 30min， 8000rpm离心 10min， 将上清转移至已知重量的干燥旋转 蒸发瓶中， 重复上述步骤直至上清无色。 合并上清后旋转蒸干， 称重并计算油脂含量。  Determination of fat: Take a certain amount of algae cells dried to constant weight in each culture stage, grind to powder form in a mortar, weigh the appropriate amount of algae powder (0.2 0.5 g), carefully transfer to a centrifuge tube, and add an appropriate amount of extraction solvent (chloroform). :Methanol = 2: 1) Ultrasonic shaking in an ultrasonic shaker for 30 min, centrifugation at 8000 rpm for 10 min, transferring the supernatant to a dry rotary evaporation bottle of known weight, and repeating the above steps until the supernatant is colorless. The supernatants were combined, evaporated to dryness, weighed and the fat content was calculated.
油脂含量 （％) 按下式计算：  Grease content (%) Calculated as follows:
油脂 （％) = (W2-W。） / Wi X 100 式中： ——为藻粉重量， g; Wo——为烘干至恒重的旋转蒸发瓶重量， g; W2—— 为油脂萃取液蒸干后蒸发瓶的重量， g。 Grease (%) = (W 2 -W.) / Wi X 100 Where: - is the weight of algae powder, g; Wo - the weight of the rotary evaporation bottle for drying to constant weight, g; W 2 - the weight of the evaporation bottle after evaporation of the oil extract, g.
碳水化合物的测定： 参照药典 2005版， 选择蒽酮-硫酸显色反应， 采用无水葡萄糖 作为对照品， 用比色法在 625nm处测定。  Determination of Carbohydrate: Refer to the Pharmacopoeia 2005 edition, select the anthrone-sulfuric acid color reaction, use anhydrous glucose as a control, and measure by colorimetry at 625 nm.
蛋白质含量测定：微藻（如小球藻）细胞中总蛋白质含量的测定采用凯氏定氮法（宁 正祥. 食品成分分析手册. 北京： 中国轻工业出版社， 1998, 76-78) 。  Determination of protein content: The total protein content in cells of microalgae (such as chlorella) was determined by Kjeldahl method (Ning Zhengxiang. Handbook of Food Composition Analysis. Beijing: China Light Industry Press, 1998, 76-78).
叶绿体色素含量测定： 微藻 （如小球藻） 叶绿体色素含量测定采用 Lichtenthaler等 对 Arnon法进行了修正后的方法 (Lichtenthaler HK, Wellburn AR. Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different sovents. Biochemical Society Transactions, 1983, 11 :591-592) 。  Determination of chloroplast pigment content: microalgae (such as chlorella) Determination of chloroplast pigment content by Lichtenthaler et al., Determination of total carotenoids and chlorophylls a and b of leaf extracts in different Sovents. Biochemical Society Transactions, 1983, 11 :591-592).
灰分测定： 微藻 （如小球藻） 灰分采用 550°C灼烧至恒重测量， 即国标 GB6438-86 法。  Ash determination: Microalgae (such as chlorella) Ash is measured by burning at 550 °C to constant weight, which is the national standard GB6438-86.
叶黄素测定：采用高效液相色谱法（HPLC)，具体操作步骤见文献（Zheng Yun Wu, Chun Lei Shi et al. Modeling of lutein production by heterotrophic Chlorella in batch and fed-batch cultures. World Journal of Microbiology and Biotechnology,2007, 23: 1233-1238) 。 与现有的能源微藻培养积累油脂的方法相比， 本发明的方法具有以下优点： 可在短 时间内获得高密度的微藻细胞， 同时可在短时间内使微藻细胞内的油脂含量快速提高。 本发明将微藻培养合成油脂分成藻细胞生长和油脂合成积累两个阶段， 即异养培养和光 诱导培养阶段。 异养培养微藻的目的是为了快速获得大量的藻细胞， 而光诱导培养的目 的是为了诱导微藻细胞快速合成积累大量的油脂，采用异养-稀释-光诱导串联培养方式培 养微藻提高了油脂的产率， 一定程度上解决了能源微藻细胞生长和油脂合成不同时进行 的矛盾， 大大降低了成本， 为解决生物燃料规模化制备过程中由于原料不足而导致的成 本过高问题提供新的技术手段。  Determination of lutein: high performance liquid chromatography (HPLC), specific procedures are described in the literature (Zheng Yun Wu, Chun Lei Shi et al. Modeling of lutein production by heterotrophic Chlorella in batch and fed-batch cultures. World Journal of Microbiology And Biotechnology, 2007, 23: 1233-1238). Compared with the existing energy microalgae culture method for accumulating oil and fat, the method of the invention has the following advantages: high-density microalgae cells can be obtained in a short time, and the oil content in the microalgae cells can be obtained in a short time. Improve quickly. The invention divides the microalgae culture synthetic oil into two stages of algae cell growth and oil fat synthesis accumulation, namely heterotrophic culture and light induction culture stage. The purpose of heterotrophic cultivation of microalgae is to obtain a large number of algae cells quickly, and the purpose of light-induced culture is to induce rapid synthesis and accumulation of large amounts of oil and fat in microalgae cells, and to promote microalgae cultivation by heterotrophic-dilution-light-induced tandem culture. The yield of oil and fat has solved the contradiction between energy microalgae cell growth and oil and fat synthesis to a certain extent, greatly reducing the cost, and providing the problem of over-costing caused by insufficient raw materials in the large-scale preparation process of biofuels. New technical means.
此外， 本申请之前， 本领域的普遍认识是只有通过光自养的模式才能够利用微藻来 大规模生产油脂。 然而， 光自养培养与本发明的光诱导培养完全不同。 光自养培养中， 培养基不含有有机碳源， 微藻利用大气中的 C02、 水、 光、 和少量无机营养盐进行光合 自养， 维持自身的生长， 一般接种密度较低 （ 0.1g/L左右） ， 生长慢 （有时候为了促进 生长， 还需要在培养过程中人工通入 C02) ， 培养周期长 （一般要一周以上， 户外光自 养培养细胞密度难以达到较高水平） ， 尤其在大规模培养的时候， 为了提供大规模培养 所需的接种藻液， 种子扩培需要的时间很长 （一般需要 1-2个月） ， 生产效率很低。 而 本申请的光诱导所用的培养基和光自养的培养基不一样， 由于异养阶段藻液中含有丰富 的无机盐和微量元素等营养成分， 所以经过稀释后， 所需的光诱导培养基比光自养培养 基要简单很多， 例如和文献上常见的微藻光自养培养基 BG-11相比， 稀释用的光诱导培 养基中不需要添加磷酸盐、 碳酸钠、 柠檬酸铁铵以及微量元素， 同时添加的硝酸盐用量 也比光自养培养基用量要少很多。 通过异养阶段的培养， 可以在短时间内积累大量藻细 胞， 规模化培养时， 通过稀释后， 进入光诱导的藻细胞密度 （以小球藻为例， 密度范围 在 2~5g/L左右） 比光自养培养方式的接种密度要高 20倍以上， 很好的解决了光自养培 养方式接种密度低， 种子扩培周期长等缺点， 同时在较短时间的光照诱导周期内 （一般 8~24小时） ， 藻细胞密度几乎维持不变 （即它已经不再生长） ， 胞内油脂含量却能够快 速积累， 含量成倍增长（由 10%增加到 20~30%左右） ， 这样相对光自养而言， 占地面积 大大缩小， 面积产率大幅提高， 整个培养周期明显缩短， 采收成本降低， 生产效率大大 提高。 Furthermore, prior to the present application, it was generally recognized in the art that microalgae could be used to produce oil on a large scale only by a mode of photoautotrophicity. However, photoautotrophic culture is completely different from the light-induced culture of the present invention. In photoautotrophic culture, the medium does not contain an organic carbon source. The microalgae utilizes C0 2 , water, light, and a small amount of inorganic nutrients in the atmosphere for photoautotrophic growth to maintain their own growth. The general seeding density is low (0.1g). /L or so), slow growth (sometimes in order to promote growth, it is also necessary to manually enter C0 2 during the culture process), the culture period is long (usually more than one week, the outdoor light autotrophic culture cell density is difficult to reach a higher level), Especially in large-scale cultivation, in order to provide the inoculated algae solution required for large-scale cultivation, it takes a long time for the seed to be expanded (generally 1-2 months), and the production efficiency is very low. However, the medium for photoinduction of the present application is different from the photoautotrophic medium. Since the heterotrophic stage is rich in nutrients such as inorganic salts and trace elements, the desired light-inducing medium is diluted. It is much simpler than the photoautotrophic culture medium, for example, compared with the microalgae photoautotrophic medium BG-11 commonly used in the literature, the light-induced culture for dilution. Phosphate, sodium carbonate, ammonium ferric citrate and trace elements are not required in the nutrient base, and the amount of nitrate added is also much less than that of the photoautotrophic medium. Through the cultivation in the heterotrophic stage, a large number of algae cells can be accumulated in a short period of time. When scaled up, the light-induced algal cell density is entered by dilution (in the case of chlorella, the density ranges from 2 to 5 g/L). The inoculation density of the photoautotrophic culture method is 20 times higher than that of the light autotrophic culture method, which solves the shortcomings such as low seeding density of the photoautotrophic culture method and long seed expansion period, and is also in a short period of light induction period (general 8~24 hours), the density of algae cells is almost unchanged (that is, it no longer grows), the intracellular fat content can accumulate rapidly, and the content increases exponentially (from 10% to 20~30%). In terms of self-supporting, the area is greatly reduced, the area yield is greatly improved, the entire culture period is significantly shortened, the harvesting cost is reduced, and the production efficiency is greatly improved.
同样地， 迄今， 微藻培养产叶黄素的最高体积产率 （145mg/L/d) 和胞内叶黄素的 最高含量（7.6mg/g)均是在连续光照的混合营养培养条件下获得的（Martin, Lucia. Process for obtaining lutein from algae. EP180843A1, 2007)。但该培养模式只能在可蒸汽灭菌的封 闭式光生物反应器中进行， 且培养过程必须保证绝对的无菌， 同时需要光源的合理配置， 这在实际生产中无法实现。 因此， 利用混合营养模式培养微藻生产叶黄素不具有产业化 价值。  Similarly, the highest volume yield (145 mg/L/d) of lutein produced by microalgae and the highest content of intracellular lutein (7.6 mg/g) have been under continuous light-mixed nutrient culture conditions. (Martin, Lucia. Process for obtaining lutein from algae. EP180843A1, 2007). However, the culture mode can only be carried out in a steam-sterilizable closed photobioreactor, and the culture process must ensure absolute sterility, and a reasonable configuration of the light source is required, which cannot be achieved in actual production. Therefore, the use of a mixed nutrient model to culture microalgae to produce lutein does not have industrial value.
本发明将微藻培养法生产叶黄素分成藻体生长和产物 （叶黄素）积累两个阶段， 即 异养培养和光诱导培养。通过异养培养可在短时间内获得大量的可产叶黄素的微藻细胞， 藻液稀释后转入光诱导培养， 藻体内叶黄素含量迅速提升至初始的两倍甚至两倍以上。 本发明具有如下优势： （1 ) 光诱导的藻细胞密度很高 （2~10g/L) ， 是常规光自养培养 藻细胞密度 （约 0.2~lg /L) 的 10倍左右； （2) 光诱导时间很短 （约 ld~2d) ， 而微藻 光自养培养时间很长 （约 7d~14d) ， 因此， 叶黄素的生产效率大为提高； （3 ) 相对于 微藻光自养培养， 光诱导时较高的藻细胞密度使得光诱导所需的占地面积很小， 同时高 细胞密度使得采收成本大幅度降低； （4)异养培养几乎不受气候、 天气的影响， 光诱导 培养可以在玻璃房内， 自然光照或人工光照条件下进行， 因而， 采用本发明的方法可以 实现叶黄素的连续生产； （5 )对富含油脂和叶黄素的藻体进行分步提取， 实现微藻能源 和高附加值产品开发的耦合和藻体的资源化利用， 可获得额外的产品和经济效益， 降低 了微藻能源的生产成本。  The invention divides the production of lutein by microalgae culture into two stages of algae growth and product accumulation (lutein), namely heterotrophic culture and light-induced culture. Through heterotrophic culture, a large number of lutein-producing microalgae cells can be obtained in a short time. The algae solution is diluted and transferred to light-induced culture, and the lutein content in the algae is rapidly increased to twice or more than the initial amount. The invention has the following advantages: (1) The light-induced algal cell density is very high (2~10g/L), which is about 10 times of the conventional photoautotrophic culture algae cell density (about 0.2~lg / L); (2) The light induction time is very short (about ld~2d), while the microalgae self-cultivation time is very long (about 7d~14d), so the production efficiency of lutein is greatly improved; (3) relative to microalgae light self In culture, the higher density of algae cells in light induction makes the area required for light induction small, while the high cell density makes the harvesting cost greatly reduced. (4) Heterotrophic culture is almost independent of climate and weather. The light-induced culture can be carried out in a glass room under natural light or artificial light. Therefore, the continuous production of lutein can be achieved by the method of the invention; (5) the algae rich in oil and lutein are carried out. Step-by-step extraction, coupling of microalgae energy and high value-added product development and resource utilization of algae, can obtain additional products and economic benefits, and reduce the production cost of microalgae energy.
以下将通过实施例对本发明的有关内容作进一步的说明。 除非另有所述， 本发明采 用的培养基中各组分含量均用克 /升 (g/L)表示。 应理解， 本申请中 "含有" 、 "包含"也 包括 "由……组成"、 "由……构成" 的含义。 实施例 1  The relevant content of the present invention will be further described below by way of examples. Unless otherwise stated, the content of each component in the medium used in the present invention is expressed in grams per liter (g/L). It should be understood that "contains" and "comprises" in this application also includes the meaning of "consisting of" and "consisting of." Example 1
在 50L生物反应器中加入下述异养培养基和自来水至 25L后灭菌， 当温度降至 30 °。时按工作体积的 12%接入蛋白核小球藻， 开始异养培养。 异养培养条件： 温度为 30°C， 初始转速为 150r/min， 空气流量为 lvvm， pH小于 8.0， 培养过程中通过调节转速控制溶氧 15%以上。 The following heterotrophic medium and tap water were added to a 50 L bioreactor to 25 L and then sterilized when the temperature was lowered to 30 °. At the time of 12% of the working volume, access to Chlorella pyrenoidosa, start heterotrophic culture. Heterotrophic culture conditions: The temperature is 30 ° C, the initial rotation speed is 150 r / min, the air flow rate is lvvm, the pH is less than 8.0, and the dissolved oxygen is controlled by 15% or more by adjusting the rotation speed during the cultivation.
将异养培养过程中葡萄糖耗完的高密度藻液稀释到 2.70g/L左右， 并加入下述光诱 导培养基， 转入 3L平板式光反应器中进行光诱导培养， 温度 30°C， 光强 8000k， 空气 流量为 lvvm。光诱导培养 12h， 细胞密度从 2.70 g/L降低到 2.65 g/L, 油脂含量从 9.70% 上升至 19.70% (见图 1 ) 。  The high-density algal solution depleted of glucose in the heterotrophic culture process was diluted to about 2.70 g/L, and the following light-inducing medium was added, and transferred to a 3 L flat-plate photoreactor for light-induced culture at a temperature of 30 ° C. The light intensity is 8000k, and the air flow is lvvm. After light-induced culture for 12 h, the cell density decreased from 2.70 g/L to 2.65 g/L, and the oil content increased from 9.70% to 19.70% (see Figure 1).
异养培养基： 葡萄糖 60.0克， 尿素 8.0克， MgS04 · 7H20 2.0克， KH2P04 1.1克， Na2HP04 · 12H20 9.0克， CaCl2 0.02克，柠檬酸三钠 1.8克， Fe-EDTA溶液 1.0ml 微 量元素溶液 4.5ml， 和水 1000ml; 其中 Fe-EDTA溶液配方为 FeS04 · 7H20 15克 /升和 EDTA1.4 克 /升， 微量元素溶液配方为 H3B03 2.11 克 /升， MnCl2 · 4¾0 0.81 克 /升， ZnS04 · 7H20 0.11克 /升， CuS04 · 5H20 10.0克 /升， Na2Mo04 0.05克 /升。 Heterotrophic medium: glucose 60.0 g, urea 8.0 g, MgS0 4 · 7H 2 0 2.0 g, KH 2 P0 4 1.1 g, Na 2 HP0 4 · 12H 2 0 9.0 g, CaCl 2 0.02 g, trisodium citrate 1.8克, Fe-EDTA solution 1.0ml trace element solution 4.5ml, and water 1000ml; where Fe-EDTA solution is FeS0 4 · 7H 2 0 15 g / liter and EDTA 1.4 g / liter, trace element solution formula is H 3 B0 3 2.11 g/l, MnCl 2 · 43⁄40 0.81 g/l, ZnS0 4 · 7H 2 0 0.11 g/l, CuS0 4 · 5H 2 0 10.0 g/l, Na 2 Mo 0 4 0.05 g/l.
光诱导培养基： 尿素 0.5克， MgS04'7H20 1.0克， 柠檬酸三钠 0.05克， CaCl2 0.01 克， Fe-EDTA溶液 0.4ml， 和水 1000ml; 其中 Fe-EDTA溶液配方为 15克 /升和 EDTA1.4 克 /升。 实施例 2 Light-induced medium: 0.5 g of urea, 1.0 g of MgS0 4 '7H 2 0, 0.05 g of trisodium citrate, 0.01 g of CaCl 2 , 0.4 ml of Fe-EDTA solution, and 1000 ml of water; the formulation of Fe-EDTA solution is 15 g. / liter and EDTA 1.4 g / liter. Example 2
在 5 T发酵罐中加入异养培养基和自来水至 2.5 T后灭菌， 当温度降至 30°C时， 以 500 L发酵罐作为种子罐培养的普通小球藻， 培养过程中根据细胞生长情况、发酵液中溶 氧水平来调节罐压、 通气量和搅拌转速， 维持培养液中充足的氧含量， 确保藻细胞的快 速生长。  Adding heterotrophic medium and tap water to the 5 T fermentor to 2.5 T and then sterilizing. When the temperature is lowered to 30 ° C, the 500 L fermenter is used as a seed tank to culture the common chlorella, and the cells grow according to the culture process. The situation, the dissolved oxygen level in the fermentation broth to adjust the tank pressure, aeration and agitation speed, maintain sufficient oxygen content in the culture solution to ensure the rapid growth of algae cells.
将前期补料分批异养培养过程中葡萄糖耗完的高密度藻液经光诱导培养基稀释后， 转至 30 L平板式光反应器及 60 L鼓泡式大盆中在户外进行光诱导培养。光诱导培养条件: 自然温度， 温度在 13~30°C， 自然光照， 光强在 0~36 kk (见图 2) ， 空气流量为 1 vvm。 光诱导培养 8 h后， 30L平板式反应器中藻细胞密度从 1.60 g/L降低到 1.46 g/L, 油脂含 量从 7.87%上升至 13.27%，后进入夜晚，整个晚上藻细胞密度从 1.46 g/L降低至 1.16 g/L, 降幅较白天明显， 油脂含量也有小幅下降， 从 13.27%下降至 12.93%， 第二天白天经过 4h的短暂光诱导后， 藻细胞密度略有上升， 从 1.16 g/L上升至 1.18 g/L， 同时油脂含量 迅速上升， 从 12.93%升高至 18.58% (见图 3 ) ， 图 4为 60 L鼓泡式大盆中光诱导情况， 数据显示变化规律与 30 L平板式光反应器的规律相类似。  The high-density algae solution depleted of glucose in the pre-feeding batch heterotrophic culture process was diluted with light-inducing medium, and then transferred to a 30 L flat-plate photoreactor and a 60 L bubbling large-scale pot for outdoor light induction. to cultivate. Light-induced culture conditions: natural temperature, temperature between 13 and 30 ° C, natural light, light intensity between 0 and 36 kk (see Figure 2), air flow of 1 vvm. After light-induced culture for 8 h, the density of algae cells in the 30 L flat reactor decreased from 1.60 g/L to 1.46 g/L, and the oil content increased from 7.87% to 13.27%. After entering the night, the density of algae cells from the night was 1.46 g. /L decreased to 1.16 g / L, the decrease was more obvious than during the day, and the oil content also decreased slightly, from 13.27% to 12.93%. After 4 hours of short-day light induction during the day, the algal cell density increased slightly from 1.16 g. /L rose to 1.18 g/L, and the oil content increased rapidly from 12.93% to 18.58% (see Figure 3). Figure 4 shows the light induction in a 60 L bubbling large basin. The data shows the variation law and 30 The law of the L plate type photoreactor is similar.
将半连续异养培养结束后的高密度普通小球藻藻液， 经光诱导培养基稀释后， 转入 3 T跑道池及 3.14m2斜叶搅拌式圆池在户外进行光诱导培养。 光诱导培养条件： 较低自 然温度， 温度在 5~18°C， 自然光照， 光强在 0~34 kk (见图 5 ) ， 空气流量为 l vvm。 因 该批次 5 T发酵罐半连续培养结束时间在傍晚， 所以藻细胞转入光诱导后即进入夜晚， 光诱导阶段共进行了 90 h， 前 2天天气晴好， 但是气温较低， 放入藻液的当晚， 最低气 温只有 5 °C， 后面两天最高气温为 14°C， 第三天开始天气转阴有小雨， 气温开始小幅回 升， 第三天傍晚开始闷热潮湿， 整个晚上伴有雷阵雨， 第四天上午阴有小雨， 午后雨停， 结束培养并采收藻细胞。 The high-density common chlorella algae solution after the semi-continuous heterotrophic culture was diluted with the light-inducing medium, transferred to a 3 T runway pool and a 3.14 m 2 oblique-leaf stirred round cell for outdoor light-induced culture. Light-induced culture conditions: lower natural temperature, temperature between 5 and 18 ° C, natural light, light intensity between 0 and 34 kk (see Figure 5), air flow of l vvm. Because the semi-continuous culture of the batch 5 T fermenter ended in the evening, the algae cells entered the night after the light was induced, and the light induction stage was carried out for 90 h. The weather was fine in the first 2 days, but the temperature was lower. The evening of the algae liquid, the lowest gas The temperature is only 5 °C, and the maximum temperature is 14 °C in the next two days. On the third day, the weather turns cloudy with light rain, and the temperature starts to rise slightly. The third day starts to be hot and humid in the evening, with thunderstorms throughout the night, and the fourth day is cloudy. There is light rain, rain stops in the afternoon, and the cultivation and harvesting of algae cells is finished.
由图 6、 7可以看出， 一方面由于气温较低， 不利于藻细胞的生长代谢； 另一方面 由于第三天开始陆续下雨， 雨水稀释了藻液， 造成了大池内细胞内浓度下降， 所以整个 光诱导过程藻细胞密度下降明显。  It can be seen from Fig. 6 and Fig. 7 that on the one hand, due to the lower temperature, it is not conducive to the growth and metabolism of algae cells; on the other hand, due to the rain on the third day, the rainwater dilutes the algae liquid, causing the intracellular concentration in the large pool to decrease. Therefore, the density of algae cells decreased significantly during the entire light-induced process.
由于接种后的前 12 小时， 正好是晚上， 光合作用不能进行， 所以色素和碳水化合 物均大幅下降， 油脂含量却显著升高， 从 12%升至 18%左右， 当晚户外温度较低， 低温 胁迫可能诱导藻细胞内油脂的合成积累， 这也是藻细胞油脂代谢对外界环境变化的一种 响应机制。后面两天温度都较低， 最高气温不超过 14°C， 藻细胞胞内成分没有明显变化， 第三天开始， 虽然有雨水， 但是气温显著回升， 并且闷热潮湿， 从实验数据来看， 这种 户外实际气候情况反而有利于光诱导过程藻体品质的提升， 但是上升速度比较缓慢。 在 整个 90h的户外光诱导过程中， 尽管出现低温、 阴雨及雷雨天气， 但通过延长光诱导时 间， 藻体的品质也能得到大幅提升， 最终油脂含量均大幅提升至 20%左右。  Since the first 12 hours after inoculation, just in the evening, photosynthesis could not be carried out, so the pigment and carbohydrates decreased drastically, but the oil content increased significantly, from 12% to 18%. The outdoor temperature was low, low temperature stress. It may induce the synthesis and accumulation of oil in algae cells, which is also a response mechanism of algae cell lipid metabolism to external environmental changes. In the next two days, the temperature was lower, the maximum temperature did not exceed 14 °C, and the intracellular composition of algae cells did not change significantly. On the third day, although there was rain, the temperature rose significantly and was hot and humid. From the experimental data, this The outdoor actual climatic conditions are conducive to the improvement of algae quality during light induction, but the rate of increase is relatively slow. During the entire 90-hour outdoor light induction process, despite the occurrence of low temperature, rain and thunderstorms, the quality of the algae can be greatly improved by prolonging the light-inducing time, and the final oil content is greatly increased to about 20%.
其中，异养培养基为：葡萄糖 60.0克，硝酸钾 10.0克， MgS04 ·7Η20 0.2克， KH2P04 0.3克， Na2HP04 ·12Η20 8.8克， CaC12 0.02克， Fe-EDTA溶液 1.0ml，微量元素溶液 3.5ml， 水 1000ml; 其中 Fe-EDTA溶液配方为 FeS04 · 7H20 15克 /升和 EDTA 1.4克 /升， 微量元 素溶液配方为 H3B03 2.86克 /升， MnCl2 · 4H20 0.11克 /升， ZnS04 · 7H20 9.22克 /升， CuS04 · 5H20 1.00克 /升， （ Η4)6Μο7024·4Η20 0.1克 /升， Co(N03)2'6H20 0.9克 /升。 Among them, the heterotrophic medium is: glucose 60.0 g, potassium nitrate 10.0 g, MgS0 4 ·7Η 2 0 0.2 g, KH 2 P0 4 0.3 g, Na 2 HP0 4 ·12Η 2 0 8.8 g, CaC12 0.02 g, Fe- 1.0ml of EDTA solution, 3.5ml of trace element solution, 1000ml of water; the formulation of Fe-EDTA solution is FeS0 4 · 7H 2 0 15g / liter and EDTA 1.4g / liter, the formula of trace element solution is H 3 B0 3 2.86 g / Liter, MnCl 2 · 4H 2 0 0.11 g / liter, ZnS0 4 · 7H 2 0 9.22 g / liter, CuS0 4 · 5H 2 0 1.00 g / liter, ( Η 4 ) 6 Μο 7 0 24 · 4 Η 2 0 0.1 g /L, Co(N0 3 ) 2 '6H 2 0 0.9 g / liter.
光诱导培养基为： 硝酸钾 0.5克， MgS04 · 7H20 0.6克， CaCl2 0.03克， Fe-EDTA 溶液 1.5ml， 水 1000ml; 其中 Fe-EDTA溶液配方为 8克 /升和 EDTA10.4克 /升。 实施例 3 : 异养 -稀释 -光诱导串联培养过程中椭圆小球藻主要生化成分变化规律的 研究 The light-inducing medium is: potassium nitrate 0.5 g, MgS0 4 · 7H 2 0 0.6 g, CaCl 2 0.03 g, Fe-EDTA solution 1.5 ml, water 1000 ml; wherein Fe-EDTA solution is 8 g/L and EDTA 10.4 G/L. Example 3: Study on the Change of Main Biochemical Compositions of Chlorella ellipses during Heterotrophic-Dilution-Light Induced Tandem Culture
本实施例在 500mL摇瓶 /3L圆柱式光生物反应器水平测定椭圆小球藻异养-稀释-光 诱导串联培养过程中主要生化成分的变化。  In this example, the changes of main biochemical components in the heterotrophic-dilution-light-induced tandem culture of Chlorella ellipsoid were determined at the level of a 500 mL shake flask / 3 L cylindrical photobioreactor.
采用与实施例 1和 2类似的方式以异养 -稀释 -光诱导串联培养技术培养椭圆小球藻， 500mL摇瓶， 装液量 200mL， 28 °C , 150rpm， 待藻细胞密度达到 10g/L以上且葡萄糖耗 尽时， 转入 3L圆柱式光生物反应器进行光诱导培养， 藻细胞密度约 2g/L， 温度 30°C， 光强 10Kk。异养和光诱导所采用的培养基和蛋白核小球藻的相应培养基一致。异养结束 时藻细胞内油脂含量为 13.94%， 转入光诱导后 12h， 藻细胞油脂含量快速增加到 23.91% (见图 8 ) 。 实施例 4 在 5L生物反应器中加入下述异养培养基和水至 2.8L后进行蒸汽灭菌， 然后当温度 降到 30°C时接入蛋白核小球藻， 开始异养培养。 异养培养条件： 温度为 30士 1 °C， 空气 流量为 lvvm， pH小于 8.0， 控制溶氧 15%以上。 In a similar manner to Examples 1 and 2, the ellipsoidal culture was cultured in a heterotrophic-dilution-light-induced tandem culture technique, a 500 mL shake flask, a liquid volume of 200 mL, 28 ° C, 150 rpm, and the algal cell density reached 10 g/L. Above and when glucose is depleted, it is transferred to a 3L cylindrical photobioreactor for light-induced culture. The algae cell density is about 2g/L, the temperature is 30°C, and the light intensity is 10Kk. The culture medium used for heterotrophic and light induction is consistent with the corresponding medium of Chlorella pyrenoidosa. At the end of heterotrophy, the oil content in the algae cells was 13.94%, and 12 hours after the light induction, the algae cell oil content increased rapidly to 23.91% (see Figure 8). Example 4 The following heterotrophic medium and water were added to a 5 L bioreactor to 2.8 L, followed by steam sterilization, and then when the temperature was lowered to 30 ° C, Chlorella pyrenoidosa was introduced to start heterotrophic culture. Heterotrophic culture conditions: The temperature is 30 ± 1 °C, the air flow is lvvm, the pH is less than 8.0, and the dissolved oxygen is controlled by more than 15%.
在接种后 53.9h后第一次补料，之后每隔 5~8h进行补料，共补加 4次，培养至 88.40h 细胞干重达到 132.2g/L (见图 9) ， 此时异养培养结束转入光诱导培养。  The first feeding was carried out 53.9 hours after inoculation, and then the feeding was carried out every 5~8h, supplemented 4 times, and the dry weight of the cells reached 88.40h reached 132.2g/L (see Figure 9). The culture is transferred to light-induced culture.
将异养培养结束后的高密度藻液稀释到 2.55g/L， 并加入下述光诱导培养基， 转入到 3L平板式光生物反应器中进行光诱导培养。 光诱导培养条件： 温度维持在 28~33 °C， 空 气流量为 lvvm， 双侧光照， 每侧光强为 15kk。 光诱导培养 27h后， 细胞干重从 2.55g/L 降低到 1.82g/L， 叶黄素从 1.57mg/gDcw上升至 3.64mg/gDcw， 光诱导 27h时叶黄素的产 率为 5.88mg/L/d; 若光诱导 12h时叶黄素的产率可达 12.1mg/L/d (为目前微藻光自养生产 叶黄素最高产率 4.8mg/L/d 的 2.5倍) (见图 10)。  The high-density algal solution after the heterotrophic culture was diluted to 2.55 g/L, and the following light-inducing medium was added, and transferred to a 3 L flat photobioreactor for light-induced culture. Light-induced culture conditions: The temperature was maintained at 28-33 °C, the air flow was lvvm, and the illumination was bilateral, with a light intensity of 15 kk per side. After 27 h of light-induced culture, the dry weight of the cells decreased from 2.55 g/L to 1.82 g/L, lutein increased from 1.57 mg/g Dcw to 3.64 mg/g Dcw, and the yield of lutein was 5.88 mg at 27 h after light induction. L/d; If the light induces 12h, the yield of lutein can reach 12.1mg/L/d (for the current microalgae photoautotrophic production of lutein, the highest yield of 4.8mg/L/d is 2.5 times) (see Figure 10).
异养及补料培养基为：葡萄糖 60.0克，尿素 8.0克， MgS(V7H20 2.0克， KH2P04 1.1 克， Na2HP04- 12H20 9.0克， CaCl2 0.02克， 柠檬酸三钠 1.8克， Fe-EDTA溶液 1.0ml, 微量元素溶液 4.5ml， 水 1000ml; 其中 Fe-EDTA溶液配方为 FeS(V7H20 15 克 /升和 EDTA1.4 克 /升， 微量元素溶液配方为 H3B03 2.11 克 /升， MnCl24H20 0.81 克 /升， ZnS04-7H20 0.11克 /升， CuS04-5H20 10.0克 /升， Na2Mo04 0.05克 /升。 The heterotrophic and feeding medium was: glucose 60.0 g, urea 8.0 g, MgS (V7H 2 0 2.0 g, KH 2 P0 4 1.1 g, Na 2 HP0 4 - 12H 2 0 9.0 g, CaCl 2 0.02 g, citric acid 1.8 g of trisodium, 1.0 ml of Fe-EDTA solution, 4.5 ml of trace element solution, 1000 ml of water; the formulation of Fe-EDTA solution is FeS (V7H 2 0 15 g / liter and EDTA 1.4 g / liter, the formulation of trace element solution is H 3 B0 3 2.11 g / liter, MnCl 2 4H 2 0 0.81 g / liter, ZnS0 4 -7H 2 0 0.11 g / liter, CuS0 4 -5H 2 0 10.0 g / liter, Na 2 Mo0 4 0.05 g / liter.
光诱导培养基为：尿素 0.5克， MgS04-7H20 1.0克，柠檬酸三钠 0.05克， CaCl2 0.01 克， Fe-EDTA溶液 0.4ml， 水 1000ml; 其中 Fe-EDTA溶液配方为 FeS04'7H20 15克 /升 禾口 EDTA1.4克 /升。 实施例 5 The light-inducing medium is: urea 0.5 g, MgS0 4 -7H 2 0 1.0 g, trisodium citrate 0.05 g, CaCl 2 0.01 g, Fe-EDTA solution 0.4 ml, water 1000 ml; wherein the Fe-EDTA solution is FeS0 4 '7H 2 0 15 g / liter and EDTA 1.4 g / liter. Example 5
在 50L生物反应器中加入下述异养培养基和自来水至 25L后在 121 °C灭菌 20min， 然后当温度降至 30°C左右时按工作体积的 10%接入蛋白核小球藻， 开始异养培养。  Add the following heterotrophic medium and tap water to 25L in a 50L bioreactor and sterilize at 121 °C for 20 min. Then, when the temperature drops to about 30 °C, access the chlorella chlorella at 10% of the working volume. Start heterotrophic culture.
异养培养条件：温度为 30°C， 空气流量为 lvvm， pH在 6.0 8.0，控制溶氧 15%以上。 在培养过程中， 当葡萄糖消耗完后， 进行补加碳源， 当尿素消耗完时， 补加氮源。 通过 6 次补加碳源， 3次补加氮源后在 98.89h藻细胞密度达 130.5g/L (见图 11 ) 。  Heterotrophic culture conditions: temperature is 30 ° C, air flow is lvvm, pH is 6.0 8.0, and dissolved oxygen is controlled by more than 15%. During the cultivation process, when the glucose is consumed, a supplementary carbon source is added, and when the urea is consumed, a nitrogen source is added. After 6 times of supplemental carbon source, the density of algae cells reached 130.5g/L at 98.89h after supplementing the nitrogen source three times (see Figure 11).
将异养培养结束后的高密度藻稀释到 2.01g/L， 并加入光诱导培养基， 转入到 10L圆 柱光生物反应器内进行光诱导。  The high-density algae after the heterotrophic culture was diluted to 2.01 g/L, and the light-inducing medium was added, and transferred to a 10 L cylindrical photobioreactor for light induction.
光诱导培养条件： 自然温度， 温度在 30°C， 光强在 3kk， 空气流量为 lvvm。 光诱 导培养 30h后， 细胞干重从 2.01g/L 降低到 1.66g/L， 叶黄素从 2.55mg/gDcw 上升至 3.47mg/gDcw, 光诱导 30h， 叶黄素产率为 4.61mg/L/d; 若只光诱导 24h， 叶黄素的产率 达 5.73mg/L/d (见图 12)。 培养基与实施例 4的培养基一致。 实施例 6 在 50L生物反应器中加入下述异养培养基和自来水至 25L后在 121 °C灭菌 20min， 然后当温度降至 30°C左右时按工作体积的 13%接入普通小球藻， 开始异养培养。 Light-induced culture conditions: natural temperature, temperature at 30 ° C, light intensity at 3 kk, air flow at lvvm. After 30 h of light-induced culture, the dry weight of the cells decreased from 2.01 g/L to 1.66 g/L, lutein increased from 2.55 mg/g Dcw to 3.47 mg/g Dcw, photoinduced for 30 h, and the lutein yield was 4.61 mg/L. /d; The yield of lutein reached 5.73 mg/L/d if only light was induced for 24 h (see Figure 12). The medium was identical to the medium of Example 4. Example 6 Add the following heterotrophic medium and tap water to the 25L bioreactor to 25L and sterilize at 121 °C for 20min, then connect to the common chlorella at 13% of the working volume when the temperature drops to about 30 °C. Heterotrophic culture.
异养培养条件： 温度为 30°C， 空气流量为 lvvm， pH小于 9.0。 在培养过程中， 当碳 源消耗完后， 进行补加葡萄糖， 当氮源消耗完时， 补加硝酸钾。 通过 5次补加碳源和氮 源， 58.20h藻细胞密度最高达 54.5g/L (见图 13 ) 。  Heterotrophic culture conditions: temperature is 30 ° C, air flow is lvvm, pH is less than 9.0. During the cultivation process, when the carbon source is consumed, glucose is added, and when the nitrogen source is consumed, potassium nitrate is added. By adding carbon and nitrogen sources five times, the cell density of 58.20h algae is up to 54.5g/L (see Figure 13).
将异养培养的高密度藻稀释到 3.2g/L左右， 并加入光诱导培养基， 转入到 30L平板 式光生物反应器在户外进行光诱导培养。 光诱导培养条件： 自然温度， 自然光照， 空气 流量为 1.0wm。 光诱导 28h， 叶黄素从初始的 1.10mg/gDcw上升至 1.82mg/gDcw; 光诱 导 23h时， 叶黄素含量为 1.67mg/gDcw， 叶黄素产率达 5.23mg/L/dC见图 15)。  The heterotrophic cultured high-density algae was diluted to about 3.2 g/L, and light-inducing medium was added, and transferred to a 30-liter flat photobioreactor for outdoor light-induced culture. Light-induced culture conditions: natural temperature, natural light, and air flow rate of 1.0 wm. After light induction for 28h, lutein increased from the initial 1.10mg/gDcw to 1.82mg/gDcw; when photoinduced for 23h, the lutein content was 1.67mg/gDcw, and the lutein yield reached 5.23mg/L/dC. 15).
异养和补料培养基为：葡萄糖 60.0克，硝酸钾 10.0克， MgS(V7H20 0.2克， KH2P04 0.3克， Na2HP(V 12H20 8.8克， CaC12 0.02克， Fe-EDTA溶液 1.0ml,微量元素溶液 3.5ml， 水 1000ml; 其中 Fe-EDTA溶液配方为 FeS(V7H20 15克 /升和 EDTA1.4克 /升，微量元素 溶液配方为 H3B03 2.86 克 /升、 MnCl2-4H20 0.11 克 /升、 ZnS04-7H20 9.22 克 /升、 CuS04-5H20 1.00克 /升、 （ Η4)6Μο7024·4Η20 0.1克 /升和 Co(N03)2'6H20 0.9克 /升。 The heterotrophic and feeding medium was: glucose 60.0 g, potassium nitrate 10.0 g, MgS (V7H 2 0 0.2 g, KH 2 P0 4 0.3 g, Na 2 HP (V 12H 2 0 8.8 g, CaC12 0.02 g, Fe-) 1.0ml of EDTA solution, 3.5ml of trace element solution, 1000ml of water; The formula of Fe-EDTA solution is FeS (V7H 2 15 15g / liter and EDTA 1.4g / liter, the formula of trace element solution is H 3 B0 3 2.86 g / Liter, MnCl 2 -4H 2 0 0.11 g / liter, ZnS0 4 -7H 2 0 9.22 g / liter, CuS0 4 -5H 2 0 1.00 g / liter, ( Η 4 ) 6 Μο 7 0 24 · 4 Η 2 0 0.1 g /L and Co(N0 3 ) 2 '6H 2 0 0.9 g / liter.
光诱导培养基为： 硝酸钾 0.5克， MgS(V7H20 0.6克， CaCl2 0.03克， Fe-EDTA溶 液 1.5ml， 水 1000ml; 其中 Fe-EDTA溶液配方为 FeS04'7H20 8克 /升和 EDTA 10.4克 / 升。 实施例 7 The light-inducing medium was: potassium nitrate 0.5 g, MgS (V7H 2 0.6 g, CaCl 2 0.03 g, Fe-EDTA solution 1.5 ml, water 1000 ml; wherein the Fe-EDTA solution formulation was FeS0 4 '7H 2 0 8 g/ l and EDTA 10.4 g / l. Example 7
在 5L生物反应器中加入下述异养培养基和水至 2.8L后进行蒸汽灭菌， 然后当温度 降到 30°C时按工作体积的 8%接入椭圆小球藻， 开始异养培养。 异养培养条件： 温度为 30士 1 °C， 空气流量为 lvvm， pH小于 8.5， 控制溶氧 5%以上。 补料两次后在 66h， 藻细 胞密度为 53.0g/L (见图 14) ， 此时异养培养结束转入光诱导培养。  Add the following heterotrophic medium and water to a 2.8L in a 5L bioreactor, then steam sterilize, then connect ellipsoids to 8% of the working volume when the temperature drops to 30 °C, and start heterotrophic culture. . Heterotrophic culture conditions: temperature is 30 ± 1 °C, air flow is lvvm, pH is less than 8.5, and dissolved oxygen is controlled at 5% or more. At 66 h after feeding twice, the algal cell density was 53.0 g/L (see Figure 14), at which point the heterotrophic culture was transferred to light-induced culture.
将异养培养结束后的高密度藻液稀释到 4.0g/L， 并加入下述光诱导培养基， 转入到 3L平板式光生物反应器中进行光诱导培养。 光诱导培养条件： 温度维持在 28~33 °C， 空 气流量为 l .Ovvm, 双侧光照， 每侧光强为 15klx。 光诱导培养 48h后， 细胞干重从 4.0g/L 降低到 2.8g/L， 叶黄素从 1.5mg/gDcw上升至 3.6mg/gDcw， 叶黄素产率为 5.04mg/L/d; 光诱导 24h， 叶黄素产率可达 9.24mg/L/d (见图 16) 。 培养基与实施例 6的培养基一致。 尽管上面已经描述了本发明的具体例子， 但是有一点对于本领域技术人员来说是明 显的， 即在不脱离本发明的精神和范围的前提下可对本发明作各种变化和改动。 因此， 所附权利要求覆盖了所有这些在本发明范围内的变动。  The high-density algal solution after the heterotrophic culture was diluted to 4.0 g/L, and the following light-inducing medium was added, and transferred to a 3 L flat photobioreactor for light-induced culture. Light-induced culture conditions: the temperature was maintained at 28~33 °C, the air flow rate was l.Ovvm, and the bilateral light intensity was 15klx per side. After 48 h of light-induced culture, the dry weight of the cells decreased from 4.0 g/L to 2.8 g/L, lutein increased from 1.5 mg/g Dcw to 3.6 mg/g Dcw, and the lutein yield was 5.04 mg/L/d. After 24 h induction, the lutein yield reached 9.24 mg/L/d (see Figure 16). The medium was identical to the medium of Example 6. While the invention has been described with respect to the specific embodiments of the present invention, it will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Therefore, the appended claims are intended to cover all such modifications within the scope of the invention.

Claims

权 利 要 书 Essentials
1 . 一种微藻培养方法， 其特征在于， 该方法包括微藻异养培养的步骤， 和将异养培 养所获得的微藻培养液稀释后进行光诱导培养的步骤。 A method for cultivating a microalgae, comprising the steps of heterotrophic culture of microalgae, and the step of diluting the microalgae culture liquid obtained by heterotrophic culture to perform light-induced culture.
2. 一种快速积累微藻胞内油脂和 /或叶黄素和 /或其他胞内物质 （如蛋白质、 叶绿素 等）的方法， 其特征在于， 该方法包括微藻的异养培养步骤， 将异养培养的微藻藻液稀释 后进行光诱导培养的步骤， 以及任选的藻细胞采收、 油脂和 /或叶黄素和 /或其他胞内物质 分离提取的步骤。  2. A method for rapidly accumulating microalgae intracellular lipids and/or lutein and/or other intracellular substances (such as proteins, chlorophyll, etc.), characterized in that the method comprises a heterotrophic culture step of microalgae, which will be heterotrophic The step of light-inducing culture after dilution of the cultured microalgal algae solution, and optionally the step of extracting and extracting algae cells, oil and/or lutein and/or other intracellular substances.
3 . 如权利要求 1或 2所述方法， 其特征在于， 所述的微藻选自：  3. The method according to claim 1 or 2, wherein the microalgae is selected from the group consisting of:
绿藻门小球藻属中的蛋白核小球藻 CMorella pyrenoidosa ， 普通小球藻 i Chlorella vulgaris)， #||l| ^J^¾c¾ i Chlorella ellipsoidea)， Chlorella emersonii, Chlorella sorokiniana, Chlorella saccharophila, Chlorella regular is, Chlorella minutissima, Chlorella protothecoides, Chlorella zofingiensis， 以及绿藻门中的 Brachiomonas submarina， Chlamydobonas reinhardtii， Chlamydomonas acidophila, Haematococcus pluvialis , Haematococcus lacustris, Scenedesmus obliquus , Spongiococcum exetriccium， Tetraselmis suecica, Tetraselmis chuii， Tetraselmis tetrathele, Tetraselmis verrucosa, Micractinium pusillum;  CMorella pyrenoidosa, Chlorella vulgaris, Chlorella ellipsoidea, Chlorella emersonii, Chlorella sorokiniana, Chlorella saccharophila, Chlorella Regular is, Chlorella minutissima, Chlorella protothecoides, Chlorella zofingiensis, and Brachiomonas submarina, Chlamydobonas reinhardtii, Chlamydomonas acidophila, Haematococcus pluvialis, Haematococcus lacustris, Scenedesmus obliquus, Spongiococcum exetriccium, Tetraselmis suecica, Tetraselmis chuii, Tetraselmis tetrathele, Tetraselmis verrucosa , Micractinium pusillum;
藻门的 Cylindrotheca fusiformis, Nitzschia laevis , Nitzschia alba, Nitzschia fonticola, Navicula incerta, Navicula pelliculosa;  Cylindrotheca fusiformis, Nitzschia laevis, Nitzschia alba, Nitzschia fonticola, Navicula incerta, Navicula pelliculosa;
蓝藻门的 Anabaena variabilis;  Anabaena variabilis
金藻门的 Poterioochromonas malhamemis  Poterioochromonas malhamemis
甲藻门的 Amphidinium carter ae, Crypthecodinium cohnii;  Amphidinium carter ae, Crypthecodinium cohnii;
裸藻门的 Euglena grid lis; 禾口  Euglena grid lis;
红藻门的 Galdieria sulphuraria。  Galdieria sulphuraria of the red algae gate.
4. 如权利要求 1一 3中任一项所述的方法， 其特征在于， 所述异养微藻的步骤包括 在生物反应器中进行异养培养： 在生物反应器中加入 pH为 4.0 10.0的培养基， 按工作体 积的 0.1~30%接入微藻藻种进行分批培养、补料分批培养、 半连续培养或连续培养， 培养 温度为 10~50°C， 控制 pH小于 10.0， 控制溶氧在 1 %以上。  The method according to any one of claims 1 to 3, wherein the step of heterotrophic microalgae comprises heterotrophic culture in a bioreactor: adding a pH of 4.0 10.0 to the bioreactor The culture medium is connected to the microalgae algae according to the working volume of 0.1~30% for batch culture, fed-batch batch culture, semi-continuous culture or continuous culture. The culture temperature is 10~50 °C, and the control pH is less than 10.0. Control dissolved oxygen above 1%.
5. 如权利要求 1一 4中任一项所述的方法， 其特征在于， 所述藻液稀释包括用培养 基将异养培养获得的藻液稀释至细胞密度为 0.1 50克 /升， 所述培养基不含有机碳源， 其 pH为 4.0 10.0。  The method according to any one of claims 1 to 4, wherein the diluting the algae liquid comprises diluting the algal liquid obtained by the heterotrophic culture with a medium to a cell density of 0.150 g/liter. The medium does not contain an organic carbon source and has a pH of 4.0 10.0.
6. 如权利要求 1一 5中任一项所述的方法， 其特征在于， 所述光诱导培养包括将稀 释后的藻液转入光诱导装置中进行光诱导， 培养温度为 5〜50°C， 连续光照或间歇光照， 光照强度为 0.1〜150kk， 光诱导培养周期为 1〜150小时。 The method according to any one of claims 1 to 5, wherein the photoinduced culture comprises transferring the diluted algal liquid into a light-inducing device for light induction, and the culture temperature is 5 to 50°. C, continuous lighting or intermittent lighting, The light intensity is 0.1 to 150 kk, and the light-induced culture period is 1 to 150 hours.
7. 如权利要求 1一 6中任一项所述的方法， 其特征在于， 异养培养基由氮源、 有机 碳源以及少量无机盐、 微量元素和水组成； 光诱导培养基由氮源、 无机盐和水组成。  The method according to any one of claims 1 to 6, wherein the heterotrophic medium is composed of a nitrogen source, an organic carbon source, and a small amount of inorganic salts, trace elements and water; the light-inducing medium is composed of a nitrogen source. , inorganic salt and water.
8. 如权利要求 1一 7中任一项所述的方法， 其特征在于， 所述异养步骤在摇瓶、 机 械搅拌式、气升式或鼓泡式可异养培养的生物反应器中进行，所述光诱导培养步骤在摇瓶 或选自敞开式的跑道池或圆池、封闭式的平板式光生物反应器或管道式光生物反应器或柱 式光生物反应器或薄膜立袋与吊袋用于微藻光自养培养的装置中进行，光源为自然光或各 种人工光。  The method according to any one of claims 1 to 7, wherein the heterotrophic step is in a shake flask, mechanical agitation, airlift or bubbling heterotrophic culture bioreactor Performing, the light-inducing culture step is carried out in a shake flask or selected from an open runway or round pool, a closed flat photobioreactor or a tubular photobioreactor or a column photobioreactor or a film stand pouch The bag is used in a device for self-cultivation of microalgae light, and the light source is natural light or various artificial light.
9. 如权利要求 1一 8中任一项所述的方法， 其特征在于， 当小球藻为普通小球藻时， 异养所使用的培养基基本上由以下成分组成： KN03 5-15 克 /升、 葡萄糖 10 60 克 /升、 KH2P04 0.3-0.9克 /升、 Na2HP04'12H20 1.0-10.0克 /升、 MgS04'7H20 0.2-1.0克 /升、 CaCl2 0.05~0.3克 /升、 FeS(V7H20 0.01 0.05克 /升； 微量元素 0.5~4ml， 其中微量元素的组成为 H3B03 5-15 克 /升， ZnS04'7H20 5.0-10.0 克 /升， MnCl2'H20 1.0-2.0 克 /升， ( Η4)6Μο7024·4Η20 0.5~1.5克 /升， CuS04'5H20 1.0~2.0克 /升， Co(N03)2-6H20 0.1~0.9克 / 升； 水。 The method according to any one of claims 1 to 8, wherein when the chlorella is a common chlorella, the medium used for heterotrophy consists essentially of the following components: KN0 3 5- 15 g / liter, glucose 10 60 g / liter, KH 2 P0 4 0.3-0.9 g / liter, Na 2 HP0 4 '12H 2 0 1.0-10.0 g / liter, MgS0 4 '7H 2 0 0.2-1.0 g / liter , CaCl 2 0.05~0.3 g / liter, FeS (V7H 2 0 0.01 0.05 g / liter; trace element 0.5~4ml, wherein the composition of trace elements is H 3 B0 3 5-15 g / liter, ZnS0 4 '7H 2 0 5.0-10.0 g/L, MnCl 2 'H 2 0 1.0-2.0 g/L, ( Η 4 ) 6 Μο 7 0 24 ·4Η 2 0 0.5~1.5 g/L, CuS0 4 '5H 2 0 1.0~2.0 g /L, Co(N0 3 ) 2 -6H 2 0 0.1~0.9 g / l; water.
10. 如权利要求 1一 8中任一项所述的方法， 其特征在于， 当小球藻为蛋白核小球藻 时， 异养所使用的培养基基本上由以下成分组成： 葡萄糖 10 60克 /升， 尿素 2~8克 /升， KH2P04 1-2克 /升， Na2HP04'12H20 1.0-10.0克 /升， MgS04'7H20 1-2克 /升， CaCl2 0.05-0.1 克 /升，柠檬酸三钠 0.1~2.0克 /升， Fe-EDTA溶液 0.5~1 mL， A5溶液 l~5mL;其中 Fe-EDTA 溶液配方为 FeS04'7H20 20-30克 /升和 EDTA 20-40克 /升； A5溶液配方为 H3B03 2.5-4.0 克 /升， MnCl2'4H20 1.0~2.0克 /升， ZnS04'7H20 0. 〜 0.6克 /升， CuS04'5H20 5~10克 /升， Na2MoO4 0.01~0.05克 /升; 水。 The method according to any one of claims 1 to 8, wherein when the chlorella is Chlorella pyrenoidosa, the medium used for heterotrophy consists essentially of the following components: Glucose 10 60 Gram / liter, urea 2 ~ 8 g / liter, KH 2 P0 4 1-2 g / liter, Na 2 HP0 4 '12H 2 0 1.0-10.0 g / liter, MgS0 4 '7H 2 0 1-2 g / liter , CaCl 2 0.05-0.1 g / l, trisodium citrate 0.1 ~ 2.0 g / l, Fe-EDTA solution 0.5 ~ 1 mL, A5 solution l ~ 5mL; Fe-EDTA solution formula is FeS0 4 '7H 2 0 20 -30 g / liter and EDTA 20-40 g / liter; A5 solution formula is H 3 B0 3 2.5-4.0 g / liter, MnCl 2 '4H 2 0 1.0 ~ 2.0 g / liter, ZnS0 4 '7H 2 0 0. ~ 0.6 g / liter, CuS0 4 '5H 2 0 5 ~ 10 g / liter, Na 2 MoO 4 0.01 ~ 0.05 g / liter; water.
11. 一种油脂和 /或叶黄素和 /或其他胞内物质（如蛋白质、 叶绿素等）生产方法， 其 特征在于，所述方法包括异养培养微藻的步骤，将异养培养的微藻藻液稀释后进行光诱导 培养的步骤， 以及藻细胞采收、 油脂和 /或叶黄素和 /或其他胞内物质分离提取的步骤。  A method for producing oil and/or lutein and/or other intracellular substances (such as protein, chlorophyll, etc.), characterized in that the method comprises the step of heterotrophic cultivation of microalgae, and the heterotrophic culture of microalgae The step of performing light-induced culture after liquid dilution, and the step of separating and extracting algae cells, oil and/or lutein and/or other intracellular substances.
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